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Larry H. Bernstein, MD, FCAP, Curator

Chief Scientific Officer, Leaders in Pharmaceutical Intelligence

Cholesterol and Regulation of Liver Synthetic Pathways

SREBPs: activators of the complete program of cholesterol and fatty acid synthesis in the liver

Jay D. Horton1,2, Joseph L. Goldstein1 and Michael S. Brown1

1Department of Molecular Genetics, and
2Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas, USA

J Clin Invest. 2002;109(9):1125–1131.
http://dx.doi.org:/10.1172/JCI15593
Lipid homeostasis in vertebrate cells is regulated by a family of membrane-bound transcription factors designated sterol regulatory element–binding proteins (SREBPs). SREBPs directly activate the expression of more than 30 genes dedicated to the synthesis and uptake of cholesterol, fatty acids, triglycerides, and phospholipids, as well as the NADPH cofactor required to synthesize these molecules (14). In the liver, three SREBPs regulate the production of lipids for export into the plasma as lipoproteins and into the bile as micelles. The complex, interdigitated roles of these three SREBPs have been dissected through the study of ten different lines of gene-manipulated mice. These studies form the subject of this review.

SREBPs: activation through proteolytic processing

SREBPs belong to the basic helix-loop-helix–leucine zipper (bHLH-Zip) family of transcription factors, but they differ from other bHLH-Zip proteins in that they are synthesized as inactive precursors bound to the endoplasmic reticulum (ER) (1, 5). Each SREBP precursor of about 1150 amino acids is organized into three domains: (a) an NH2-terminal domain of about 480 amino acids that contains the bHLH-Zip region for binding DNA; (b) two hydrophobic transmembrane–spanning segments interrupted by a short loop of about 30 amino acids that projects into the lumen of the ER; and (c) a COOH-terminal domain of about 590 amino acids that performs the essential regulatory function described below.

In order to reach the nucleus and act as a transcription factor, the NH2-terminal domain of each SREBP must be released from the membrane proteolytically (Figure 1). Three proteins required for SREBP processing have been delineated in cultured cells, using the tools of somatic cell genetics (see ref. 5for review). One is an escort protein designated SREBP cleavage–activating protein (SCAP). The other two are proteases, designated Site-1 protease (S1P) and Site-2 protease (S2P). Newly synthesized SREBP is inserted into the membranes of the ER, where its COOH-terminal regulatory domain binds to the COOH-terminal domain of SCAP (Figure 1).

Figure 1

Model for the sterol-mediated proteolytic release of SREBPs from membranes JCI0215593.f1

Model for the sterol-mediated proteolytic release of SREBPs from membranes JCI0215593.f1

Model for the sterol-mediated proteolytic release of SREBPs from membranes. SCAP is a sensor of sterols and an escort of SREBPs. When cells are depleted of sterols, SCAP transports SREBPs from the ER to the Golgi apparatus, where two proteases, Site-1 protease (S1P) and Site-2 protease (S2P), act sequentially to release the NH2-terminal bHLH-Zip domain from the membrane. The bHLH-Zip domain enters the nucleus and binds to a sterol response element (SRE) in the enhancer/promoter region of target genes, activating their transcription. When cellular cholesterol rises, the SCAP/SREBP complex is no longer incorporated into ER transport vesicles, SREBPs no longer reach the Golgi apparatus, and the bHLH-Zip domain cannot be released from the membrane. As a result, transcription of all target genes declines. Reprinted from ref. 5 with permission.

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SCAP is both an escort for SREBPs and a sensor of sterols. When cells become depleted in cholesterol, SCAP escorts the SREBP from the ER to the Golgi apparatus, where the two proteases reside. In the Golgi apparatus, S1P, a membrane-bound serine protease, cleaves the SREBP in the luminal loop between its two membrane-spanning segments, dividing the SREBP molecule in half (Figure 1). The NH2-terminal bHLH-Zip domain is then released from the membrane via a second cleavage mediated by S2P, a membrane-bound zinc metalloproteinase. The NH2-terminal domain, designated nuclear SREBP (nSREBP), translocates to the nucleus, where it activates transcription by binding to nonpalindromic sterol response elements (SREs) in the promoter/enhancer regions of multiple target genes.

When the cholesterol content of cells rises, SCAP senses the excess cholesterol through its membranous sterol-sensing domain, changing its conformation in such a way that the SCAP/SREBP complex is no longer incorporated into ER transport vesicles. The net result is that SREBPs lose their access to S1P and S2P in the Golgi apparatus, so their bHLH-Zip domains cannot be released from the ER membrane, and the transcription of target genes ceases (1, 5). The biophysical mechanism by which SCAP senses sterol levels in the ER membrane and regulates its movement to the Golgi apparatus is not yet understood. Elucidating this mechanism will be fundamental to understanding the molecular basis of cholesterol feedback inhibition of gene expression.

SREBPs: two genes, three proteins

The mammalian genome encodes three SREBP isoforms, designated SREBP-1a, SREBP-1c, and SREBP-2. SREBP-2 is encoded by a gene on human chromosome 22q13. Both SREBP-1a and -1c are derived from a single gene on human chromosome 17p11.2 through the use of alternative transcription start sites that produce alternate forms of exon 1, designated 1a and 1c (1). SREBP-1a is a potent activator of all SREBP-responsive genes, including those that mediate the synthesis of cholesterol, fatty acids, and triglycerides. High-level transcriptional activation is dependent on exon 1a, which encodes a longer acidic transactivation segment than does the first exon of SREBP-1c. The roles of SREBP-1c and SREBP-2 are more restricted than that of SREBP-1a. SREBP-1c preferentially enhances transcription of genes required for fatty acid synthesis but not cholesterol synthesis. Like SREBP-1a, SREBP-2 has a long transcriptional activation domain, but it preferentially activates cholesterol synthesis (1). SREBP-1a and SREBP-2 are the predominant isoforms of SREBP in most cultured cell lines, whereas SREBP-1c and SREBP-2 predominate in the liver and most other intact tissues (6).

When expressed at higher than physiologic levels, each of the three SREBP isoforms can activate all enzymes indicated in Figure 2, which shows the biosynthetic pathways used to generate cholesterol and fatty acids. However, at normal levels of expression, SREBP-1c favors the fatty acid biosynthetic pathway and SREBP-2 favors cholesterologenesis. SREBP-2–responsive genes in the cholesterol biosynthetic pathway include those for the enzymes HMG-CoA synthase, HMG-CoA reductase, farnesyl diphosphate synthase, and squalene synthase. SREBP-1c–responsive genes include those for ATP citrate lyase (which produces acetyl-CoA) and acetyl-CoA carboxylase and fatty acid synthase (which together produce palmitate [C16:0]). Other SREBP-1c target genes encode a rate-limiting enzyme of the fatty acid elongase complex, which converts palmitate to stearate (C18:0) (ref.7); stearoyl-CoA desaturase, which converts stearate to oleate (C18:1); and glycerol-3-phosphate acyltransferase, the first committed enzyme in triglyceride and phospholipid synthesis (3). Finally, SREBP-1c and SREBP-2 activate three genes required to generate NADPH, which is consumed at multiple stages in these lipid biosynthetic pathways (8) (Figure 2).

Figure 2

major metabolic intermediates in the pathways for synthesis of cholesterol, fatty acids, and triglycerides JCI0215593.f2

major metabolic intermediates in the pathways for synthesis of cholesterol, fatty acids, and triglycerides JCI0215593.f2

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Genes regulated by SREBPs. The diagram shows the major metabolic intermediates in the pathways for synthesis of cholesterol, fatty acids, and triglycerides. In vivo, SREBP-2 preferentially activates genes of cholesterol metabolism, whereas SREBP-1c preferentially activates genes of fatty acid and triglyceride metabolism. DHCR, 7-dehydrocholesterol reductase; FPP, farnesyl diphosphate; GPP, geranylgeranyl pyrophosphate synthase; CYP51, lanosterol 14α-demethylase; G6PD, glucose-6-phosphate dehydrogenase; PGDH, 6-phosphogluconate dehydrogenase; GPAT, glycerol-3-phosphate acyltransferase.

Genes regulated by SREBPs. The diagram shows the major metabolic intermediates in the pathways for synthesis of cholesterol, fatty acids, and triglycerides. In vivo, SREBP-2 preferentially activates genes of cholesterol metabolism, whereas SREBP-1c preferentially activates genes of fatty acid and triglyceride metabolism. DHCR, 7-dehydrocholesterol reductase; FPP, farnesyl diphosphate; GPP, geranylgeranyl pyrophosphate synthase; CYP51, lanosterol 14α-demethylase; G6PD, glucose-6-phosphate dehydrogenase; PGDH, 6-phosphogluconate dehydrogenase; GPAT, glycerol-3-phosphate acyltransferase.

Knockout and transgenic mice

Ten different genetically manipulated mouse models that either lack or overexpress a single component of the SREBP pathway have been generated in the last 6 years (916). The key molecular and metabolic alterations observed in these mice are summarized in Table 1.

