Archive for the ‘Evidence-based decision-making’ Category

Reporter and Curator: Dr. Sudipta Saha, Ph.D.

Researchers have classified a brand-new organ inside human body. Known as the mesentery, the new organ is found in our digestive systems, and was long thought to be made up of fragmented, separate structures. But recent research has shown that it’s actually one, continuous organ. The evidence for the organ’s reclassification is now published in The Lancet Gastroenterology & Hepatology. Although we now know about the structure of this new organ, its function is still poorly understood, and studying it could be the key to better understanding and treatment of abdominal and digestive disease.


J Calvin Coffey, a researcher from the University Hospital Limerick in Ireland, who first discovered that the mesentery was an organ. In 2012, Coffey and his colleagues showed through detailed microscopic examinations that the mesentery is actually a continuous structure. Over the past four years, they’ve gathered further evidence that the mesentery should actually be classified as its own distinct organ, and the latest paper makes it official. Mesentery is a double fold of peritoneum – the lining of the abdominal cavity – that holds our intestine to the wall of our abdomen. It was described by the Italian polymath Leanardo da Vinci in 1508, but it has been ignored throughout the centuries, until now. Although there are generally considered to be five organs in the human body, there are in fact now 79, including the mesentery. The heart, brain, liver, lungs and kidneys are the vital organs, but there are another 74 that play a role in keeping us healthy. The distinctive anatomical and functional features of mesentery have been revealed that justify designation of the mesentery as an organ. Accordingly, the mesentery should be subjected to the same investigatory focus that is applied to other organs and systems. This provides a platform from which to direct future scientific investigation of the human mesentery in health and disease.








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From the NIH Website

New NIH breast cancer research to focus on prevention

A new phase of the Breast Cancer and the Environment Research Program (BCERP), focused on prevention, is being launched at the National Institutes of Health. Grant-funded researchers will now work across scientific disciplines, involve new racially and ethnically diverse communities, and expand the study of risk factors that precede breast cancer, such as breast density.

These new directions reflect recommendations made by the Interagency Breast Cancer and Environmental Research Coordinating Committee (IBCERCC) in 2013. IBCERCC was congressionally mandated to review the state of the science around breast cancer and environmental influences by the Breast Cancer and Environmental Research Act. Recommendations included prioritizing prevention, involving transdisciplinary research teams, engaging public stakeholders, collaborating across federal agencies, and communicating the science to the public.

This broadened research focus will add to the growing knowledge of environmental and genetic factors that may influence breast cancer risk across the lifespan. The six new BCERP projects, plus a new coordinating center promoting cross-project collaboration, are jointly funded by the National Institute of Environmental Health Sciences (NIEHS) and the National Cancer Institute. All projects involve strong partnerships between researchers and organizations focused on breast cancer prevention or environmental health.

The new research will be conducted at the following institutions

  • Brigham and Women’s Hospital, Boston
  • City of Hope/Beckman Research Institute, Duarte, California
  • Columbia University, New York City
  • Georgetown Lombardi Comprehensive Cancer Center, Washington, D.C.
  • Michigan State University, Lansing
  • University of Massachusetts, Amherst
  • University of Wisconsin – Madison (Coordinating Center)

“The beauty of this research is that scientific discoveries and community observations inform each other, in order to dive deeper into the complex causes of breast cancer,” said Gwen Collman, Ph.D., director of NIEHS Division of Extramural Research and Training.

The focus on minority and socio-economically disadvantaged women is an important step in addressing disparities in breast cancer outcomes. Although African-American women are diagnosed with breast cancer less often than white women, more aggressive cancers and breast cancer deaths are more common among African-American women.

Another new direction for BCERP is research on the role of breast density as a possible intermediate risk factor for breast cancer. Dense breast tissue is one of the most common risk factors for breast cancer. Identifying links between environmental exposures and high breast density may provide new insights into prevention.

“These priorities reflect our continued commitment to breast cancer prevention,” noted Caroline Dilworth, Ph.D., BCERP program lead at NIEHS. “Our goal is to build on the high quality science we’ve been funding for more than a decade, while also being responsive to the expert recommendations of the IBCERCC report.”

Grant Numbers: U01ES026130, U01ES026137, U01ES026122, U01ES026132, U01ES026119, U01ES026140, U01ES026127

NIEHS supports research to understand the effects of the environment on human health and is part of NIH. For more information on environmental health topics, visit www.niehs.nih.gov. Subscribe to one or more of the NIEHS news lists to stay current on NIEHS news, press releases, grant opportunities, training, events, and publications.

The National Cancer Institute leads the National Cancer Program and the NIH’s efforts to dramatically reduce the prevalence of cancer and improve the lives of cancer patients and their families, through research into prevention and cancer biology, the development of new interventions, and the training and mentoring of new researchers. For more information about cancer, please visit the NCI website at http://www.cancer.gov or call NCI’s Cancer Information Service at 1-800-4-CANCER.

About the National Institutes of Health (NIH): NIH, the nation’s medical research agency, includes 27 Institutes and Centers and is a component of the U.S. Department of Health and Human Services. NIH is the primary federal agency conducting and supporting basic, clinical, and translational medical research, and is investigating the causes, treatments, and cures for both common and rare diseases. For more information about NIH and its programs, visit www.nih.gov.

Other posts on this site on  Cancer and Early Detection  include

Early Detection of Prostate Cancer: American Urological Association (AUA) Guideline

Mechanism involved in Breast Cancer Cell Growth: Function in Early Detection & Treatment

Warning signs may lead to better early detection of ovarian cancer

‘Mosaicism’ is Associated with Aging and Chronic Diseases like Cancer: detection of genetic mosaicism could be an early marker for detecting cancer.

CDC Findings: Due to Aging Population, Actual Number of Cancer Deaths is Rising while Risk of Dying From Cancer is Falling in the US

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Treatments other than Chemotherapy for Leukemias and Lymphomas

Author, Curator, Editor: Larry H. Bernstein, MD, FCAP

2.5.1 Radiation Therapy 


Radiation therapy, also called radiotherapy or irradiation, can be used to treat leukemia, lymphoma, myeloma and myelodysplastic syndromes. The type of radiation used for radiotherapy (ionizing radiation) is the same that’s used for diagnostic x-rays. Radiotherapy, however, is given in higher doses.

Radiotherapy works by damaging the genetic material (DNA) within cells, which prevents them from growing and reproducing. Although the radiotherapy is directed at cancer cells, it can also damage nearby healthy cells. However, current methods of radiotherapy have been improved upon, minimizing “scatter” to nearby tissues. Therefore its benefit (destroying the cancer cells) outweighs its risk (harming healthy cells).

When radiotherapy is used for blood cancer treatment, it’s usually part of a treatment plan that includes drug therapy. Radiotherapy can also be used to relieve pain or discomfort caused by an enlarged liver, lymph node(s) or spleen.

Radiotherapy, either alone or with chemotherapy, is sometimes given as conditioning treatment to prepare a patient for a blood or marrow stem cell transplant. The most common types used to treat blood cancer are external beam radiation (see below) and radioimmunotherapy.
External Beam Radiation

External beam radiation is the type of radiotherapy used most often for people with blood cancers. A focused radiation beam is delivered outside the body by a machine called a linear accelerator, or linac for short. The linear accelerator moves around the body to deliver radiation from various angles. Linear accelerators make it possible to decrease or avoid skin reactions and deliver targeted radiation to lessen “scatter” of radiation to nearby tissues.

The dose (total amount) of radiation used during treatment depends on various factors regarding the patient, disease and reason for treatment, and is established by a radiation oncologist. You may receive radiotherapy during a series of visits, spread over several weeks (from two to 10 weeks, on average). This approach, called dose fractionation, lessens side effects. External beam radiation does not make you radioactive.

2.5.2  Bone marrow (BM) transplantation


There are three kinds of bone marrow transplants:

Autologous bone marrow transplant: The term auto means self. Stem cells are removed from you before you receive high-dose chemotherapy or radiation treatment. The stem cells are stored in a freezer (cryopreservation). After high-dose chemotherapy or radiation treatments, your stems cells are put back in your body to make (regenerate) normal blood cells. This is called a rescue transplant.

Allogeneic bone marrow transplant: The term allo means other. Stem cells are removed from another person, called a donor. Most times, the donor’s genes must at least partly match your genes. Special blood tests are done to see if a donor is a good match for you. A brother or sister is most likely to be a good match. Sometimes parents, children, and other relatives are good matches. Donors who are not related to you may be found through national bone marrow registries.

Umbilical cord blood transplant: This is a type of allogeneic transplant. Stem cells are removed from a newborn baby’s umbilical cord right after birth. The stem cells are frozen and stored until they are needed for a transplant. Umbilical cord blood cells are very immature so there is less of a need for matching. But blood counts take much longer to recover.

Before the transplant, chemotherapy, radiation, or both may be given. This may be done in two ways:

Ablative (myeloablative) treatment: High-dose chemotherapy, radiation, or both are given to kill any cancer cells. This also kills all healthy bone marrow that remains, and allows new stem cells to grow in the bone marrow.

Reduced intensity treatment, also called a mini transplant: Patients receive lower doses of chemotherapy and radiation before a transplant. This allows older patients, and those with other health problems to have a transplant.

A stem cell transplant is usually done after chemotherapy and radiation is complete. The stem cells are delivered into your bloodstream usually through a tube called a central venous catheter. The process is similar to getting a blood transfusion. The stem cells travel through the blood into the bone marrow. Most times, no surgery is needed.

Donor stem cells can be collected in two ways:

  • Bone marrow harvest. This minor surgery is done under general anesthesia. This means the donor will be asleep and pain-free during the procedure. The bone marrow is removed from the back of both hip bones. The amount of marrow removed depends on the weight of the person who is receiving it.
  • Leukapheresis. First, the donor is given 5 days of shots to help stem cells move from the bone marrow into the blood. During leukapheresis, blood is removed from the donor through an IV line in a vein. The part of white blood cells that contains stem cells is then separated in a machine and removed to be later given to the recipient. The red blood cells are returned to the donor.

Why the Procedure is Performed

A bone marrow transplant replaces bone marrow that either is not working properly or has been destroyed (ablated) by chemotherapy or radiation. Doctors believe that for many cancers, the donor’s white blood cells can attach to any remaining cancer cells, similar to when white cells attach to bacteria or viruses when fighting an infection.

Your doctor may recommend a bone marrow transplant if you have:

Certain cancers, such as leukemia, lymphoma, and multiple myeloma

A disease that affects the production of bone marrow cells, such as aplastic anemia, congenital neutropenia, severe immunodeficiency syndromes, sickle cell anemia, thalassemia

Had chemotherapy that destroyed your bone

2.5.3 Autologous stem cell transplantation

Phase II trial of 131I-B1 (anti-CD20) antibody therapy with autologous stem cell transplantation for relapsed B cell lymphomas

O.W Press,  F Appelbaum,  P.J Martin, et al.

25 patients with relapsed B-cell lymphomas were evaluated with trace-labelled doses (2·5 mg/kg, 185-370 MBq [5-10 mCi]) of 131I-labelled anti-CD20 (B1) antibody in a phase II trial. 22 patients achieved 131I-B1 biodistributions delivering higher doses of radiation to tumor sites than to normal organs and 21 of these were treated with therapeutic infusions of 131I-B1 (12·765-29·045 GBq) followed by autologous hemopoietic stem cell reinfusion. 18 of the 21 treated patients had objective responses, including 16 complete remissions. One patient died of progressive lymphoma and one died of sepsis. Analysis of our phase I and II trials with 131I-labelled B1 reveal a progression-free survival of 62% and an overall survival of 93% with a median follow-up of 2 years. 131I-anti-CD20 (B1) antibody therapy produces complete responses of long duration in most patients with relapsed B-cell lymphomas when given at maximally tolerated doses with autologous stem cell rescue.

Autologous (Self) Transplants


An autologous transplant (or rescue) is a type of transplant that uses the person’s own stem cells. These cells are collected in advance and returned at a later stage. They are used to replace stem cells that have been damaged by high doses of chemotherapy, used to treat the person’s underlying disease.

In most cases, stem cells are collected directly from the bloodstream. While stem cells normally live in your marrow, a combination of chemotherapy and a growth factor (a drug that stimulates stem cells) called Granulocyte Colony Stimulating Factor (G-CSF) is used to expand the number of stem cells in the marrow and cause them to spill out into the circulating blood. From here they can be collected from a vein by passing the blood through a special machine called a cell separator, in a process similar to dialysis.

Most of the side effects of an autologous transplant are caused by the conditioning therapy used. Although they can be very unpleasant at times it is important to remember that most of them are temporary and reversible.

Procedure of Hematopoietic Stem Cell Transplantation

Hematopoietic stem cell transplantation (HSCT) is the transplantation of multipotent hematopoietic stem cells, usually derived from bone marrow, peripheral blood, or umbilical cord blood. It may be autologous (the patient’s own stem cells are used) or allogeneic (the stem cells come from a donor).

Hematopoietic Stem Cell Transplantation

Author: Ajay Perumbeti, MD, FAAP; Chief Editor: Emmanuel C Besa, MD

Hematopoietic stem cell transplantation (HSCT) involves the intravenous (IV) infusion of autologous or allogeneic stem cells to reestablish hematopoietic function in patients whose bone marrow or immune system is damaged or defective.

The image below illustrates an algorithm for typically preferred hematopoietic stem cell transplantation cell source for treatment of malignancy.

An algorithm for typically preferred hematopoietic stem cell transplantation cell source for treatment of malignancy: If a matched sibling donor is not available, then a MUD is selected; if a MUD is not available, then choices include a mismatched unrelated donor, umbilical cord donor(s), and a haploidentical donor.

Supportive Therapies

2.5.4  Blood transfusions – risks and complications of a blood transfusion

  • Allogeneic transfusion reaction (acute or delayed hemolytic reaction)
  • Allergic reaction
  • Viruses Infectious Diseases

The risk of catching a virus from a blood transfusion is very low.

HIV. Your risk of getting HIV from a blood transfusion is lower than your risk of getting killed by lightning. Only about 1 in 2 million donations might carry HIV and transmit HIV if given to a patient.

Hepatitis B and C. The risk of having a donation that carries hepatitis B is about 1 in 205,000. The risk for hepatitis C is 1 in 2 million. If you receive blood during a transfusion that contains hepatitis, you’ll likely develop the virus.

Variant Creutzfeldt-Jakob disease (vCJD). This disease is the human version of Mad Cow Disease. It’s a very rare, yet fatal brain disorder. There is a possible risk of getting vCJD from a blood transfusion, although the risk is very low. Because of this, people who may have been exposed to vCJD aren’t eligible blood donors.

  • Fever
  • Iron Overload
  • Lung Injury
  • Graft-Versus-Host Disease

Graft-versus-host disease (GVHD) is a condition in which white blood cells in the new blood attack your tissues.

2.5.5 Erythropoietin

Erythropoietin, (/ɨˌrɪθrɵˈpɔɪ.ɨtɨn/UK /ɛˌrɪθr.pˈtɪn/) also known as EPO, is a glycoprotein hormone that controls erythropoiesis, or red blood cell production. It is a cytokine (protein signaling molecule) for erythrocyte (red blood cell) precursors in the bone marrow. Human EPO has a molecular weight of 34 kDa.

Also called hematopoietin or hemopoietin, it is produced by interstitial fibroblasts in the kidney in close association with peritubular capillary and proximal convoluted tubule. It is also produced in perisinusoidal cells in the liver. While liver production predominates in the fetal and perinatal period, renal production is predominant during adulthood. In addition to erythropoiesis, erythropoietin also has other known biological functions. For example, it plays an important role in the brain’s response to neuronal injury.[1] EPO is also involved in the wound healing process.[2]

Exogenous erythropoietin is produced by recombinant DNA technology in cell culture. Several different pharmaceutical agents are available with a variety ofglycosylation patterns, and are collectively called erythropoiesis-stimulating agents (ESA). The specific details for labelled use vary between the package inserts, but ESAs have been used in the treatment of anemia in chronic kidney disease, anemia in myelodysplasia, and in anemia from cancer chemotherapy. Boxed warnings include a risk of death, myocardial infarction, stroke, venous thromboembolism, and tumor recurrence.[3]

2.5.6  G-CSF (granulocyte-colony stimulating factor)

Granulocyte-colony stimulating factor (G-CSF or GCSF), also known as colony-stimulating factor 3 (CSF 3), is a glycoprotein that stimulates the bone marrow to produce granulocytes and stem cells and release them into the bloodstream.

There are different types, including

  • Lenograstim (Granocyte)
  • Filgrastim (Neupogen, Zarzio, Nivestim, Ratiograstim)
  • Long acting (pegylated) filgrastim (pegfilgrastim, Neulasta) and lipegfilgrastim (Longquex)

Pegylated G-CSF stays in the body for longer so you have treatment less often than with the other types of G-CSF.

2.5.7  Plasma Exchange (plasmapheresis)


Plasmapheresis is a term used to refer to a broad range of procedures in which extracorporeal separation of blood components results in a filtered plasma product.[1, 2] The filtering of plasma from whole blood can be accomplished via centrifugation or semipermeable membranes.[3] Centrifugation takes advantage of the different specific gravities inherent to various blood products such as red cells, white cells, platelets, and plasma.[4] Membrane plasma separation uses differences in particle size to filter plasma from the cellular components of blood.[3]

Traditionally, in the United States, most plasmapheresis takes place using automated centrifuge-based technology.[5] In certain instances, in particular in patients already undergoing hemodialysis, plasmapheresis can be carried out using semipermeable membranes to filter plasma.[4]

In therapeutic plasma exchange, using an automated centrifuge, filtered plasma is discarded and red blood cells along with replacement colloid such as donor plasma or albumin is returned to the patient. In membrane plasma filtration, secondary membrane plasma fractionation can selectively remove undesired macromolecules, which then allows for return of the processed plasma to the patient instead of donor plasma or albumin. Examples of secondary membrane plasma fractionation include cascade filtration,[6] thermofiltration, cryofiltration,[7] and low-density lipoprotein pheresis.

