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Happy 80th Birthday: Radioiodine (RAI) Theranostics: Collaboration between Physics and Medicine, the Utilization of Radionuclides to Diagnose and Treat: Radiation Dosimetry by Discoverer Dr. Saul Hertz, the early history of RAI in diagnosing and treating Thyroid diseases and Theranostics
Both authors contributed to the development, drafting and final editing of this manuscript and are responsible for its content.
Abstract
March 2021 will mark the eightieth anniversary of targeted radionuclide therapy, recognizing the first use of radioactive iodine to treat thyroid disease by Dr. Saul Hertz on March 31, 1941. The breakthrough of Dr. Hertz and collaborator physicist Arthur Roberts was made possible by rapid developments in the fields of physics and medicine in the early twentieth century. Although diseases of the thyroid gland had been described for centuries, the role of iodine in thyroid physiology had been elucidated only in the prior few decades. After the discovery of radioactivity by Henri Becquerel in 1897, rapid advancements in the field, including artificial production of radioactive isotopes, were made in the subsequent decades. Finally, the diagnostic and therapeutic use of radioactive iodine was based on the tracer principal that was developed by George de Hevesy. In the context of these advancements, Hertz was able to conceive the potential of using of radioactive iodine to treat thyroid diseases. Working with Dr. Roberts, he obtained the experimental data and implemented it in the clinical setting. Radioiodine therapy continues to be a mainstay of therapy for hyperthyroidism and thyroid cancer. However, Hertz struggled to gain recognition for his accomplishments and to continue his work and, with his early death in 1950, his contributions have often been overlooked until recently. The work of Hertz and others provided a foundation for the introduction of other radionuclide therapies and for the development of the concept of theranostics.
Dr. Saul Hertz was Director of The Massachusetts General Hospital’s Thyroid Unit, when he heard about the development of artificial radioactivity. He conceived and brought from bench to bedside the successful use of radioiodine (RAI) to diagnose and treat thyroid diseases. Thus was born the science of theragnostics used today for neuroendocrine tumors and prostate cancer. Dr. Hertz’s work set the foundation of targeted precision medicine.
Keywords: Dr. Saul Hertz, nuclear medicine, radioiodine
How to cite this article:
Hertz B. A tribute to Dr. Saul Hertz: The discovery of the medical uses of radioiodine. World J Nucl Med 2019;18:8-12
How to cite this URL:
Hertz B. A tribute to Dr. Saul Hertz: The discovery of the medical uses of radioiodine. World J Nucl Med [serial online] 2019 [cited 2021 Mar 2];18:8-12. Available from: http://www.wjnm.org/text.asp?2019/18/1/8/250309
Dr Saul Hertz (1905-1950) discovers the medical uses of radioiodine
Barbara Hertz, Pushan Bharadwaj, Bennett Greenspan»
Thyroid practitioners and patients are acutely aware of the enormous benefit nuclear medicine has made to mankind. This month we celebrate the 80th anniversary of the early use of radioiodine(RAI).
Dr. Saul Hertz predicted that radionuclides “…would hold the key to the larger problem of cancer in general,” and may just be the best hope for diagnosing and treating cancer successfully. Yes, RAI has been used for decades to diagnose and treat disease. Today’s “theranostics,” a term that is a combination of “therapy” and “diagnosis” is utilized in the treatment of thyroid disease and cancer.
This short note is to celebrate Dr. Saul Hertz who conceived and brought from bench to bedside the medical uses of RAI; then in the form of 25 minute iodine-128.
On March 31st 1941, Massachusetts General Hospital’s Dr. Saul Hertz (1905-1950) administered the first therapeutic use of Massachusetts Institute of Technology (MIT) cyclotron produced RAI. This landmark case was the first in Hertz’s clinical studies conducted with MIT, physicist Arthur Roberts, Ph.D.
[Photo – Courtesy of Dr Saul Hertz Archives ]
Dr Saul Hertz demonstrating RAI Uptake Testing
Dr. Hertz’s research and successful utilization of radionuclides to diagnose and treat diseases and conditions, established the use of radiation dosimetry and the collaboration between physics and medicine and other significant practices. Sadly, Saul Hertz (a WWII veteran) died at a very young age.
About Dr. Saul Hertz
Dr. Saul Hertz (1905 – 1950) discovered the medical uses of radionuclides. His breakthrough work with radioactive iodine (RAI) created a dynamic paradigym change integrating the sciences. Radioactive iodine (RAI) is the first and Gold Standard of targeted cancer therapies. Saul Hertz’s research documents Hertz as the first and foremost person to conceive and develop the experimental data on RAI and apply it in the clinical setting.
Dr. Hertz was born to Orthodox Jewish immigrant parents in Cleveland, Ohio on April 20, 1905. He received his A.B. from the University of Michigan in 1925 with Phi Beta Kappa honors. He graduated from Harvard Medical School in 1929 at a time of quotas for outsiders. He fulfilled his internship and residency at Mt. Sinai Hospital in Cleveland. He came back to Boston in 1931 as a volunteer to join The Massachusetts General Hospital serving as the Chief of the Thyroid Unit from 1931 – 1943.
Two years after the discovery of artifically radioactivity, on November 12, 1936 Dr. Karl Compton, president of the Massachusetts Institute of Technology (MIT), spoke at Harvard Medical School. President Compton’s topic was What Physics can do for Biology and Medicine. After the presentation Dr. Hertz spontaneously asked Dr. Compton this seminal question, “Could iodine be made radioactive artificially?” Dr. Compton responded in writing on December 15, 1936 that in fact “iodine can be made artificially radioactive.”
Shortly thereafter, a collaboration between Dr. Hertz (MGH) and Dr. Arthur Roberts, a physicist of MIT, was established. In late 1937, Hertz and Roberts created and produced animal studies involving 48 rabbits that demonstrated that the normal thyroid gland concentrated Iodine 128 (non cyclotron produced), and the hyperplastic thyroid gland took up even more Iodine. This was a GIANT step for Nuclear Medicine.
In early 1941, Dr. Hertz administer the first therapeutic treatment of MIT Markle Cyclotron produced radioactive iodine (RAI) at the Massachusetts General Hospital. This led to the first series of twenty-nine patients with hyperthyroidism being treated successfully with RAI. ( see “Research” RADIOACTIVE IODINE IN THE STUDY OF THYROID PHYSIOLOGY VII The use of Radioactive Iodine Therapy in Hyperthyroidism, Saul Hertz and Arthur Roberts, JAMA Vol. 31 Number 2).
In 1937, at the time of the rabbit studies Dr Hertz conceived of RAI in therapeutic treatment of thyroid carsonoma. In 1942 Dr Hertz gave clinical trials of RAI to patients with thyroid carcinoma.
After serving in the Navy during World War II, Dr. Hertz wrote to the director of the Mass General Hospital in Boston, Dr. Paxon on March 12, 1946, “it is a coincidence that my new research project is in Cancer of the Thyroid, which I believe holds the key to the larger problem of cancer in general.”
Dr. Hertz established the Radioactive Isotope Research Institute, in September, 1946 with a major focus on the use of fission products for the treatment of thyroid cancer, goiter, and other malignant tumors. Dr Samuel Seidlin was the Associate Director and managed the New York City facilities. Hertz also researched the influence of hormones on cancer.
Dr. Hertz’s use of radioactive iodine as a tracer in the diagnostic process, as a treatment for Graves’ disease and in the treatment of cancer of the thyroid remain preferred practices. Saul Hertz is the Father of Theranostics.
Saul Hertz passed at 45 years old from a sudden death heart attack as documented by an autopsy. He leaves an enduring legacy impacting countless generations of patients, numerous institutions worldwide and setting the cornerstone for the field of Nuclear Medicine. A cancer survivor emailed, The cure delivered on the wings of prayer was Dr Saul Hertz’s discovery, the miracle of radioactive iodine. Few can equal such a powerful and precious gift.
To read and hear more about Dr. Hertz and the early history of RAI in diagnosing and treating thyroid diseases and theranostics see –
Hertz S, Roberts A. Radioactive iodine in the study of thyroid physiology. VII The use of radioactive iodine therapy in hyperthyroidism. J Am Med Assoc 1946;131:81-6.
Hertz S. A plan for analysis of the biologic factors involved in experimental carcinogenesis of the thyroid by means of radioactive isotopes. Bull New Engl Med Cent 1946;8:220-4.
Thrall J. The Story of Saul Hertz, Radioiodine and the Origins of Nuclear Medicine. Available from: http://www.youtube.com/watch?v=34Qhm8CeMuc. [Last accessed on 2018 Dec 01].
Krolicki L, Morgenstern A, Kunikowska J, Koiziar H, Krolicki B, Jackaniski M, et al. Glioma Tumors Grade II/III-Local Alpha Emitters Targeted Therapy with 213 Bi-DOTA-Substance P, Endocrine Abstracts. Vol. 57. Society of Nuclear Medicine and Molecular Imaging; 2016. p. 632.
Baum RP, Kulkarni HP. Duo PRRT of neuroendocrine tumours using concurrent and sequential administration of Y-90- and Lu-177-labeled somatostatin analogues. In: Hubalewska-Dydejczyk A, Signore A, de Jong M, Dierckx RA, Buscombe J, Van de Wiel CJ, editors. Somatostatin Analogues from Research to Clinical Practice. New York: Wiley; 2015.
Research about marijuana and fertility is limited but some previous studies suggested that it might harm semen quality. Smoking of any type is also known to be a risk factor for male infertility. So, men who have smoked cannabis are expected to have worse measures of fertility but the data from a recent study suggested the opposite. The finding contradicts all conventional knowledge on how weed affects sperm. This may be because previous research typically focused on men with drug abuse history but this present study simply asked men if they had smoked more than two joints in their life.
Analysis of 1,143 semen samples from 662 men collected between 2000 and 2017 at the Fertility Clinic at Massachusetts General Hospital showed that those who had smoked weed at some point in their life had a mean sperm concentration of 62.7 million sperm per milliliter (mL) of ejaculate, while men who avoided marijuana entirely had mean concentrations of 45.4 million/mL. Added to this only 5% of weed smokers had sperm concentrations below the 15 million/mL threshold the World Health Organization has set for a “normal” sperm count, versus 12% of men who never smoked marijuana.
The study has some imperfections such as the participants are not necessarily representative of the general population. They were predominantly college educated men with a mean age of 36, and were all seeking treatment at a fertility center. Further research is needed to support the findings. Two possibilities are put forward by the researchers as the reason behind such data. The first is that low levels of marijuana could have a positive effect on the endocannabinoid system, the neurotransmitters in the nervous system that bind to cannabinoid receptors, and are known to regulate fertility. The second is that may be weed-smokers are just bigger risk takers and men with higher testosterone levels and thus have better sperm count.
But, there’s certainly no medical recommendation to smoke weed as a fertility treatment but this study, at least, suggests that a little marijuana doesn’t hurt and might benefit sperm production in some way. But, the researchers specified that their finding does not necessarily mean that smoking cannabis increases the chances of fatherhood.
Hypertriglyceridemia: Evaluation and Treatment Guideline
Reporter and Curator: Dr. Sudipta Saha, Ph.D.
Severe and very severe hypertriglyceridemia increase the risk for pancreatitis, whereas mild or moderate hypertriglyceridemia may be a risk factor for cardiovascular disease. Individuals found to have any elevation of fasting triglycerides should be evaluated for secondary causes of hyperlipidemia including endocrine conditions and medications. Patients with primary hypertriglyceridemia must be assessed for other cardiovascular risk factors, such as central obesity, hypertension, abnormalities of glucose metabolism, and liver dysfunction. The aim of this study was to develop clinical practice guidelines on hypertriglyceridemia.