Table 1
Alterations in hepatic lipid metabolism in gene-manipulated mice overexpressing or lacking SREBPs

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Knockout mice that lack all nSREBPs die early in embryonic development. For instance, a germline deletion of S1p, which prevents the processing of all SREBP isoforms, results in death before day 4 of development (15, 17). Germline deletion of Srebp2 leads to 100% lethality at a later stage of embryonic development than does deletion of S1p (embryonic day 7–8). In contrast, germline deletion of Srebp1, which eliminates both the 1a and the 1c transcripts, leads to partial lethality, in that about 15–45% of Srebp1–/– mice survive (13). The surviving homozygotes manifest elevated levels of SREBP-2 mRNA and protein (Table 1), which presumably compensates for the loss of SREBP-1a and -1c. When the SREBP-1c transcript is selectively eliminated, no embryonic lethality is observed, suggesting that the partial embryonic lethality in the Srebp1–/– mice is due to the loss of the SREBP-1a transcript (16).

To bypass embryonic lethality, we have produced mice in which all SREBP function can be disrupted in adulthood through induction of Cre recombinase. For this purpose, loxP recombination sites were inserted into genomic regions that flank crucial exons in the Scap or S1p genes (so-called floxed alleles) (14, 15). Mice homozygous for the floxed gene and heterozygous for a Cre recombinase transgene, which is under control of an IFN-inducible promoter (MX1-Cre), can be induced to delete Scap or S1p by stimulating IFN expression. Thus, following injection with polyinosinic acid–polycytidylic acid, a double-stranded RNA that provokes antiviral responses, the Cre recombinase is produced in liver and disrupts the floxed gene by recombination between the loxP sites.

Cre-mediated disruption of Scap or S1p dramatically reduces nSREBP-1 and nSREBP-2 levels in liver and diminishes expression of all SREBP target genes in both the cholesterol and the fatty acid synthetic pathways (Table 1). As a result, the rates of synthesis of cholesterol and fatty acids fall by 70–80% in Scap- and S1p-deficient livers.

In cultured cells, the processing of SREBP is inhibited by sterols, and the sensor for this inhibition is SCAP (5). To learn whether SCAP performs the same function in liver, we have produced transgenic mice that express a mutant SCAP with a single amino acid substitution in the sterol-sensing domain (D443N) (12). Studies in tissue culture show that SCAP(D443N) is resistant to inhibition by sterols. Cells that express a single copy of this mutant gene overproduce cholesterol (18). Transgenic mice that express this mutant version of SCAP in the liver exhibit a similar phenotype (12). These livers manifest elevated levels of nSREBP-1 and nSREBP-2, owing to constitutive SREBP processing, which is not suppressed when the animals are fed a cholesterol-rich diet. nSREBP-1 and -2 increase the expression of all SREBP target genes shown in Figure 2, thus stimulating cholesterol and fatty acid synthesis and causing a marked accumulation of hepatic cholesterol and triglycerides (Table 1). This transgenic model provides strong in vivo evidence that SCAP activity is normally under partial inhibition by endogenous sterols, which keeps the synthesis of cholesterol and fatty acids in a partially repressed state in the liver.

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Function of individual SREBP isoforms in vivo

To study the functions of individual SREBPs in the liver, we have produced transgenic mice that overexpress truncated versions of SREBPs (nSREBPs) that terminate prior to the membrane attachment domain. These nSREBPs enter the nucleus directly, bypassing the sterol-regulated cleavage step. By studying each nSREBP isoform separately, we could determine their distinct activating properties, albeit when overexpressed at nonphysiologic levels.

Overexpression of nSREBP-1c in the liver of transgenic mice produces a triglyceride-enriched fatty liver with no increase in cholesterol (10). mRNAs for fatty acid synthetic enzymes and rates of fatty acid synthesis are elevated fourfold in this tissue, whereas the mRNAs for cholesterol synthetic enzymes and the rate of cholesterol synthesis are not increased (8). Conversely, overexpression of nSREBP-2 in the liver increases the mRNAs only fourfold. This increase in cholesterol synthesis is even more remarkable when encoding all cholesterol biosynthetic enzymes; the most dramatic is a 75-fold increase in HMG-CoA reductase mRNA (11). mRNAs for fatty acid synthesis enzymes are increased to a lesser extent, consistent with the in vivo observation that the rate of cholesterol synthesis increases 28-fold in these transgenic nSREBP-2 livers, while fatty acid synthesis increases one considers the extent of cholesterol overload in this tissue, which would ordinarily reduce SREBP processing and essentially abolish cholesterol synthesis (Table 1).

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We have also studied the consequences of overexpressing SREBP-1a, which is expressed only at low levels in the livers of adult mice, rats, hamsters, and humans (6). nSREBP-1a transgenic mice develop a massive fatty liver engorged with both cholesterol and triglycerides (9), with heightened expression of genes controlling cholesterol biosynthesis and, still more dramatically, fatty acid synthesis (Table 1). The preferential activation of fatty acid synthesis (26-fold increase) relative to cholesterol synthesis (fivefold increase) explains the greater accumulation of triglycerides in their livers. The relative representation of the various fatty acids accumulating in this tissue is also unusual. Transgenic nSREBP-1a livers contain about 65% oleate (C18:1), markedly higher levels than the 15–20% found in typical wild-type livers (8) — a result of the induction of fatty acid elongase and stearoyl-CoA desaturase-1 (7). Considered together, the overexpression studies indicate that both SREBP-1 isoforms show a relative preference for activating fatty acid synthesis, whereas SREBP-2 favors cholesterol.

The phenotype of animals lacking the Srebp1 gene, which encodes both the SREBP-1a and -1c transcripts, also supports the notion of distinct hepatic functions for SREBP-1 and SREBP-2 (13). Most homozygous SREBP-1 knockout mice die in utero. The surviving Srebp1–/– mice show reduced synthesis of fatty acids, owing to reduced expression of mRNAs for fatty acid synthetic enzymes (Table 1). Hepatic nSREBP-2 levels increase in these mice, presumably in compensation for the loss of nSREBP-1. As a result, transcription of cholesterol biosynthetic genes increases, producing a threefold increase in hepatic cholesterol synthesis (Table 1).

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The studies in genetically manipulated mice clearly show that, as in cultured cells, SCAP and S1P are required for normal SREBP processing in the liver. SCAP, acting through its sterol-sensing domain, mediates feedback regulation of cholesterol synthesis. The SREBPs play related but distinct roles: SREBP-1c, the predominant SREBP-1 isoform in adult liver, preferentially activates genes required for fatty acid synthesis, while SREBP-2 preferentially activates the LDL receptor gene and various genes required for cholesterol synthesis. SREBP-1a and SREBP-2, but not SREBP-1c, are required for normal embryogenesis.

Transcriptional regulation of SREBP genes

Regulation of SREBPs occurs at two levels — transcriptional and posttranscriptional. The posttranscriptional regulation discussed above involves the sterol-mediated suppression of SREBP cleavage, which results from sterol-mediated suppression of the movement of the SCAP/SREBP complex from the ER to the Golgi apparatus (Figure 1). This form of regulation is manifest not only in cultured cells (1), but also in the livers of rodents fed cholesterol-enriched diets (19).

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The transcriptional regulation of the SREBPs is more complex. SREBP-1c and SREBP-2 are subject to distinct forms of transcriptional regulation, whereas SREBP-1a appears to be constitutively expressed at low levels in liver and most other tissues of adult animals (6). One mechanism of regulation shared by SREBP-1c and SREBP-2 involves a feed-forward regulation mediated by SREs present in the enhancer/promoters of each gene (20, 21). Through this feed-forward loop, nSREBPs activate the transcription of their own genes. In contrast, when nSREBPs decline, as in Scap or S1p knockout mice, there is a secondary decline in the mRNAs encoding SREBP-1c and SREBP-2 (14, 15).

Three factors selectively regulate the transcription of SREBP-1c: liver X-activated receptors (LXRs), insulin, and glucagon. LXRα and LXRβ, nuclear receptors that form heterodimers with retinoid X receptors, are activated by a variety of sterols, including oxysterol intermediates that form during cholesterol biosynthesis (2224). An LXR-binding site in the SREBP-1c promoter activates SREBP-1c transcription in the presence of LXR agonists (23). The functional significance of LXR-mediated SREBP-1c regulation has been confirmed in two animal models. Mice that lack both LXRα and LXRβ express reduced levels of SREBP-1c and its lipogenic target enzymes in liver and respond relatively weakly to treatment with a synthetic LXR agonist (23). Because a similar blunted response is found in mice that lack SREBP-1c, it appears that LXR increases fatty acid synthesis largely by inducing SREBP-1c (16). LXR-mediated activation of SREBP-1c transcription provides a mechanism for the cell to induce the synthesis of oleate when sterols are in excess (23). Oleate is the preferred fatty acid for the synthesis of cholesteryl esters, which are necessary for both the transport and the storage of cholesterol.