The Apheresis Applications Committee of the American Society for Apheresis periodically evaluates potential indications for apheresis and categorizes them from I to IV based on the available medical literature. The following are some of the indications, and their categorization, from the society’s 2010 guidelines.[2]

  • The only Category I indication for hemopoietic malignancy is Hyperviscosity in monoclonal gammopathies

2.5.8  Platelet Transfusions

Indications for platelet transfusion in children with acute leukemia

Scott Murphy, Samuel Litwin, Leonard M. Herring, Penelope Koch, et al.
Am J Hematol Jun 1982; 12(4): 347–356

In an attempt to determine the indications for platelet transfusion in thrombocytopenic patients, we randomized 56 children with acute leukemia to one of two regimens of platelet transfusion. The prophylactic group received platelets when the platelet count fell below 20,000 per mm3 irrespective of clinical events. The therapeutic group was transfused only when significant bleeding occurred and not for thrombocytopenia alone. The time to first bleeding episode was significantly longer and the number of bleeding episodes were significantly reduced in the prophylactic group. The survival curves of the two groups could not be distinguished from each other. Prior to the last month of life, the total number of days on which bleeding was present was significantly reduced by prophylactic therapy. However, in the terminal phase (last month of life), the duration of bleeding episodes was significantly longer in the prophylactic group. This may have been due to a higher incidence of immunologic refractoriness to platelet transfusion. Because of this terminal bleeding, comparison of the two groups for total number of days on which bleeding was present did not show a significant difference over the entire study period.

Clinical and Laboratory Aspects of Platelet Transfusion Therapy
Yuan S, Goldfinger D

INTRODUCTION — Hemostasis depends on an adequate number of functional platelets, together with an intact coagulation (clotting factor) system. This topic covers the logistics of platelet use and the indications for platelet transfusion in adults. The approach to the bleeding patient, refractoriness to platelet transfusion, and platelet transfusion in neonates are discussed elsewhere.

Pooled Platelets – A single unit of platelets can be isolated from every unit of donated blood, by centrifuging the blood within the closed collection system to separate the platelets from the red blood cells (RBC). The number of platelets per unit varies according to the platelet count of the donor; a yield of 7 x 1010 platelets is typical [1]. Since this number is inadequate to raise the platelet count in an adult recipient, four to six units are pooled to allow transfusion of 3 to 4 x 1011 platelets per transfusion [2]. These are called whole blood-derived or random donor pooled platelets.

Advantages of pooled platelets include lower cost and ease of collection and processing (a separate donation procedure and pheresis equipment are not required). The major disadvantage is recipient exposure to multiple donors in a single transfusion and logistic issues related to bacterial testing.

Apheresis (single donor) Platelets – Platelets can also be collected from volunteer donors in the blood bank, in a one- to two-hour pheresis procedure. Platelets and some white blood cells are removed, and red blood cells and plasma are returned to the donor. A typical apheresis platelet unit provides the equivalent of six or more units of platelets from whole blood (ie, 3 to 6 x 1011 platelets) [2]. In larger donors with high platelet counts, up to three units can be collected in one session. These are called apheresis or single donor platelets.

Advantages of single donor platelets are exposure of the recipient to a single donor rather than multiple donors, and the ability to match donor and recipient characteristics such as HLA type, cytomegalovirus (CMV) status, and blood type for certain recipients.

Both pooled and apheresis platelets contain some white blood cells (WBC) that were collected along with the platelets. These WBC can cause febrile non-hemolytic transfusion reactions (FNHTR), alloimmunization, and transfusion-associated graft-versus-host disease (ta-GVHD) in some patients.

Platelet products also contain plasma, which can be implicated in adverse reactions including transfusion-related acute lung injury (TRALI) and anaphylaxis. (See ‘Complications of platelet transfusion’ .)

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Classification of Microbiota –

An Overview of Clinical Microbiology, Classification, and Antimicrobial Resistance

Author and Curator: Larry H. Bernstein, MD, FCAP

Classification of Microbiota

Introduction to Overview of Microbiology

This is a contribution to a series of pieces on the history of biochemistry, molecular biology, physiology and medicine in the 20th century.  Here I describe the common microbial organisms encountered in the clinical laboratory, the method of their collection, plating, culture and identification, and antibiotic sensitivity testing and resistant strains.

I may begin with the recognition that there are common strains in the environment that are not pathogenic, and there are pathogenic bacteria.
In addition, there are bacteria that coexist in the body habitat under specific conditions so that we are able to map the types expected to location, such as, skin, mouth and nasal cavities, the colon, the vagina and urinary system.  Meningitides occur as a result of extension from the nasal cavity to the brain.  When bacteria invade the circulation, it is referred to as septicemia, and the bacteria can cause valvular heart damage.

Bacteriology can be traced to origins in the 19th century.  The clinical features of localized infection are classically referred to as redness, heat, a raised lesion (pustule), and exudate (serous or purulent – watery or cellular).  This not only holds for a focal lesion (as skin), but also for pneumonia, urinary infection, and genital. It may be accompanied by cough, or bloody cough and wheezing, or by an unclear urine. In the case of septicemia, there is fever, and there may be seizures or delirium.

Collection and handling of specimens

Specimens are collected by sterile technique by a nurse or physician and sent to a lab as a swab, or as a blood specimen.  In the case of a febrile illness, blood cultures may be obtained from opposite arms, and another an hour later.  This is related to the possible cyclical seeding of bacteria into the circulation.  If the specimen is collected from a site of infection, a swab may be put onto a glass slide for gram staining.  The specimen collected is sent to the laboratory.

We may consider syphilis and tuberculosis special cases that I’ll set aside.  I shall not go into virology either, although I may referred to smallpox, influenza, polio, HIV under epidemic.  The first step in identification is the Gram stain, developed in the 19th century.  Organisms of the skin are Gram positive and appear blue on staining.  They are cocci, or circular, organized in characteristic clusters (staphylococcus, streptococcus) or in pairs (diplococci, eg. Pneumococcus), and if from the intestine (enterococcus).  If they are elongated rods, they might be coliform.  If they stain red, they are Gram negative.  Gram negative rods are coliform, and are enterobacteriaceae. Meningococci are Gram negative cocci.  So we have certain information about these organisms before we plate them for growth.

Laboratory growth characteristics

The specimen is applied to an agar plate with a metal rod applicator, or perhaps onto more than one agar plate.  The agar plate contains a growth media or a growth inhibitor that is more favorable to certain species than to others.  The bacteria are grown at 37o C in an incubator and colonies develop that are white or nonwhite, and they are smooth or wrinkled.  The appearance of the colonies is characteristic for certain strains.  If there is no contamination, all of the colonies look the same.  The next step is to:

  • Gram stain from a colony
  • Transfer samples from the colony to a series of growth media that identify presence or absence of specific nutrient requirements for growth (which is presumed from the prior findings).

In addition, the colony samples are grown on an agar to which is applied antibiotic tabs.  The tabs either allow or repress growth.  It wa some 50 years ago that the infectious disease physician and microbiologist Abraham Braude would culture the bacteria on agar plates that had a gradient of antibiotic to check for concentration that would inhibit growth.

Principles of Diagnosis (Extracts)

By John A. Washington

The clinical presentation of an infectious disease reflects the interaction between the host and the microorganism. This interaction is affected by the host immune status and microbial virulence factors. Signs and symptoms vary according to the site and severity of infection. Diagnosis requires a composite of information, including history, physical examination, radiographic findings, and laboratory data.

Microbiologic Examination

Direct Examination and Techniques: Direct examination of specimens reveals gross pathology. Microscopy may identify microorganisms. Immunofluorescence, immuno-peroxidase staining, and other immunoassays may detect specific microbial antigens. Genetic probes identify genus- or species-specific DNA or RNA sequences.

Culture: Isolation of infectious agents frequently requires specialized media. Nonselective (noninhibitory) media permit the growth of many microorganisms. Selective media contain inhibitory substances that permit the isolation of specific types of microorganisms.

Microbial Identification: Colony and cellular morphology may permit preliminary identification. Growth characteristics under various conditions, utilization of carbohydrates and other substrates, enzymatic activity, immunoassays, and genetic probes are also used.

Serodiagnosis: A high or rising titer of specific IgG antibodies or the presence of specific IgM antibodies may suggest or confirm a diagnosis.

Antimicrobial Susceptibility: Microorganisms, particularly bacteria, are tested in vitro to determine whether they are susceptible to antimicrobial agents.

Diagnostic medical microbiology is the discipline that identifies etiologic agents of disease. The job of the clinical microbiology laboratory is to test specimens from patients for microorganisms that are, or may be, a cause of the illness and to provide information (when appropriate) about the in vitro activity of antimicrobial drugs against the microorganisms identified (Fig. 1).

Laboratory procedures used in confirming a clinical diagnosis of infectious disease with a bacterial etiology


A variety of microscopic, immunologic, and hybridization techniques have been developed for rapid diagnosis

techniques have been developed for rapid diagnosis

techniques have been developed for rapid diagnosis

From: Chapter 10, Principles of Diagnosis
Medical Microbiology. 4th edition.
Baron S, editor.
Galveston (TX): University of Texas Medical Branch at Galveston; 1996.

For immunologic detection of microbial antigens, latex particle agglutination, coagglutination, and enzyme-linked immunosorbent assay (ELISA) are the most frequently used techniques in the clinical laboratory. Antibody to a specific antigen is bound to latex particles or to a heat-killed and treated protein A-rich strain of Staphylococcus aureus to produce agglutination (Fig. 10-2). There are several approaches to ELISA; the one most frequently used for the detection of microbial antigens uses an antigen-specific antibody that is fixed to a solid phase, which may be a latex or metal bead or the inside surface of a well in a plastic tray. Antigen present in the specimen binds to the antibody as inFig. 10-2. The test is then completed by adding a second antigen-specific antibody bound to an enzyme that can react with a substrate to produce a colored product. The initial antigen antibody complex forms in a manner similar to that shown inFigure 10-2. When the enzyme-conjugated antibody is added, it binds to previously unbound antigenic sites, and the antigen is, in effect, sandwiched between the solid phase and the enzyme-conjugated antibody. The reaction is completed by adding the enzyme substrate.

agglutination test ch10f2

agglutination test ch10f2

Figure 2 Agglutination test in which inert particles (latex beads or heat-killed S aureus Cowan 1 strain with protein A) are coated with antibody to any of a variety of antigens and then used to detect the antigen in specimens or in isolated bacteria


Genetic probes are based on the detection of unique nucleotide sequences with the DNA or RNA of a microorganism. Once such a unique nucleotide sequence, which may represent a portion of a virulence gene or of chromosomal DNA, is found, it is isolated and inserted into a cloning vector (plasmid), which is then transformed into Escherichia coli to produce multiple copies of the probe. The sequence is then reisolated from plasmids and labeled with an isotope or substrate for diagnostic use. Hybridization of the sequence with a complementary sequence of DNA or RNA follows cleavage of the double-stranded DNA of the microorganism in the specimen.

The use of molecular technology in the diagnoses of infectious diseases has been further enhanced by the introduction of gene amplication techniques, such as the polymerase chain reaction (PCR) in which DNA polymerase is able to copy a strand of DNA by elongating complementary strands of DNA that have been initiated from a pair of closely spaced oligonucleotide primers. This approach has had major applications in the detection of infections due to microorganisms that are difficult to culture (e.g. the human immunodeficiency virus) or that have not as yet been successfully cultured (e.g. the Whipple’s disease bacillus).

Solid media, although somewhat less sensitive than liquid media, provide isolated colonies that can be quantified if necessary and identified. Some genera and species can be recognized on the basis of their colony morphologies.

In some instances one can take advantage of differential carbohydrate fermentation capabilities of microorganisms by incorporating one or more carbohydrates in the medium along with a suitable pH indicator. Such media are called differential media (e.g., eosin methylene blue or MacConkey agar) and are commonly used to isolate enteric bacilli. Different genera of the Enterobacteriaceae can then be presumptively identified by the color as well as the morphology of colonies.

Culture media can also be made selective by incorporating compounds such as antimicrobial agents that inhibit the indigenous flora while permitting growth of specific microorganisms resistant to these inhibitors. One such example is Thayer-Martin medium, which is used to isolate Neisseria gonorrhoeae. This medium contains vancomycin to inhibit Gram-positive bacteria, colistin to inhibit most Gram-negative bacilli, trimethoprim-sulfamethoxazole to inhibit Proteus species and other species that are not inhibited by colistin and anisomycin to inhibit fungi. The pathogenic Neisseria species, N gonorrhoeae and N meningitidis, are ordinarily resistant to the concentrations of these antimicrobial agents in the medium.

Infection of the bladder (cystitis) or kidney (pyelone-phritis) is usually accompanied by bacteriuria of about ≥ 104 CFU/ml. For this reason, quantitative cultures (Fig. 10-3) of urine must always be performed. For most other specimens a semiquantitative streak method (Fig. 10-3) over the agar surface is sufficient. For quantitative cultures, a specific volume of specimen is spread over the agar surface and the number of colonies per milliliter is estimated.

Identification of bacteria (including mycobacteria) is based on growth characteristics (such as the time required for growth to appear or the atmosphere in which growth occurs), colony and microscopic morphology, and biochemical, physiologic, and, in some instances, antigenic or nucleotide sequence characteristics. The selection and number of tests for bacterial identification depend upon the category of bacteria present (aerobic versus anaerobic, Gram-positive versus Gram-negative, cocci versus bacilli) and the expertise of the microbiologist examining the culture. Gram-positive cocci that grow in air with or without added CO2 may be identified by a relatively small number of tests. The identification of most Gram-negative bacilli is far more complex and often requires panels of 20 tests for determining biochemical and physiologic characteristics.

Antimicrobial susceptibility tests are performed by either disk diffusion or a dilution method. In the former, a standardized suspension of a particular microorganism is inoculated onto an agar surface to which paper disks containing various antimicrobial agents are applied. Following overnight incubation, any zone diameters of inhibition about the disks are measured. An alternative method is to dilute on a log2 scale each antimicrobial agent in broth to provide a range of concentrations and to inoculate each tube or, if a microplate is used, each well containing the antimicrobial agent in broth with a standardized suspension of the microorganism to be tested. The lowest concentration of antimicrobial agent that inhibits the growth of the microorganism is the minimal inhibitory concentration.

Classification Principles

This Week’s Citation Classic®_______ Sneath P H A & Sokal R R.
Numerical taxonomy: the principles and practice of
numerical classification. San Francisco: Freeman, 1973. 573 p.
[Medical Research Council Microbial Systematics Unit, Univ. Leicester, England
and Dept. Ecology and Evolution, State Univ. New York, Stony Brook, NY]
Numerical taxonomy establishes classification
of organisms based on their similarities. It utilizes
many equally weighted characters and employs
clustering and similar algorithms to yield
objective groupings. It can beextended to give
phylogenetic or diagnostic systems and can be
applied to many other fields of endeavour.

Mathematical Foundations of Computer Science 1998
Lecture Notes in Computer Science Volume 1450, 1998, pp 474-482
Date: 28 May 2006
Positive Turing and truth-table completeness for NEXP are incomparable 1998
Levke Bentzien

The truth-table method [matrix method] is one of the decision procedures for sentence logic (q.v., §3.2). The method is based on the fact that the truth value of a compound formula of sentence logic, construed as a truth-function, is determined by the truth values of its arguments (cf. “Sentence logic” §2.2). To decide whether a formula A is a tautology or not, we list all possible combinations of truth values to the variables in A: A is a tautology if it takes the value truth under each assignment.

Using ideas introduced by Buhrman et al. ([2], [3]) to separate various completeness notions for NEXP = NTIME (2poly), positive Turing complete sets for NEXP are studied. In contrast to many-one completeness and bounded truth-table completeness with norm 1 which are known to coincide on NEXP ([3]), whence any such set for NEXP is positive Turing complete, we give sets A and B such that

A is ≤ bT(2) P -complete but not ≤ posT P -complete for NEXP

B is ≤ posT P -complete but not ≤ tt P -complete for NEXP. These results come close to optimality since a further strengthening of (1), as was done by Buhrman in [1] for EXP = DTIME(2poly), seems to require the assumption NEXP = co-NEXP.

Computability and Models
The University Series in Mathematics 2003, pp 1-10
Truth-Table Complete Computably Enumerable Sets
Marat M. Arslanov

We prove a truth-table completeness criterion for computably enumerable sets.
The authors research was partially supported by Russian Foundation of Basic Research, Project 99-01-00830, and RFBR-INTAS, Project 97-91-71991.

Department of Microbiology, Lovelace Foundation for Medical Education and Research,
Albuquerque, N.M. 87108, U.S.A.
Space life sciences 1971-12-1; 3(2): pp 135-156
(Received 15 July, 1971)
Abstract. A logical basis for classification is that elements grouped together and higher categories of elements should have a high degree of similarity with the provision that all groups and categories be disjoint to some degree. A methodology has been developed for constructing classifications automatically that gives
nearly instantaneous correlations of character patterns of organisms with time and clusters with apparent similarity. This means that automatic numerical identification will always construct schemes from which disjoint answers can be obtained if test sensitivities for characters are correct. Unidentified organisms are recycled through continuous classification with reconstruction of identification schemes. This process is
cyclic and self-correcting. The method also accumulates and analyzes data which updates and presents a more accurate biological picture.

Syndromic classification: A process for amplifying information using S-clustering

Eugene W. Rypka, PHD


Optimal classification/Rypka < Optimal classification>


1 Rypka’s Method

1.1 Equations

1.2 Examples

2 Notes and References

Rypka’s Method

Rypka’s[1] method[2] utilizes the theoretical and empirical separatory equations shown below to perform the task of optimal classification. The method finds the optimal order of the fewest attributes, which in combination define a bounded class of elements.