The diagnosis of hypertriglyceridemia should be based on fasting levels, that mild and moderate hypertriglyceridemia (triglycerides of 150–999 mg/dl) be diagnosed to aid in the evaluation of cardiovascular risk, and that severe and very severe hypertriglyceridemia (triglycerides of >1000 mg/dl) be considered a risk for pancreatitis. The patients with hypertriglyceridemia must be evaluated for secondary causes of hyperlipidemia and that subjects with primary hypertriglyceridemia be evaluated for family history of dyslipidemia and cardiovascular disease.
The treatment goal in patients with moderate hypertriglyceridemia should be a non-high-density lipoprotein cholesterol level in agreement with National Cholesterol Education Program Adult Treatment Panel guidelines. The initial treatment should be lifestyle therapy; a combination of diet modification, physical activity and drug therapy may also be considered. In patients with severe or very severe hypertriglyceridemia, a fibrate can be used as a first-line agent for reduction of triglycerides in patients at risk for triglyceride-induced pancreatitis.
Three drug classes (fibrates, niacin, n-3 fatty acids) alone or in combination with statins may be considered as treatment options in patients with moderate to severe triglyceride levels. Statins are not be used as monotherapy for severe or very severe hypertriglyceridemia. However, statins may be useful for the treatment of moderate hypertriglyceridemia when indicated to modify cardiovascular risk.
Changes in Levels of Sex Hormones and N-Terminal Pro–B-Type Natriuretic Peptide as Biomarker for Cardiovascular Diseases
Reporter and Curator: Dr. Sudipta Saha, Ph.D.
Considerable differences exist in the prevalence and manifestation of atherosclerotic cardiovascular disease (CVD) and heart failure (HF) between men and women. Premenopausal women have a lower risk of CVD and HF compared with men; however, this risk increases after menopause. Sex hormones, particularly androgens, are associated with CVD risk factors and events and have been postulated to mediate the observed sex differences in CVD.
B-type natriuretic peptides (BNPs) are secreted from cardiomyocytes in response to myocardial wall stress. BNP plays an important role in cardiovascular remodelling and volume homeostasis. It exerts numerous cardioprotective effects by promoting vasodilation, natriuresis, and ventricular relaxation and by antagonizing fibrosis and the effects of the renin-angiotensin-aldosterone system. Although the physiological role of BNP is cardioprotective, pathologically elevated N-terminal pro–BNP (NT-proBNP) levels are used clinically to indicate left ventricular hypertrophy, dysfunction, and myocardial ischemia. Higher NT-proBNP levels among individuals free of clinical CVD are associated with an increased risk of incident CVD, HF, and cardiovascular mortality.
BNP and NT-proBNP levels are higher in women than men in the general population. Several studies have proposed the use of sex- and age-specific reference ranges for BNP and NT-proBNP levels, in which reference limits are higher for women and older individuals. The etiology behind this sex difference has not been fully elucidated, but prior studies have demonstrated an association between sex hormones and NT-proBNP levels. Recent studies measuring endogenous sex hormones have suggested that androgens may play a larger role in BNP regulation by inhibiting its production.
Data were collected from a large, multiethnic community-based cohort of individuals free of CVD and HF at baseline to analyze both the cross-sectional and longitudinal associations between sex hormones [total testosterone (T), bioavailable T, freeT, dehydroepiandrosterone (DHEA), SHBG, and estradiol] and NT-proBNP, separately for women and men. It was found that a more androgenic pattern of sex hormones was independently associated with lower NT-proBNP levels in cross-sectional analyses in men and postmenopausal women.
This association may help explain sex differences in the distribution of NT-proBNP and may contribute to the NP deficiency in men relative to women. In longitudinal analyses, a more androgenic pattern of sex hormones was associated with a greater increase in NT-proBNP levels in both sexes, with a more robust association among women. This relationship may reflect a mechanism for the increased risk of CVD and HF seen in women after menopause.
Additional research is needed to further explore whether longitudinal changes in NT-proBNP levels seen in our study are correlated with longitudinal changes in sex hormones. The impact of menopause on changes in NT-proBNP levels over time should also be explored. Furthermore, future studies should aim to determine whether sex hormones directly play a role in biological pathways of BNP synthesis and clearance in a causal fashion. Lastly, the dual role of NTproBNP as both
a cardioprotective hormone and
a biomarker of CVD and HF, as well as
the role of sex hormones in delineating these processes,
should be further explored. This would provide a step toward improved clinical CVD risk stratification and prognostication based on
Over the past 20 years, studies have shown that girls and possibly boys have been experiencing puberty at progressively younger ages. This is troubling news, as earlier age at puberty has been linked with increased risk of mental illness, breast and ovarian cancer in girls and testicular cancer in boys. Researchers found that daughters of mothers who had higher levels of diethyl phthalate and triclosan in their bodies during pregnancy experienced puberty at younger ages. The same trend was not observed in boys. So, researchers suspected that girls exposed to chemicals commonly found in toothpaste, makeup, soap and other personal care products before birth may hit puberty earlier.
Diethyl phthalate is often used as a stabilizer in fragrances and cosmetics. The antimicrobial agent triclosan — which the FDA banned from use in hand soap in 2017 because it was shown to be ineffective — is still used in some toothpastes. Researchers suspected that many chemicals in personal care products can interfere with natural hormones in human bodies, and studies have shown that exposure to these chemicals can alter reproductive development in rats. Chemicals that have been implicated include phthalates, which are often found in scented products like perfumes, soaps and shampoos; parabens, which are used as preservatives in cosmetics; and phenols, which include triclosan.
However, few studies have looked at how these chemicals might affect the growth of human children. This present study at UC Berkeley, USA recruited pregnant women living in the farm-working, primarily Latino communities of Central California’s Salinas Valley between 1999 and 2000. While the primary aim of the study was to examine the impact of pesticide exposure on childhood development, the researchers used the opportunity to examine the effects of other chemicals as well. The scientists measured concentrations of phthalates, parabens and phenols in urine samples taken from mothers twice during pregnancy, and from children at the age of 9. They then followed the growth of the children — 159 boys and 179 girls — between the ages of 9 and 13 to track the timing of developmental milestones marking different stages of puberty.
The vast majority — more than 90 percent — of urine samples of both mothers and children showed detectable concentrations of all three classes of chemicals, with the exception of triclosan which was present in approximately 70 percent of samples. The researchers found that every time the concentrations of diethyl phthalate and triclosan in the mother’s urine doubled, the timing of developmental milestones in girls shifted approximately one month earlier. Girls who had higher concentrations of parabens in their urine at age 9 also experienced puberty at younger ages. However, it is unclear if the chemicals were causing the shift, or if girls who reached puberty earlier were more likely to start using personal care products at younger ages.
The limitations are that these chemicals are quickly metabolized and one to two urinary measurements per developmental point may not accurately reflect usual exposure. The study population was limited to Latino children of low socioeconomic status living in a farmworker community and may not be widely generalizable. But, this study contributes to a growing literature that suggests that exposure to certain endocrine disrupting chemicals may impact timing of puberty in children.
Anti-Müllerian Hormone (AMH), is secreted by growing follicles that contains the egg or ovum. According to regular practice low AMH and high Follicle Stimulating Hormone (FSH) are generally considered as indicators of diminished egg quantity in a female. But, there are several cases the female conceived absolutely normally without any support even after low AMH was reported.
Therefore, a new research published in the Journal of the American Medical Association declares that AMH doesn’t dictate a woman’s reproductive potential. Although AMH testing is one of the most common ways that doctors assess a woman’s fertility. Present research says that all it takes is one egg each cycle and AMH is not a marker of whether a female can or cannot become pregnant. So, for women who haven’t yet tried to get pregnant and who are wondering whether they are fertile, an AMH value isn’t going to be helpful in that context. In addition, AMH is not necessarily a good marker to predict that whether one has to cryopreserve her eggs. So, practically doctors don’t yet have a way to definitively predict egg quality or a woman’s long-term ability to conceive, but age is obviously one of the most important factors.
The above mentioned study followed 750 women between the ages of 30 and 44 who had been trying to conceive for three months or less. During the 12-month observation period, those with low AMH values of less than 0.7 were not less likely to conceive than those who had normal AMH values. The study had various limitations, however, that are worth noting. The researchers only included women who did not have a history of infertility. Women who sought fertility treatments (about 6 percent) were withdrawn. And only 12 percent of the women were in the 38-to-44 age range. In addition, the number of live births was unavailable.
Among women aged 30 to 44 years without a history of infertility who had been trying to conceive for 3 months or less, biomarkers indicating diminished ovarian reserve compared with normal ovarian reserve were not associated with reduced fertility. These findings do not support the use of urinary or blood FSH tests or AMH levels to assess natural fertility for women with these characteristics. The researchers’ next want to see whether low AMH is associated with a higher risk of miscarriage among the women who conceived.
Although AMH testing isn’t designed to be an overall gauge of a woman’s fertility, it can still provide valuable information, especially for women who are infertile and seeking treatment. It can assist in diagnosing polycystic ovarian syndrome, and identify when a woman is getting closer to menopause. Previous research also showed that AMH is good predictor of a woman’s response to ovarian stimulation for in vitro fertilization and therefore it can predict the probability of conceiving via in vitro fertilization (I.V.F.).
An American research group designed a new estrogen molecule, PaPE-1, and found that PaPE-1 might have beneficial effects on metabolism and vascular health, while not enhancing proliferation in uterine and breast cells, therefore not increasing cancer risk.
After menopause, estrogen therapy is proved to help prevent metabolic and vascular diseases. However, estrogen (a female sex hormone) also has proliferative effects on the uterus and breasts, increasing cancer risk. Estrogens operate through two pathways: the nuclear-initiated and extranuclear-initiated pathways. The extranuclear-initiated pathway is responsible for the beneficial effects of estrogens, while activation of the nuclear-initiated pathway may result in adverse effects, such as cell proliferation of the uterus and breasts. It seems that estrogen molecules that bind to estrogen receptors (ER) with high affinity activate both pathways, while an estrogen molecule with a low-affinity binding capability can only activate the extranuclear-initiated pathway, and therefore have only beneficial effects.
In an article recently published in Science Signalling, an American research group investigated the effects of pathway preferential estrogens (PaPEs) compared to estradiol (E2, a form of estrogen and a common supplement for post-menopausal women). They altered the structure of E2 which reduced the binding affinity of the new molecule (PaPE-1) by 50000 times compared to E2, but could still form a competent complex with the receptor. They found that PaPE-1 was selective in activating the extranuclear-initiated pathway and it did not stimulate proliferation of human breast cancer cells, but activated the mTOR pathway that modulates metabolic functions. Both E2 and PaPE-1 treatment resulted in increased gene expression, however E2 activated more genes overall and more genes related to cell proliferation.
To test the new molecule in vivo, researchers used ovariectomized female mice (mice with ovaries removed) and treated them with a control vehicle, E2, or PaPE-1. Compared to E2, PaPE-1 did not have any effect on uterine weight, mammary ductal growth and thymus size. However, both E2 and PaPE-1 reduced the mammary gland fat deposits that developed after ovariectomy and they both decreased body weight. Both hormones reduced fat mass, triglyceride (fat) concentrations in blood and liver lipid content, but only E2 increased total lean mass and water mass.
Investigators further examined the gene expression and found that both PaPE-1 and E2 induced gene expression changes in liver and skeletal muscles, while only E2 altered the gene expression in the uterus.
When researchers used mice that had no α-type ER, E2 did not stimulate uterine growth and neither PaPE-1 nor E2 decreased blood triglycerides.
Researchers also investigated the beneficial vascular effects of E2 and PaPE-1 compared to the control vehicle. They treated mice with one of the three substances, injured their carotid artery and then continued the therapy for 3 more days. They found that both E2 and PaPE-1 treatment caused similar vascular repair and that the effect could be prevented by administering anti-estrogens.
The research group designed 3 more molecules, PaPE-2, 3 and 4, that also activated the extranuclear-initiated pathway and therefore showed similar tissue-selective effects.