LXR-mediated regulation of SREBP-1c appears also to be one mechanism by which unsaturated fatty acids suppress SREBP-1c transcription and thus fatty acid synthesis. Rodents fed diets enriched in polyunsaturated fatty acids manifest reduced SREBP-1c mRNA expression and low rates of lipogenesis in liver (25). In vitro, unsaturated fatty acids competitively block LXR activation of SREBP-1c expression by antagonizing the activation of LXR by its endogenous ligands (26). In addition to LXR-mediated transcriptional inhibition, polyunsaturated fatty acids lower SREBP-1c levels by accelerating degradation of its mRNA (27). These combined effects may contribute to the long-recognized ability of polyunsaturated fatty acids to lower plasma triglyceride levels.

SREBP-1c and the insulin/glucagon ratio

The liver is the organ responsible for the conversion of excess carbohydrates to fatty acids to be stored as triglycerides or burned in muscle. A classic action of insulin is to stimulate fatty acid synthesis in liver during times of carbohydrate excess. The action of insulin is opposed by glucagon, which acts by raising cAMP. Multiple lines of evidence suggest that insulin’s stimulatory effect on fatty acid synthesis is mediated by an increase in SREBP-1c. In isolated rat hepatocytes, insulin treatment increases the amount of mRNA for SREBP-1c in parallel with the mRNAs of its target genes (28, 29). The induction of the target genes can be blocked if a dominant negative form of SREBP-1c is expressed (30). Conversely, incubating primary hepatocytes with glucagon or dibutyryl cAMP decreases the mRNAs for SREBP-1c and its associated lipogenic target genes (30, 31).

In vivo, the total amount of SREBP-1c in liver and adipose tissue is reduced by fasting, which suppresses insulin and increases glucagon levels, and is elevated by refeeding (32, 33). The levels of mRNA for SREBP-1c target genes parallel the changes in SREBP-1c expression. Similarly, SREBP-1c mRNA levels fall when rats are treated with streptozotocin, which abolishes insulin secretion, and rise after insulin injection (29). Overexpression of nSREBP-1c in livers of transgenic mice prevents the reduction in lipogenic mRNAs that normally follows a fall in plasma insulin levels (32). Conversely, in livers of Scap knockout mice that lack all nSREBPs in the liver (14) or knockout mice lacking either nSREBP-1c (16) or both SREBP-1 isoforms (34), there is a marked decrease in the insulin-induced stimulation of lipogenic gene expression that normally occurs after fasting/refeeding. It should be noted that insulin and glucagon also exert a posttranslational control of fatty acid synthesis though changes in the phosphorylation and activation of acetyl-CoA carboxylase. The posttranslational regulation of fatty acid synthesis persists in transgenic mice that overexpress nSREBP-1c (10). In these mice, the rates of fatty acid synthesis, as measured by [3H]water incorporation, decline after fasting even though the levels of the lipogenic mRNAs remain high (our unpublished observations).

Taken together, the above evidence suggests that SREBP-1c mediates insulin’s lipogenic actions in liver. Recent in vitro and in vivo studies involving adenoviral gene transfer suggest that SREBP-1c may also contribute to the regulation of glucose uptake and glucose synthesis. When overexpressed in hepatocytes, nSREBP-1c induces expression of glucokinase, a key enzyme in glucose utilization. It also suppresses phosphoenolpyruvate carboxykinase, a key gluconeogenic enzyme (35, 36).

SREBPs in disease

Many individuals with obesity and insulin resistance also have fatty livers, one of the most commonly encountered liver abnormalities in the US (37). A subset of individuals with fatty liver go on to develop fibrosis, cirrhosis, and liver failure. Evidence indicates that the fatty liver of insulin resistance is caused by SREBP-1c, which is elevated in response to the high insulin levels. Thus, SREBP-1c levels are elevated in the fatty livers of obese (ob/ob) mice with insulin resistance and hyperinsulinemia caused by leptin deficiency (38, 39). Despite the presence of insulin resistance in peripheral tissues, insulin continues to activate SREBP-1c transcription and cleavage in the livers of these insulin-resistant mice. The elevated nSREBP-1c increases lipogenic gene expression, enhances fatty acid synthesis, and accelerates triglyceride accumulation (31, 39). These metabolic abnormalities are reversed with the administration of leptin, which corrects the insulin resistance and lowers the insulin levels (38).

Metformin, a biguanide drug used to treat insulin-resistant diabetes, reduces hepatic nSREBP-1 levels and dramatically lowers the lipid accumulation in livers of insulin-resistant ob/ob mice (40). Metformin stimulates AMP-activated protein kinase (AMPK), an enzyme that inhibits lipid synthesis through phosphorylation and inactivation of key lipogenic enzymes (41). In rat hepatocytes, metformin-induced activation of AMPK also leads to decreased mRNA expression of SREBP-1c and its lipogenic target genes (41), but the basis of this effect is not understood.

The incidence of coronary artery disease increases with increasing plasma LDL-cholesterol levels, which in turn are inversely proportional to the levels of hepatic LDL receptors. SREBPs stimulate LDL receptor expression, but they also enhance lipid synthesis (1), so their net effect on plasma lipoprotein levels depends on a balance between opposing effects. In mice, the plasma levels of lipoproteins tend to fall when SREBPs are either overexpressed or underexpressed. In transgenic mice that overexpress nSREBPs in liver, plasma cholesterol and triglycerides are generally lower than in control mice (Table 1), even though these mice massively overproduce fatty acids, cholesterol, or both. Hepatocytes of nSREBP-1a transgenic mice overproduce VLDL, but these particles are rapidly removed through the action of LDL receptors, and they do not accumulate in the plasma. Indeed, some nascent VLDL particles are degraded even before secretion by a process that is mediated by LDL receptors (42). The high levels of nSREBP-1a in these animals support continued expression of the LDL receptor, even in cells whose cholesterol concentration is elevated. In LDL receptor–deficient mice carrying the nSREBP-1a transgene, plasma cholesterol and triglyceride levels rise tenfold (43).

Mice that lack all SREBPs in liver as a result of disruption of Scap or S1p also manifest lower plasma cholesterol and triglyceride levels (Table 1).

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In these mice, hepatic cholesterol and triglyceride synthesis is markedly reduced, and this likely causes a decrease in VLDL production and secretion. LDL receptor mRNA and LDL clearance from plasma is also significantly reduced in these mice, but the reduction in LDL clearance is less than the overall reduction in VLDL secretion, the net result being a decrease in plasma lipid levels (15). However, because

humans and mice differ substantially with regard to LDL receptor expression, LDL levels, and other aspects of lipoprotein metabolism,

it is difficult to predict whether human plasma lipids will rise or fall when the SREBP pathway is blocked or activated.

SREBPs in liver: unanswered questions

The studies of SREBPs in liver have exposed a complex regulatory system whose individual parts are coming into focus. Major unanswered questions relate to the ways in which the transcriptional and posttranscriptional controls on SREBP activity are integrated so as to permit independent regulation of cholesterol and fatty acid synthesis in specific nutritional states. A few clues regarding these integration mechanisms are discussed below.

Whereas cholesterol synthesis depends almost entirely on SREBPs, fatty acid synthesis is only partially dependent on these proteins. This has been shown most clearly in cultured nonhepatic cells such as Chinese hamster ovary cells. In the absence of SREBP processing, as when the Site-2 protease is defective, the levels of mRNAs encoding cholesterol biosynthetic enzymes and the rates of cholesterol synthesis decline nearly to undetectable levels, whereas the rate of fatty acid synthesis is reduced by only 30% (44). Under these conditions, transcription of the fatty acid biosynthetic genes must be maintained by factors other than SREBPs. In liver, the gene encoding fatty acid synthase (FASN) can be activated transcriptionally by upstream stimulatory factor, which acts in concert with SREBPs (45). The FASN promoter also contains an LXR element that permits a low-level response to LXR ligands even when SREBPs are suppressed (46). These two transcription factors may help to maintain fatty acid synthesis in liver when nSREBP-1c is low.

Another mechanism of differential regulation is seen in the ability of cholesterol to block the processing of SREBP-2, but not SREBP-1, under certain metabolic conditions. This differential regulation has been studied most thoroughly in cultured cells such as human embryonic kidney (HEK-293) cells. When these cells are incubated in the absence of fatty acids and cholesterol, the addition of sterols blocks processing of SREBP-2, but not SREBP-1, which is largely produced as SREBP-1a in these cells (47). Inhibition of SREBP-1 processing requires an unsaturated fatty acid, such as oleate or arachidonate, in addition to sterols (47). In the absence of fatty acids and in the presence of sterols, SCAP may be able to carry SREBP-1 proteins, but not SREBP-2, to the Golgi apparatus. Further studies are necessary to document this apparent independent regulation of SREBP-1 and SREBP-2 processing and to determine its mechanism.