Application of the method begins with construction of an attribute-valued system in truth table[3] or spreadsheet form with elements listed in the left most column beginning in the second row. Characteristics[4] are listed in the first row beginning in the second column with the code name of the data in the upper left most cell. The values which connect each characteristic with each element are placed in the intersecting cells. Selecting appropriate characteristics to universally define the class of elements may be the most difficult part for the classifier of utilizing this method.

The elements are first sorted in descending order according to their truth table value, which is calculated from the existing sequence and value of characteristics for each element. Duplicate truth table values or multisets for the entire bounded class reveal either the need to eliminate duplicate elements or the need to include additional characteristics.

An empirical separatory value is calculated for each characteristic in the set and the characteristic with the greatest empirical separatory value is exchanged with the characteristic which occupies the most significant attribute position.

Next the second most significant characteristic is found by calculating an empirical separatory value for each remaining characteristic in combination with the first characteristic. The characteristic which produces the greatest separatory value is then exchanged with the characteristic which occupies the second most significant attribute position.

Next the third most significant characteristic is found by calculating an empirical separatory value for each remaining characteristic in combination with the first and second characteristics. The characteristic which produces the greatest empirical separatory value is then exchanged with the characteristic which occupies the third most significant attribute position. This procedure may continue until all characteristics have been processed or until one hundred percent separation of the elements has been achieved.

A larger radix will allow faster identification by excluding a greater percentage of elements per characteristic. A binary radix for instance excludes only fifty percent of the elements per characteristic whereas a five-valued radix excludes eighty percent of the elements per characteristic.[5] What follows is an elucidation of the matrix and separatory equations.[6]

Computational Example
Bounded Class Data

bounded class data

Bounded Class Dimensions

G = 28 – 28 elements – i = 0…G-1[1]

C = 10 – 10 characteristics or attributes – j = 0…C-1

V = 5 – 5 valued logic – l = 0…V-1

Order of Elements

order of elements

Count multisets

count multisets

Squared multiset Counts

squared multiset counts

Separatory Values

separatory values


max(T) = 309 = S8 = highest initial separatory value


Mathcad’s ORIGIN function applies to all arrays such that if more than one array is being used and one array requires a zero origin then the other arrays must use a zero origin with all variables being adapted as well.

Rypka’s Method Edit

Rypka’s[1] method[2] utilizes the theoretical and empirical separatory equations shown below to perform the task of optimal classification. The method finds the optimal order of the fewest attributes, which in combination define a bounded class of elements.

Application of the method begins with construction of an attribute-valued system in truth table[3] or spreadsheet form with elements listed in the left most column beginning in the second row. Characteristics[4] are listed in the first row beginning in the second column with the title of the attributes in the upper left most cell. Normally the file name of the data is given the title of the element class. The values which connect each characteristic with each element are placed in the intersecting cells. Selecting characteristics which all elements share may be the most difficult part of creating a database which can utilizing this method.

The elements are first sorted in descending order according to their truth table value, which is calculated from the existing sequence and value of characteristics for each element. Duplicate truth table values or multisets for the entire bounded class reveal either the need to eliminate duplicate elements or the need to include additional characteristics.

An empirical separatory value is calculated for each characteristic in the set and the characteristic with the greatest empirical separatory value is exchanged with the characteristic which occupies the most significant attribute position.

Next the second most significant characteristic is found by calculating an empirical separatory value for each remaining characteristic in combination with the first characteristic. The characteristic which produces the greatest separatory value is then exchanged with the characteristic which occupies the second most significant attribute position.

Next the third most significant characteristic is found by calculating an empirical separatory value for each remaining characteristic in combination with the first and second characteristics. The characteristic which produces the greatest empirical separatory value is then exchanged with the characteristic which occupies the third most significant attribute position. This procedure may continue until all characteristics have been processed or until one hundred percent separation of the elements has been achieved.

A larger radix will allow faster identification by excluding a greater percentage of elements per characteristic. A binary radix for instance excludes only fifty percent of the elements per characteristic whereas a five-valued radix excludes eighty percent of the elements per characteristic.[5] What follows is an elucidation of the matrix and separatory equations.[6]

Syndromic Classification: A Process for Amplifying Information Using S-Clustering

Eugene W. Rypka, PhD
University of New Mexico, Albuquerque, New Mexico, USA
Statistics Editor: Marcello Pagano, PhD
Harvard School of Public Health, Boston, Massachusetts, USA
Nutrition 1996; 12(11/12): 827-829

In a previous issue of Nutrition, Drs. Bernstein and Pleban’ use the method of S-clustering to aid in nutritional classification of patients directly on-line. Classification of this type is called primary or syndromic classification.* It is created by a process called separatory (S-) clustering (E. Rypka, unpublished observations). The authors use S-clustering in Table I.  S-clustering extracts features (analytes, variables) from endogenous data that amplify or maximize structural information to create classes of patients (pathophysiologic events) which are the most disjointed or separable. S-clustering differs from other classificatory methods because it finds in a database a theoretic- or more- number of variables with the required variety that map closest to an ideal, theoretic, or structural information standard. In Table I of their article, Bernstein and Pleban’ indicate there would have to be 3 ’ = 243 rows to show all possible patterns. In Table II of this article, I have used a 33 = 27 row truth table to convey the notion of mapping amplified information to an ideal, theoretic standard using just the first three columns. Variables are scaled for use in S-clustering.

A Survey of Binary Similarity and Distance Measures
Seung-Seok Choi, Sung-Hyuk Cha, Charles C. Tappert
The binary feature vector is one of the most common
representations of patterns and measuring similarity and
distance measures play a critical role in many problems
such as clustering, classification, etc. Ever since Jaccard
proposed a similarity measure to classify ecological
species in 1901, numerous binary similarity and distance
measures have been proposed in various fields. Applying
appropriate measures results in more accurate data
analysis. Notwithstanding, few comprehensive surveys
on binary measures have been conducted. Hence we
collected 76 binary similarity and distance measures used
over the last century and reveal their correlations through
the hierarchical clustering technique.

This paper is organized as follows. Section 2 describes
the definitions of 76 binary similarity and dissimilarity
measures. Section 3 discusses the grouping of those
measures using hierarchical clustering. Section 4
concludes this work.

Historically, all the binary measures observed above have
had a meaningful performance in their respective fields.
The binary similarity coefficients proposed by Peirce,
Yule, and Pearson in 1900s contributes to the evolution
of the various correlation based binary similarity
measures. The Jaccard coefficient proposed at 1901 is
still widely used in the various fields such as ecology and
biology. The discussion of inclusion or exclusion of
negative matches was actively arisen by Sokal & Sneath
in during 1960s and by Goodman & Kruskal in 1970s.

Polyphasic Taxonomy of the Genus Vibrio: Numerical Taxonomy of Vibrio cholerae, Vibrio
parahaemolyticus, and Related Vibrio Species
JOURNAL OF BACTERIOLOGY, Oct. 1970;  104(1): 410-433
A set of 86 bacterial cultures, including 30 strains of Vibrio cholerae, 35 strains of
V. parahaemolyticus, and 21 representative strains of Pseudomonas, Spirillum,
Achromobacter, Arthrobacter, and marine Vibrio species were tested for a total of 200
characteristics. Morphological, physiological, and biochemical characteristics were
included in the analysis. Overall deoxyribonucleic acid (DNA) base compositions
and ultrastructure, under the electron microscope, were also examined. The taxonomic
data were analyzed by computer by using numerical taxonomy programs
designed to sort and cluster strains related phenetically. The V. cholerae strains
formed an homogeneous cluster, sharing overall S values of >75%. Two strains,
V. cholerae NCTC 30 and NCTC 8042, did not fall into the V. cholerae species
group when tested by the hypothetical median organism calculation. No separation
of “classic” V. cholerae, El Tor vibrios, and nonagglutinable vibrios was observed.
These all fell into a single, relatively homogeneous, V. cholerae species cluster.
PJ. parahaemolyticus strains, excepting 5144, 5146, and 5162, designated members
of the species V. alginolyticus, clustered at S >80%. Characteristics uniformly
present in all the Vibrio species examined are given, as are also characteristics and
frequency of occurrence for V. cholerae and V. parahaemolyticus. The clusters formed
in the numerical taxonomy analyses revealed similar overall DNA base compositions,
with the range for the Vibrio species of 40 to 48% guanine plus cytosine. Generic
level of relationship of V. cholerae and V. parahaemolyticus is considered
dubious. Intra- and intergroup relationships obtained from the numerical taxonomy
studies showed highly significant correlation with DNA/DNA reassociation data.

A Numerical Classification of the Genus Bacillus
Journal of General Microbiology (1988), 134, 1847-1882.

Three hundred and sixty-eight strains of aerobic, endospore-forming bacteria which included type and reference cultures of Bacillus and environmental isolates were studied. Overall similarities of these strains for 118 unit characters were determined by the SSMS,, and Dp coefficients and clustering achieved using the UPGMA algorithm. Test error was within acceptable limits. Six cluster-groups were defined at 70% SSM which corresponded to 69% Sp and 48-57% SJ.G roupings obtained with the three coefficients were generally similar but there were some changes in the definition and membership of cluster-groups and clusters, particularly with the SJ coefficient. The Bacillus strains were distributed among 31 major (4 or more strains), 18 minor (2 or 3 strains) and 30 single-member clusters at the 83% SsMle vel. Most of these clusters can be regarded as taxospecies. The heterogeneity of several species, including Bacillus breuis, B. circulans, B. coagulans, B. megateriun, B . sphaericus and B . stearothermophilus, has been indicated  and the species status of several taxa of hitherto uncertain validity confirmed. Thus on the basis of the numerical phenetic and appropriate (published) molecular genetic data, it is proposed
that the following names be recognized; BacillusJlexus (Batchelor) nom. rev., Bacillus fusiformis (Smith et al.) comb. nov., Bacillus kaustophilus (Prickett) nom. rev., Bacilluspsychrosaccharolyticus (Larkin & Stokes) nom. rev. and Bacillus simplex (Gottheil) nom. rev. Other phenetically well-defined taxospecies included ‘ B. aneurinolyticus’, ‘B. apiarius’, ‘B. cascainensis’, ‘B. thiaminolyticus’ and three clusters of environmental isolates related to B . firmus and previously described as ‘B. firmus-B. lentus intermediates’. Future developments in the light of the numerical phenetic data are discussed.

Numerical Classification of Bacteria
Part II. * Computer Analysis of Coryneform Bacteria (2)
Comparison of Group-Formations Obtained on Two
Different Methods of Scoring Data
By Eitaro MASUOan d Toshio NAKAGAWA
[Agr. Biol. Chem., 1969; 33(8): 1124-1133.
Sixty three organisms selected from 12 genera of bacteria were subjected to numerical analysis. The purpose of this work is to examine the relationships among 38 coryneform bacteria included in the test organisms by two coding methods-Sneath’s and Lockhart’s systems-, and to compare the results with conventional classification. In both cases of codification, five groups and one or two single item(s) were found in the resultant classifications. Different codings brought, however, a few distinct differences in some groups , especially in a group of sporogenic bacilli or lactic-acid bacteria. So far as the present work concerns, the result obtained on Lockhart’s coding rather than that obtained on Sneath’s coding resembled the conventional classification. The taxonomic positions of corynebacteria were quite different from those of the conventional classification, regardless
of which coding method was applied.
Though animal corynebacteria have conventionally been considered to occupy the
taxonomic position neighboring to genera Arthrobacter and Cellulornonas and regarded to be the nucleus of so-called “coryneform bacteria,’ the present work showed that many of the corynebacteria are akin to certain mycobacteria rather than to the organisms belonging to the above two genera.

Numerical Classification of Bacteria
Part III. Computer Analysis of “Coryneform Bacteria” (3)
Classification Based on DNA Base Compositions
By EitaroM ASUaOnd ToshioN AKAGAWA
Agr. Biol. Chem., 1969; 33(11): 1570-1576
It has been known that the base compositions of deoxyribonucleic acids (DNA) are
quite different from organism to organism. A pertinent example of this diversity is
found in bacterial species. The base compositions of DNA isolated from a wide variety
of bacteria are distributed in a range from 25 to 75 GC mole-percent (100x(G+C)/
(A+T+G+C)).1) The usefulness of the information of DNA base composition for
the taxonomy of bacteria has been emphasized by several authors. Lee et al.,” Sueoka,” and Freese) have speculated on the evolutionary significance of microbial DNA base composition. They pointed out that closely related microorganisms generally showed similar base compositions of DNA, and suggested that phylogenetic relationship should be reflected in the GC content.
In the present paper are compared the results of numerical classifications of 45
bacteria based on the two different similarity matrices: One representing the overall
similarities of phenotypic properties, the other representing the similarities of GC contents.

Advanced computational algorithms for microbial community analysis using massive 16S rRNA
sequence data
Y Sun, Y Cai, V Mai, W Farmerie, F Yu, J Li and S Goodison
Nucleic Acids Research, 2010; 38(22): e205

With the aid of next-generation sequencing technology, researchers can now obtain millions of microbial signature sequences for diverse applications ranging from human epidemiological studies to global ocean surveys. The development of advanced computational strategies to maximally extract pertinent information from massive nucleotide data has become a major focus of the bioinformatics community. Here, we describe a novel analytical strategy including discriminant and topology analyses that enables researchers to deeply investigate the hidden world of microbial communities, far beyond basic microbial diversity estimation. We demonstrate the utility of our
approach through a computational study performed on a previously published massive human gut 16S rRNA data set. The application of discriminant and
topology analyses enabled us to derive quantitative disease-associated microbial signatures and describe microbial community structure in far more detail than previously achievable. Our approach provides rigorous statistical tools for sequence based studies aimed at elucidating associations between known or unknown organisms and a variety of physiological or environmental conditions.

What is Drug Resistance?

Antimicrobial resistance is the ability of microbes, such as bacteria, viruses, parasites, or fungi, to grow in the presence of a chemical (drug) that would normally kill it or limit its growth.

Diagram showing the difference between non-resistant bacteria and drug resistant bacteria.

Credit: NIAID

DrugResistance difference between non-resistant bacteria and drug resistant bacteria

DrugResistance difference between non-resistant bacteria and drug resistant bacteria


Diagram showing the difference between non-resistant bacteria and drug resistant bacteria. Non-resistant bacteria multiply, and upon drug treatment, the bacteria die. Drug resistant bacteria multiply as well, but upon drug treatment, the bacteria continue to spread.

Between 5 and 10 percent of all hospital patients develop an infection. About 90,000 of these patients die each year as a result of their infection, up from 13,300 patient deaths in 1992.

According to the Centers for Disease Control and Prevention (April 2011), antibiotic resistance in the United States costs an estimated $20 billion a year in excess health care costs, $35 million in other societal costs and more than 8 million additional days that people spend in the hospital.

Resistance to Antibiotics: Are We in the Post-Antibiotic Era?

Alfonso J. Alanis
Archives of Medical Research 36 (2005) 697–705

Serious infections caused by bacteria that have become resistant to commonly used antibiotics have become a major global healthcare problem in the 21st century. They not only are more severe and require longer and more complex treatments, but they are also significantly more expensive to diagnose and to treat. Antibiotic resistance, initially a problem of the hospital setting associated with an increased number of hospital acquired infections usually in critically ill and immunosuppressed patients, has now extended into the community causing severe infections difficult to diagnose and treat. The molecular mechanisms by which bacteria have become resistant to antibiotics are diverse and complex. Bacteria have developed resistance to all different classes of antibiotics discovered to date. The most frequent type of resistance is acquired and transmitted horizontally via the conjugation of a plasmid. In recent times new mechanisms of resistance have resulted in the simultaneous development of resistance to several antibiotic classes creating very dangerous multidrug-resistant (MDR) bacterial strains, some also known as ‘‘superbugs’’. The indiscriminate and inappropriate use of antibiotics in outpatient clinics, hospitalized patients and in the food industry is the single largest factor leading to antibiotic resistance. The pharmaceutical industry, large academic institutions or the government are not investing the necessary resources to produce the next generation of newer safe and effective antimicrobial drugs. In many cases, large pharmaceutical companies have terminated their anti-infective research programs altogether due to economic reasons. The potential negative consequences of all these events are relevant because they put society at risk for the spread of potentially serious MDR bacterial infections.

Targeting the Human Macrophage with Combinations of Drugs and Inhibitors of Ca2+ and K+ Transport to Enhance the Killing of Intracellular Multi-Drug Resistant M. tuberculosis (MDR-TB) – a Novel, Patentable Approach to Limit the Emergence of XDR-TB

Marta Martins
Recent Patents on Anti-Infective Drug Discovery, 2011, 6, 000-000

The emergence of resistance in Tuberculosis has become a serious problem for the control of this disease. For that reason, new therapeutic strategies that can be implemented in the clinical setting are urgently needed. The design of new compounds active against mycobacteria must take into account that Tuberculosis is mainly an intracellular infection of the alveolar macrophage and therefore must maintain activity within the host cells. An alternative therapeutic approach will be described in this review, focusing on the activation of the phagocytic cell and the subsequent killing of the internalized bacteria. This approach explores the combined use of antibiotics and phenothiazines, or Ca2+ and K+ flux inhibitors, in the infected macrophage. Targeting the infected macrophage and not the internalized bacteria could overcome the problem of bacterial multi-drug resistance. This will potentially eliminate the appearance of new multi-drug resistant tuberculosis (MDR-TB) cases and subsequently prevent the emergence of extensively-drug resistant tuberculosis (XDR-TB). Patents resulting from this novel and innovative approach could be extremely valuable if they can be implemented in the clinical setting. Other patents will also be discussed such as the treatment of TB using immunomodulator compounds (for example: betaglycans).