After menopause, estrogen supplementation can have beneficial cardiovascular and metabolic effects. However, estrogen therapy may result in proliferation of uterine and breast cells and it increases uterus and breast cancer risk. PaPE-1 seems to be an estrogen molecule that combines all the beneficial effects of estrogen supplementation without increasing cancer risk. Furthermore, the process used to develop these new molecules can be applied to other hormones, such as glucocorticoids and Vitamin D, which may result in drugs with more selective activity.
Scientists at the Swiss Federal Institute of Technology (ETH) in Zurich have found an exciting new use for the cells that reside in the undesirable flabby tissue—creating pancreatic beta cells. The ETH researchers extracted stem cells from a 50-year-old test subject’s fatty tissue and reprogrammed them into mature, insulin-producing beta cells.
The findings from this study were published recently in Nature Communications in an article entitled “A Programmable Synthetic Lineage-Control Network That Differentiates Human IPSCs into Glucose-Sensitive Insulin-Secreting Beta-Like Cells.”
The investigators added a highly complex synthetic network of genes to the stem cells to recreate precisely the key growth factors involved in this maturation process. Central to the process were the growth factors Ngn3, Pdx1, and MafA; the researchers found that concentrations of these factors change during the differentiation process.
For instance, MafA is not present at the start of maturation. Only on day 4, in the final maturation step, does it appear, its concentration rising steeply and then remaining at a high level. The changes in the concentrations of Ngn3 and Pdx1, however, are very complex: while the concentration of Ngn3 rises and then falls again, the level of Pdx1 rises at the beginning and toward the end of maturation.
Senior study author Martin Fussenegger, Ph.D., professor of biotechnology and bioengineering at ETH Zurich’s department of biosystems science and engineering stressed that it was essential to reproduce these natural processes as closely as possible to produce functioning beta cells, stating that “the timing and the quantities of these growth factors are extremely important.”
The ETH researchers believe that their work is a real breakthrough, in that a synthetic gene network has been used successfully to achieve genetic reprogramming that delivers beta cells. Until now, scientists have controlled such stem cell differentiation processes by adding various chemicals and proteins exogenously.
“It’s not only really hard to add just the right quantities of these components at just the right time, but it’s also inefficient and impossible to scale up,” Dr. Fussenegger noted.
While the beta cells not only looked very similar to their natural counterparts—containing dark spots known as granules that store insulin—the artificial beta cells also functioned in a very similar manner. However, the researchers admit that more work needs to be done to increase the insulin output.
“At the present time, the quantities of insulin they secrete are not as great as with natural beta cells,” Dr. Fussenegger stated. Yet, the key point is that the researchers have for the first time succeeded in reproducing the entire natural process chain, from stem cell to differentiated beta cell.
In future, the ETH scientists’ novel technique might make it possible to implant new functional beta cells in diabetes sufferers that are made from their adipose tissue. While beta cells have been transplanted in the past, this has always required subsequent suppression of the recipient’s immune system—as with any transplant of donor organs or tissue.
“With our beta cells, there would likely be no need for this action since we can make them using endogenous cell material taken from the patient’s own body,” Dr. Fussenegger said. “This is why our work is of such interest in the treatment of diabetes.”
A programmable synthetic lineage-control network that differentiates human IPSCs into glucose-sensitive insulin-secreting beta-like cells
Synthetic biology has advanced the design of standardized transcription control devices that programme cellular behaviour. By coupling synthetic signalling cascade- and transcription factor-based gene switches with reverse and differential sensitivity to the licensed food additive vanillic acid, we designed a synthetic lineage-control network combining vanillic acid-triggered mutually exclusive expression switches for the transcription factors Ngn3 (neurogenin 3; OFF-ON-OFF) and Pdx1 (pancreatic and duodenal homeobox 1; ON-OFF-ON) with the concomitant induction of MafA (V-maf musculoaponeurotic fibrosarcoma oncogene homologue A; OFF-ON). This designer network consisting of different network topologies orchestrating the timely control of transgenic and genomic Ngn3, Pdx1 and MafA variants is able to programme human induced pluripotent stem cells (hIPSCs)-derived pancreatic progenitor cells into glucose-sensitive insulin-secreting beta-like cells, whose glucose-stimulated insulin-release dynamics are comparable to human pancreatic islets. Synthetic lineage-control networks may provide the missing link to genetically programme somatic cells into autologous cell phenotypes for regenerative medicine.
Cell-fate decisions during development are regulated by various mechanisms, including morphogen gradients, regulated activation and silencing of key transcription factors, microRNAs, epigenetic modification and lateral inhibition. The latter implies that the decision of one cell to adopt a specific phenotype is associated with the inhibition of neighbouring cells to enter the same developmental path. In mammals, insights into the role of key transcription factors that control development of highly specialized organs like the pancreas were derived from experiments in mice, especially various genetically modified animals1, 2, 3, 4. Normal development of the pancreas requires the activation of pancreatic duodenal homeobox protein (Pdx1) in pre-patterned cells of the endoderm. Inactivating mutations of Pdx1 are associated with pancreas agenesis in mouse and humans5, 6. A similar cell fate decision occurs later with the activation of Ngn3 that is required for the development of all endocrine cells in the pancreas7. Absence of Ngn3 is associated with the loss of pancreatic endocrine cells, whereas the activation of Ngn3 not only allows the differentiation of endocrine cells but also induces lateral inhibition of neighbouring cells—via Delta-Notch pathway—to enter the same pancreatic endocrine cell fate8. This Ngn3-mediated cell-switch occurs at a specific time point and for a short period of time in mice9. Thereafter, it is silenced and becomes almost undetectable in postnatal pancreatic islets. Conversely, Pdx1-positive Ngn3-positive cells reduce Pdx1 expression, as Ngn3-positive cells are Pdx1 negative10. They re-express Pdx1, however, as they go on their path towards glucose-sensitive insulin-secreting cells with parallel induction of MafA that is required for proper differentiation and maturation of pancreatic beta cells11. Data supporting these expression dynamics are derived from mice experiments1, 11, 12. A synthetic gene-switch governing cell fate decision in human induced pluripotent stem cells (hIPSCs) could facilitate the differentiation of glucose-sensitive insulin-secreting cells.
In recent years, synthetic biology has significantly advanced the rational design of synthetic gene networks that can interface with host metabolism, correct physiological disturbances13 and provide treatment strategies for a variety of metabolic disorders, including gouty arthritis14, obesity15 and type-2 diabetes16. Currently, synthetic biology principles may provide the componentry and gene network topologies for the assembly of synthetic lineage-control networks that can programme cell-fate decisions and provide targeted differentiation of stem cells into terminally differentiated somatic cells. Synthetic lineage-control networks may therefore provide the missing link between human pluripotent stem cells17 and their true impact on regenerative medicine18, 19, 20. The use of autologous stem cells in regenerative medicine holds great promise for curing many diseases, including type-1 diabetes mellitus (T1DM), which is characterized by the autoimmune destruction of insulin-producing pancreatic beta cells, thus making patients dependent on exogenous insulin to control their blood glucose21, 22. Although insulin therapy has changed the prospects and survival of T1DM patients, these patients still suffer from diabetic complications arising from the lack of physiological insulin secretion and excessive glucose levels23. The replacement of the pancreatic beta cells either by pancreas transplantation or by transplantation of pancreatic islets has been shown to normalize blood glucose and even improve existing complications of diabetes24. However, insulin independence 5 years after islet transplantation can only be achieved in up to 55% of the patients even when using the latest generation of immune suppression strategies25, 26. Transplantation of human islets or the entire pancreas has allowed T1DM patients to become somewhat insulin independent, which provides a proof-of-concept for beta-cell replacement therapies27, 28. However, because of the shortage of donor pancreases and islets, as well as the significant risk associated with transplantation and life-long immunosuppression, the rational differentiation of stem cells into functional beta-cells remains an attractive alternative29, 30. Nevertheless, a definitive cure for T1DM should address both the beta-cell deficit and the autoimmune response to cells that express insulin. Any beta-cell mimetic should be able to store large amounts of insulin and secrete it on demand, as in response to glucose stimulation29, 31. The most effective protocols for the in vitro generation of bonafide insulin-secreting beta-like cells that are suitable for transplantation have been the result of sophisticated trial-and-error studies elaborating timely addition of complex growth factor and small-molecule compound cocktails to human pancreatic progenitor cells32, 33, 34. The differentiation of pancreatic progenitor cells to beta-like cells is the most challenging part as current protocols provide inconsistent results and limited success in programming pancreatic progenitor cells into glucose-sensitive insulin-secreting beta-like cells35, 36, 37. One of the reasons for these observations could be the heterogeneity in endocrine differentiation and maturation towards a beta cell phenotype. Here we show that a synthetic lineage-control network programming the dynamic expression of the transcription factors Ngn3, Pdx1 and MafA enables the differentiation of hIPSC-derived pancreatic progenitor cells to glucose-sensitive insulin-secreting beta-like cells (Supplementary Fig. 1).
The differentiation pathway from pancreatic progenitor cells to glucose-sensitive insulin-secreting pancreatic beta-cells combines the transient mutually exclusive expression switches of Ngn3 (OFF-ON-OFF) and Pdx1 (ON-OFF-ON) with the concomitant induction of MafA (OFF-ON) expression10,11. Since independent control of the pancreatic transcription factors Ngn3, Pdx1 and MafA by different antibiotic transgene control systems responsive to tetracycline, erythromycin and pristinamycin did not result in the desired differential control dynamics (Supplementary Fig. 2), we have designed a vanillic acid-programmable synthetic lineage-control network that programmes hIPSC-derived pancreatic progenitor cells to specifically differentiate into glucose-sensitive insulin-secreting beta-like cells in a seamless and self-sufficient manner. The timely coordination of mutually exclusive Ngn3 and Pdx1 expression with MafA induction requires the trigger-controlled execution of a complex genetic programme that orchestrates two overlapping antagonistic band-pass filter expression profiles (OFF-ON-OFF and ON-OFF-ON), a positive band-pass filter for Ngn3 (OFF-ON-OFF) and a negative band-pass filter, also known as band-stop filter, for Pdx1 (ON-OFF-ON), the ramp-up expression phase of which is linked to a graded induction of MafA (OFF-ON).
The core of the synthetic lineage-control network consists of two transgene control devices that are sensitive to the food component and licensed food additive vanillic acid. These devices are a synthetic vanillic acid-inducible (ON-type) signalling cascade that is gradually induced by increasing the vanillic acid concentration and a vanillic acid-repressible (OFF-type) gene switch that is repressed in a vanillic acid dose-dependent manner (Fig. 1a,b). The designer cascade consists of the vanillic acid-sensitive mammalian olfactory receptor MOR9-1, which sequentially activates the G protein Sα (GSα) and adenylyl cyclase to produce a cyclic AMP (cAMP) second messenger surge38 that is rewired via the cAMP-responsive protein kinase A-mediated phospho-activation of the cAMP-response element-binding protein 1 (CREB1) to the induction of synthetic promoters (PCRE) containing CREB1-specific cAMP response elements (CRE; Fig. 1a). The co-transfection of pCI-MOR9-1 (PhCMV-MOR9-1-pASV40) and pCK53 (PCRE-SEAP-pASV40) into human mesenchymal stem cells (hMSC-TERT) confirmed the vanillic acid-adjustable secreted alkaline phosphatase (SEAP) induction of the designer cascade (>10nM vanillic acid; Fig. 1a). The vanillic acid-repressible gene switch consists of the vanillic acid-dependent transactivator (VanA1), which binds and activates vanillic acid-responsive promoters (for example, P1VanO2) at low and medium vanillic acid levels (<2μM). At high vanillic acid concentrations (>2μM), VanA1 dissociates from P1VanO2, which results in the dose-dependent repression of transgene expression39 (Fig. 1b). The co-transfection of pMG250 (PSV40-VanA1-pASV40) and pMG252 (P1VanO2-SEAP-pASV40) into hMSC-TERT corroborated the fine-tuning of the vanillic acid-repressible SEAP expression (Fig. 1b).