Acknowledgments

Support for the research cited from the authors’ laboratories was provided by grants from the NIH (HL-20948), the Moss Heart Foundation, the Keck Foundation, and the Perot Family Foundation. J.D. Horton is a Pew Scholar in the Biomedical Sciences and is the recipient of an Established Investigator Grant from the American Heart Association and a Research Scholar Award from the American Digestive Health Industry.

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Essential Fatty Acids


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

Chief Scientific Officer, Leaders in Pharmaceutical Intelligence

Essential Fatty Acids

http://pharmaceuticalintelligence.com/10/25/2014/larryhbern/Essential_Fatty_Acids

The Recognition of Essential Fatty Acids

Dietary fat has long been recognized as an important source of energy for mammals, but in the late 1920s, researchers demonstrated the dietary requirement for particular fatty acids, which came to be called essential fatty acids. It was not until the advent of intravenous feeding, however, that the importance of essential fatty acids was widely accepted: Clinical signs of essential fatty acid deficiency are generally observed only in patients on total parenteral nutrition who received mixtures devoid of essential fatty acids or in those with malabsorption syndromes.

These signs include dermatitis and changes in visual and neural function. Over the past 40 years, an increasing number of physiological functions, such as immunomodulation, have been attributed to the essential fatty acids and their metabolites, and this area of research remains quite active.1, 2

Fatty Acid Nomenclature

The fat found in foods consists largely of a heterogeneous mixture of triacylglycerols (triglycerides)–glycerol molecules that are each combined with three fatty acids. The fatty acids can be divided into two categories, based on chemical properties: saturated fatty acids, which are usually solid at room temperature, and unsaturated fatty acids, which are liquid at room temperature. The term “saturation” refers to a chemical structure in which each carbon atom in the fatty acyl chain is bound to (saturated with) four other atoms, these carbons are linked by single bonds, and no other atoms or molecules can attach; unsaturated fatty acids contain at least one pair of carbon atoms linked by a double bond, which allows the attachment of additional atoms to those carbons (resulting in saturation). Despite their differences in structure, all fats contain approximately the same amount of energy (37 kilojoules/gram, or 9 kilocalories/gram).

The class of unsaturated fatty acids can be further divided into monounsaturated and polyunsaturated fatty acids. Monounsaturated fatty acids (the primary constituents of olive and canola oils) contain only one double bond. Polyunsaturated fatty acids (PUFAs) (the primary constituents of corn, sunflower, flax seed and many other vegetable oils) contain more than one double bond. Fatty acids are often referred to using the number of carbon atoms in the acyl chain, followed by a colon, followed by the number of double bonds in the chain (e.g., 18:1 refers to the 18-carbon monounsaturated fatty acid, oleic acid; 18:3 refers to any 18-carbon PUFA with three double bonds).

PUFAs are further categorized on the basis of the location of their double bonds. An omega or n notation indicates the number of carbon atoms from the methyl end of the acyl chain to the first double bond. Thus, for example, in the omega-3 (n-3) family of PUFAs, the first double bond is 3 carbons from the methyl end of the molecule. The trivial names, chemical names and abbreviations for the omega-3 fatty acids are detailed in Table 1.1.  Finally, PUFAs can be categorized according to their chain length. The 18-carbon n-3 and n-6 short-chain PUFAs are precursors to the longer 20- and 22-carbon PUFAs, called long-chain PUFAs (LCPUFAs).

Fatty Acid Metabolism

Mammalian cells can introduce double bonds into all positions on the fatty acid chain except the n-3 and n-6 position. Thus, the short-chain alpha- linolenic acid (ALA, chemical abbreviation: 18:3n-3) and linoleic acid (LA, chemical abbreviation: 18:2n-6) are essential fatty acids.

No other fatty acids found in food are considered ‘essential’ for humans, because they can all be synthesized from the short chain fatty acids.

Following ingestion, ALA and LA can be converted in the liver to the long chain, more unsaturated n-3 and n-6 LCPUFAs by a complex set of synthetic pathways that share several enzymes (Figure 1). LC PUFAs retain the original sites of desaturation (including n-3 or n-6). The omega-6 fatty acid LA is converted to gamma-linolenic acid (GLA, 18:3n-6), an omega- 6 fatty acid that is a positional isomer of ALA. GLA, in turn, can be converted to the longerchain omega-6 fatty acid, arachidonic acid (AA, 20:4n-6). AA is the precursor for certain classes of an important family of hormone- like substances called the eicosanoids (see below).

The omega-3 fatty acid ALA (18:3n-3) can be converted to the long-chain omega-3 fatty acid, eicosapentaenoic acid (EPA; 20:5n-3). EPA can be elongated to docosapentaenoic acid (DPA 22:5n-3), which is further desaturated to docosahexaenoic acid (DHA; 22:6n-3). EPA and DHA are also precursors of several classes of eicosanoids and are known to play several other critical roles, some of which are discussed further below.

The conversion from parent fatty acids into the LC PUFAs – EPA, DHA, and AA – appears to occur slowly in humans. In addition, the regulation of conversion is not well understood, although it is known that ALA and LA compete for entry into the metabolic pathways.

Physiological Functions of EPA and AA

As stated earlier, fatty acids play a variety of physiological roles. The specific biological functions of a fatty acid are determined by the number and position of double bonds and the length of the acyl chain.

Both EPA (20:5n-3) and AA (20:4n-6) are precursors for the formation of a family of hormone- like agents called eicosanoids. Eicosanoids are rudimentary hormones or regulating – molecules that appear to occur in most forms of life. However, unlike endocrine hormones, which travel in the blood stream to exert their effects at distant sites, the eicosanoids are autocrine or paracrine factors, which exert their effects locally – in the cells that synthesize them or adjacent cells. Processes affected include the movement of calcium and other substances into and out of cells, relaxation and contraction of muscles, inhibition and promotion of clotting, regulation of secretions including digestive juices and hormones, and control of fertility, cell division, and growth.3

The eicosanoid family includes subgroups of substances known as prostaglandins, leukotrienes, and thromboxanes, among others. As shown in Figure 1.1, the long-chain omega-6 fatty acid, AA (20:4n-6), is the precursor of a group of eicosanoids that include series-2 prostaglandins and series-4 leukotrienes. The omega-3 fatty acid, EPA (20:5n-3), is the precursor to a group of eicosanoids that includes series-3 prostaglandins and series-5 leukotrienes. The AA-derived series-2 prostaglandins and series-4 leukotrienes are often synthesized in response to some emergency such as injury or stress, whereas the EPA-derived series-3 prostaglandins and series-5 leukotrienes appear to modulate the effects of the series-2 prostaglandins and series-4 leukotrienes (usually on the same target cells). More specifically, the series-3 prostaglandins are formed at a slower rate and work to attenuate the effects of excessive levels of series-2 prostaglandins. Thus, adequate production of the series-3 prostaglandins seems to protect against heart attack and stroke as well as certain inflammatory diseases like arthritis, lupus, and asthma.3.

EPA (22:6 n-3) also affects lipoprotein metabolism and decreases the production of substances – including cytokines, interleukin 1ß (IL-1ß), and tumor necrosis factor a (TNF-a) – that have pro-inflammatory effects (such as stimulation of collagenase synthesis and the expression of adhesion molecules necessary for leukocyte extravasation [movement from the circulatory system into tissues]).2 The mechanism responsible for the suppression of cytokine production by omega-3 LC PUFAs remains unknown, although suppression of omega-6-derived eicosanoid production by omega-3 fatty acids may be involved, because the omega-3 and omega-6 fatty acids compete for a common enzyme in the eicosanoid synthetic pathway, delta-6 desaturase.

DPA (22:5n-3) (the elongation product of EPA) and its metabolite DHA (22:6n-3) are frequently referred to as very long chain n-3 fatty acids (VLCFA). Along with AA, DHA is the major PUFA found in the brain and is thought to be important for brain development and function. Recent research has focused on this role and the effect of supplementing infant formula with DHA (since DHA is naturally present in breast milk but not in formula).

Dietary Sources and Requirements

Both ALA and LA are present in a variety of foods. LA is present in high concentrations in many commonly used oils, including safflower, sunflower, soy, and corn oil. ALA is present in some commonly used oils, including canola and soybean oil, and in some leafy green vegetables. Thus, the major dietary sources of ALA and LA are PUFA-rich vegetable oils. The proportion of LA to ALA as well as the proportion of those PUFAs to others varies considerably by the type of oil. With the exception of flaxseed, canola, and soybean oil, the ratio of LA to ALA in vegetable oils is at least 10 to 1. The ratios of LA to ALA for flaxseed, canola, and soy are approximately 1: 3.5, 2:1, and 8:1, respectively; however, flaxseed oil is not typically consumed in the North American diet. It is estimated that on average in the U.S., LA accounts for 89% of the total PUFAs consumed, and ALA accounts for 9%. Another estimate suggests that Americans consume 10 times more omega-6 than omega-3 fatty acids.4 Table 1.2 shows the proportion of omega 3 fatty acids for a number of foods.