Six Epigenetic Faces of Streptococcus

Kevin Mayer

Medical illustration of Streptococcus pneumonia. [CDC]

Streptococcus pneumonia

Streptococcus pneumonia

It appears that S. pneumoniae has even more personalities, each associated with a different proclivity toward invasive, life-threatening disease. In fact, any of six personalities may emerge depending on the action of a single genetic switch.

To uncover the switch, an international team of scientists conducted a study in genomics, but they looked beyond nucleotide polymorphisms or accessory regions as possible phenotype-shifting mechanisms. Instead, they focused on the potential of restriction-modification (RM) systems to mediate gene regulation via epigenetic changes.

Scientists representing the University of Leicester, Griffith University’s Institute for Glycomics, theUniversity of Adelaide, and Pacific Biosciences realized that the S. pneumoniae genome contains two Type I, three Type II, and one Type IV RM systems. Of these, only the DpnI Type II RM system had been described in detail. Switchable Type I systems had been described previously, but these reports did not provide evidence for differential methylation or for phenotypic impact.

As it turned out, the Type I system embodied a mechanism capable of randomly changing the bacterium’s characteristics into six alternative states. The mechanism’s details were presented September 30 in Nature Communications, in an article entitled, “A random six-phase switch regulates pneumococcal virulence via global epigenetic changes.”

“The underlying mechanism for such phase variation consists of genetic rearrangements in a Type I restriction-modification system (SpnD39III),” wrote the authors. “The rearrangements generate six alternative specificities with distinct methylation patterns, as defined by single-molecule, real-time (SMRT) methylomics.”

Eradication of multidrug-resistant A. baumanniii in burn wounds by antiseptic pulsed electric field.

A Golberg, GF Broelsch, D Vecchio,S Khan, MR Hamblin, WG Austen, Jr, RL Sheridan,  ML Yarmush.

Emerging bacterial resistance to multiple drugs is an increasing problem in burn wound management. New non-pharmacologic interventions are needed for wound disinfection. Here we report on a novel physical method for disinfection: antiseptic pulsed electric field (PEF) applied externally to the infected wounds.  In an animal model, we show that PEF can reduce the load of multidrug resistant Acinetobacter baumannii present in a full thickness burn wound by more than four orders of magnitude, as detected by bioluminescence imaging. Furthermore, using a finite element numerical model, we demonstrate that PEF provides non-thermal, homogeneous, full thickness treatment for the burn wound, thus, overcoming the limitation of treatment depth for many topical antimicrobials. These modeling tools and our in vivo results will be extremely useful for further translation of the PEF technology to the clinical setting. We believe that PEF, in combination with systemic antibiotics, will synergistically eradicate multidrug-resistant burn wound infections, prevent biofilm formation and restore natural skin microbiome. PEF provides a new platform for infection combat in patients, therefore it has a potential to significantly decreasing morbidity and mortality.

Golberg, A. & Yarmush, M. L. Nonthermal irreversible electroporation: fundamentals, applications, and challenges. IEEE Trans Biomed Eng 60, 707-14 (2013).

Mechanisms Of Antibiotic Resistance In Salmonella: Efflux Pumps, Genetics, Quorum Sensing And Biofilm Formation.

Martins M, McCusker M, Amaral L, Fanning S
Perspectives in Drug Discovery and Design 02/2011; 8:114-123.

In Salmonella the main mechanisms of antibiotic resistance are mutations in target genes (such as DNA gyrase and topoisomerase IV) and the over-expression of efflux pumps. However, other mechanisms such as changes in the cell envelope; down regulation of membrane porins; increased lipopolysaccharide (LPS) component of the outer cell membrane; quorum sensing and biofilm formation can also contribute to the resistance seen in this microorganism. To overcome this problem new therapeutic approaches are urgently needed. In the case of efflux-mediated multidrug resistant isolates, one of the treatment options could be the use of efflux pump inhibitors (EPIs) in combination with the antibiotics to which the bacteria is resistant. By blocking the efflux pumps resistance is partly or wholly reversed, allowing antibiotics showing no activity against the MDR strains to be used to treat these infections. Compounds that show potential as an EPI are therefore of interest, as well as new strategies to target the efflux systems. Quorum sensing (QS) and biofilm formation are systems also known to be involved in antibiotic resistance. Consequently, compounds that can disrupt or inhibit these bacterial “communication systems” will be of use in the treatment of these infections.

Role of Phenothiazines and Structurally Similar Compounds of Plant Origin in the Fight against Infections by Drug Resistant Bacteria

SG Dastidar, JE Kristiansen, J Molnar and L Amaral
Antibiotics 2013, 2, 58-71;

Phenothiazines have their primary effects on the plasma membranes of prokaryotes and eukaryotes. Among the components of the prokaryotic plasma membrane affected are efflux pumps, their energy sources and energy providing enzymes, such as ATPase, and genes that regulate and code for the permeability aspect of a bacterium. The response of multidrug and extensively drug resistant tuberculosis to phenothiazines shows an alternative therapy for its treatment. Many phenothiazines have shown synergistic activity with several antibiotics thereby lowering the doses of antibiotics administered for specific bacterial infections. Trimeprazine is synergistic with trimethoprim. Flupenthixol (Fp) has been found to be synergistic with penicillin and chlorpromazine (CPZ); in addition, some antibiotics are also synergistic. Along with the antibacterial action described in this review, many phenothiazines possess plasmid curing activities, which render the bacterial carrier of the plasmid sensitive to antibiotics. Thus, simultaneous applications of a phenothiazine like TZ would not only act as an additional antibacterial agent but also would help to eliminate drug resistant plasmid from the infectious bacterial cells.

Multidrug Efflux Pumps Described for Staphylococcus aureus

Efflux Pump  Family Regulator(s) Substrate Specificity  References 
Chromosomally-encoded Efflux Systems 
NorA MFS MgrA, NorG(?) Hydrophilic fluoroquinolones (ciprofloxacin, norfloxacin)QACs (tetraphenylphosphonium, benzalkonium chloride)

Dyes (e.g. ethidium bromide, rhodamine)

NorB MFS MgrA, NorG Fluoroquinolones (e.g. hydrophilic: ciprofloxacin, norfloxacin and hydrophobic: moxifloxacin,
sparfloxacin)TetracyclineQACs (e.g. tetraphenylphosphonium, cetrimide)Dyes (e.g. ethidium bromide)
NorC MFS MgrA(?), NorG Fluoroquinolones (e.g. hydrophilic: ciprofloxacin and hydrophobic: moxifloxacin)Dyes (e.g. rhodamine) [35,36]
MepA MATE MepR Fluoroquinolones (e.g. hydrophilic: ciprofloxacin, norfloxacin and hydrophobic: moxifloxacin,
sparfloxacin)Glycylcyclines (e.g. tigecycline)QACs (e.g. tetraphenylphosphonium, cetrimide, benzalkonium chloride)Dyes (e.g. ethidium bromide)
MdeA MFS n.i. Hydrophilic fluoroquinolones (e.g. ciprofloxacin, norfloxacin)Virginiamycin, novobiocin, mupirocin, fusidic acid

QACs (e.g. tetraphenylphosphonium, benzalkonium chloride, dequalinium)

Dyes (e.g. ethidium bromide)

SepA n.d. n.i. QACs (e.g. benzalkonium chloride)Biguanidines (e.g. chlorhexidine)

Dyes (e.g. acriflavine)

SdrM MFS n.i. Hydrophilic fluoroquinolones (e.g. norfloxacin)Dyes (e.g. ethidium bromide, acriflavine) [42]
LmrS MFS n.i. Oxazolidinone (linezolid)Phenicols (e.g. choramphenicol, florfenicol)

Trimethoprim, erythromycin, kanamycin, fusidic acid

QACs (e.g. tetraphenylphosphonium)

Detergents (e.g. sodium docecyl sulphate)

Dyes (e.g. ethidium bromide)


Plasmid-encoded Efflux Systems

QacA MFS QacR QACs (e.g. tetraphenylphosphonium, benzalkonium chloride, dequalinium)Biguanidines (e.g. chlorhexidine)

Diamidines (e.g. pentamidine)

Dyes (e.g. ethidium bromide, rhodamine, acriflavine)

QacB MFS QacR QACs (e.g. tetraphenylphosphonium, benzalkonium chloride)Dyes (e.g. ethidium bromide, rhodamine, acriflavine) [53]
Smr SMR n.i. QACs (e.g. benzalkonium chloride, cetrimide)Dyes (e.g. ethidium bromide) [58,61]
QacG SMR n.i. QACs (e.g. benzalkonium chloride, cetyltrymethylammonium)Dyes (e.g. ethidium bromide) [67]
QacH SMR n.i. QACs (e.g. benzalkonium chloride, cetyltrymethylammonium)Dyes (e.g. ethidium bromide) [68]
QacJ SMR n.i. QACs (e.g. benzalkonium chloride, cetyltrymethylammonium)Dyes (e.g. ethidium bromide) [69]

a n.d.: The family of transporters to which SepA belongs is not elucidated to date.
b n.i.: The transporter has no regulator identified to date.
QACs: quaternary ammonium compounds
The importance of efflux pumps in bacterial antibiotic resistance

  1. A. Webber and L. J. V. Piddock
    Journal of Antimicrobial Chemotherapy (2003) 51, 9–11
    http://dx.doi.org:/10.1093/jac/dkg050Efflux pumps are transport proteins involved in the extrusion of toxic substrates (including virtually all classes of clinically relevant antibiotics) from within cells into the external environment. These proteins are found in both Gram-positive and -negative bacteria as well as in eukaryotic organisms. Pumps may be specific for one substrate or may transport a range of structurally dissimilar compounds (including antibiotics of multiple classes); such pumps can be associated with multiple drug resistance (MDR). In the prokaryotic kingdom there are five major families of efflux transporter: MF (major facilitator), MATE (multidrug and toxic efflux), RND (resistance-nodulation-division), SMR (small multidrug resistance) and ABC (ATP binding cassette). All these systems utilize the proton motive force as an energy source. Advances in DNA technology have led to the identification of members of the above families. Transporters that efflux multiple substrates, including antibiotics, have not evolved in response to the stresses of the antibiotic era. All bacterial genomes studied contain efflux pumps that indicate their ancestral origins. It has been estimated that ∼5–10% of all bacterial genes are involved in transport and a large proportion of these encode efflux pumps.
The efflux pump

The efflux pump

Multidrug-resistance efflux pumps — not just for resistance

Laura J. V. Piddock
Nature Reviews | Microbiology | Aug 2006; 4: 629

It is well established that multidrug-resistance efflux pumps encoded by bacteria can confer clinically relevant resistance to antibiotics. It is now understood that these efflux pumps also have a physiological role(s). They can confer resistance to natural substances produced by the host, including bile, hormones and host defense molecules. In addition, some efflux pumps of the resistance nodulation division (RND) family have been shown to have a role in the colonization and the persistence of bacteria in the host. Here, I present the accumulating evidence that multidrug-resistance efflux pumps have roles in bacterial pathogenicity and propose that these pumps therefore have greater clinical relevance than is usually attributed to them.

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Larry H. Bernstein, MD, FCAP, Reporter, Reposted

Leaders in Pharmaceutical Intelligence




MK-966, MK-0966, Vioxx


Percent Composition: C 64.95%, H 4.49%, O 20.36%, S 10.20%
LitRef: Selective cyclooxygenase-2 (COX-2) inhibitor. Prepn: Y. Ducharme et al., WO 9500501; eidem, US5474995 (both 1995 to Merck Frosst).
Therap-Cat: Anti-inflammatory; analgesic.

Rofecoxib /ˌrɒfɨˈkɒksɪb/ is a nonsteroidal anti-inflammatory drug (NSAID) that has now been withdrawn over safety concerns. It was marketed by Merck & Co. to treat osteoarthritisacute pain conditions, and dysmenorrhoea. Rofecoxib was approved by the Food and Drug Administration (FDA) on May 20, 1999, and was marketed under the brand names VioxxCeoxx, and Ceeoxx.



Rofecoxib gained widespread acceptance among physicians treating patients with arthritis and other conditions causing chronic or acute pain. Worldwide, over 80 million people were prescribed rofecoxib at some time.[1]

On September 30, 2004, Merck withdrew rofecoxib from the market because of concerns about increased risk of heart attack and stroke associated with long-term, high-dosage use. Merck withdrew the drug after disclosures that it withheld information about rofecoxib’s risks from doctors and patients for over five years, resulting in between 88,000 and 140,000 cases of serious heart disease.[2] Rofecoxib was one of the most widely used drugs ever to be withdrawn from the market. In the year before withdrawal, Merck had sales revenue of US$2.5 billion from Vioxx.[3] Merck reserved $970 million to pay for its Vioxx-related legal expenses through 2007, and have set aside $4.85bn for legal claims from US citizens.

Rofecoxib was available on prescription in both tablet-form and as an oral suspension. It was available by injection for hospital use.


 Mode of action
 Cyclooxygenase (COX) has two well-studied isoforms, called COX-1 and COX-2.
  • COX-1 mediates the synthesis of prostaglandins responsible for protection of the stomach lining, while
  • COX-2 mediates the synthesis of prostaglandins responsible for pain and inflammation.
prostaglandin PGE2

prostaglandin PGE2

By creating “selective” NSAIDs that inhibit COX-2, but not COX-1, the same pain relief as traditional NSAIDs is offered, but with greatly reduced risk of fatal or debilitating peptic ulcers. Rofecoxib is a selective COX-2 inhibitor, or “coxib”.

Others include Merck’s etoricoxib (Arcoxia), Pfizer’s celecoxib (Celebrex) and valdecoxib (Bextra). Interestingly, at the time of its withdrawal, rofecoxib was the only coxib with clinical evidence of its superior gastrointestinal adverse effect profile over conventional NSAIDs. This was largely based on the VIGOR (Vioxx GI Outcomes Research) study, which compared the efficacy and adverse effect profiles of rofecoxib and naproxen.[4]


The therapeutic recommended dosages were 12.5, 25, and 50 mg with an approximate bioavailability of 93%.[5][6][7] Rofecoxib crossed the placenta and blood–brain barrier,[5][6][8]and took 1–3 hours to reach peak plasma concentration with an effective half-life (based on steady-state levels) of approximately 17 hours.[5][7][9] The metabolic products are cis-dihydro and trans-dihydro derivatives of rofecoxib[5][9] which are primarily excreted through urine.

Fabricated efficacy studies

On March 11, 2009, Scott S. Reuben, former chief of acute pain at Baystate Medical Center, Springfield, Mass., revealed that data for 21 studies he had authored for the efficacy of the drug (along with others such as celecoxib) had been fabricated in order to augment the analgesic effects of the drugs. There is no evidence that Reuben colluded with Merck in falsifying his data. Reuben was also a former paid spokesperson for the drug company Pfizer (which owns the intellectual property rights for marketing celecoxib in the United States). The retracted studies were not submitted to either the FDA or the European Union’s regulatory agencies prior to the drug’s approval. Drug manufacturer Merckhad no comment on the disclosure.[10]

Adverse drug reactions

VIOXX sample blister pack.jpg

Aside from the reduced incidence of gastric ulceration, rofecoxib exhibits a similar adverse effect profile to other NSAIDs.

Prostaglandin is a large family of lipids. Prostaglandin I2/PGI2/prostacyclin is just one member of it. Prostaglandins other than PGI2 (such as PGE2) also play important roles in vascular tone regulation. Prostacyclin/thromboxane are produced by both COX-1 and COX-2, and rofecoxib suppresses just COX-2 enzyme, so there is no reason to believe that prostacyclin levels are significantly reduced by the drug. And there is no reason to believe that only the balance between quantities of prostacyclin and thromboxane is the determinant factor for vascular tone.[11] Indeed Merck has stated that there was no effect on prostacyclin production in blood vessels in animal testing.[12] Other researchers have speculated that the cardiotoxicity may be associated with maleic anhydride metabolites formed when rofecoxib becomes ionized under physiological conditions. (Reddy & Corey, 2005)

 Adverse cardiovascular events

VIGOR study and publishing controversy

The VIGOR (Vioxx GI Outcomes Research) study, conducted by Bombardier, et al., which compared the efficacy and adverse effect profiles of rofecoxib and naproxen, had indicated a significant 4-fold increased risk of acute myocardial infarction (heart attack) in rofecoxib patients when compared with naproxen patients (0.4% vs 0.1%, RR 0.25) over the 12 month span of the study. The elevated risk began during the second month on rofecoxib. There was no significant difference in the mortality from cardiovascular events between the two groups, nor was there any significant difference in the rate of myocardial infarction between the rofecoxib and naproxen treatment groups in patients without high cardiovascular risk. The difference in overall risk was by the patients at higher risk of heart attack, i.e. those meeting the criteria for low-dose aspirin prophylaxis of secondary cardiovascular events (previous myocardial infarction, angina, cerebrovascular accidenttransient ischemic attack, or coronary artery bypass).

Merck’s scientists interpreted the finding as a protective effect of naproxen, telling the FDA that the difference in heart attacks “is primarily due to” this protective effect (Targum, 2001). Some commentators have noted that naproxen would have to be three times as effective as aspirin to account for all of the difference (Michaels 2005), and some outside scientists warned Merck that this claim was implausible before VIGOR was published.[13] No evidence has since emerged for such a large cardioprotective effect of naproxen, although a number of studies have found protective effects similar in size to those of aspirin.[14][15] Though Dr. Topol’s 2004 paper criticized Merck’s naproxen hypothesis, he himself co-authored a 2001 JAMA article stating “because of the evidence for an antiplatelet effect of naproxen, it is difficult to assess whether the difference in cardiovascular event rates in VIGOR was due to a benefit from naproxen or to a prothrombotic effect from rofecoxib.” (Mukherjee, Nissen and Topol, 2001.)