Figure 1: Design of a vanillic acid-responsive positive band-pass filter providing an OFF-ON-OFF expression profile.
a) Vanillic acid-inducible transgene expression. The constitutively expressed vanillic acid-sensitive olfactory G protein-coupled receptor MOR9-1 (pCI-MOR9-1; PhCMV-MOR9-1-pA) senses extracellular vanillic acid levels and triggers G protein (Gs)-mediated activation of the membrane-bound adenylyl cyclase (AC) that converts ATP into cyclic AMP (cAMP). The resulting intracellular cAMP surge activates PKA (protein kinase A), whose catalytic subunits translocate into the nucleus to phosphorylate cAMP response element-binding protein 1 (CREB1). Activated CREB1 binds to synthetic promoters (PCRE) containing cAMP-response elements (CRE) and induces PCRE-driven expression of human placental secreted alkaline phosphatase (SEAP; pCK53, PCRE-SEAP-pA). Co-transfection of pCI-MOR9-1 and pCK53 into human mesenchymal stem cells (hMSC-TERT) grown for 48h in the presence of increasing vanillic acid concentrations results in a dose-inducible SEAP expression profile. (b) Vanillic acid-repressible transgene expression. The constitutively expressed, vanillic acid-dependent transactivator VanA1(pMG250, PSV40-VanA1-pA, VanA1, VanR-VP16) binds and activates the chimeric promoter P1VanO2 (pMG252, P1VanO2-SEAP-pA) in the absence of vanillic acid. In the presence of increasing vanillic acid concentrations, VanA1 is released from P1VanO2, and transgene expression is shut down. Co-transfection of pMG250 and pMG252 into hMSC-TERT grown for 48h in the presence of increasing vanillic acid concentrations results in a dose-repressible SEAP expression profile. (c) Positive band-pass expression filter. Serial interconnection of the synthetic vanillic acid-inducible signalling cascade (a) with the vanillic acid-repressible transcription factor-based gene switch (b) by PCRE-mediated expression of VanA1 (pSP1, PCRE-VanA1-pA) results in a two-level feed-forward cascade. Owing to the opposing responsiveness and differential sensitivity to vanillic acid, this synthetic gene network programmes SEAP expression with a positive band-pass filter profile (OFF-ON-OFF) as vanillic acid levels are increased. Medium vanillic acid levels activate MOR9-1, which induces PCRE-driven VanA1 expression. VanA1remains active and triggers P1VanO2-mediated SEAP expression in feed-forward manner, which increases to maximum levels. At high vanillic acid concentrations, MOR9-1 maintains PCRE-driven VanA1 expression, but the transactivator dissociates from P1VanO2, which shuts SEAP expression down. Co-transfection of pCI-MOR9-1, pSP1 and pMG252 into hMSC-TERT grown for 48h in the presence of increasing vanillic acid concentrations programmes SEAP expression with a positive band-pass profile (OFF-ON-OFF). Data are the means±s.d. of triplicate experiments (n=9).
The opposing responsiveness and differential sensitivity of the control devices to vanillic acid are essential to programme band-pass filter expression profiles. Upon daisy-chaining the designer cascade (pCI-MOR9-1; PhCMV-MOR9-1-pASV40; pSP1, PCRE-VanA1-pASV40) and the gene switch (pSP1, PCRE-VanA1-pASV40; pMG252, P1VanO2-SEAP-pASV40) in the same cell, the network executes a band-pass filter SEAP expression profile when exposed to increasing concentrations of vanillic acid (Fig. 1c). Medium vanillic acid levels (10nM to 2μM) activate MOR9-1, which induces PCRE-driven VanA1 expression. VanA1 remains active within this concentration range and, in a feed-forward amplifier manner, triggers P1VanO2-mediated SEAP expression, which gradually increases to maximum levels (Fig. 1c). At high vanillic acid concentrations (2μM to 400μM), MOR9-1 maintains PCRE-driven VanA1 expression, but the transactivator is inactivated and dissociates from P1VanO2, which results in the gradual shutdown of SEAP expression (Fig. 1c).
For the design of the vanillic acid-programmable synthetic lineage-control network, constitutive MOR9-1 expression and PCRE-driven VanA1 expression were combined with pSP12 (pASV40-Ngn3cm←P3VanO2mFT-miR30Pdx1g-shRNA-pASV40) for endocrine specification and pSP17(PCREm-Pdx1cm-2A-MafAcm-pASV40) for maturation of developing beta-cells (Fig. 2a,b). ThepSP12-encoded expression unit enables the VanA1-controlled induction of the optimized bidirectional vanillic acid-responsive promoter (P3VanO2) that drives expression of a codon-modified Ngn3cm, the nucleic acid sequence of which is distinct from its genomic counterpart (Ngn3g) to allow for quantitative reverse transcription–PCR (qRT–PCR)-based discrimination. In the opposite direction, P3VanO2 transcribes miR30Pdx1g-shRNA, which exclusively targets genomicPdx1 (Pdx1g) transcripts for RNA interference-based destruction and is linked to the production of a blue-to-red medium fluorescent timer40 (mFT) for precise visualization of the unit’s expression dynamics in situ.pSP17 contains a dicistronic expression unit in which the modified high-tightness and lower-sensitivity PCREm promoter (see below) drives co-cistronic expression of Pdx1cm andMafAcm, which are codon-modified versions producing native transcription factors that specifically differ from their genomic counterparts (Pdx1g, MafAg) in their nucleic acid sequence. After individual validation of the vanillic acid-controlled expression and functionality of all network components (Supplementary Figs 2–9), the lineage-control network was ready to be transfected into hIPSC-derived pancreatic progenitor cells. These cells are characterized by high expression of Pdx1g and Nkx6.1 levels and the absence of Ngn3g and MafAg production32, 33, 34 (day 0:Supplementary Figs 10–16).
(a) Schematic of the synthetic lineage-control network. The constitutively expressed, vanillic acid-sensitive olfactory G protein-coupled receptor MOR9-1 (pCI-MOR9-1; PhCMV-MOR9-1-pA) senses extracellular vanillic acid levels and triggers a synthetic signalling cascade, inducing PCRE-driven expression of the transcription factor VanA1 (pSP1, PCRE-VanA1-pA). At medium vanillic acid concentrations (purple arrows), VanA1 binds and activates the bidirectional vanillic acid-responsive promoter P3VanO2 (pSP12, pA-Ngn3cm←P3VanO2mFT-miR30Pdx1g-shRNA-pA), which drives the induction of codon-modified Neurogenin 3 (Ngn3cm) as well as the coexpression of both the blue-to-red medium fluorescent timer (mFT) for precise visualization of the unit’s expression dynamics and miR30pdx1g-shRNA (a small hairpin RNA programming the exclusive destruction of genomic pancreatic and duodenal homeobox 1 (Pdx1g) transcripts). Consequently, Ngn3cm levels switch from low to high (OFF-to-ON), and Pdx1g levels toggle from high to low (ON-to-OFF). In addition, Ngn3cm triggers the transcription of Ngn3g from its genomic promoter, which initiates a positive-feedback loop. At high vanillic acid levels (orange arrows), VanA1 is inactivated, and both Ngn3cm and miR30pdx1g-shRNA are shut down. At the same time, the MOR9-1-driven signalling cascade induces the modified high-tightness and lower-sensitivity PCREm promoter that drives the co-cistronic expression of the codon-modified variants of Pdx1 (Pdx1cm) and V-maf musculoaponeurotic fibrosarcoma oncogene homologue A (MafAcm; pSP17, PCREm-Pdx1cm-2A-MafAcm-pA). Consequently, Pdx1cm and MafAcm become fully induced. As Pdx1cm expression ramps up, it initiates a positive-feedback loop by inducing the genomic counterparts Pdx1g and MafAg. Importantly, Pdx1cm levels are not affected by miR30Pdx1g-shRNA because the latter is specific for genomic Pdx1g transcripts and because the positive feedback loop-mediated amplification of Pdx1gexpression becomes active only after the shutdown of miR30Pdx1g-shRNA. Overall, the synthetic lineage-control network provides vanillic acid-programmable, transient, mutually exclusive expression switches for Ngn3 (OFF-ON-OFF) and Pdx1 (ON-OFF-ON) as well as the concomitant induction of MafA (OFF-ON) expression, which can be followed in real time (Supplementary Movies 1 and 2). (b) Schematic illustrating the individual differentiation steps from human IPSCs towards beta-like cells. The colours match the cell phenotypes reached during the individual differentiation stages programmed by the lineage-control network shown in a.
Following the co-transfection of pCI-MOR9-1 (PhCMV-MOR9-1-pASV40), pSP1 (PCRE-VanA1-pASV40), pSP12 (pASV40-Ngn3cm←P3VanO2mFT-miR30Pdx1g-shRNA-pASV40) and pSP17(PCREm-Pdx1cm-2A-MafAcm-pASV40) into hIPSC-derived pancreatic progenitor cells, the synthetic lineage-control network should override random endogenous differentiation activities and execute the pancreatic beta-cell-specific differentiation programme in a vanillic acid remote-controlled manner. To confirm that the lineage-control network operates as programmed, we cultivated network-containing and pEGFP-N1-transfected (negative-control) cells for 4 days at medium (2μM) and then 7 days at high (400μM) vanillic acid concentrations and profiled the differential expression dynamics of all of the network components and their genomic counterparts as well as the interrelated transcription factors and hormones in both whole populations and individual cells at days 0, 4, 11 and 14 (Figs 2 and 3 and Supplementary Figs 11–17).
Figure 3: Dynamics of the lineage-control network.
(a,b) Quantitative RT–PCR-based expression profiling of the pancreatic transcription factors Ngn3cm/g, Pdx1cm/g and MafAcm/g in hIPSC-derived pancreatic progenitor cells containing the synthetic lineage-control network at days 4 and 11. Data are the means±s.d. of triplicate experiments (n=9). (c–g) Immunocytochemistry of pancreatic transcription factors Ngn3cm/g, Pdx1cm/g and MafAcm/g in hIPSC-derived pancreatic progenitor cells containing the synthetic lineage-control network at days 4 and 11. hIPSC-derived pancreatic progenitor cells were co-transfected with the lineage-control vectors pCI-MOR9-1 (PhCMV-MOR9-1-pA), pSP1 (PCRE-VanA1-pA), pSP12 (pA-Ngn3cm←P3VanO2mFT-miR30Pdx1g-shRNA-pA) and pSP17 (PCREm-Pdx1cm-2A-MafAcm) and immunocytochemically stained for (c) VanA1 and Pdx1 (day 4), (d) VanA1 and Ngn3 (day 4), (e) VanA1 and Pdx1 (day 11), (f) MafA and Pdx1 (day 11) as well as (g) VanA1 and insulin (C-peptide) (day 11). The cells staining positive for VanA1 are containing the lineage-control network. DAPI, 4′,6-diamidino-2-phenylindole. Scale bar, 100μm.
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Multicellular organisms, including humans, consist of a highly structured assembly of a multitude of specialized cell phenotypes that originate from the same zygote and have traversed a preprogrammed multifactorial developmental plan that orchestrates sequential differentiation steps with high precision in space and time19, 51. Because of the complexity of terminally differentiated cells, the function of damaged tissues can for most medical indications only be restored via the transplantation of donor material, which is in chronically short supply52.