Evidence Report/Technology Assessment   Number 89

 Effects of Omega-3 Fatty Acids on Lipids and Glycemic Control in Type II Diabetes and the Metabolic Syndrome and on Inflammatory Bowel Disease, Rheumatoid Arthritis, Renal Disease, Systemic Lupus Erythematosus, and Osteoporosis

 Prepared for:

Agency for Healthcare Research and Quality

U.S. Department of Health and Human Services

540 Gaither Road

Rockville, MD 20850

http://www.ahrq.gov

Contract No. 290-02-0003

 Chapter 1. Introduction

This report is one of a group of evidence reports prepared by three Agency for Healthcare Research and Quality (AHRQ)-funded Evidence-Based Practice Centers (EPCs) on the role of omega-3 fatty acids (both from food sources and from dietary supplements) in the prevention or treatment of a variety of diseases. These reports were requested and funded by the Office of Dietary Supplements, National Institutes of Health. The three EPCs – the Southern California EPC (SCEPC, based at RAND), the Tufts-New England Medical Center (NEMC) EPC, and the University of Ottawa EPC – have each produced evidence reports. To ensure consistency of approach, the three EPCs collaborated on selected methodological elements, including literature search strategies, rating of evidence, and data table design.

The aim of these reports is to summarize the current evidence on the effects of omega-3 fatty acids on prevention and treatment of cardiovascular diseases, cancer, child and maternal health, eye health, gastrointestinal/renal diseases, asthma, immune- mediated diseases, tissue/organ transplantation, mental health, and neurological diseases and conditions. In addition to informing the research community and the public on the effects of omega-3 fatty acids on various health conditions, it is anticipated that the findings of the reports will also be used to help define the agenda for future research.

This report focuses on the effects of omega-3 fatty acids on immune- mediated diseases, bone metabolism, and gastrointestinal/renal diseases. Subsequent reports from the SCEPC will focus on cancer and neurological diseases and conditions.

This chapter provides a brief review of the current state of knowledge about the metabolism, physiological functions, and sources of omega-3 fatty acids.

fatty acid metabolism

fatty acid metabolism

Inositol lipid regulation of lipid transfer in specialized membrane domains

Inositol lipid regulation of lipid transfer in specialized membrane domains

Fatty acid oxidation and ETC 11306_2014_721_Fig3_HTML

major metabolic intermediates in the pathways for synthesis of cholesterol, fatty acids

major metabolic intermediates in the pathways for synthesis of cholesterol, fatty acids

Arachidonate pathways

Arachidonate pathways

arachidonic acid derivatives

arachidonic acid derivatives

benefits of omega 3s

benefits of omega 3s

flowchart of food energy

flowchart of food energy

Fatty acid synthase

Fatty acid synthase

Elongation and Desaturation of Fatty Acids

Elongation and Desaturation of Fatty Acids


Larry H. Bernstein, MD, FCAP, Author and Curator
Chief Scientific Officer, Leaders in Pharmaceutical Intelligence

Oxidation and Synthesis of Fatty Acids

http://pharmaceuticalintelligence.com/Oxidation and Synthesis of Fatty Acids/

Lipid Metabolism

http://www.elmhurst.edu/~chm/vchembook/622overview.html

Overview of Lipid Catabolism:

The major aspects of lipid metabolism are involved with

  • Fatty Acid Oxidation to produce energy or
  • the synthesis of lipids which is called Lipogenesis.

The metabolism of lipids and carbohydrates are related by the conversion of lipids from carbohydrates. This can be seen in the diagram. Notice the link through actyl-CoA, the seminal discovery of Fritz Lipmann. The metabolism of both is upset by diabetes mellitus, which results in the release of ketones (2/3 betahydroxybutyric acid) into the circulation.

metabolism of fats

metabolism of fats

http://www.elmhurst.edu/~chm/vchembook/images/590metabolism.gif

The first step in lipid metabolism is the hydrolysis of the lipid in the cytoplasm to produce glycerol and fatty acids.

Since glycerol is a three carbon alcohol, it is metabolized quite readily into an intermediate in glycolysis, dihydroxyacetone phosphate. The last reaction is readily reversible if glycerol is needed for the synthesis of a lipid.

The hydroxyacetone, obtained from glycerol is metabolized into one of two possible compounds. Dihydroxyacetone may be converted into pyruvic acid, a 3-C intermediate at the last step of glycolysis to make energy.

In addition, the dihydroxyacetone may also be used in gluconeogenesis (usually dependent on conversion of gluconeogenic amino acids) to make glucose-6-phosphate for glucose to the blood or glycogen depending upon what is required at that time.

Fatty acids are oxidized to acetyl CoA in the mitochondria using the fatty acid spiral. The acetyl CoA is then ultimately converted into ATP, CO2, and H2O using the citric acid cycle and the electron transport chain.

There are two major types of fatty acids – ω-3 and ω-6.  There are also saturated and unsaturated with respect to the existence of double bonds, and monounsaturated and polyunsatured.  Polyunsaturated fatty acids (PUFAs) are important in long term health, and it will be seen that high cardiovascular risk is most associated with a low ratio of ω-3/ω-6, the denominator being from animal fat. Ω-3 fatty acids are readily available from fish, seaweed, and flax seed. More can be said of this later.

Fatty acids are synthesized from carbohydrates and occasionally from proteins. Actually, the carbohydrates and proteins have first been catabolized into acetyl CoA. Depending upon the energy requirements, the acetyl CoA enters the citric acid cycle or is used to synthesize fatty acids in a process known as LIPOGENESIS.

The relationships between lipid and carbohydrate metabolism are
summarized in Figure 2.

fattyacidspiral

fattyacidspiral

http://www.elmhurst.edu/~chm/vchembook/images/620fattyacidspiral.gif

 Energy Production Fatty Acid Oxidation:

Visible” ATP:

In the fatty acid spiral, there is only one reaction which directly uses ATP and that is in the initiating step. So this is a loss of ATP and must be subtracted later.

A large amount of energy is released and restored as ATP during the oxidation of fatty acids. The ATP is formed from both the fatty acid spiral and the citric acid cycle.

 

Connections to Electron Transport and ATP:

One turn of the fatty acid spiral produces ATP from the interaction of the coenzymes FAD (step 1) and NAD+ (step 3) with the electron transport chain. Total ATP per turn of the fatty acid spiral is:

Electron Transport Diagram – (e.t.c.)

Step 1 – FAD into e.t.c. = 2 ATP
Step 3 – NAD+ into e.t.c. = 3 ATP
Total ATP per turn of spiral = 5 ATP

In order to calculate total ATP from the fatty acid spiral, you must calculate the number of turns that the spiral makes. Remember that the number of turns is found by subtracting one from the number of acetyl CoA produced. See the graphic on the left bottom.

Example with Palmitic Acid = 16 carbons = 8 acetyl groups

Number of turns of fatty acid spiral = 8-1 = 7 turns

ATP from fatty acid spiral = 7 turns and 5 per turn = 35 ATP.

This would be a good time to remember that single ATP that was needed to get the fatty acid spiral started. Therefore subtract it now.

NET ATP from Fatty Acid Spiral = 35 – 1 = 34 ATP

Review ATP Summary for Citric Acid Cycle:The acetyl CoA produced from the fatty acid spiral enters the citric acid cycle. When calculating ATP production, you have to show how many acetyl CoA are produced from a given fatty acid as this controls how many “turns” the citric acid cycle makes.Starting with acetyl CoA, how many ATP are made using the citric acid cycle? E.T.C = electron transport chain

 Step  ATP produced
7  1
Step 4 (NAD+ to E.T.C.) 3
Step 6 (NAD+ to E.T.C.)  3
Step10 (NAD+ to E.T.C.)  3
Step 8 (FAD to E.T.C.) 2
 NET 12 ATP
 ATP Summary for Palmitic Acid – Complete Metabolism:The phrase “complete metabolism” means do reactions until you end up with carbon dioxide and water. This also means to use fatty acid spiral, citric acid cycle, and electron transport as needed.Starting with palmitic acid (16 carbons) how many ATP are made using fatty acid spiral? This is a review of the above panel E.T.C = electron transport chain

 Step  ATP (used -) (produced +)
Step 1 (FAD to E.T.C.) +2
Step 4 (NAD+ to E.T.C.) +3
Total ATP  +5
 7 turns  7 x 5 = 35
initial step  -1
 NET  34 ATP

The fatty acid spiral ends with the production of 8 acetyl CoA from the 16 carbon palmitic acid.

Starting with one acetyl CoA, how many ATP are made using the citric acid cycle? Above panel gave the answer of 12 ATP per acetyl CoA.