The results of the VIGOR study were submitted to the United States Food and Drug Administration (FDA) in February 2001. In September 2001, the FDA sent a warning letter to the CEO of Merck, stating, “Your promotional campaign discounts the fact that in the VIGOR study, patients on Vioxx were observed to have a four to five fold increase in myocardial infarctions (MIs) compared to patients on the comparator non-steroidal anti-inflammatory drug (NSAID), Naprosyn (naproxen).”[16] This led to the introduction, in April 2002, of warnings on Vioxx labeling concerning the increased risk of cardiovascular events (heart attack and stroke).

Months after the preliminary version of VIGOR was published in the New England Journal of Medicine, the journal editors learned that certain data reported to the FDA were not included in the NEJM article. Several years later, when they were shown a Merck memo during the depositions for the first federal Vioxx trial, they realized that these data had been available to the authors months before publication. The editors wrote an editorial accusing the authors of deliberately withholding the data.[17] They released the editorial to the media on December 8, 2005, before giving the authors a chance to respond. NEJM editor Gregory Curfman explained that the quick release was due to the imminent presentation of his deposition testimony, which he feared would be misinterpreted in the media. He had earlier denied any relationship between the timing of the editorial and the trial. Although his testimony was not actually used in the December trial, Curfman had testified well before the publication of the editorial.[18]

The editors charged that “more than four months before the article was published, at least two of its authors were aware of critical data on an array of adverse cardiovascular events that were not included in the VIGOR article.” These additional data included three additional heart attacks, and raised the relative risk of Vioxx from 4.25-fold to 5-fold. All the additional heart attacks occurred in the group at low risk of heart attack (the “aspirin not indicated” group) and the editors noted that the omission “resulted in the misleading conclusion that there was a difference in the risk of myocardial infarction between the aspirin indicated and aspirin not indicated groups.” The relative risk for myocardial infarctions among the aspirin not indicated patients increased from 2.25 to 3 (although it remained statitistically insignificant). The editors also noted a statistically significant (2-fold) increase in risk for serious thromboembolic events for this group, an outcome that Merck had not reported in the NEJM, though it had disclosed that information publicly in March 2000, eight months before publication.[19]

The authors of the study, including the non-Merck authors, responded by claiming that the three additional heart attacks had occurred after the prespecified cutoff date for data collection and thus were appropriately not included. (Utilizing the prespecified cutoff date also meant that an additional stroke in the naproxen population was not reported.) Furthermore, they said that the additional data did not qualitatively change any of the conclusions of the study, and the results of the full analyses were disclosed to the FDA and reflected on the Vioxx warning label. They further noted that all of the data in the “omitted” table were printed in the text of the article. The authors stood by the original article.[20]

NEJM stood by its editorial, noting that the cutoff date was never mentioned in the article, nor did the authors report that the cutoff for cardiovascular adverse events was before that for gastrointestinal adverse events. The different cutoffs increased the reported benefits of Vioxx (reduced stomach problems) relative to the risks (increased heart attacks).[19]

Some scientists have accused the NEJM editorial board of making unfounded accusations.[21][22] Others have applauded the editorial. Renowned research cardiologist Eric Topol,[23] a prominent Merck critic, accused Merck of “manipulation of data” and said “I think now the scientific misconduct trial is really fully backed up”.[24] Phil Fontanarosa, executive editor of the prestigious Journal of the American Medical Association, welcomed the editorial, saying “this is another in the long list of recent examples that have generated real concerns about trust and confidence in industry-sponsored studies”.[25]

On May 15, 2006, the Wall Street Journal reported that a late night email, written by an outside public relations specialist and sent to Journal staffers hours before the Expression of Concern was released, predicted that “the rebuke would divert attention to Merck and induce the media to ignore the New England Journal of Medicine‘s own role in aiding Vioxx sales.”[26]

“Internal emails show the New England Journal’s expression of concern was timed to divert attention from a deposition in which Executive Editor Gregory Curfman made potentially damaging admissions about the journal’s handling of the Vioxx study. In the deposition, part of the Vioxx litigation, Dr. Curfman acknowledged that lax editing might have helped the authors make misleading claims in the article.” The Journal stated that NEJM‘s “ambiguous” language misled reporters into incorrectly believing that Merck had deleted data regarding the three additional heart attacks, rather than a blank table that contained no statistical information; “the New England Journal says it didn’t attempt to have these mistakes corrected.”[26]

APPROVe study

In 2001, Merck commenced the APPROVe (Adenomatous Polyp PRevention On Vioxx) study, a three-year trial with the primary aim of evaluating the efficacy of rofecoxib for theprophylaxis of colorectal polypsCelecoxib had already been approved for this indication, and it was hoped to add this to the indications for rofecoxib as well. An additional aim of the study was to further evaluate the cardiovascular safety of rofecoxib.

The APPROVe study was terminated early when the preliminary data from the study showed an increased relative risk of adverse thrombotic cardiovascular events (includingheart attack and stroke), beginning after 18 months of rofecoxib therapy. In patients taking rofecoxib, versus placebo, the relative risk of these events was 1.92 (rofecoxib 1.50 events vs placebo 0.78 events per 100 patient years). The results from the first 18 months of the APPROVe study did not show an increased relative risk of adverse cardiovascular events. Moreover, overall and cardiovascular mortality rates were similar between the rofecoxib and placebo populations.[28]

In summary, the APPROVe study suggested that long-term use of rofecoxib resulted in nearly twice the risk of suffering a heart attack or stroke compared to patients receiving a placebo.

Other studies

Several very large observational studies have also found elevated risk of heart attack from rofecoxib. For example, a recent retrospective study of 113,000 elderly Canadians suggested a borderline statistically significant increased relative risk of heart attacks of 1.24 from Vioxx usage, with a relative risk of 1.73 for higher-dose Vioxx usage. (Levesque, 2005). Another study, using Kaiser Permanente data, found a 1.47 relative risk for low-dose Vioxx usage and 3.58 for high-dose Vioxx usage compared to current use of celecoxib, though the smaller number was not statistically significant, and relative risk compared to other populations was not statistically significant. (Graham, 2005).

Furthermore, a more recent meta-study of 114 randomized trials with a total of 116,000+ participants, published in JAMA, showed that Vioxx uniquely increased risk of renal (kidney) disease, and heart arrhythmia.[31]

Other COX-2 inhibitors

Any increased risk of renal and arrhythmia pathologies associated with the class of COX-2 inhibitors, e.g. celecoxib (Celebrex), valdecoxib (Bextra), parecoxib (Dynastat),lumiracoxib, and etoricoxib is not evident,[31] although smaller studies[32][33] had demonstrated such effects earlier with the use of celecoxib, valdecoxib and parecoxib.

Nevertheless, it is likely that trials of newer drugs in the category will be extended in order to supply additional evidence of cardiovascular safety. Examples are some more specific COX-2 inhibitors, including etoricoxib (Arcoxia) and lumiracoxib (Prexige), which are currently (circa 2005) undergoing Phase III/IV clinical trials.

Besides, regulatory authorities worldwide now require warnings about cardiovascular risk of COX-2 inhibitors still on the market. For example, in 2005, EU regulators required the following changes to the product information and/or packaging of all COX-2 inhibitors:[34]

  • Contraindications stating that COX-2 inhibitors must not be used in patients with established ischaemic heart disease and/or cerebrovascular disease (stroke), and also in patients with peripheral arterial disease
  • Reinforced warnings to healthcare professionals to exercise caution when prescribing COX-2 inhibitors to patients with risk factors for heart disease, such as hypertension, hyperlipidaemia (high cholesterol levels), diabetes and smoking
  • Given the association between cardiovascular risk and exposure to COX-2 inhibitors, doctors are advised to use the lowest effective dose for the shortest possible duration of treatment

Other NSAIDs

Since the withdrawal of Vioxx it has come to light that there may be negative cardiovascular effects with not only other COX-2 inhibitiors, but even the majority of other NSAIDs. It is only with the recent development of drugs like Vioxx that drug companies have carried out the kind of well executed trials that could establish such effects and these sort of trials have never been carried out in older “trusted” NSAIDs such as ibuprofendiclofenac and others. The possible exceptions may be aspirin and naproxen due to their anti-platelet aggregation properties.


Due to the findings of its own APPROVe study, Merck publicly announced its voluntary withdrawal of the drug from the market worldwide on September 30, 2004.[35]

In addition to its own studies, on September 23, 2004 Merck apparently received information about new research by the FDA that supported previous findings of increased risk of heart attack among rofecoxib users (Grassley, 2004). FDA analysts estimated that Vioxx caused between 88,000 and 139,000 heart attacks, 30 to 40 percent of which were probably fatal, in the five years the drug was on the market.[36]

On November 5, the medical journal The Lancet published a meta-analysis of the available studies on the safety of rofecoxib (Jüni et al., 2004). The authors concluded that, owing to the known cardiovascular risk, rofecoxib should have been withdrawn several years earlier. The Lancet published an editorial which condemned both Merck and the FDA for the continued availability of rofecoxib from 2000 until the recall. Merck responded by issuing a rebuttal of the Jüni et al. meta-analysis that noted that Jüni omitted several studies that showed no increased cardiovascular risk. (Merck & Co., 2004).

In 2005, advisory panels in both the U.S. and Canada encouraged the return of rofecoxib to the market, stating that rofecoxib’s benefits outweighed the risks for some patients. The FDA advisory panel voted 17-15 to allow the drug to return to the market despite being found to increase heart risk. The vote in Canada was 12-1, and the Canadian panel noted that the cardiovascular risks from rofecoxib seemed to be no worse than those from ibuprofen—though the panel recommended that further study was needed for all NSAIDs to fully understand their risk profiles. Notwithstanding these recommendations, Merck has not returned rofecoxib to the market.[37]

In 2005, Merck retained Debevoise & Plimpton LLP to investigate Vioxx study results and communications conducted by Merck. Through the report, it was found that Merck’s senior management acted in good faith, and that the confusion over the clinical safety of Vioxx was due to the sales team’s overzealous behavior. The report that was filed gave a timeline of the events surrounding Vioxx and showed that Merck intended to operate honestly throughout the process. Any mistakes that were made regarding the mishandling of clinical trial results and withholding of information was the result of oversight, not malicious behavior….The report was published in February 2006, and Merck was satisfied with the findings of the report and promised to consider the recommendations contained in the Martin Report. Advisers to the US Food and Drug Administration (FDA) have voted, by a narrow margin, that it should not ban Vioxx — the painkiller withdrawn by drug-maker Merck.

They also said that Pfizer’s Celebrex and Bextra, two other members of the family of painkillers known as COX-2 inhibitors, should remain available, despite the fact that they too boost patients’ risk of heart attack and stroke. url = http://www.nature.com/drugdisc/news/articles/433790b.html The recommendations of the arthritis and drug safety advisory panel offer some measure of relief to the pharmaceutical industry, which has faced a barrage of criticism for its promotion of the painkillers. But the advice of the panel, which met near Washington DC over 16–18 February, comes with several strings attached.

For example, most panel members said that manufacturers should be required to add a prominent warning about the drugs’ risks to their labels; to stop direct-to-consumer advertising of the drugs; and to include detailed, written risk information with each prescription. The panel also unanimously stated that all three painkillers “significantly increase the risk of cardiovascular events”.

External links

For more details and references.. they are provided in the entirety in the original post

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Metformin, thyroid-pituitary axis, diabetes mellitus, and metabolism

Metformin, thyroid-pituitary axis, diabetes mellitus, and metabolism

Larry H, Bernstein, MD, FCAP, Author and Curator
and Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/9/27/2014/Metformin,_thyroid-pituitary_ axis,_diabetes_mellitus,_and_metabolism

The following article is a review of the central relationship between the action of
metformin as a diabetic medication and its relationship to AMPK, the important and
essential regulator of glucose and lipid metabolism under normal activity, stress, with
its effects on skeletal muscle, the liver, the action of T3 and more.

We start with a case study and a publication in the J Can Med Assoc.  Then we shall look
into key literature on these metabolic relationships.

Part I.  Metformin , Diabetes Mellitus, and Thyroid Function

Hypothyroidism, Insulin resistance and Metformin
May 30, 2012   By Janie Bowthorpe
The following was written by a UK hypothyroid patient’s mother –
Sarah Wilson.

My daughter’s epilepsy is triggered by unstable blood sugars. And since taking
Metformin to control her blood sugar, she has significantly reduced the number of
seizures. I have been doing research and read numerous academic medical journals,
which got me thinking about natural thyroid hormone and Hypothyroidism. My hunch
was that when patients develop hypothyroid symptoms, they are actually becoming
insulin resistant (IR). There are many symptoms in common between women with
polycystic ovaries and hypothyroidism–the hair loss, the weight gain, etc.

A hypothyroid person’s body behaves as if it’s going into starvation mode and so, to
preserve resources and prolong life, the metabolism changes. If hypothyroid is prolonged
or pronounced, then perhaps, chemical preservation mode becomes permanent even
with the reintroduction of thyroid hormones. To get back to normal, they need
a “jump-start” reinitiate a higher rate of metabolism. The kick start is initiated through
AMPK, which is known as the “master metabolic regulating enzyme.”
(http://en.wikipedia.org/wiki/AMP-activated protein kinase).

Guess what? This is exactly what happens to Diabetes patients when Metformin is
introduced. http://en.wikipedia.org/wiki/Metformin
Suggested articles: http://www.springerlink.com/content/r81606gl3r603167/  and

Note the following comments/partial statements:
“Hypothyroidism is characterized by decreased insulin responsiveness”;
“the pivotal regulatory role of T3 in major metabolic pathways”.

The community knows that T3/NTH (natural thyroid hormone [Armour]) makes
hypothyroid patients feel better – but the medical establishment is averse to T3/NTH
(treating subclinical hypoT (T3/T4 euthyroid) with natural dessicated thyroid (NDT).
The medical establishment might find an alternative view about impaired metabolism
more if shown real proof that the old NDT **was/is** having the right result –i.e., the
T3 is jump-starting the metabolism by re-activating

If NDT also can be used for hypothyroidism without the surmised “dangers” of NTH,
then they should consider it. [The reality in the choice is actually recombinant TH
(Synthroid)]. Metformin is cheap, stable and has very few serious side effects. I use the
car engine metaphor, and refer to glucose as our petrol, AMPK as the spark plug and
both T3 and Metformin as the ignition switches. Sometimes if you have flat batteries in
the car, it doesn’t matter how much you turn the ignition switch or pump the petrol
pedal, all it does is flatten the battery and flood the engine.

Dr. Skinner in the UK has been treating “pre-hypothyroidism” the way that some
doctors treat “pre-diabetes”. Those hypothyroid patients who get treated early
might not have had their AMPK pathways altered and the T4-T3 conversion still works.
There seems to be no reason why thyroid hormone replacement therapy shouldn’t
logically be given to ward off a greater problem down the line.

It’s my belief that there is clear and abundant academic evidence that the AMPK/
Metformin research should branch out to also look at thyroid disease.

Point – direct T3 is kicking the closed -down metabolic process back into life,
just like Metformin does for insulin resistance.
There is serotonin resistance! http://www.ncbi.nlm.nih.gov/pubmed/17250776

Metformin Linked to Risk of Low Levels of Thyroid Hormone

CMAJ (Canadian Medical Association Journal) 09/22/2014

Metformin, the drug commonly for treating type 2 diabetes,

  • is linked to an increased risk of low thyroid-stimulating hormone
    (TSH) levels
  • in patients with underactive thyroids (hypothyroidism),

according to a study in CMAJ (Canadian Medical Association Journal).

Metformin is used to lower blood glucose levels

  • by reducing glucose production in the liver.

previous studies have raised concerns that

  • metformin may lower thyroid-stimulating hormone levels.

Study characteristics:

  1. Retrospective  long-term
  2. 74 300 patient who received metformin and sulfonylurea
  3. 25-year study period.
  4. 5689 had treated hypothyroidism
  5. 59 937 had normal thyroid function.

Metformin and low levels of thyroid-stimulating hormone in
patients with type 2 diabetes mellitus

Jean-Pascal Fournier,  Hui Yin, Oriana Hoi Yun Yu, Laurent Azoulay  +
Centre for Clinical Epidemiology (Fournier, Yin, Yu, Azoulay), Lady Davis Institute,
Jewish General Hospital; Department of Epidemiology, Biostatistics and Occupational
Health (Fournier), McGill University; Division of Endocrinology (Yu), Jewish General
Hospital; Department of Oncology (Azoulay), McGill University, Montréal, Que., Cananda

CMAJ Sep 22, 2014,   http://dx.doi.org:/10.1503/cmaj.140688


  • metformin may lower thyroid-stimulating hormone (TSH) levels.


  • determine whether the use of metformin monotherapy, when compared with
    sulfonylurea monotherapy,
  • is associated with an increased risk of low TSH levels(< 0.4 mIU/L)
  • in patients with type 2 diabetes mellitus.


  • Used the Clinical Practice Research Datalink,
  • identified patients who began receiving metformin or sulfonylurea monotherapy
    between Jan. 1, 1988, and Dec. 31, 2012.
  • 2 subcohorts of patients with treated hypothyroidism or euthyroidism,

followed them until Mar. 31, 2013.

  • Used Cox proportional hazards models to evaluate the association of low TSH
    levels with metformin monotherapy, compared with sulfonylurea monotherapy,
    in each subcohort.


  • 5689 patients with treated hypothyroidism and 59 937 euthyroid patients were
    included in the subcohorts.

For patients with treated hypothyroidism:

  1. 495 events of low TSH levels were observed (incidence rate 0.1197/person-years).
  2. 322 events of low TSH levels were observed (incidence rate 0.0045/person-years)
    in the euthyroid group.
  • metformin monotherapy was associated with a 55% increased risk of low TSH
    in patients with treated hypothyroidism (incidence rate 0.0795/person-years
    vs.0.1252/ person-years, adjusted hazard ratio [HR] 1.55, 95% confidence
    interval [CI] 1.09– 1.20), compared with sulfonylurea monotherapy,
  • the highest risk in the 90–180 days after initiation (adjusted HR 2.30, 95% CI
  • No association was observed in euthyroid patients (adjusted HR 0.97, 95% CI 0.69–1.36).