Despite significant progress in regenerative medicine and the availability of stem cells, the design of protocols that replicate natural differentiation programmes and provide fully functional cell mimetics remains challenging29, 53. For example, efforts to generate beta-cells from human embryonic stem cells (hESCs) have led to reliable protocols involving the sequential administration of growth factors (activin A, bone morphogenetic protein 4 (BMP-4), basic fibroblast growth factor (bFGF), FGF-10, Noggin, vascular endothelial growth factor (VEGF) and Wnt3A) and small-molecule compounds (cyclopamine, forskolin, indolactam V, IDE1, IDE2, nicotinamide, retinoic acid, SB−431542 and γ-secretase inhibitor) that modulate differentiation-specific signalling pathways31, 54, 55. In vitro differentiation of hESC-derived pancreatic progenitor cells into beta-like cells is more challenging and has been achieved recently by a complex media formulation with chemicals and growth factors32, 33, 34.
hIPSCs have become a promising alternative to hESCs; however, their use remains restricted in many countries56. Most hIPSCs used for directed differentiation studies were derived from a juvenescent cell source that is expected to show a higher degree of differentiation potential compared with older donors that typically have a higher need for medical interventions37, 57, 58. We previously succeeded in producing mRNA-reprogrammed hIPSCs from adipose tissue-derived mesenchymal stem cells of a 50-year-old donor, demonstrating that the reprogramming of cells from a donor of advanced age is possible in principle59.
Recent studies applying similar hESC-based differentiation protocols to hIPSCs have produced cells that release insulin in response to high glucose32, 33, 34. This observation suggests that functional beta-like cells can eventually be derived from hIPSCs32, 33. In our hands, the growth-factor/chemical-based technique for differentiating human IPSCs resulted in beta-like cells with poor glucose responsiveness. Recent studies have revealed significant variability in the lineage specification propensity of different hIPSC lines35, 60 and substantial differences in the expression profiles of key transcription factors in hIPSC-derived beta-like cells33. Therefore, the growth-factor/chemical-based protocols may require further optimization and need to be customized for specific hIPSC lines35. Synthetic lineage-control networks providing precise dynamic control of transcription factor expression may overcome the challenges associated with the programming of beta-like cells from different hIPSC lines.
Rather than exposing hIPSCs to a refined compound cocktail that triggers the desired differentiation in a fraction of the stem cell population, we chose to design a synthetic lineage-control network to enable single input-programmable differentiation of hIPSC-derived pancreatic progenitor cells into glucose-sensitive insulin-secreting beta-like cells. In contrast with the use of growth-factor/chemical-based cocktails, synthetic lineage-control networks are expected to (i) be more economical because of in situ production of the required transcription factors, (ii) enable simultaneous control of ectopic and chromosomally encoded transcription factor variants, (iii) tap into endogenous pathways and not be limited to cell-surface input, (iv) display improved reversibility that is not dependent on the removal of exogenous growth factors via culture media replacement, (v) provide lateral inhibition, thereby reducing the random differentiation of neighbouring cells and (vi) enable trigger-programmable and (vii) precise differential transcription factor expression switches.
The synthetic lineage-control network that precisely replicates the endogenous relative expression dynamics of the transcription factors Pdx-1, Ngn3 and MafA required the design of a new network topology that interconnects a synthetic signalling cascade and a gene switch with differential and opposing sensitivity to the food additive vanillic acid. This differentiation device provides different band-pass filter, time-delay and feed-forward amplifier topologies that interface with endogenous positive-feedback loops to orchestrate the timely expression and repression of heterologous and chromosomally encoded Ngn3, Pdx1 and MafA variants. The temporary nature of the engineering intervention, which consists of transient transfection of the genetic lineage-control components in the absence of any selection, is expected to avoid stable modification of host chromosomes and alleviate potential safety concerns. In addition, the resulting beta-cell mass could be encapsulated inside vascularized microcontainers28, a proven containment strategy in prototypic cell-based therapies currently being tested in animal models of prominent human diseases14, 15, 16, 61, 62 as well as in human clinical trials28.
The hIPSC-derived beta-like cells resulting from this trigger-induced synthetic lineage-control network exhibited glucose-stimulated insulin-release dynamics and capacity matching the human physiological range and transcriptional profiling, flow cytometric analysis and electron microscopy corroborated the lineage-controlled stem cells reached a mature beta-cell phenotype. In principle, the combination of hIPSCs derived from the adipose tissue of a 50-year-old donor59 with a synthetic lineage-control network programming glucose-sensitive insulin-secreting beta-like cells closes the design cycle of regenerative medicine63. However, hIPSCs that are derived from T1DM patients, differentiated into beta-like cells and transplanted back into the donor would still be targeted by the immune system, as demonstrated in the transplantation of segmental pancreatic grafts from identical twins64. Therefore, any beta-cell-replacement therapy will require complementary modulation of the immune system either via drugs30, 65, engineering or cell-based approaches66, 67 or packaging inside vascularizing, semi-permeable immunoprotective microcontainers28.
Capitalizing on the design principles of synthetic biology, we have successfully constructed and validated a synthetic lineage-control network that replicates the differential expression dynamics of critical transcription factors and mimicks the native differentiation pathway to programme hIPSC-derived pancreatic progenitor cells into glucose-sensitive insulin-secreting beta-like cells that compare with human pancreatic islets at a high level. The design of input-triggered synthetic lineage-control networks that execute a preprogrammed sequential differentiation agenda coordinating the timely induction and repression of multiple genes could provide a new impetus for the advancement of developmental biology and regenerative medicine.
Other related articles published in this Open Access Online Scientific Journal include the following:
Adipocyte Derived Stroma Cells: Their Usage in Regenerative Medicine and Reprogramming into Pancreatic Beta-Like Cells
Cellular switch molecule for sperm motility control: a novel target for male contraception and infertility treatments
Reporter and Curator: Sudipta Saha, Ph.D.
Researchers have discovered the cellular switch that boosts the activity of sperm cells so that they can travel to the egg. The finding may lead to new options for male contraception as well as treatments for infertility resulting from problems with sperm mobility.
Inside the male reproductive tract, mature sperm are capable of limited movement. This limited movement, however, is not enough to propel them toward the egg when they enter the female reproductive tract. To begin their journey, they must first be activated by the hormone progesterone, which is released by the egg.
The researchers reported that the molecule to which progesterone must bind is the enzyme alpha/beta hydrolase domain containing protein 2 (ABHD2), found in the sperm cell’s outer membrane. Similarly, strategies to bypass or enhance the enzyme might provide therapies for treating infertility resulting from sperm that lack movement capability.
Before a sperm can transition to the hyper-active phase, calcium must pass through the cell’s outer membrane and enter the flagella, the tail-like appendage the cell uses to propel itself. The sperm protein known as CatSper joins with similar proteins in the flagella to allow the entry of calcium.
When the researchers undertook the current study, it was not known whether progesterone interacted directly with CatSper to trigger the calcium influx, or acted on some other molecule (which, in turn, acted on CatSper). Before treating sperm with progesterone, the researchers exposed them to a chemical that inhibits a particular class of enzymes that they believed could include the candidate molecule that acted on CatSper. The hunch proved correct: the treated cells remained inactive after progesterone exposure, indicating that CatSper was not directly involved.
Working with modified progesterone, the researchers eventually isolated ABHD2 from the sperm tails. When the researchers inactivated ABHD2, exposure to progesterone failed to activate the sperm cells, confirming that ABHD2 is the molecular target for progesterone.
All of the technical terminology aside, this means that the researchers have pinned down the cellular switch that boosts the sperm along to the egg, so by blocking the ABHD2 activity, new male birth control methods could be on the way. Conversely, enhancing the enzyme could lead to new treatments for male infertility.
It will be interesting to see how this discovery impacts future research concerning male birth control and infertility treatments. Perhaps it’s the missing piece of information that will quickly yield an effective new male contraception option.
(Reuters Health) – – People taking metformin, a common type 2 diabetes medication, for several years may be at heightened risk of vitamin B12 deficiency and anemia, according to a new analysis of long-term data.
Metformin helps to control the amount of sugar, or glucose, in the blood by reducing how much glucose is absorbed from food and produced by the liver, and by increasing the body’s response to the hormone insulin, according to the National Institutes of Health.
“Metformin is the most commonly used drug to treat type 2 diabetes, so many millions of people are taking it, usually for a prolonged period (many years),” said senior study author Dr. Jill P Crandall of Albert Einstein College of Medicine in New York City, by email.
“Smaller numbers of people take metformin for prevention of diabetes or treatment of polycystic ovary syndrome,” Crandall told Reuters Health.
The researchers used data from the Diabetes Prevention Program and the Diabetes Prevention Program Outcomes Study, which followed participants at high risk for type 2 diabetes for more than 10 years.
The study began with more than 3,000 people age 25 years and older with high blood sugar. The participants were randomly assigned to receive either 850 milligrams of metformin twice daily, placebo medication or an intensive lifestyle program than did not include medication. For the new analysis only those taking placebo or metformin were considered, and about 50 participants were excluded after having weight-loss surgery, which would affect their diabetes outcomes.
During follow-up, the participants provided blood samples at the five- and 13-year points.
Using these blood samples, the researchers found that at year five, average B12 levels were lower in the metformin group than the placebo group, and B12 deficiency was more common, affecting 4 percent of those on metformin compared to 2 percent of those not taking the drug.
Borderline low B12 levels affected almost 20 percent of those on metformin and 10 percent of those taking placebo.
Average vitamin B12 levels were higher by year 13 than in year five, but B12 deficiency was also more common in both the metformin and placebo groups, as reported in the Journal of Clinical Endocrinology and Metabolism.
Vitamin B12 deficiency may lead to nerve damage which can be severe and may be irreversible, Crandall said.
“Severe and prolonged B12 deficiency has also been linked to impaired cognition and dementia,” she said. “It can also cause anemia (low red blood cell count) – fortunately, this condition is reversible with treatment.”
More people in the metformin group were also anemic at year five than in the placebo group.
There are more than three million cases of vitamin B12 deficiency each year in the U.S., which can be caused by diet or certain medical conditions. Symptoms include fatigue, and numbness or tingling.
Humans do not make vitamin B12 and need to consume it from animal sources or supplements. Vegetarians may get enough from eating eggs and dairy products, but vegans need to rely on supplements or fortified grains.
Doctors who prescribe metformin to patients long-term for type 2 diabetes, gestational diabetes, polycystic ovarian syndrome or other indications should consider routine measurement of vitamin B12 levels, the authors conclude.
“The FDA and organizations such as the American Diabetes Association do not have any formal recommendations for B12 monitoring for people taking metformin,” Crandall said. “That said, our study (and others) suggests that a small but significant number of people may develop deficiency.”
“People who are taking metformin should ask their doctor about measuring their B12 level,” she said.
Restoring healthy B12 levels is easy to accomplish with pills or monthly injections, she added.
“The risk of B12 deficiency should not be considered a reason to avoid taking metformin,” Crandall said.
SOURCE: bit.ly/1SWRxed Journal of Clinical Endocrinology and Metabolism, online February 22, 2016.
Long-term Metformin Use and Vitamin B12 Deficiency in the Diabetes Prevention Program Outcomes Study
Vitamin B12 deficiency may occur with metformin treatment, but few studies have assessed risk with long-term use.
Objective:
To assess risk of B12 deficiency with metformin use in the DPP/DPPOS
Design:
Secondary analysis from DPP/DPPOS. Participants were assigned to placebo (n=1082, PLA) or metformin (n=1073, MET) for 3.2 years; MET received open-label metformin for an additional 9 years.
Setting:
27 study centers in the US
Patients:
DPP eligibility: elevated fasting glucose, impaired glucose tolerance, and overweight/obesity. Analytic population: participants with available stored samples. B12 levels were assessed at 5 (n=857, n=858) and 13 years [n=756, n=764] in PLA and MET, respectively.