E.T.C = electron transport chain

 Step  ATP produced
One acetyl CoA per turn C.A.C. +12 ATP
8 Acetyl CoA = 8 turns C.A.C. 8 x 12 = 96 ATP
Fatty Acid Spiral 34 ATP
GRAND TOTAL  130 ATP

Fyodor Lynen

Feodor Lynen was born in Munich on 6 April 1911, the son of Wilhelm Lynen, Professor of Mechanical Engineering at the Munich Technische Hochschule. He received his Doctorate in Chemistry from Munich University under Heinrich Wieland, who had won the Nobel Prize for Chemistry in 1927, in March 1937 with the work: «On the Toxic Substances in Amanita». in 1954 he became head of the Max-Planck-Institut für Zellchemie, newly created for him as a result of the initiative of Otto Warburg and Otto Hahn, then President of the Max-Planck-Gesellschaft zur Förderung der Wissenschaften.

Lynen’s work was devoted to the elucidation of the chemical details of metabolic processes in living cells, and of the mechanisms of metabolic regulation. The problems tackled by him, in conjunction with German and other workers, include the Pasteur effect, acetic acid degradation in yeast, the chemical structure of «activated acetic acid» of «activated isoprene», of «activated carboxylic acid», and of cytohaemin, degradation of fatty acids and formation of acetoacetic acid, degradation of tararic acid, biosynthesis of cysteine, of terpenes, of rubber, and of fatty acids.

In 1954 Lynen received the Neuberg Medal of the American Society of European Chemists and Pharmacists, in 1955 the Liebig Commemorative Medal of the Gesellschaft Deutscher Chemiker, in 1961 the Carus Medal of the Deutsche Akademie der Naturforscher «Leopoldina», and in 1963 the Otto Warburg Medal of the Gesellschaft für Physiologische Chemie. He was also a member of the U>S> National Academy of Sciences, and shared the Nobel Prize in Physiology and Medicine with Konrad Bloch in 1964, and was made President of the Gesellschaft Deutscher Chemiker (GDCh) in 1972.

This biography was written at the time of the award and first published in the book series Les Prix Nobel. It was later edited and republished in Nobel Lectures, and shortened by myself.

The Pathway from “Activated Acetic Acid” to the Terpenes and Fatty Acids

My first contact with dynamic biochemistry in 1937 occurred at an exceedingly propitious time. The remarkable investigations on the enzyme chain of respiration, on the oxygen-transferring haemin enzyme of respiration, the cytochromes, the yellow enzymes, and the pyridine proteins had thrown the first rays of light on the chemical processes underlying the mystery of biological catalysis, which had been recognised by your famous countryman Jöns Jakob Berzelius. Vitamin B2 , which is essential to the nourishment of man and of animals, had been recognised by Hugo Theorell in the form of the phosphate ester as the active group of an important class of enzymes, and the fermentation processes that are necessary for Pasteur’s “life without oxygen”

had been elucidated as the result of a sequence of reactions centered around “hydrogen shift” and “phosphate shift” with adenosine triphosphate as the phosphate-transferring coenzyme. However, 1,3-diphosphoglyceric acid, the key substance to an understanding of the chemical relation between oxidation and phosphorylation, still lay in the depths of the unknown. Never-

theless, Otto Warburg was on its trail in the course of his investigations on the fermentation enzymes, and he was able to present it to the world in 1939.

This was the period in which I carried out my first independent investigation, which was concerned with the metabolism of yeast cells after freezing in liquid air, and which brought me directly into contact with the mechanism of alcoholic fermentation. This work taught me a great deal, and yielded two important pieces of information.

  • The first was that in experiments with living cells, special attention must be given to the permeability properties of the cell membranes, and
  • the second was that the adenosine polyphosphate system plays a vital part in the cell,
    • not only in energy transfer, but
    • also in the regulation of the metabolic processes.

.

This investigation aroused by interest in problems of metabolic regulation, which led me to the investigation of the Pasteur effects, and has remained with me to the present day.

My subsequent concern with the problem of the acetic acid metabolism arose from my stay at Heinrich Wieland’s laboratory. Workers here had studied the oxidation of acetic acid by yeast cells, and had found that though most of the acetic acid undergoes complete oxidation, some remains in the form of succinic and citric acids.

The explanation of these observations was provided-by the Thunberg-Wieland process, according to which two molecules of acetic acid are dehydrogenated to succinic acid, which is converted back into acetic acid via oxaloacetic acid, pyruvic acid, and acetaldehyde, or combines at the oxaloacetic acid stage with a further molecule of acetic acid to form citric acid (Fig. 1). However, an experimental check on this view by a Wieland’s student Robert Sonderhoffs brought a surprise. The citric acid formed when trideuteroacetic acid was supplied to yeast cells contained the expected quantity of deuterium, but the succinic acid contained only half of the four deuterium atoms required by Wieland’s scheme.

This investigation aroused by interest in problems of metabolic regulation, which led me to the investigation of the Pasteur effects, and has remained with me to the present day. My subsequent concern with the problem of the acetic acid metabolism arose from my stay at Heinrich Wieland’s laboratory. Workers here had studied the oxidation of acetic acid by yeast cells, and had found that though most of the acetic acid undergoes complete oxidation, some remains in the form of succinic and citric acid

The answer provided by Martius was that citric acid  is in equilibrium with isocitric acid and is oxidised to cr-ketoglutaric acid, the conversion of which into succinic acid had already been discovered by Carl Neuberg (Fig. 1).

It was possible to assume with fair certainty from these results that the succinic acid produced by yeast from acetate is formed via citric acid. Sonderhoff’s experiments with deuterated acetic acid led to another important discovery.

In the analysis of the yeast cells themselves, it was found that while the carbohydrate fraction contained only insignificant quantities of deuterium, large quantities of heavy hydrogen were present in the fatty acids formed and in the sterol fraction. This showed that

  • fatty acids and sterols were formed directly from acetic acid, and not indirectly via the carbohydrates.

As a result of Sonderhoff’s early death, these important findings were not pursued further in the Munich laboratory.

  • This situation was elucidated only by Konrad Bloch’s isotope experiments, on which he reports.

My interest first turned entirely to the conversion of acetic acid into citric acid, which had been made the focus of the aerobic degradation of carbohydrates by the formulation of the citric acid cycle by Hans Adolf Krebs. Unlike Krebs, who regarded pyruvic acid as the condensation partner of acetic acid,

  • we were firmly convinced, on the basis of the experiments on yeast, that pyruvic acid is first oxidised to acetic acid, and only then does the condensation take place.

Further progress resulted from Wieland’s observation that yeast cells that had been “impoverished” in endogenous fuels by shaking under oxygen were able to oxidise added acetic acid only after a certain “induction period” (Fig. 2). This “induction period” could be shortened by addition of small quantities of a readily oxidisable substrate such as ethyl alcohol, though propyl and butyl alcohol were also effective. I explained this by assuming that acetic acid is converted, at the expense of the oxidation of the alcohol, into an “activated acetic acid”, and can only then condense with oxalacetic acid.

In retrospect, we find that I had come independently on the same group of problems as Fritz Lipmann, who had discovered that inorganic phosphate is indispensable to the oxidation of pyruvic acid by lactobacilli, and had detected acetylphosphate as an oxidation product. Since this anhydride of acetic acid and phosphoric acid could be assumed to be the “activated acetic acid”.

I learned of the advances that had been made in the meantime in the investigation of the problem of “activated acetic acid”. Fritz Lipmann has described the development at length in his Nobel Lecture’s, and I need not repeat it. The main advance was the recognition that the formation of “activated acetic acid” from acetate involved not only ATP as an energy source, but also the newly discovered coenzyme A, which contains the vitamin pantothenic acid, and that “activated acetic acid” was probably an acetylated coenzyme  A.

http://www.nobelprize.org/nobel_prizes/medicine/laureates/1964/lynen-bio.html

http://onlinelibrary.wiley.com/store/10.1002/anie.201106003/asset/image_m/mcontent.gif?v=1&s=1e6dc789dfa585fe48947e92cc5dfdcabd8e2677

Fyodor Lynen

Lynen’s most important research at the University of Munich focused on intermediary metabolism, cholesterol synthesis, and fatty acid biosynthesis. Metabolism involves all the chemical processes by which an organism converts matter and energy into forms that it can use. Metabolism supplies the matter—the molecular building blocks an organism needs for the growth of new tissues. These building blocks must either come from the breakdown of molecules of food, such as glucose (sugar) and fat, or be built up from simpler molecules within the organism.

Cholesterol is one of the fatty substances found in animal tissues. The human body produces cholesterol, but this substance also enters the body in food. Meats, egg yolks, and milk products, such as butter and cheese, contain cholesterol. Such organs as the brain and liver contain much cholesterol. Cholesterol is a type of lipid, one of the classes of chemical compounds essential to human health. It makes up an important part of the membranes of each cell in the body. The body also uses cholesterol to produce vitamin D and certain hormones.

All fats are composed of an alcohol called glycerol and substances called fatty acids. A fatty acid consists of a long chain of carbon atoms, to which hydrogen atoms are attached. There are three types of fatty acids: saturated, monounsaturated, and polyunsaturated.