Interpretation: The clinical consequences of this needs further investigation.


Crude and adjusted hazard ratios for suppressed thyroid-stimulating hormone
levels (< 0.1 mIU/L) associated with the use metformin monotherapy, compared
with sulfonylurea monotherapy, in patients with treated hypothyroidism or
euthyroidism and type 2 diabetes
Variable No. events
TSH levels
Person-years of
Incidence rate,
per 1000 person-years (95% CI)
Adjusted HR*(95% CI)
Patients with treated hypothyroidism, = 5689
= 762
18 503 35.8
1.00 1.00
= 4927
130 3 633 35.8
1.05 0.99
Euthyroid patients, = 59 937
= 7980
12 8 576 1.4
1.00 1.00
= 51 957
75 63 047 1.2
0.85 1.03


Part II. Metabolic Underpinning 
(Source: Wikipedia, AMPK and thyroid)

5′ AMP-activated protein kinase or AMPK or 5′ adenosine monophosphate-activated protein kinase
is an enzyme that plays a role in cellular energy homeostasis.
It consists of three proteins (subunits) that

  1. together make a functional enzyme, conserved from yeast to humans.
  2. It is expressed in a number of tissues, including the liver, brain, and skeletal
  3. The net effect of AMPK activation is stimulation of
    1. hepatic fatty acid oxidation and ketogenesis,
    2. inhibition of cholesterol synthesis,
    3. lipogenesis, and triglyceride synthesis,
    4. inhibition of adipocyte lipolysis and lipogenesis,
    5. stimulation of skeletal muscle fatty acid oxidation and muscle
      glucose uptake, and
    6. modulation of insulin secretion by pancreatic beta-cells.

The heterotrimeric protein AMPK is formed by α, β, and γ subunits. Each of these three
subunits takes on a specific role in both the stability and activity of AMPK.

  • the γ subunit includes four particular Cystathionine beta synthase (CBS) domains
    giving AMPK its ability to sensitively detect shifts in the AMP:ATP ratio.
  • The four CBS domains create two binding sites for AMP commonly referred to as
    Bateman domains. Binding of one AMP to a Bateman domain cooperatively
    increases the binding affinity of the second AMP to the other Bateman domain.
  • As AMP binds both Bateman domains the γ subunit undergoes a conformational
    change which exposes the catalytic domain found on the α subunit.
  • It is in this catalytic domain where AMPK becomes activated when
    phosphorylation takes place at threonine-172by an upstream AMPK kinase
    (AMPKK). The α, β, and γ subunits can also be found in different isoforms.

AMPK acts as a metabolic master switch regulating several intracellular systems

  1. the cellular uptake of glucose,
  2. the β-oxidation of fatty acids and
  3. the biogenesis of glucose transporter 4 (GLUT4) and
  4. mitochondria

The energy-sensing capability of AMPK can be attributed to

  • its ability to detect and react to fluctuations in the AMP:ATP ratio that take
    place during rest and exercise (muscle stimulation).

During muscle stimulation,

  • AMP increases while ATP decreases, which changes AMPK into a good substrate
    for activation.
  • AMPK activity increases while the muscle cell experiences metabolic stress
    brought about by an extreme cellular demand for ATP.
  • Upon activation, AMPK increases cellular energy levels by
    • inhibiting anabolic energy consuming pathways (fatty acid synthesis,
      protein synthesis, etc.) and
    • stimulating energy producing, catabolic pathways (fatty acid oxidation,
      glucose transport, etc.).

A recent JBC paper on mice at Johns Hopkins has shown that when the activity of brain
AMPK was pharmacologically inhibited,

  • the mice ate less and lost weight.

When AMPK activity was pharmacologically raised (AICAR see below)

  • the mice ate more and gained weight.

Research in Britain has shown that the appetite-stimulating hormone ghrelin also
affects AMPK levels.

The antidiabetic drug metformin (Glucophage) acts by stimulating AMPK, leading to

  1. reduced glucose production in the liver and
  2. reduced insulin resistance in the muscle.

(Metformin usually causes weight loss and reduced appetite, not weight gain and
increased appetite, ..opposite of expected from the Johns Hopkins mouse study results.)

Triggering the activation of AMPK can be carried out provided two conditions are met.

First, the γ subunit of AMPK

  • must undergo a conformational change so as to
  • expose the active site(Thr-172) on the α subunit.

The conformational change of the γ subunit of AMPK can be accomplished

  • under increased concentrations of AMP.

Increased concentrations of AMP will

  • give rise to the conformational change on the γ subunit of AMPK
  • as two AMP bind the two Bateman domains located on that subunit.
  • It is this conformational change brought about by increased concentrations
    of  AMP that exposes the active site (Thr-172) on the α subunit.

This critical role of AMP is further substantiated in experiments that demonstrate

  • AMPK activation via an AMP analogue 5-amino-4-imidazolecarboxamide
    ribotide (ZMP) which is derived fromthe familiar
  • 5-amino-4-imidazolecarboxamide riboside (AICAR)

AMPK is a good substrate for activation via an upstream kinase complex, AMPKK
AMPKK is a complex of three proteins,

  1. STE-related adaptor (STRAD),
  2. mouse protein 25 (MO25), and
  3. LKB1 (a serine/threonine kinase).

The second condition that must be met is

  • the phosphorylation/activation of AMPK on its activating loop at
    Thr-172of the α subunit
  • brought about by an upstream kinase (AMPKK).

The complex formed between LKB1 (STK 11), mouse protein 25 (MO25), and the
pseudokinase STE-related adaptor protein (STRAD) has been identified as

  • the major upstream kinase responsible for phosphorylation of AMPK
    on its activating loop at Thr-172

Although AMPK must be phosphorylated by the LKB1/MO25/STRAD complex,

  • it can also be regulated by allosteric modulators which
  • directly increase general AMPK activity and
  • modify AMPK to make it a better substrate for AMPKK
  • and a worse substrate for phosphatases.

It has recently been found that 3-phosphoglycerate (glycolysis intermediate)

  • acts to further pronounce AMPK activation via AMPKK

Muscle contraction is the main method carried out by the body that can provide
the conditions mentioned above needed for AMPK activation

  • As muscles contract, ATP is hydrolyzed, forming ADP.
  • ADP then helps to replenish cellular ATP by donating a phosphate group to
    another ADP,

    • forming an ATP and an AMP.
  • As more AMP is produced during muscle contraction,
    • the AMP:ATP ratio dramatically increases,
  • leading to the allosteric activation of AMPK

For over a decade it has been known that calmodulin-dependent protein kinase
kinase-beta (CaMKKbeta) can phosphorylate and thereby activate AMPK,

  • but it was not the main AMPKK in liver.

CaMKK inhibitors had no effect on 5-aminoimidazole-4-carboxamide-1-beta-4-
ribofuranoside (AICAR) phosphorylation and activation of AMPK.

  • AICAR is taken into the celland converted to ZMP,
  • an AMP analogthat has been shown to activate AMPK.

Recent LKB1 knockout studies have shown that without LKB1,

  • electrical and AICAR stimulation of muscleresults in very little
    phosphorylation of AMPK and of ACC, providing evidence that
  • LKB1-STRAD-MO25 is the major AMPKK in muscle.

Two particular adipokines, adiponectin and leptin, have even been demonstrated
to regulate AMPK. A main functions of leptin in skeletal muscle is

  • the upregulation of fatty acid oxidation.

Leptin works by way of the AMPK signaling pathway, and adiponectin also
stimulates the oxidation of fatty acids via the AMPK pathway, and

  • Adiponectin also stimulates the uptake of glucose in skeletal muscle.

An increase in enzymes which specialize in glucose uptake in cells such as GLUT4
and hexokinase II are thought to be mediated in part by AMPK when it is activated.
Increases in AMPK activity are brought about by increases in the AMP:ATP ratio
during single bouts of exercise and long-term training.

One of the key pathways in AMPK’s regulation of fatty acid oxidation is the

  • phosphorylation and inactivation of acetyl-CoA carboxylase.
  1. Acetyl-CoA carboxylase (ACC) converts acetyl-CoA (ACA) to malonyl-CoA
    (MCA), an inhibitor of carnitine palmitoyltransferase 1 (CPT-1).
  2. CPT-1 transports fatty acids into the mitochondria for oxidation.
  3. Inactivation of ACC results in increased fatty acid transport and oxidation.
  4. the AMPK induced ACC inactivation  and reduced conversion to MCA
    may occur as a result of malonyl-CoA decarboxylase (MCD)
  5. MCD as an antagonist to ACC, decarboxylatesmalonyl-CoA to acetyl-CoA
    (reversal of ACC conversion of ACA to MCA)
  6. This resultsin decreased malonyl-CoA and increased CPT-1 and fatty acid oxidation.

AMPK also plays an important role in lipid metabolism in the liver. It has long been
known that hepatic ACC has been regulated in the liver.

  1. It phosphorylates and inactivates 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR)
  2. acetyl-CoA(ACA) is converted to mevalonic acid (MVA) by ACC
    with inhibition of CPT-1
  3. HMGR converts 3-hydroxy-3-methylglutaryl-CoA, which is made from MVA
  4. which then travels down several more metabolic steps to become cholesterol.

Insulin facilitates the uptake of glucose into cells via increased expression and
translocation of glucose transporter GLUT-4. In addition, glucose is phosphorylated
by hexokinase wheni iot enters the cell. The phosphorylated form keeps glucose from
leaving the cell,

  • The decreasedthe concentration of glucose molecules creates a gradient for more
    glucose to be transported into the cell.
AMPK and thyroid hormone regulate some similar processes. Knowing these similarities,
Winder and Hardie et al. designed an experiment to see if AMPK was influenced by thyroid
hormone. They found that all of the subunits of AMPK were increased in skeletal muscle,
especially in the soleus and red quadriceps, with thyroid hormone treatment. There was
also an increase in phospho-ACC, a marker of AMPK activity.
  •  Winder WW, Hardie DG (July 1999). “AMP-activated protein kinase,
    a metabolic master switch: possible roles in type 2 diabetes”. J. Physiol. 277
    (1 Pt 1): E1–10. PMID 10409121.
  • Winder WW, Hardie DG (February 1996). “Inactivation of acetyl-CoA
    carboxylase and activation of AMP-activated protein kinase in muscle
    during exercise”. J. Physiol. 270 (2 Pt 1): E299–304. PMID 8779952.
  • Hutber CA, Hardie DG, Winder WW (February 1997). “Electrical stimulation
    inactivates muscle acetyl-CoA carboxylase and increases AMP-activated
    protein kinase”. Am. J. Physiol. 272 (2 Pt 1): E262–6. PMID 9124333
  • Durante PE, Mustard KJ, Park SH, Winder WW, Hardie DG (July 2002).
    “Effects of endurance training on activity and expression of AMP-activated
    protein kinase isoforms in rat muscles”. Am. J. Physiol. Endocrinol.
    Metab. 283 (1): E178–86. doi:10.1152/ajpendo.00404.2001. PMID 12067859
  • Corton JM, Gillespie JG, Hardie DG (April 1994). “Role of the AMP-activated
    protein kinase in the cellular stress response”. Curr. Biol. 4 (4):
    315–24. doi:10.1016/S0960-9822(00)00070-1. PMID 7922340
  • Winder WW (September 2001). “Energy-sensing and signaling by
    AMP-activated protein kinase in skeletal muscle”. J. Appl. Physiol. 91 (3):
    1017–28. PMID 11509493
  • Suter M, Riek U, Tuerk R, Schlattner U, Wallimann T, Neumann D (October
    2006). “Dissecting the role of 5′-AMP for allosteric stimulation, activation,
    and deactivation of AMP-activated protein kinase”.  J. Biol. Chem.
    281 (43): 32207–6. doi:10.1074/jbc.M606357200. PMID 16943194


Part III. Pituitary-thyroid axis and diabetes mellitus
The Interface Between Thyroid and Diabetes Mellitus

Leonidas H. Duntas, Jacques Orgiazzi, Georg Brabant   Clin Endocrinol. 2011;75(1):1-9.
Interaction of Metformin and Thyroid Function

Metformin acts primarily by

  • suppressing hepatic gluconeogenesis via activation of AMPK
  • It has the opposite effects on hypothalamic AMPK,
    • inhibiting activity of the enzyme.
  • the metformin effects on hypothalamic AMPK activity will
    • counteractT3 effects at the hypothalamic level.
  • AMPK therefore represents a direct target for dual regulation
    • in the hypothalamic partitioning of energy homeostasis.
  • metformin crossesthe blood–brain barrier and
    • levels in the pituitary gland are substantially increased.
  • It convincinglysuppresses TSH

A recent study recruiting 66 patients with benign thyroid nodules furthermore
demonstrated that metformin significantly decreases nodule size in patients with
insulin resistance.[76] The effect of metformin, which was produced over a
6-month treatment period, parallelled a fall in TSH concentrations and achieved a
shrinkage amounting to 30% of the initial nodule size when metformin was
administered alone and up to 55% when it was added to ongoing LT4 treatment.

These studies reveal a

  • suppressive effect of metformin on TSH secretion patterns in
    hypothyroid patients, an effect that is apparently
  • independent of T4 treatment and does not alter the TH profile.
  • A rebound of TSH secretion occurs at about 3 months following metformin

It appears that recommendations for more frequent testing, on an annual to
biannual basis, seems justified in higher risk groups like patients over 50 or 55,
particularly with suggestive symptoms, raised antibody titres or dylipidaemia.
We thus would support the suggestion of an initial TSH and TPO antibody testing
which, as discussed, will help to predict the development of hypothyroidism in
patients with diabetes.

Hypothalamic AMPK and fatty acid metabolism mediate thyroid
regulation of energy 
M López,  L Varela,  MJ Vázquez,  S Rodríguez-Cuenca, CR González, …, & Vidal-Puig
Nature Medicine  29 Aug 2010; 16: 1001–1008 http://dx.doi.org:/10.1038/nm.2207

Thyroid hormones have widespread cellular effects; however it is unclear whether
their effects on the central nervous system (CNS) contribute to global energy balance.
Here we demonstrate that either

  • whole-body hyperthyroidism or central administration of triiodothyronine
    (T3) decreases

    • the activity of hypothalamic AMP-activated protein kinase (AMPK),
    • increases sympathetic nervous system (SNS) activity and
    • upregulates thermogenic markers in brown adipose tissue (BAT).

Inhibition of the lipogenic pathway in the ventromedial nucleus of the hypothalamus
(VMH) prevents CNS-mediated activation of BAT by thyroid hormone and reverses
the weight loss associated with hyperthyroidism. Similarly, inhibition of thyroid
hormone receptors in the VMH reverses the weight loss associated with hyperthyroidism.

This regulatory mechanism depends on AMPK inactivation, as genetic inhibition of this
enzyme in the VMH of euthyroid rats induces feeding-independent weight loss and
increases expression of thermogenic markers in BAT. These effects are reversed by
pharmacological blockade of the SNS. Thus, thyroid hormone–induced modulation
of AMPK activity and lipid metabolism in the hypothalamus is a major regulator of
whole-body energy homeostasis.

Metabolic Basis for Thyroid Hormone Liver Preconditioning:
Upregulation of AMP-Activated Protein Kinase Signaling
LA Videla,1 V Fernández, P Cornejo, and R Vargas
1Molecular and Clinical Pharmacology Program, Institute of Biomedical Sciences,
Faculty of Medicine, University of Chile, 2Faculty of Medicine, Diego Portales University,
Santiago, Chile
Academic Editors: H. M. Abu-Soud and D. Benke
The Scientific World Journal 2012; 2012, ID 475675, 10 pp

The liver is a major organ responsible for most functions of cellular metabolism and

  • a mediator between dietary and endogenous sources of energy for extrahepatic tissues.
  • In this context, adenosine-monophosphate- (AMP-) activated protein kinase (AMPK)
    constitutes an intrahepatic energy sensor
  • regulating physiological energy dynamics by limiting anabolism and stimulating
    catabolism, thus increasing ATP availability.
  • This is achieved by mechanisms involving direct allosteric activation and
    reversible phosphorylation of AMPK, in response to signals such as

    • energy status,
    • serum insulin/glucagon ratio,
    • nutritional stresses,
    • pharmacological and natural compounds, and
    • oxidative stress status.

Reactive oxygen species (ROS) lead to cellular AMPK activation and

  • downstream signaling under several experimental conditions.

Thyroid hormone (L-3,3′,5-triiodothyronine, T3) administration, a condition
that enhances liver ROS generation,

  • triggers the redox upregulation of cytoprotective proteins
    • affording preconditioning against ischemia-reperfusion (IR) liver injury.

Data discussed in this work suggest that T3-induced liver activation of AMPK

  • may be of importance in the promotion of metabolic processes
  • favouring energy supply for the induction and operation of preconditioning

These include

  1. antioxidant,
  2. antiapoptotic, and
  3. anti-inflammatory mechanisms,
  4. repair or resynthesis of altered biomolecules,
  5. induction of the homeostatic acute-phase response, and
  6. stimulation of liver cell proliferation,

which are required to cope with the damaging processes set in by IR.

The liver functions as a mediator between dietary and endogenous sources
of energy and extrahepatic organs that continuously require energy, mainly
the brain and erythrocytes, under cycling conditions between fed and fasted states.

In the fed state, where insulin action predominates, digestion-derived glucose is
converted to pyruvate via glycolysis, which is oxidized to produce energy, whereas
fatty acid oxidation is suppressed. Excess glucose can be either stored as hepatic
glycogen or channelled into de novo lipogenesis.

In the fasted state, considerable liver fuel metabolism changes occur due to decreased
serum insulin/glucagon ratio, with higher glucose production as a consequence of
stimulated glycogenolysis and gluconeogenesis (from alanine, lactate, and glycerol).