Interventions:
Metformin 850 mg twice daily versus placebo (DPP); open-label metformin in MET (DPPOS)
Main Outcome Measures:
B12 deficiency, anemia, peripheral neuropathy
Results:
Low B12 (≤203 pg/ml) occurred more often in MET than PLA at 5 years (4.3% vs 2.3%, p=0.02) but not at 13 years (7.4% vs. 5.4%, p=0.12). Combined low and borderline-low B12 (≤298 pg/ml) was more common in MET at 5 (19.1% vs 9.5%, p<0.01) and 13 years (20.3% vs 15.6%, p=0.02). Years of metformin use was associated with increased risk of B12 deficiency (OR B12 deficiency/year metformin use, 1.13, 95% CI 1.06–1.20). Anemia prevalence was higher in MET, but did not differ by B12 status. Neuropathy prevalence was higher in MET with low B12 levels.
Conclusions:
Long-term use of metformin in DPPOS was associated with biochemical B12 deficiency and anemia. Routine testing of vitamin B12 levels in metformin-treated patients should be considered.
Secondary analysis of data from the Diabetes Prevention Program Outcomes Study (DPPOS), one of the largest and longest studies of metformin treatment in patients at high risk of developing type 2 diabetes mellitus, shows that long-term use of metformin is associated with vitamin B12deficiency.
Aroda, V. R.et al. Long-term metformin use and vitamin B12 deficiency in the Diabetes Prevention Program Outcomes Study. J. Clin. Endocrinol. Metab.http://dx.doi.org/10.1210/jc.2015-3754 (2016)
Long-term Follow-up of Diabetes Prevention Program Shows Continued Reduction in Diabetes Development
Treatments used to decrease the development of type 2 diabetes continue to be effective an average of 15 years later, according to the latest findings of the Diabetes Prevention Program Outcomes Study, a landmark study funded by the National Institutes of Health (NIH).
The results, presented at the American Diabetes Association’s 74th Scientific Sessions®, come more than a decade after the Diabetes Prevention Program, or DPP, reported its original findings. In 2001, after an average of three years of study, the DPP announced that the study’s two interventions, a lifestyle program designed to reduce weight and increase activity levels and the diabetes medicinemetformin, decreased the development of type 2 diabetes in a diverse group of people, all of whom were at high risk for the disease, by 58 and 31 percent, respectively, compared with a group taking placebo.
The Diabetes Prevention Program Outcomes Study, or DPPOS, was conducted as an extension of the DPP to determine the longer-term effects of the two interventions, including further reduction in diabetes development and whether delaying diabetes would reduce the development of the diabetes complications that can lead to blindness, kidney failure, amputations and heart disease. Funded largely by the NIH’s National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), the new findings show that the lifestyle intervention and metformin treatment have beneficial effects, even years later, but did not reduce microvascular complications.
Delaying Type 2 Diabetes
Participants in the study who were originally assigned to the lifestyle intervention and metformin during DPP continued to have lower rates of type 2 diabetes development than those assigned to placebo, with 27 percent and 17 percent reductions, respectively, after 15 years.
“What we’re finding is that we can prevent or delay the onset of type 2 diabetes, a chronic disease, through lifestyle intervention or with metformin, over a very long period of time,” said David M. Nathan, MD, Chairman of the DPP/DPPOS and Professor of Medicine at Harvard Medical School. “After the initial randomized treatment phase in DPP, all participants were offered lifestyle intervention and the rates of diabetes development fell in the metformin and former placebo groups, leading to a reduction in the treatment group differences over time. However, the lifestyle intervention and metformin are still quite effective at delaying, if not preventing, type 2 diabetes,” Dr. Nathan said. Currently, an estimated 79 million American adults are at high-risk for developing type 2 diabetes.
Microvascular Complications
The DPPOS investigators followed participants for an additional 12 years after the end of the DPP to determine both the extent of diabetes prevention over time and whether the study treatments would also decrease the small vessel -or microvascular- complications, such as eye, nerve and kidney disease. These long-term results did not demonstrate significant differences among the lifestyle intervention, metformin or placebo groups on the microvascular complications, reported Kieren Mather, MD, Professor of Medicine at Indiana University School of Medicine and a study investigator.
“However, regardless of type of initial treatment, participants who didn’t develop diabetes had a 28 percent lower occurrence of the microvascular complications than those participants who did develop diabetes. These findings show that intervening in the prediabetes phase is important in reducing early stage complications,” Dr. Mather noted. The absence of differences in microvascular complications among the intervention groups may be explained by the small differences in average glucose levels among the groups at this stage of follow-up.
Risk for Cardiovascular Disease
The DPP population was relatively young and healthy at the beginning of the study, and few participants had experienced any severe cardiovascular events, such as heart attack or stroke, 15 years later. The relatively small number of events meant that the DPPOS researchers could not test the effects of interventions on cardiovascular disease. However, the research team did examine whether the study interventions, or a delay in the onset of type 2 diabetes, improved cardiovascular risk factors.
“We found that cardiovascular risk factors, such as hypertension, are generally improved by the lifestyle intervention and somewhat less by metformin,” said Ronald Goldberg, MD, Professor of Medicine at the University of Miami and one of the DPPOS investigators. “We know that people with type 2 diabetes are at much higher risk for heart disease and stroke than those who do not have diabetes, so a delay in risk factor development or improvement in risk factors may prove to be beneficial.”
Long-term Results with Metformin
The DPP/DPPOS is the largest and longest duration study to examine the effects of metformin, an inexpensive, well-known and generally safe diabetes medicine, in people who have not been diagnosed with diabetes. For DPPOS participants, metformin treatment was associated with a modest degree of long-term weight loss. “Other than a small increase in vitamin B-12 deficiency, which is a recognized consequence of metformin therapy, it has been extremely safe and well-tolerated over the 15 years of our study,” said Jill Crandall, MD, Professor of Medicine at Albert Einstein College of Medicine and a DPPOS investigator. “Further study will help show whether metformin has beneficial effects on heart disease and cancer, which are both increased in people with type 2 diabetes.”
Looking to the Future
In addition to the current findings, the DPPOS includes a uniquely valuable population that can help researchers understand the clinical course of type 2 diabetes. Since the participants did not have diabetes at the beginning of the DPP, for those who have developed diabetes, the data show precisely when they developed the disease, which is rare in previous studies. “The DPP and DPPOS have given us an incredible wealth of information by following a very diverse group of people with regard to race and age as they have progressed from prediabetes to diabetes,” said Judith Fradkin, MD, Director of the NIDDK Division of Diabetes, Endocrinology and Metabolic Diseases. “The study provides us with an opportunity to make crucial discoveries about the clinical course of type 2 diabetes.”
Dr. Fradkin noted that the study population held promise for further analyses because researchers would now be able to examine how developing diabetes at different periods of life may cause the disease to progress differently. “We can look at whether diabetes behaves differently if you develop it before the age of 50 or after the age of 60,” she said. “Thanks to the large and diverse population of DPPOS that has remained very loyal to the study, we will be able to see how and when complications first develop and understand how to intervene most effectively.”
She added that NIDDK had invited the researchers to submit an application for a grant to follow the study population for an additional 10 years.
The Diabetes Prevention Program Outcomes Study was funded under NIH grant U01DK048489 by the NIDDK; National Institute on Aging; National Cancer Institute; National Heart, Lung, and Blood Institute; National Eye Institute; National Center on Minority Health and Health Disparities; and the Office of the NIH Director; Eunice Kennedy Shriver National Institute of Child Health and Human Development; Office of Research on Women’s Health; and Office of Dietary Supplements, all part of the NIH, as well as the Indian Health Service, Centers for Disease Control and Prevention and American Diabetes Association. Funding in the form of supplies was provided by Merck Sante, Merck KGaA and LifeScan.
The American Diabetes Association is leading the fight to Stop Diabetes® and its deadly consequences and fighting for those affected by diabetes. The Association funds research to prevent, cure and manage diabetes; delivers services to hundreds of communities; provides objective and credible information; and gives voice to those denied their rights because of diabetes. Founded in 1940, our mission is to prevent and cure diabetes and to improve the lives of all people affected by diabetes. For more information please call the American Diabetes Association at 1-800-DIABETES (1-800-342-2383) or visit http://www.diabetes.org. Information from both these sources is available in English and Spanish.
Association of Biochemical B12Deficiency With Metformin Therapy and Vitamin B12Supplements
The National Health and Nutrition Examination Survey, 1999–2006
OBJECTIVE To describe the prevalence of biochemical B12deficiency in adults with type 2 diabetes taking metformin compared with those not taking metformin and those without diabetes, and explore whether this relationship is modified by vitamin B12supplements.
RESEARCH DESIGN AND METHODS Analysis of data on U.S. adults ≥50 years of age with (n = 1,621) or without type 2 diabetes (n = 6,867) from the National Health and Nutrition Examination Survey (NHANES), 1999–2006. Type 2 diabetes was defined as clinical diagnosis after age 30 without initiation of insulin therapy within 1 year. Those with diabetes were classified according to their current metformin use. Biochemical B12 deficiency was defined as serum B12concentrations ≤148 pmol/L and borderline deficiency was defined as >148 to ≤221 pmol/L.
RESULTS Biochemical B12 deficiency was present in 5.8% of those with diabetes using metformin compared with 2.4% of those not using metformin (P = 0.0026) and 3.3% of those without diabetes (P = 0.0002). Among those with diabetes, metformin use was associated with biochemical B12 deficiency (adjusted odds ratio 2.92; 95% CI 1.26–6.78). Consumption of any supplement containing B12 was not associated with a reduction in the prevalence of biochemical B12deficiency among those with diabetes, whereas consumption of any supplement containing B12 was associated with a two-thirds reduction among those without diabetes.
CONCLUSIONS Metformin therapy is associated with a higher prevalence of biochemical B12 deficiency. The amount of B12recommended by the Institute of Medicine (IOM) (2.4 μg/day) and the amount available in general multivitamins (6 μg) may not be enough to correct this deficiency among those with diabetes.
It is well known that the risks of both type 2 diabetes and B12deficiency increase with age (1,2). Recent national data estimate a 21.2% prevalence of diagnosed diabetes among adults ≥65 years of age and a 6 and 20% prevalence of biochemical B12 deficiency (serum B12<148 pmol/L) and borderline deficiency (serum B12 ≥148–221 pmol/L) among adults ≥60 years of age (3,4).
The diabetes drug metformin has been reported to cause a decrease in serum B12 concentrations. In the first efficacy trial, DeFronzo and Goodman (5) demonstrated that although metformin offers superior control of glycosylated hemoglobin levels and fasting plasma glucose levels compared with glyburide, serum B12 concentrations were lowered by 22% compared with placebo, and 29% compared with glyburide therapy after 29 weeks of treatment. A recent, randomized control trial designed to examine the temporal relationship between metformin and serum B12 found a 19% reduction in serum B12 levels compared with placebo after 4 years (6). Several other randomized control trials and cross-sectional surveys reported reductions in B12ranging from 9 to 52% (7–16). Although classical B12 deficiency presents with clinical symptoms such as anemia, peripheral neuropathy, depression, and cognitive impairment, these symptoms are usually absent in those with biochemical B12 deficiency (17).
Several researchers have made recommendations to screen those with type 2 diabetes on metformin for serum B12 levels (6,7,14–16,18–21). However, no formal recommendations have been provided by the medical community or the U.S. Prevention Services Task Force. High-dose B12 injection therapy has been successfully used to correct the metformin-induced decline in serum B12 (15,21,22). The use of B12supplements among those with type 2 diabetes on metformin in a nationally representative sample and their potentially protective effect against biochemical B12 deficiency has not been reported. It is therefore the aim of the current study to use the nationally representative National Health and Nutrition Examination Survey (NHANES) population to determine the prevalence of biochemical B12deficiency among those with type 2 diabetes ≥50 years of age taking metformin compared with those with type 2 diabetes not taking metformin and those without diabetes, and to explore how these relationships are modified by B12 supplement consumption.