Living cells manufacture complicated chemical compounds from simpler substances through a process called biosynthesis. For example, simple molecules called amino acids are put together to make proteins. The biosynthesis of both fatty acids and cholesterol begins with a chemically active form of acetate, a two-carbon molecule. Lynen discovered that the active form of acetate is a coenzyme, a heat-stabilized, water-soluble portion of an enzyme, called acetyl coenzyme A. Lynen and his colleagues demonstrated that the formation of cholesterol begins with the condensation of two molecules of acetyl coenzyme A to form acetoacetyl coenzyme A, a four-carbon molecule.

http://science.howstuffworks.com/dictionary/famous-scientists/biologists/feodor-lynen-info.htm

Fyodor Lynen

Fyodor Lynen


Larry H. Bernstein, MD, FCAP, Author, Curator
Chief Scientific Officer, Leaders in Pharmaceutical Intelligence

Introduction to Lipid Metabolism

http://pharmaceuticalintelligence.com/10/25/2014/larryhbern/Introduction_
to_Lipid_Metabolism/

This series of articles is concerned with lipid metabolism. These discussions lay
the groundwork to proceed to discussions that will take on a somewhat different
approach, but they are critical to developing a more complete point of view of life
processes.  I have indicated that there are protein-protein interactions or protein-membrane interactions and associated regulatory features, but the focus of the
discussion or points made were different, and will be returned to.  The role of
lipids in circulating plasma proteins as biomarkers for coronary vascular disease
can be traced to the early work of Frederickson and the classification of lipid disorders.  The very critical role of lipids in membrane structure in health and
disease has had much less attention, despite the enormous importance,
especially in the nervous system.

This portion of the discussions of metabolism will have several topics on lipid
metabolism.  The first is concerned with the basic types of lipids -which are defined structurally and have different carbon chain length, and have
two basic types of indispensible fatty acid derivations – along pro-inflammatory
and anti-inflammatory pathways:

  1. ALA and LA, LCPUFAs (EPA, DHA, and AA), eicosanoids,
    delta-3-desaturase, prostaglandins, and leukotrienes.
  2. the role of the mitochondrial electron transport chain in hydrogen transfers
    and oxidative phosphorylation with respect to the oxidation of fatty acids
    and fatty acid synthesis.
  3. The membrane structures of the cell, including
  • the cytoskeleton, essential organelles, and the intercellular matrix, which
    is a critical consideration for
  • cell motility, membrane conductivity, flexibility, and  signaling.
  • The membrane structure involves aggregation of lipids with proteins,
  • and is associated with hydrophobicity.
  1. The pathophysiology of systemic circulating lipid disorders.
  2. The fifth is the pathophysiology of cell structures under oxidative
    stress.
  3. Lipid disposal and storage diseases.

Originally posted on What's The Big Data?:

michaelijordanGreat Interview in IEEE Spectrum with machine learning expert, UC Berkeley Professor, and IEEE Fellow Michael Jordan:

“…people continue to infer… that deep learning is taking advantage of an understanding of how the brain processes information, learns, makes decisions, or copes with large amounts of data. And that is just patently false.”

“There is progress at the very lowest levels of neuroscience. But for issues of higher cognition—how we perceive, how we remember, how we act—we have no idea how neurons are storing information, how they are computing, what the rules are, what the algorithms are, what the representations are, and the like. So we are not yet in an era in which we can be using an understanding of the brain to guide us in the construction of intelligent systems.”

“…with big data, it will take decades, I suspect, to get a real engineering approach, so that you…

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Diabetes Mellitus


Larry H. Bernstein, MD, FCAP, Writer, Curator

Chief Scientific Officer, Leaders in Pharmaceutical Intelligence

Diabetes Mellitus

Diabetes mellitus (DM) is a group of metabolic diseases defined by high blood glucose levels, which, depending on the fasting blood glucose, may be pre-diabetes or overt diabetes (110 mg/dl. 124 mg/dl). This blood glucose level reflects a disorder of control of glucose metabolism, which is mediated through the pituitary growth hormone acting on the liver, which produces insulin growth factor 1 (IGF1).  Diabetes is due to either the pancreas not producing enough insulin, or the cells of the body not responding properly to the insulin produced. That said, there is much to be understood about the long term systemic effects of this disorder, a multisystem disease. The presence of pre-diabetes glucose levels is sufficient to proactively take measures to reduce the circulating glucose.

Globally, as of 2013, an estimated 382 million people have diabetes worldwide, with type 2 diabetes making up about 90% of the cases. This is equal to 8.3% of the adults population, with equal rates in both women and men. Worldwide in 2012 and 2013 diabetes resulted in 1.5 to 5.1 million deaths per year, making it the 8th leading cause of death. Diabetes overall at least doubles the risk of death. The number of people with diabetes is expected to rise to 592 million by 2035. The economic costs of diabetes globally was estimated in 2013 at $548 billion and in the United States in 2012 $245 billion.

The observation of symptoms of frequent urination, increased thirst, and increased hunger is symptomatic of overt DM, and is seen with diabetic ketoacidosis, with very high hyperglycemia and glucosuria, particularly in Type 1 DM. Untreated, diabetes leads to serious complications. Acute complications include diabetic ketoacidosis. Serious long-term complications include heart disease, stroke, kidney failure, foot ulcers and damage to the eyes.

There are three main types of diabetes mellitus:

  • Type 1 DM results from the body’s failure to produce enough insulin. This form was previously referred to as “insulin-dependent diabetes mellitus” (IDDM) or “juvenile diabetes”. The cause is unknown.
  • Type 2 DM begins with insulin resistance, a condition in which cells fail to respond to insulin properly. As the disease progresses a lack of insulin may also develop. This form was previously referred to as “non insulin-dependent diabetes mellitus” (NIDDM) or “adult-onset diabetes”. The primary cause is excessive body weight and not enough exercise.
  • Gestational diabetes, the third, occurs when pregnant women without a previous history of diabetes develop a high blood glucose level.

Type 1 DM, which presents suddenly in children or young adults, is possibly an as yet unidentified post-translational or epigenetic form, unrelated to Type 2, which is becoming more common in children.  It results in the destruction of islet beta cells that then have no capacity to produce insulin.  A family history of the disease would be a signal to raise a child with great care to not stress the pancreas.  Even though I raised the possibility of an epigenetic factor, it is important to keep in mind that the regulation of glucose is responsive to a number of stresses, even in a healthy person.  These are:

  • Corticosteroids
  • Glucagon
  • Growth hormone
  • Catecholamines
  • Proinflammatory cytokines
  • Anxiety disorder
  • Eating disorder

Gestational diabetes is perhaps Type 2 diabetes in a pregnant woman initiated by the condition of pregnancy. Whether these women were not diabetic, with a glucose level between 100-110 prior to pregnancy, is an open question. However, the pregnant state is accompanied by large effects by hormone levels.

Type 2 diabetes has been increasing worldwide, not only in western nations.  However, in non-western countries that have large populations of underserved, there is still a major problem with protein energy malnutrition (PEM). Globally, as of 2013, an estimated 382 million people have diabetes worldwide, with type 2 diabetes making up about 90% of the cases. This is equal to 8.3% of the adults population, with equal rates in both women and men. Worldwide in 2012 and 2013 diabetes resulted in 1.5 to 5.1 million deaths per year, making it the 8th leading cause of death. Diabetes overall at least doubles the risk of death. The number of people with diabetes is expected to rise to 592 million by 2035. The economic costs of diabetes globally was estimated in 2013 at $548 billion and in the United States in 2012 $245 billion.

The major long-term complications relate to damage to blood vessels. Diabetes doubles the risk of cardiovascular disease and about 75% of deaths in diabetics are due to coronary artery disease. Other “macrovascular” diseases are stroke, and peripheral vascular disease. The primary microvascular complications of diabetes include damage to the eyes, kidneys, and nerves. Damage to the eyes, known as diabetic retinopathy, is caused by damage to the blood vessels in the retina of the eye, and can result in gradual vision loss and potentially blindness. Damage to the kidneys, known as diabetic nephropathy, can lead to tissue scarring, urine protein loss, and eventually chronic kidney disease, sometimes requiring dialysis or kidney transplant. Damage to the nerves of the body, known as diabetic neuropathy, is the most common complication of diabetes.

Prevention and treatment involves a healthy diet, physical exercise, not using tobacco and being a normal body weight. Blood pressure control and proper foot care are also important for people with the disease. Type 1 diabetes must be managed with insulin injections. Type 2 diabetes may be treated with medications with or without insulin. Insulin and some oral medications can cause low blood sugar. Weight loss surgery in those with obesity is an effective measure in those with type 2 DM. Gestational diabetes usually resolves after the birth of the baby.

A number of articles in http://pharmaceuticalintelligence,com (this journal) have presented the relationship of DM to heart and vascular disease. The complexity of the disease is not to be underestimated, and there havr been serious controversies with adverse consequences over the use of the class of drugs that includes rosiglitazone and piaglitazone, which has opened serious issues about how clinical trials are conducted, and how the data obtained in studies may be compromised.