Major enhancement in fatty acid oxidation also occurs to provide energy for liver
processes and ketogenesis to supply metabolic fuels for extrahepatic tissues. For these
reasons, the liver is considered as the metabolic processing organ of the body, and
alterations in liver functioning affect whole-body metabolism and energy homeostasis.

In this context, adenosine-monophosphate- (AMP-) activated protein kinase (AMPK)
is the downstream component of a protein kinase cascade acting as an

  • intracellular energy sensor regulating physiological energy dynamics by
  • limiting anabolic pathways, to prevent excessive adenosine triphosphate (ATP)
    utilization, and
  • by stimulating catabolic processes, to increase ATP production.

Thus, the understanding of the mechanisms by which liver AMPK coordinates hepatic
energy metabolism represents a crucial point of convergence of regulatory signals
monitoring systemic and cellular energy status

Liver AMPK: Structure and Regulation

AMPK, a serine/threonine kinase, is a heterotrimeric complex comprising

  1. a catalytic subunit α and
  2. two regulatory subunits β and γ .

The α subunit has a threonine residue (Thr172) within the activation loop of the kinase
domain, with the C-terminal region being required for association with β and γ subunits.
The β subunit associates with α and γ by means of its C-terminal region , whereas

  • the γ subunit has four cystathionine β-synthase (CBS) motifs, which
  • bind AMP or ATP in a competitive manner.

75675.fig.001 (not shown)

Figure 1: Regulation of AMP-activated protein kinase (AMPK) by
(A) direct allosteric activation and
(B) reversible phosphorylation and downstream responses maintaining
intracellular energy balance.

Regulation of liver AMPK activity involves both direct allosteric activation and
reversible phosphorylation. AMPK is allosterically activated by AMP through

  • binding to the regulatory subunit-γ, which induces a conformational change in
    the kinase domain of subunit α that protects AMPK from dephosphorylation
    of Thr172, probably by protein phosphatase-2C.

Activation of AMPK requires phosphorylation of Thr172 in its α subunit, which can be
attained by either

(i) tumor suppressor LKB1 kinase following enhancement in the AMP/ATP ratio, a
kinase that plays a crucial role in AMPK-dependent control of liver glucose and
lipid metabolism;

(ii) Ca2+-calmodulin-dependent protein kinase kinase-β (CaMKKβ) that
phosphorylates AMPK in an AMP-independent, Ca2+-dependent manner;

(iii) transforming growth-factor-β-activated kinase-1 (TAK1), an important
kinase in hepatic Toll-like receptor 4 signaling in response to lipopolysaccharide.

Among these kinases, the relevance of CaMKKβ and TAK1 in liver AMPK activation
remains to be established in metabolic stress conditions. Both allosteric and
phosphorylation mechanisms are able to elicit

  • over 1000-fold increase in AMPK activity, thus allowing
  • the liver to respond to small changes in energy status in a highly sensitive fashion.

In addition to rapid AMPK regulation through allosterism and reversible phosphorylation

  • long-term effects of AMPK activation induce changes in hepatic gene expression.

This was demonstrated for

(i) the transcription factor carbohydrate-response element-binding protein (ChREBP),

  • whose Ser568 phosphorylation by activated AMPK
  • blocks its DNA binding capacity and glucose-induced gene transcription
  • under hyperlipidemic conditions;(ii) liver sterol regulatory element-binding
    protein-1c (SREBP-1c), whose mRNA and protein expression and those of
    its target gene for fatty acid synthase (FAS)
  • are reduced by metformin-induced AMPK activation,
  • decreasing lipogenesis and increasing fatty acid oxidation due to
    malonyl-CoA depletion;

(iii) transcriptional coactivator transducer of regulated CREB activity-2 (TORC2),
a crucial component of the hepatic gluconeogenic program, was reported
to be phosphorylated by activated AMPK.

This modification leads to subsequent cytoplasmatic sequestration of TORC2 and
inhibition of gluconeogenic gene expression, a mechanism underlying

  • the plasma glucose-lowering effects of adiponectin and metformin
  • through AMPK activation by upstream LKB1.

Activation of AMPK in the liver is a key regulatory mechanism controlling glucose
and lipid metabolism,

  1. inhibiting anabolic processes, and
  2. enhancing catabolic pathways in response to different signals, including
    1. energy status,
    2. serum insulin/glucagon ratio,
    3. nutritional stresses,
    4. pharmacological and natural compounds, and
    5. oxidative stress status

Reactive Oxygen Species (ROS) and AMPK Activation

The high energy demands required to cope with all the metabolic functions
of the liver are met by

  • fatty acid oxidation under conditions of both normal blood glucose levels and
    hypoglycemia, whereas
  • glucose oxidation is favoured in hyperglycemic states, with consequent
    generation of ROS.

Under normal conditions, ROS occur at relatively low levels due to their fast processing
by antioxidant mechanisms, whereas at acute or prolonged high ROS levels, severe
oxidation of biomolecules and dysregulation of signal transduction and gene expression
is achieved, with consequent cell death through necrotic and/or apoptotic-signaling

Thyroid Hormone (L-3,3′,5-Triiodothyronine, T3), Metabolic Regulation,
and ROS Production

T3 is important for the normal function of most mammalian tissues, with major actions
on O2 consumption and metabolic rate, thus

  • determining enhancement in fuel consumption for oxidation processes
  • and ATP repletion.

T3 acts predominantly through nuclear receptors (TR) α and β, forming

  • functional complexes with retinoic X receptor that
  • bind to thyroid hormone response elements (TRE) to activate gene expression.

T3 calorigenesis is primarily due to the

  • induction of enzymes related to mitochondrial electron transport and ATP
    synthesis, catabolism, and
  • some anabolic processes via upregulation of genomic mechanisms.

The net result of T3 action is the enhancement in the rate of O2 consumption of target
tissues such as liver, which may be effected by secondary processes induced by T3

(i) energy expenditure due to higher active cation transport,

(ii) energy loss due to futile cycles coupled to increase in catabolic and anabolic pathways, and

(iii) O2 equivalents used in hepatic ROS generation both in hepatocytes and Kupffer cells

In addition, T3-induced higher rates of mitochondrial oxidative phosphorylation are
likely to induce higher levels of ATP, which are partially balanced by intrinsic uncoupling
afforded by induction of uncoupling proteins by T3. In agreement with this view, the
cytosolic ATP/ADP ratio is decreased in hyperthyroid tissues, due to simultaneous
stimulation of ATP synthesis and consumption.

Regulation of fatty acid oxidation is mainly attained by carnitine palmitoyltransferase Iα (CPT-Iα),

  • catalyzing the transport of fatty acids from cytosol to mitochondria for β-oxidation,
    and acyl-CoA oxidase (ACO),
  • catalyzing the first rate-limiting reaction of peroxisomal β-oxidation, enzymes that are
    induced by both T3 and peroxisome proliferator-activated receptor α (PPAR-α).

Furthermore, PPAR-α-mediated upregulation of CPT-Iα mRNA is enhanced by PPAR-γ
coactivator 1α (PGC-1α), which in turn

  • augments T3 induction of CPT-Iα expression.

Interestingly, PGC-1α is induced by

  1. T3,
  2. AMPK activation, and
  3. ROS,

thus establishing potential links between

  • T3 action, ROS generation, and AMPK activation

with the onset of mitochondrial biogenesis and fatty acid β-oxidation.

Liver ROS generation leads to activation of the transcription factors

  1. nuclear factor-κB (NF-κB),
  2. activating protein 1 (AP-1), and
  3. signal transducer and activator of transcription 3 (STAT3)

at the Kupffer cell level, with upregulation of cytokine expression (TNF-α, IL-1, IL-6),
which upon interaction with specific receptors in hepatocytes trigger the expression of

  1. cytoprotective proteins (Figure 3(A)).

These responses and the promotion of hepatocyte and Kupffer-cell proliferation
represent hormetic effects reestablishing

  1. redox homeostasis,
  2. promoting cell survival, and
  3. protecting the liver against ischemia-reperfusion injury.

T3 liver preconditioning also involves the activation of the

  1. Nrf2-Keap1 defense pathway
  • upregulating antioxidant proteins,
  • phase-2 detoxifying enzymes, and
  • multidrug resistance proteins, members of the ATP binding cassette (ABC)
    superfamily of transporters (Figure 3(B))

In agreement with T3-induced liver preconditioning, T3 or L-thyroxin afford
preconditioning against IR injury in the heart, in association with

  • activation of protein kinase C and
  • attenuation of p38 and
  • c-Jun-N-terminal kinase activation ,

and in the kidney, in association with

  • heme oxygenase-1 upregulation.



Figure 2: Calorigenic response of thyroid hormone (T3) and its relationship with O2
consumption, reactive oxygen species (ROS) generation, and antioxidant depletion in the liver.
Abbreviations: CYP2E1, cytochrome P450 isoform 2E1; GSH, reduced glutathione; QO2, rate
of O2 consumption; SOD, superoxide dismutase.


genomic signaling in T3 calorigenesis and ROS production 475675.fig.003

genomic signaling in T3 calorigenesis and ROS production 475675.fig.003


Figure 3: Genomic signaling mechanisms in T3 calorigenesis and liver reactive oxygen
species (ROS) production leading to
(A) upregulation of cytokine expression in Kupffer cells and hepatocyte activation of genes
conferring cytoprotection,
(B) Nrf2 activation controling expression of antioxidant and detoxication proteins, and
(C) activation of the AMPK cascade regulating metabolic functions.

Abbreviations: AP-1, activating protein 1; ARE, antioxidant responsive element; CaMKKβ,
Ca2+-calmodulin-dependent kinase kinase-β; CBP, CREB binding protein; CRC, chromatin
remodelling complex; EH, epoxide hydrolase; HO-1, hemoxygenase-1; GC-Ligase,
glutamate cysteine ligase; GPx, glutathione peroxidase; G-S-T, glutathione-S-transferase;
HAT, histone acetyltransferase; HMT, histone arginine methyltransferase; IL1,
interleukin 1; iNOS, inducible nitric oxide synthase; LKB1, tumor suppressor LKB1 kinase;
MnSOD, manganese superoxide dismutase; MRPs, multidrug resistance proteins; NF-κB,
nuclear factor-κB; NQO1, NADPH-quinone oxidoreductase-1; NRF-1, nuclear respiratory
factor-1; Nrf2, nuclear receptor-E2-related factor 2; PCAF, p300/CBP-associated
factor; RXR, retinoic acid receptor; PGC-1, peroxisome proliferator-activated receptor-γ
coactivator-1; QO2, rate of O2 consumption; STAT3, signal transducer and activator
of transcription 3; TAK1, transforming-growth-factor-β-activated kinase-1; TNF-α, tumor
necrosis factor-α; TR, T 3 receptor; TRAP, T3-receptor-associated protein; TRE,  T3 responsive element; UCP, uncoupling proteins; (—), reported mechanisms;
(- - - -), proposed mechanisms.


T3 is a key metabolic regulator coordinating short-term and long-term energy needs,
with major actions on liver metabolism. These include promotion of

(i) gluconeogenesis and hepatic glucose production, and

(ii) fatty acid oxidation coupled to enhanced adipose tissue lipolysis, with

  • higher fatty acid flux to the liver and
  • consequent ROS production (Figure 2) and
  • redox upregulation of cytoprotective proteins

affording liver preconditioning (Figure 3).

Thyroid Hormone and AMPK Activation: Skeletal Muscle and Heart

In skeletal muscle, T3 increases the levels of numerous proteins involved in

  1. glucose uptake (GLUT4),
  2. glycolysis (enolase, pyruvate kinase, triose phosphate isomerase),
  3. fatty acid oxidation (carnitine palmitoyl transferase-1, mitochondrial thioesterase I),
    and uncoupling protein-3,

effects that are achieved through enhanced transcription of TRE-containing genes

Skeletal muscle AMPK activation is characterized by

(i) being a rapid and transient response,

(ii) upstream activation by Ca2+-induced mobilization and CaMKKβ activation,

(iii) upstream upregulation of LKB1 expression, which requires association with STRAD
and MO25 for optimal phosphorylation/activation of AMPK, and

(iv) stimulation of mitochondrial fatty acid β-oxidation.

T3-induced muscle AMPK activation was found to trigger two major downstream

signaling pathways, namely,

(i) peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) mRNA
expression and phosphorylation, a transcriptional regulator for genes related to

  • mitochondrial biogenesis,
  • fatty acid oxidation, and
  • gluconeogenesis and

(ii) cyclic AMP response element binding protein (CREB) phosphorylation, which

  • in turn induces PGC-1α expression in liver tissue, thus
  • reinforcing mechanism (i).

These data indicate that AMPK phosphorylation of PGC-1α initiates many of the
important gene regulatory functions of AMPK in skeletal muscle.

In heart, hyperthyroidism increased glycolysis and sarcolemmal GLUT4 levels by the
combined effects of AMPK activation and insulin stimulation, with concomitant increase
in fatty acid oxidation proportional to enhanced cardiac mass and contractile function.

Thyroid Hormone, AMPK Activation, and Liver Preconditioning

Recent studies by our group revealed that administration of a single dose of 0.1 mg T3/kg
to rats activates liver AMPK (Figure 4; unpublished work).

  1. enhancement in phosphorylated AMPK/nonphosphorylated AMPK ratios in T3-
    treated rats over control values thatis significant in the time period of 1 to 48
    hours after hormone treatment
  2. Administration of a substantially higher dose (0.4 mg T3/kg) resulted in
    decreased liver AMPK activation at 4 h to return to control values at 6 h
    after treatment

Activation of liver AMPK by T3 may be of relevance in terms of

  • promotion of fatty acid oxidation for ATP supply,
  • supporting hepatoprotection against IR injury (Figure 3(C)).

This proposal is based on the high energy demands underlying effective liver
preconditioning for full operation of hepatic

  • antioxidant, antiapoptotic, and anti-inflammatory mechanisms,
  • oxidized biomolecules repair or resynthesis,
  • induction of the homeostatic acute-phase response, and
  • promotion of hepatocyte and Kupffer cell proliferation,

mechanisms that are needed to cope with the damaging processes set in by IR.
T3 liver preconditioning , in addition to that afforded by

  • n-3 long-chain polyunsaturated fatty acids given alone or
  • combined with T3 at lower dosages, or
  • by iron supplementation,

constitutes protective strategies against hepatic IR injury.

Studies on the molecular mechanisms underlying T3-induced liver AMPK
activation (Figure 4) are currently under assessment in our laboratory.


Fernández and L. A. Videla, “Kupffer cell-dependent signaling in thyroid hormone
calorigenesis: possible applications for liver preconditioning,” Current Signal
Transduction Therapy 2009; 4(2): 144–151.

Viollet, B. Guigas, J. Leclerc et al., “AMP-activated protein kinase in the regulation
of  hepatic energy metabolism: from physiology to therapeutic perspectives,” Acta
Physiologica 2009; 196(1): 81–98.

Carling, “The AMP-activated protein kinase cascade – A unifying system
for energy control,” Trends in Biochemical Sciences, 2004;. 29(1): 18–24.

E. Kemp, D. Stapleton, D. J. Campbell et al., “AMP-activated protein kinase,
metabolic regulator,” Biochemical Society Transactions 2003; 31(1):

G. Hardie, “AMP-activated protein kinase-an energy sensor that
regulates all ;aspects of cell function,” Genes and Development,
2011; 25(18): 1895–1908.

Woods, P. C. F. Cheung, F. C. Smith et al., “Characterization of AMP-activated
protein kinase βandγ subunits Assembly of the heterotrimeric complex in vitro,”
Journal of Biological Chemistry 1996;271(17): 10282–10290.

Xiao, R. Heath, P. Saiu et al., “Structural basis for AMP binding to mammalian AMP-
activated protein kinase,” Nature 2007; 449(7161): 496–500.


Impact of Metformin and compound C on NIS expression and iodine uptake in vitro and in vivo: a role for CRE in AMPK modulation of thyroid function.
Abdulrahman RM1, Boon MRSips HCGuigas BRensen PCSmit JWHovens GC.
Author information 
Thyroid. 2014 Jan;24(1):78-87.  Epub 2013 Sep 25.  PMID: 23819433

Although adenosine monophosphate activated protein kinase (AMPK) plays a crucial role
in energy metabolism, a direct effect of AMPK modulation on thyroid function has only
recently been reported, and much of its function in the thyroid is currently unknown.

The aim of this study was

  1. to investigate the mechanism of AMPK modulation in iodide uptake.
  2. to investigate the potential of the AMPK inhibitor compound C as an enhancer of
    iodide uptake by thyrocytes.

Metformin reduced NIS promoter activity (0.6-fold of control), whereas compound C
stimulated its activity (3.4-fold) after 4 days. This largely coincides with

  • CRE activation (0.6- and 3.0-fold).

These experiments show that AMPK exerts its effects on iodide uptake, at least partly,
through the CRE element in the NIS promoter. Furthermore, we have used AMPK-alpha1
knockout mice to determine the long-term effects of AMPK inhibition without chemical compounds.
These mice have a less active thyroid, as shown by reduced colloid volume and reduced
responsiveness to thyrotropin.

NIS expression and iodine uptake in thyrocytes

  • can be modulated by metformin and compound C.

These compounds exert their effect by

  • modulation of AMPK, which, in turn, regulates
  • the activation of the CRE element in the NIS promoter.

Overall, this suggests that AMPK modulating compounds may be useful for the
enhancement of iodide uptake by thyrocytes, which could be useful for the
treatment of thyroid cancer patients with radioactive iodine.