Design overview
NHANES is a nationally representative sample of the noninstitutionalized U.S. population with targeted oversampling of U.S. adults ≥60 years of age, African Americans, and Hispanics. Details of these surveys have been described elsewhere (23). All participants gave written informed consent, and the survey protocol was approved by a human subjects review board.
Setting and participants
Our study included adults ≥50 years of age from NHANES 1999–2006. Participants with positive HIV antibody test results, high creatinine levels (>1.7 mg/dL for men and >1.5 mg/dL for women), and prescription B12 injections were excluded from the analysis. Participants who reported having prediabetes or borderline diabetes (n = 226) were removed because they could not be definitively grouped as having or not having type 2 diabetes. We also excluded pregnant women, those with type 1 diabetes, and those without diabetes taking metformin. Based on clinical aspects described by the American Diabetes Association and previous work in NHANES, those who were diagnosed before the age of 30 and began insulin therapy within 1 year of diagnosis were classified as having type 1 diabetes (24,25). Type 2 diabetes status in adults was dichotomized as yes/no. Participants who reported receiving a physician’s diagnosis after age 30 (excluding gestational diabetes) and did not initiate insulin therapy within 1 year of diagnosis were classified as having type 2 diabetes.
Outcomes and follow-up
The primary outcome was biochemical B12 deficiency determined by serum B12 concentrations. Serum B12 levels were quantified using the Quantaphase II folate/vitamin B12 radioassay kit from Bio-Rad Laboratories (Hercules, CA). We defined biochemical B12 deficiency as serum levels ≤148 pmol/L, borderline deficiency as serum B12 >148 to ≤221 pmol/L, and normal as >221 pmol/L (26).
The main exposure of interest was metformin use. Using data collected in the prescription medicine questionnaire, those with type 2 diabetes were classified as currently using metformin therapy (alone or in combination therapy) versus those not currently using metformin. Length of metformin therapy was used to assess the relationship between duration of metformin therapy and biochemical B12 deficiency. In the final analysis, two control groups were used to allow the comparison of those with type 2 diabetes taking metformin with those with type 2 diabetes not taking metformin and those without diabetes.
To determine whether the association between metformin and biochemical B12 deficiency is modified by supplemental B12 intake, data from the dietary supplement questionnaire were used. Information regarding the dose and frequency was used to calculate average daily supplemental B12 intake. We categorized supplemental B12 intake as 0 μg (no B12 containing supplement), >0–6 μg, >6–25 μg, and >25 μg. The lower intake group, >0–6 μg, includes 6 μg, the amount of vitamin B12 typically found in over-the-counter multivitamins, and 2.4 μg, the daily amount the IOM recommends for all adults ≥50 years of age to consume through supplements or fortified food (1). The next group, >6–25 μg, includes 25 μg, the amount available in many multivitamins marketed toward senior adults. The highest group contains the amount found in high-dose B-vitamin supplements.
In the final analysis, there were 575 U.S. adults ≥50 years of age with type 2 diabetes using metformin, 1,046 with type 2 diabetes not using metformin, and 6,867 without diabetes. The demographic and biological characteristics of the groups are shown in Table 1. Among metformin users, mean age was 63.4 ± 0.5 years, 50.3% were male, 66.7% were non-Hispanic white, and 40.7% used a supplement containing B12. The median duration of metformin use was 5 years. Compared with those with type 2 diabetes not taking metformin, metformin users were younger (P < 0.0001), reported a lower prevalence of insulin use (P < 0.001), and had a shorter duration of diabetes (P = 0.0207). Compared with those without diabetes, metformin users had a higher proportion of nonwhite racial groups (P< 0.0001), a higher proportion of obesity (P < 0.0001), a lower prevalence of macrocytosis (P = 0.0017), a lower prevalence of supplemental folic acid use (P = 0.0069), a lower prevalence of supplemental vitamin B12 use (P = 0.0180), and a lower prevalence of calcium supplement use (P = 0.0002). There was a twofold difference in the prevalence of anemia among those with type 2 diabetes versus those without, and no difference between the groups with diabetes.
Association of Biochemical B12Deficiency With Metformin Therapy and Vitamin B12Supplements
Demographic and biological characteristics of U.S. adults ≥50 years of age: NHANES 1999–2006
The geometric mean serum B12 concentration among those with type 2 diabetes taking metformin was 317.5 pmol/L. This was significantly lower than the geometric mean concentration in those with type 2 diabetes not taking metformin (386.7 pmol/L; P = 0.0116) and those without diabetes (350.8 pmol/L; P = 0.0011). As seen in Fig. 1, the weighted prevalence of biochemical B12 deficiency adjusted for age, race, and sex was 5.8% for those with type 2 diabetes taking metformin, 2.2% for those with type 2 diabetes not taking metformin (P = 0.0002), and 3.3% for those without diabetes (P = 0.0026). Among the three aforementioned groups, borderline deficiency was present in 16.2, 5.5, and 8.8%, respectively (P < 0.0001). Applying the Fleiss formula for calculating attributable risk from cross-sectional data (27), among all of the cases of biochemical B12 deficiency, 3.5% of the cases were attributable to metformin use; and among those with diabetes, 41% of the deficient cases were attributable to metformin use. When the prevalence of biochemical B12 deficiency among those with diabetes taking metformin was analyzed by duration of metformin therapy, there was no notable increase in the prevalence of biochemical B12 deficiency as the duration of metformin use increased. The prevalence of biochemical B12 deficiency was 4.1% among those taking metformin <1 year, 6.3% among those taking metformin ≥1–3 years, 4.1% among those taking metformin >3–10 years, and 8.1% among those taking metformin >10 years (P = 0.3219 for <1 year vs. >10 years). Similarly, there was no clear increase in the prevalence of borderline deficiency as the duration of metformin use increased (15.9% among those taking metformin >10 years vs. 11.4% among those taking metformin <1 year; P = 0.4365).
Weighted prevalence of biochemical B12 deficiency and borderline deficiency adjusted for age, race, and sex in U.S. adults ≥50 years of age: NHANES 1999–2006. Black bars are those with type 2 diabetes on metformin, gray bars are those with type 2 diabetes not on metformin, and the white bars are those without diabetes. *P = 0.0002 vs. type 2 diabetes on metformin. †P < 0.0001 vs. type 2 diabetes on metformin. ‡P = 0.0026 vs. type 2 diabetes on metformin.
Table 2 presents a stratified analysis of the weighted prevalence of biochemical B12 deficiency and borderline deficiency by B12supplement use. For those without diabetes, B12 supplement use was associated with an ∼66.7% lower prevalence of both biochemical B12deficiency (4.8 vs. 1.6%; P < 0.0001) and borderline deficiency (16.6 vs. 5.5%; P < 0.0001). A decrease in the prevalence of biochemical B12deficiency was seen at all levels of supplemental B12 intake compared with nonusers of supplements. Among those with type 2 diabetes taking metformin, supplement use was not associated with a decrease in the prevalence of either biochemical B12 deficiency (5.6 vs. 5.3%; P= 0.9137) or borderline deficiency (15.5 vs. 8.8%; P = 0.0826). Among the metformin users who also used supplements, those who consumed >0–6 μg of B12 had a prevalence of biochemical B12 deficiency of 14.1%. However, consumption of a supplement containing >6 μg of B12 was associated with a prevalence of biochemical B12 deficiency of 1.8% (P = 0.0273 for linear trend). Similar trends were seen in the association of supplemental B12 intake and the prevalence of borderline deficiency. For those with type 2 diabetes not taking metformin, supplement use was also not associated with a decrease in the prevalence of biochemical B12 deficiency (2.1 vs. 2.0%; P = 0.9568) but was associated with a 54% reduction in the prevalence of borderline deficiency (7.8 vs. 3.4%; P = 0.0057 for linear trend).
Comparison of average daily B12 supplement intake by weighted prevalence of biochemical B12 deficiency (serum B12 ≤148 pmol/L) and borderline deficiency (serum B12 >148 to ≤221 pmol/L) among U.S. adults ≥50 years of age: NHANES 1999–2006.
Table 3 demonstrates the association of various risk factors with biochemical B12 deficiency. Metformin therapy was associated with biochemical B12 deficiency (odds ratio [OR] 2.89; 95% CI 1.33–6.28) and borderline deficiency (OR 2.32; 95% CI 1.31–4.12) in a crude model (results not shown). After adjusting for age, BMI, and insulin and supplement use, metformin maintained a significant association with biochemical B12 deficiency (OR 2.92; 95% CI 1.28–6.66) and borderline deficiency (OR 2.16; 95% CI 1.22–3.85). Similar to Table 2, B12 supplements were protective against borderline (OR 0.43; 95% CI 0.23–0.81), but not biochemical, B12 deficiency (OR 0.76; 95% CI 0.34–1.70) among those with type 2 diabetes. Among those without diabetes, B12 supplement use was ∼70% protective against biochemical B12 deficiency (OR 0.26; 95% CI 0.17–0.38) and borderline deficiency (OR 0.27; 95% CI 0.21–0.35).
Polytomous logistic regression for potential risk factors of biochemical B12 deficiency and borderline deficiency among U.S. adults ≥50 years of age: NHANES 1999–2006, OR (95% CI)
The IOM has highlighted the detection and diagnosis of B12 deficiency as a high-priority topic for research (1). Our results suggest several findings that add to the complexity and importance of B12 research and its relation to diabetes, and offer new insight into the benefits of B12 supplements. Our data confirm the relationship between metformin and reduced serum B12 levels beyond the background prevalence of biochemical B12 deficiency. Our data demonstrate that an intake of >0–6 μg of B12, which includes the dose most commonly found in over-the-counter multivitamins, was associated with a two-thirds reduction of biochemical B12 deficiency and borderline deficiency among adults without diabetes. This relationship has been previously reported with NHANES and Framingham population data (4,29). In contrast, we did not find that >0–6 μg of B12 was associated with a decrease in the prevalence of biochemical B12 deficiency or borderline deficiency among adults with type 2 diabetes taking metformin. This observation suggests that metformin reduces serum B12 by a mechanism that is additive to or different from the mechanism in older adults. It is also possible that metformin may exacerbate the deficiency among older adults with low serum B12. Our sample size was too small to determine which amount >6 μg was associated with maximum protection, but we did find a dose-response trend.
We were surprised to find that those with type 2 diabetes not using metformin had the lowest prevalence of biochemical B12 deficiency. It is possible that these individuals may seek medical care more frequently than the general population and therefore are being treated for their biochemical B12 deficiency. Or perhaps, because this population had a longer duration of diabetes and a higher proportion of insulin users compared with metformin users, they have been switched from metformin to other diabetic treatments due to low serum B12 concentrations or uncontrolled glucose levels and these new treatments may increase serum B12 concentrations. Despite the observed effects of metformin on serum B12 levels, it remains unclear whether or not this reduction is a public health concern. With lifetime risks of diabetes estimated to be one in three and with metformin being a first-line intervention, it is important to increase our understanding of the effects of oral vitamin B12 on metformin-associated biochemical deficiency (20,21).
The strengths of this study include its nationally representative, population-based sample, its detailed information on supplement usage, and its relevant biochemical markers. This is the first study to use a nationally representative sample to examine the association between serum B12 concentration, diabetes status, and metformin use as well as examine how this relationship may be modified by vitamin B12 supplementation. The data available regarding supplement usage provided specific information regarding dose and frequency. This aspect of NHANES allowed us to observe the dose-response relationship in Table 2 and to compare it within our three study groups.