AMPK: Master Metabolic Regulator

© 1996–2013 themedicalbiochemistrypage.org, LLC | info
@ themedicalbiochemistrypage.org

AMPK-activating drugs metformin or phenformin might provide protection against cancer 1741-7007-11-36-5

AMPK-activating drugs metformin or phenformin might provide protection against cancer 1741-7007-11-36-5


AMPK and AMPK-related kinase (ARK) family  1741-7007-11-36-4

AMPK and AMPK-related kinase (ARK) family 1741-7007-11-36-4


central role of AMPK in the regulation of metabolism



AMP-activated protein kinase (AMPK) was first discovered as an activity that

AMPK induces a cascade of events within cells in response to the ever changing energy
charge of the cell. The role of AMPK in regulating cellular energy charge places this
enzyme at a central control point in maintaining energy homeostasis.

More recent evidence has shown that AMPK activity can also be regulated by physiological stimuli, independent of the energy charge of the cell, including hormones and nutrients.


Once activated, AMPK-mediated phosphorylation events

These events are rapidly initiated and are referred to as

  • short-term regulatory processes.

The activation of AMPK also exerts

  • long-term effects at the level of both gene expression and protein synthesis.

Other important activities attributable to AMPK are

  1. regulation of insulin synthesis and
  2. secretion in pancreatic islet β-cells and
  3. modulation of hypothalamic functions involved in the regulation of satiety.

How these latter two functions impact obesity and diabetes will be discussed below.

Regulation of AMPK

In the presence of AMP the activity of AMPK is increased approximately 5-fold.
However, more importantly is the role of AMP in regulating the level of phosphorylation
of AMPK. An increased AMP to ATP ratio leads to a conformational change in the γ-subunit
leading to increased phosphorylation and decreased dephosphorylation of AMPK.

The phosphorylation of AMPK results in activation by at least 100-fold. AMPK is
phosphorylated by at least three different upstream AMPK kinases (AMPKKs).
Phosphorylation of AMPK occurs in the α subunit at threonine 172 (T172) which

  • lies in the activation loop.

One kinase activator of AMPK is

  • Ca2+-calmodulin-dependent kinase kinase β (CaMKKβ)
  • which phosphorylates and activates AMPK in response to increased calcium.

The distribution of CaMKKβ expression is primarily in the brain with detectable levels
also found in the testes, thymus, and T cells. As described for the Ca2+-mediated
regulation of glycogen metabolism,

  • increased release of intracellular stores of Ca2+ create a subsequent demand for

Activation of AMPK in response to Ca fluxes

  • provides a mechanism for cells to anticipate the increased demand for ATP.

Evidence has also demonstrated that the serine-threonine kinase, LKB1 (also called
serine-threonine kinase 11, STK11) which is encoded by the Peutz-Jeghers syndrome
tumor suppressor gene, is required for activation of AMPK in response to stress.

The active LKB1 kinase is actually a complex of three proteins:

  1. LKB1,
  2. Ste20-related adaptor (STRAD) and
  3. mouse protein 25 (MO25).

Thus, the enzyme complex is often referred to as LKB1-STRAD-MO25. Phosphorylation
of AMPK by LKB1 also occurs on T172. Unlike the limited distribution of CaMKKβ,

  • LKB1 is widely expressed, thus making it the primary AMPK-regulating kinase.

Loss of LKB1 activity in adult mouse liver leads to

  • near complete loss of AMPK activity and
  • is associated with hyperglycemia.

The hyperglycemia is, in part, due to an increase in the transcription of gluconeogenic
genes. Of particular significance is the increased expression of

  • the peroxisome proliferator-activated receptor-γ (PPAR-γ) coactivator 1α
    (PGC-1α), which drives gluconeogenesis.
  • Reduction in PGC-1α activity results in normalized blood glucose levels in
    LKB1-deficient mice.

The third AMPK phosphorylating kinase is transforming growth factor-β-activated
kinase 1 (TAK1). However, the normal physiological conditions under which TAK1
phosphorylates AMPK are currently unclear.

The effects of AMP are two-fold:

  1. a direct allosteric activation and making AMPK a poorer substrate for

Because AMP affects both
the rate of AMPK phoshorylation in the positive direction and
dephosphorylation in the negative direction,

the cascade is ultrasensitive. This means that

  1. a very small rise in AMP levels can induce a dramatic increase in the activity of

The activity of adenylate kinase, catalyzing the reaction shown below, ensures that

  • AMPK is highly sensitive to small changes in the intracellular [ATP]/[ADP] ratio.

2 ADP ——> ATP + AMP

Negative allosteric regulation of AMPK also occurs and this effect is exerted by
phosphocreatine. As indicated above, the β subunits of AMPK have a glycogen-binding domain, GBD. In muscle, a high glycogen content

  • represses AMPK activity and
  • this is likely the result of interaction between the GBD and glycogen,
  • the GBD of AMPK allows association of the enzyme with the regulation of glycogen metabolism
  • by placing AMPK in close proximity to one of its substrates glycogen synthase.

AMPK has also been shown to be activated by receptors that are coupled to

  • phospholipase C-β (PLC-β) and by
  • hormones secreted by adipose tissue (termed adipokines) such as leptinand adiponectin (discussed below).

Targets of AMPK

The signaling cascades initiated by the activation of AMPK exert effects on

  • glucose and lipid metabolism,
  • gene expression and
  • protein synthesis.

These effects are most important for regulating metabolic events in the liver, skeletal
muscle, heart, adipose tissue, and pancreas.

Demonstration of the central role of AMPK in the regulation of metabolism in response
to events such as nutrient- or exercise-induced stress. Several of the known physiologic
targets for AMPK are included as well as several pathways whose flux is affected by
AMPK activation. Arrows indicate positive effects of AMPK, whereas, T-lines indicate
the resultant inhibitory effects of AMPK action.

The uptake, by skeletal muscle, accounts for >70% of the glucose removal from the
serum in humans. Therefore, it should be obvious that this event is extremely important
for overall glucose homeostasis, keeping in mind, of course, that glucose uptake by
cardiac muscle and adipocytes cannot be excluded from consideration. An important fact
related to skeletal muscle glucose uptake is that this process is markedly impaired in
individuals with type 2 diabetes.

The uptake of glucose increases dramatically in response to stress (such as ischemia) and
exercise and is stimulated by insulin-induced recruitment of glucose transporters
to the plasma membrane, primarily GLUT4. Insulin-independent recruitment of glucose
transporters also occurs in skeletal muscle in response to contraction (exercise).

The activation of AMPK plays an important, albeit not an exclusive, role in the induction of
GLUT4 recruitment to the plasma membrane. The ability of AMPK to stimulate
GLUT4 translocation to the plasma membrane in skeletal muscle is by a different mechanism
than that stimulated by insulin and insulin and AMPK effects are additive.

Under ischemic/hypoxic conditions in the heart the activation of AMPK leads to the
phosphorylation and activation of the kinase activity of phosphofructokinase-2, PFK-2
(6-phosphofructo-2-kinase). The product of the action of PFK-2 (fructose-2,6-bisphosphate,
F2,6BP) is one of the most potent regulators of the rate of flux through
glycolysis and gluconeogenesis.

In liver the PKA-mediated phosphorylation of PFK-2 results in conversion of the
enzyme from a kinase that generates F2,6BP to a phosphatase that removes the
2-phosphate thus reducing the levels of the potent allosteric activator of the glycolytic
enzyme 6-phosphfructo-1-kinase, PFK-1 and the potent allosteric inhibitor
of the gluconeogenic enzyme fructose-1,6-bisphosphatase (F1,-6BPase).

It is important to note that like many enzymes, there are multiple isoforms of PFK-2
(at least 4) and neither the liver or the skeletal muscle isoforms contain the AMPK
phosphorylation sites found in the cardiac and inducible (iPFK2) isoforms of PFK-2.

Inducible PFK-2 is expressed in the monocyte/macrophage lineage in response to pro-
inflammatory stimuli. The ability to activate the kinase activity by phosphorylation of
PFK-2 in cardiac tissue and macrophages in response to ischemic conditions allows these
cells to continue to have a source of ATP via anaerobic glycolysis. This phenomenon is
recognized as the Pasteur effect: an increased rate of glycolysis in response to hypoxia.

Of pathological significance is the fact that the inducible form of PFK-2 is commonly
expressed in many tumor cells and this may allow AMPK to play an important role in
protecting tumor cells from hypoxic stress. Indeed, techniques for depleting AMPK in
tumor cells have shown that these cells become sensitized to nutritional stress upon loss
of AMPK activity.

Whereas, stress and exercise are powerful inducers of AMPK activity in skeletal muscle,
additional regulators of its activity have been identified.

Insulin-sensitizing drugs of the thiazolidinedione family (activators of PPAR-γ, see
below) as well as the hypoglycemia drug metformin exert a portion of their effects
through regulation of the activity of AMPK.

As indicated above, the activity of the AMPK activating kinase, LKB1, is critical for
regulating gluconeogenic flux and consequent glucose homeostasis. The action of
metformin in reducing blood glucose levels

  • requires the activity of LKB1 in the liver for this function.

Also, several adipokines (hormones secreted by adipocytes) either stimulate or inhibit
AMPK activation:

  1. leptin and adiponectin have been shown to stimulate AMPK activation, whereas,
  2. resistininhibits AMPK activation.

Cardiac effects exerted by activation of AMPK also include

AMPK-mediated phosphorylation of eNOS leads to increased activity and consequent
NO production and provides a link between metabolic stresses and cardiac function.

In platelets, insulin action leads to an increase in eNOS activity that is

  • due to its phosphorylation by AMPK.

Activation of NO production in platelets leads to

  • a decrease in thrombin-induced aggregation, thereby,
  • limiting the pro-coagulant effects of platelet activation.

The response of platelets to insulin function clearly indicates why disruption in insulin
action is a major contributing factor in the development of the metabolic syndrome

Activation of AMPK leads to a reduction in the level of SREBP

  • a transcription factor &regulator of the expression of numerous
    lipogenic enzymes

Another transcription factor reduced in response to AMPK activation is

  • hepatocyte nuclear factor 4α, HNF4α
    • a member of the steroid/thyroid hormone superfamily.
    • HNF4α is known to regulate the expression of several liver and
      pancreatic β-cell genes such as GLUT2, L-PK and preproinsulin.
  • Of clinical significance is that mutations in HNF4α are responsible for
    • maturity-onset diabetes of the young, MODY-1.

Recent evidence indicates that the gene for the carbohydrate-response-element-
binding protein (ChREBP) is a target for AMPK-mediated transcriptional regulation
in the liver. ChREBP is rapidly being recognized as a master regulator of lipid
metabolism in liver, in particular in response to glucose uptake.

The target of the thiazolidinedione (TZD) class of drugs used to treat type 2 diabetes is
the peroxisome proliferator-activated receptor γPPARγ which

  • itself may be a target for the action of AMPK.

The transcription co-activator, p300, is phosphorylated by AMPK

  • which inhibits interaction of p300 with not only PPARγ but also
  • the retinoic acid receptor, retinoid X receptor, and
  • thyroid hormone receptor.

PPARγ is primarily expressed in adipose tissue and thus it was difficult to reconcile how
a drug that was apparently acting only in adipose tissue could lead to improved insulin
sensitivity of other tissues. The answer to this question came when it was discovered that the TZDs stimulated the expression and release of the adipocyte hormone (adipokine),
adiponectin. Adiponectin stimulates glucose uptake and fatty acid oxidation in skeletal
muscle. In addition, adiponectin stimulates fatty acid oxidation in liver while inhibiting
expression of gluconeogenic enzymes in this tissue.

These responses to adiponectin are exerted via activation of AMPK. Another
transcription factor target of AMPK is the forkhead protein, FKHR (now referred to as
FoxO1). FoxO1 is involved in the activation of glucose-6-phosphatase expression and,
therefore, loss of FoxO1 activity in response to AMPK activation will lead to reduced
hepatic output of glucose.

This concludes a very complicated perspective that ties together the thyroid hormone
activity, the hypophysis, diabetes mellitus, and AMPK tegulation of metabolism in the
liver, skeletal muscle, adipose tissue, and heart.  I also note at this time that there
nongenetic points to be made here:

  1. The tissue specificity of isoenzymes
  2. The modulatory role of AMP:ATP ratio in phosphorylation/dephosphorylation
    effects on metabolism tied to AMPK
  3. The tie in of stress or ROS with fast reactions to protect harm to tissues
  4. The relationship of cytokine activation and release to the above metabolic events
  5. The relationship of effective and commonly used diabetes medications to AMPK
    mediated processes
  6. The preceding presentation is notable for the importance of proteomic and
    metabolomic invetigations in elucidation common chronic and nongenetic diseases


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Larry H. Bernstein, MD, FCAP, Curator

Leaders in Pharmaceutical Intelligence

Early discharge using single cardiac troponin and copeptin testing in patients
with suspected 
acute coronary syndrome (ACS): a randomized, controlled
clinical process study
M Mockel, J Searle, Christian Hamm, A Slagman, S Blankenberg, et al.
EurHeartJ Apr 2014.  http://dx.doi.org:/10.1093/eurheartj/ehu178

This randomized controlled trial (RCT) evaluated whether a process with single
combined testing of copeptin and troponin at admission in patients with low-to-
intermediate risk and suspected acute coronary syndrome (ACS)  does not lead to a higher proportion of major adverse cardiac events (MACE) than
the current standard process (non-inferiority design). After clinical work-up and  single combined testing of troponin and copeptin to rule-out AMI,  early  discharge
of low- to intermediate risk patients with suspected ACS seems to be safe and has
the potential to shorten length of stay in the ED.

Diagnostic accuracy of combined cardiac troponin and copeptin
assessment for 
early rule-out of myocardial infarction: a systematic
review and meta-analysis
T Raskovalova, R Twerenbold, PO Collinson, T Keller, H Bouvaist, et al.
EurHeartJ: Acute Cardiovascular Care 2014; 3(1): 18-27.

This systematic review aimed to investigate the diagnostic accuracy of combined
cardiac troponin (cTn) and  copeptin assessment in comparison to cTn alone for
early rule-out of acute myocardial infarction (AMI).  In 15  studies totalling 8740
patients (prevalence of   AMI 16%), adding copeptin improved the sensitivity
of cTn assays  (from 0.87 to 0.96, p=0.003) at the expense of lower specificity
(from 0.84 to 0.56, p<0.001).

In 12 studies providing for 6988 patients without ST-segment elevation,
the summary sensitivity and specificity  estimates were 0.95 (95% CI 0.89 to
0.98) and 0.57 (95% CI 0.49 to   0.65) for the combined assessment of cTn
and copeptin. When a high-sensitivity cTnT assay was used in combination
with copeptin,  the summary sensitivity  and specificity estimates were 0.98
(95% CI 0.96 to 1.00) and 0.50 (95% CI 0.42 to 0.58). The result indicates
that  copeptin significantly improves baseline cTn sensitivity.

Diagnostic accuracy of copeptin sensitivity and specificity in patients with
suspected non-ST-elevation myocardial infarction with troponin I below
99th centile at presentation
J Duchenne, S Mestres, N Dublanchet, N Combaret, G Marceau, et al.
BMJ Open 2014;4:e004449.

To our knowledge, our prospective multicentric study is the only one that includes
only patients with suspected non-ST-segment elevation myocardial infarction and
high-sensitive cardiac troponin I below  the 99th centile  at presentation to the
emergency department, to limit spectrum bias. Our study included only patients
with negative ultrasensitive troponin at admission. However, this is the only group
of patients for which a multimarker rule-out strategy could add diagnostic value.
Serial clinical, electrographical and biochemical investigations were performed at
admission and after 2, 4, 6 and 12 h. Hs-cTnT was measured using an assay with
Dimension VISTA, Siemens. Copeptin was measured by the BRAHMS copeptin-us
assay on the KRYPTOR Compact Plus system. The follow-up period was 90 days.

The final diagnosis was adjudicated blinded to copeptin result. During 12 months,
102 patients were analysed. Final diagnosis was NSTEMI for 7.8% (n=8), unstable
angina for 3.9% (n=4), cardiac but non coronary artery disease for 8.8% (n=9),
non-cardiac chest pain for 52% (n=53) and unknown for 27.5% (n=28).

There was no statistical difference for copeptin values between patients with
NSTEMI and others (respectively 5.5 pmol/L IQR (3.1–7.9) and 6.5 pmol/L IQR
(3.9–12.1), p=0.49). Only one patient with NSTEMI had a copeptin value
above the cut-off
 of 95th centile at admission.

In this study, copeptin does not add a diagnostic value at admission to ED for patients
with suspected acute coronary syndrome without ST-
segment elevation and with hs-cTnT below the 99th centile.

Can a Second Measurement of Copeptin Improve Acute Myocardial
Infarction Rule Out?

N Marston, K Shah, C Mueller, Sean-Xavier Neath, R Christenson, J McCord, et al.
J Am Coll Cardiol. 2014;63(12_S):A202.  Presentation Number: 1226-242

Of the 494 patients analyzed, 378 (76.5%) had a persistently elevated copeptin at 2
hours and 116 patients (23.5%) had a copeptin that fell below the cutoff of 14 pmol/l.
The AMI rate was 6.1% when the second copeptin was elevated compared to 0%
when the second copeptin was not (p=.006), yielding a sensitivity of 100%. This
strategy identified 23.5% of patients with an intermediate risk who could be ruled
out for AMI while still in the ED. In contrast, a second troponin measurement had a
sensitivity of 74%. A negative second copeptin drawn 2 hours after presentation
demonstrated 100% sensitivity for AMI, allowing for effective rule out in this
intermediate risk cohort. This strategy identified nearly 25% of intermediate risk
patients who could be considered for discharge.

 The role of copeptin as a diagnostic and prognostic biomarker for risk
stratification in the emergency department.
Nickel CH, Bingisser R and Morgenthaler NG
BMC Medicine 2012, 10:7

Copeptin, the C-terminal part of the arginine vasopressin precursor peptide,
is a sensitive and stable surrogate marker for arginine vasopressin release.
Measurement of copeptin levels has been shown to be useful in a variety of
clinical scenarios, particularly as a prognostic marker in patients with acute
diseases such as lower respiratory tract infection, heart disease and stroke.

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