This study is also subject to limitations. First, NHANES is a cross-sectional survey and it cannot assess time as a factor, and therefore the results are associations and not causal relationships. A second limitation arises in our definition of biochemical B12 deficiency. There is no general consensus on how to define normal versus low serum B12levels. Some researchers include the functional biomarker methylmalonic acid (MMA) in the definition, but this has yet to be agreed upon (30–34). Recently, an NHANES roundtable discussion suggested that definitions of biochemical B12 deficiency should incorporate one biomarker (serum B12 or holotranscobalamin) and one functional biomarker (MMA or total homocysteine) to address problems with sensitivity and specificity of the individual biomarkers. However, they also cited a need for more research on how the biomarkers are related in the general population to prevent misclassification (34). MMA was only measured for six of our survey years; one-third of participants in our final analysis were missing serum MMA levels. Moreover, it has recently been reported that MMA values are significantly greater among the elderly with diabetes as compared with the elderly without diabetes even when controlling for serum B12 concentrations and age, suggesting that having diabetes may independently increase the levels of MMA (35). This unique property of MMA in elderly adults with diabetes makes it unsuitable as part of a definition of biochemical B12 deficiency in our specific population groups. Our study may also be subject to misclassification bias. NHANES does not differentiate between diabetes types 1 and 2 in the surveys; our definition may not capture adults with type 2 diabetes exclusively. Additionally, we used responses to the question “Have you received a physician’s diagnosis of diabetes” to categorize participants as having or not having diabetes. Therefore, we failed to capture undiagnosed diabetes. Finally, we could only assess current metformin use. We cannot determine if nonmetformin users have ever used metformin or if they were not using it at the time of the survey.
Our data demonstrate several important conclusions. First, there is a clear association between metformin and biochemical B12 deficiency among adults with type 2 diabetes. This analysis shows that 6 μg of B12 offered in most multivitamins is associated with two-thirds reduction in biochemical B12 deficiency in the general population, and that this same dose is not associated with protection against biochemical B12 deficiency among those with type 2 diabetes taking metformin. Our results have public health and clinical implications by suggesting that neither 2.4 μg, the current IOM recommendation for daily B12 intake, nor 6 μg, the amount found in most multivitamins, is sufficient for those with type 2 diabetes taking metformin.
This analysis suggests a need for further research. One research design would be to identify those with biochemical B12 deficiency and randomize them to receive various doses of supplemental B12chronically and then evaluate any improvement in serum B12concentrations and/or clinical outcomes. Another design would use existing cohorts to determine clinical outcomes associated with biochemical B12 deficiency and how they are affected by B12supplements at various doses. Given that a significant proportion of the population ≥50 years of age have biochemical B12 deficiency and that those with diabetes taking metformin have an even higher proportion of biochemical B12 deficiency, we suggest that support for further research is a reasonable priority.
Institute of Medicine. Food and Nutrition Board. Dietary Reference Intakes: Thiamin, Riboflavin, Niacin, Vitamin B6, Folate, Vitamin B12, Pantothenic Acid, Biotin, and Choline.Washington, DC, National Academy Press, 1998
Centers for Disease Control and Prevention. National Diabetes Fact Sheet: National Estimates and General Information on Diabetes and Prediabetes in the United States.Atlanta, GA, Department of Health and Human Services, 2011.
. Association between vitamin B12-containing supplement consumption and prevalence of biochemically defined B12 deficiency in adults in NHANES III (third national health and nutrition examination survey). Public Health Nutr 2010;13:25–31
. Long term treatment with metformin in patients with type 2 diabetes and risk of vitamin B-12 deficiency: randomised placebo controlled trial. BMJ2010;340:c2181
.Metformin increases total serum homocysteine levels in non-diabetic male patients with coronary heart disease. Scand J Clin Lab Invest 1997;57:521–527
.Effects of metformin or rosiglitazone on serum concentrations of homocysteine, folate, and vitamin B12 in patients with type 2 diabetes mellitus. J Diabetes Complications 2007;21:118–123
. Effects of short-term treatment with metformin on serum concentrations of homocysteine, folate and vitamin B12 in type 2 diabetes mellitus: a randomized, placebo-controlled trial. J Intern Med2003;254:455–463
. Effect of metformin on plasma homocysteine, vitamin B12 and folic acid: a cross-sectional study in patients with type 2 diabetes mellitus. J Med Assoc Thai2004;87:780–787
. Vitamin B12 status of patients treated with metformin: a cross-sectional cohort study.British Journal of Diabetes and Vascular Disease.2004;4:401–406
. Association of metformin, elevated homocysteine, and methylmalonic acid levels and clinically worsened diabetic peripheral neuropathy. Diabetes Care2010;33:156–161
One research design would be to identify those with biochemical B12 deficiency and randomize them to receive various doses of supplemental B12chronically and then evaluate any improvement in serum B12concentrations and/or clinical outcomes. Another design would use existing cohorts to determine clinical outcomes associated with biochemical B12 deficiency and how they are affected by B12supplements at various doses.
This is of considerable interest. As far as I can see, there is insufficient data presented to discern all of the variables entangled. In a study of 8000 hemograms several years ago, it was of some interest that there were a large percentage of patients who were over age 75 years having a MCV of 94 – 100, not considered indicative of macrocytic anemia. It would have been interesting to explore that set of the data further.
UPDATED 3/17/2020
Nutrients. 2019 May 7;11(5). pii: E1020. doi: 10.3390/nu11051020.
Monitoring Vitamin B12 in Women Treated with Metformin for Primary Prevention of Breast Cancer and Age-Related Chronic Diseases.
Metformin (MET) is currently being used in several trials for cancer prevention or treatment in non-diabetics. However, long-term MET use in diabetics is associated with lower serum levels of total vitamin B12. In a pilot randomized controlled trial of the Mediterranean diet (MedDiet) and MET, whose participants were characterized by different components of metabolic syndrome, we tested the effect of MET on serum levels of B12, holo transcobalamin II (holo-TC-II), and methylmalonic acid (MMA). The study was conducted on 165 women receiving MET or placebo for three years. Results of the study indicate a significant overall reduction in both serum total B12 and holo-TC-II levels according with MET-treatment. In particular, in the MET group 26 of 81 patients and 10 of the 84 placebo-treated subjects had B12 below the normal threshold (<221 pmol/L) at the end of the study. Considering jointly all B12, Holo-TC-II, and MMA, 13 of the 165 subjects (10 MET and 3 placebo-treated) had at least two deficits in the biochemical parameters at the end of the study, without reporting clinical signs. Although our results do not affect whether women remain in the trial, B12 monitoring for MET-treated individuals should be implemented.
ntroduction
Metformin (MET) is the first-line treatment for type-2 diabetes and has been used for decades to treat this chronic condition [1]. Given its favorable effects on glycemic control, weight patterns, insulin requirements, and cardiovascular outcomes, MET has been recently proposed in addition to lifestyle interventions to reduce metabolic syndrome (MS) and age-related chronic diseases [2]. Observational studies have also suggested that diabetic patients treated with MET had a significantly lower risk of developing cancer or lower cancer mortality than those untreated or treated with other drugs [3,4]. For this reason, a number of clinical trials are in progress in different solid cancers.
One of the limitations in implementing long-term use of MET to prevent chronic conditions in healthy subjects relates to its potential lowering effect on vitamin B12 (B12). The aim of the present study was to assess the effect of three years of MET treatment in a randomized, controlled trial considering both B12 levels and biomarkers of its metabolism and biological effectiveness.
Cobalamin, also known as B12, is a water-soluble, cobalt-containing vitamin. All forms of B12 are converted intracellularly into adenosyl-Cbl and methylcobalamin—the biologically active forms at the cellular level [5]. Vitamin B12 is a vital cofactor of two enzymes: methionine synthase and L-methyl-malonyl-coenzyme. A mutase in intracellular enzymatic reactions related to DNA synthesis, as well as in amino and fatty acid metabolism. Vitamin B12, under the catalysis of the enzyme l-methyl-malonyl-CoA mutase, synthesizes succinyl-CoA from methylmalonyl-CoA in the mitochondria. Deficiency of B12, thus results in elevated methylmalonic acid (MMA) levels.
Dietary B12 is normally bound to proteins. Food-bound B12 is released in the stomach under the effect of gastric acid and pepsin. The free vitamin is then bound to an R-binder, a glycoprotein in gastric fluid and saliva that protects B12 from the highly acidic stomach environment. Pancreatic proteases degrade R-binder in the duodenum and liberate B12; finally, the free vitamin is then bound by the intrinsic factor (IF)—a glycosylated protein secreted by gastric parietal cells—forming an IF-B12 complex [6]. The IF resists proteolysis and serves as a carrier for B12 to the terminal ileum where the IF-B12 complex undergoes receptor (cubilin)-mediated endocytosis [7]. The vitamin then appears in circulation bound to holo-transcobalamin-I (holo-TC-I), holo-transcobalamin-II (holo-TC-II), and holo-transcobalamin-III (holo-TC-III). It is estimated that 20–30% of the total circulating B12 is bound to holo-TC-II and only this form is available to the cells [7]. Holo-TC-I binds 70–80% of circulating B12, preventing the loss of the free unneeded portion [6]. Vitamin B12 is stored mainly in the liver and kidneys.
Many mechanisms have been proposed to explain how MET interferes with the absorption of B12: diminished absorption due to changes in bacterial flora, interference with intestinal absorption of the IF–B12 complex (and)/or alterations in IF levels. The most widely accepted current mechanism suggests that MET antagonizes the calcium cation and interferes with the calcium-dependent IF–B12 complex binding to the ileal cubilin receptor [8,9]. The recognition and treatment of B12 deficiency is important because it is a cause of bone marrow failure, macrocytic anemia, and irreversible neuropathy [10].
In general, previous studies on diabetics have observed a reduction in serum levels of B12 after both short- and long-term MET treatment [1]. A recent review on observational studies showed significantly lower levels of B12 and an increased risk of borderline or frank B12 deficiency in patients on MET than not on MET [1]. The meta-analysis of four trials (only one double-blind) found a significant overall mean B12 reducing effect of MET after six weeks to three months of use [1]. A secondary analysis (13 years after randomization) of the Diabetes Prevention Program Outcomes Study, which randomized over 3000 persons at high risk for type 2 diabetes to MET or placebo, showed a 13% increase in the risk of B12 deficiency per year of total MET use [3]. In this study, B12 levels were measured from samples obtained in years 1 and 9. Stored serum samples from other time points, including baseline, were not available, and potentially informative red blood cell indices that might have demonstrated the macrocytic anemia, typical of B12 deficiency, were not recorded [3]. The HOME (Hyperinsulinaemia: the Outcome of its Metabolic Effects) study, a large randomized controlled trial investigating the long-term effects of MET versus placebo in patients with type 2 diabetes treated with insulin, showed that the addition of MET improved glycemic control, reduced insulin requirements, prevented weight gain but lowered serum B12 over time, and raised serum homocysteine, suggesting tissue B12 deficiency [4]. A recent analysis of 277 diabetics from the same trial showed that serum levels of MMA, the specific biomarker for tissue B12 deficiency [5], were significantly higher in people treated with MET than those receiving placebo after four years (on average) [4].
The risk of MET-associated B12 deficiency may be higher in older individuals and those with poor dietary habits. Prospective studies have found negative associations between obesity and B12 in numerous ethnicities [11,12]. An energy-dense but micronutrient-insufficient diet consumed by individuals who are overweight or obese might explain this [12]. Furthermore, obesity is associated with low-grade inflammation and these physiological changes have been shown to be associated, in several studies, with elevated C-reactive protein and homocysteine and with low concentrations of B12 and other vitamins [13,14].
As part of a pilot randomized controlled trial of the Mediterranean diet (MedDiet) and MET for primary prevention of breast cancer and other chronic age-related diseases in healthy women with tracts of MS [15] we tested the effect of MET on serum levels of B12, holo-TC-II, and MMA.
Other articles of note on the Mediterranean Diet in this Online Open Access Scientific Journal Include
References in https://www.wjnm.org/article.asp?issn=1450-1147;year=2019;volume=18;issue=1;spage=8;epage=12;aulast=Hertz
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