Pharmaceutical Insights

Management of Diabetes Mellitus: Could Simultaneous Targeting of Hyperglycemia and Oxidative Stress Be a Better Panacea?

Omotayo O. Erejuwa
Int. J. Mol. Sci. 2012, 13, 2965-2972; http://www.mdpi.com/journal/ijms http://dx.doi.org:/10.3390/ijms13032965

The primary aim of the current management of diabetes mellitus is to achieve and/or maintain a glycated hemoglobin level of ≤6.5%. However, recent evidence indicates that intensive treatment of hyperglycemia is characterized by increased weight gain, severe hypoglycemia and higher mortality. Besides, evidence suggests that it is difficult to achieve and/or maintain optimal glycemic control in many diabetic patients; and that the benefits of intensively-treated hyperglycemia are restricted to microvascular complications only. Evidence also indicates that multiple drugs are required to achieve optimal glycemic target in many diabetic patients. In fact, in many diabetic patients in whom optimal glycemic goal is achieved, glycemic control deteriorates even with optimal drug therapy. It does suggest that with the current hypoglycemic or antidiabetic drugs, it is difficult to achieve and/or maintain tight glycemic control in diabetic patients. In many developing countries, the vast majority of diabetic patients have limited or lack access to quality healthcare providers and good therapeutic monitoring.

While increased weight gain could be due to some component drugs (such as sulphonylureas or insulin) of the intensive therapy regimens, hypoglycemia could be drug-induced or comorbidity-induced. Considering the evidence that associates hypoglycemia with increased mortality, higher incidence of mortality in intensive therapy group could be due to hypoglycemia or too low levels of glycosylated hemoglobin. However, it is difficult to contend that increased mortality was entirely due to hypoglycemia. The possibility of drug-induced or drug-associated toxicities could not be ruled out. For instance, rosiglitazone, which has been prohibited and withdrawn from the market in Europe, was one of the hypoglycemic drugs used to achieve intensive therapy of hyperglycemia in Action to Control Cardiovascular Risk in Diabetes (ACCORD). If these findings are anything to go by, does it not suggest that targeting hyperglycemia as the only therapeutic goal in the management of diabetes mellitus could be detrimental to diabetic patients? In addition, the current hypoglycemic drugs are characterized by limitations and adverse effects. Together with the limitations of intensive glycemic treatment (only beneficial in reducing the risk of microvascular complications, but not macrovascular disease complications), does it not imply that targeting hyperglycemia alone is not only deleterious but also limited and ineffective?

The latest figures predict that the global incidence of diabetes mellitus, which was estimated to be 366 million in 2011, will rise to 522 million by 2030. In view of these frightening statistics on the prevalence of diabetes mellitus and on the lack of adequate healthcare, together with the associated diabetic complications, morbidity and mortality, does it not suggest that there is an urgent need for a better therapeutic management of this disorder? Taken together, with these findings and statistics, it can be contended that it is high time alternative and/or complementary therapies to the currently available hypoglycemic agents (which target primarily hyperglycemia only) were sought.

All these may contribute to the unabated increase in global prevalence of diabetes mellitus and its complications In view of these adverse effects and limitations of intensive treatment of hyperglycemia in preventing diabetic complications, which is linked to oxidative stress,

  • this commentary proposes a hypothesis that “simultaneous targeting of hyperglycemia and oxidative stress” could be more effective than “intensive treatment of hyperglycemia” in the management of diabetes mellitus.

Oxidative stress is defined as

  • an “imbalance between oxidants and antioxidants in favor of the oxidants, potentially leading to damage”.

It is implicated in the pathogenesis and complications of diabetes mellitus. The role of oxidative stress is more definite in the pathogenesis of type 2 diabetes mellitus than in type 1 diabetes mellitus. In regard to diabetic complications, there is compelling evidence in support of the role of oxidative stress in both types of diabetes mellitus. Evidence suggests that elevated reactive oxygen species (ROS), which causes factor of increased ROS production, causes tissue damage or diabetic complications have been identified. These include:

  • hyperglycemia-enhanced polyol pathway;
  • hyperglycemia-enhanced formation of advanced glycation endproducts (AGEs);
  • hyperglycemia-activated protein kinase C (PKC) pathway;
  • hyperglycemia-enhanced hexosamine pathway; and
  • hyperglycemia-activated Poly-ADP ribose polymerase (PARP) pathway.

These pathways are activated or enhanced by hyperglycemia-driven mitochondrial superoxide overproduction.

Even though oxidative stress plays an important role in its pathogenesis and complications,

  • unlike other diseases characterized by oxidative stress, diabetes mellitus is unique.

Its cure (restoration of euglycemia, e.g., via pancreas transplants) does not prevent oxidative stress and diabetic complications. This is very important because hyperglycemia exacerbates oxidative stress which is linked to diabetic complications. Theoretically, restoration of euglycemia should prevent oxidative stress and diabetic complications. However, this is not the case. At present, it remains unclear why restoration of euglycemia does not automatically prevent oxidative stress and diabetic complications. The development of diabetes-related complications (both microvascular and macrovascular) may occur in diabetic patients after normoglycemia has been restored. It is a phenomenon whereby previous hyperglycemic milieu is remembered in many target organs such as heart, eyes, kidneys and nerves. This phenomenon is also documented in diabetic animals. Compelling evidence implicates the role of oxidative stress as an important mechanism by which glycemic memory causes tissue damage and diabetic complications. In view of higher incidence of diabetic complications (of which oxidative stress plays an important role) in conventionally-treated diabetic patients, targeting oxidative stress in these patients might be beneficial. In other words, it is possible that the combination of a conventional therapy of hyperglycemia and antioxidant therapy might be more effective and beneficial than intensive therapy of hyperglycemia alone, which is the gold standard at the moment.

Loss of ACE 2 Exaggerates High-Calorie Diet-Induced Insulin Resistance by Reduction of GLUT4 in Mice

M Takeda, K Yamamoto, Y Takemura, H Takeshita, K Hongyo, et al.  Diabetes 61:1–11, 2012

ACE type 2 (ACE2) functions as

  • a negative regulator of the renin angiotensin system
  • by cleaving angiotensin II (AII) into angiotensin 1–7 (A1–7).

This study assessed the role of

  • endogenous ACE2 in maintaining insulin sensitivity.

Twelve-week-old male ACE2 knockout (ACE2KO) mice had normal insulin sensitivities when fed a standard diet. AII infusion or a high-fat high-sucrose (HFHS) diet impaired glucose tolerance and insulin sensitivity more severely

  • in ACE2KO mice than in their wild-type (WT) littermates.

The strain difference in glucose tolerance

  • was not eliminated by an AII receptor type 1 (AT1) blocker
  • but was eradicated by A1–7 or an AT1 blocker combined with the A1–7 inhibitor (A779).

The expression of GLUT4 and a transcriptional factor, myocyte enhancer factor (MEF) 2A,

  • was dramatically reduced in the skeletal muscles of the standard diet–fed ACE2KO mice.

The expression of GLUT4 and MEF2A was increased

  • by A1–7 in ACE2KO mice and
  • decreased by A779 in WT mice.

A1–7 enhanced upregulation of MEF2A and GLUT4 during differentiation of myoblast cells. In conclusion,

  • ACE2 protects against high calorie diet-induced insulin resistance in mice.

This mechanism may involve the transcriptional regulation of GLUT4 via an A1–7-dependent pathway.
Modulation of the action of insulin by angiotensin-(1–7)
FP. Dominici, V Burghi, MC. Munoz, JF. Giani

Clinical Science (2014) 126, 613–630 http://dx.doi.org:/10.1042/CS20130333

The prevalence of Type 2 diabetes mellitus is predicted to increase dramatically over the coming years and the clinical implications and healthcare costs from this disease are overwhelming. In many cases, this pathological condition is linked to a cluster of metabolic disorders, such as

  1. obesity,
  2. systemic hypertension and
  3. dyslipidaemia,
  • defined as the metabolic syndrome.

Insulin resistance has been proposed as the key mediator of all of these features and contributes to the associated high cardiovascular morbidity and mortality. Although the molecular mechanisms behind insulin resistance are not completely understood, a negative cross-talk between

  • AngII (angiotensin II) and the insulin signalling pathway

has been the focus of great interest in the last decade. Indeed,

substantial evidence has shown that

  • anti-hypertensive drugs that block the RAS (renin–angiotensin system) may also act to prevent diabetes.

Despite its long history, new components within the RAS continue to be discovered.

Among them, Ang-(1–7) [angiotensin-(1–7)] has gained special attention as a counter-regulatory hormone

  • opposing many of the AngII-related deleterious effects.

Specifically, we and others have demonstrated that Ang-(1–7) improves the action of insulin and opposes the negative effect that AngII exerts at this level. In the present review, we provide evidence showing that

  • insulin and Ang-(1–7) share a common intracellular signalling pathway.

We also address the molecular mechanisms behind the beneficial effects of Ang-(1–7) on

  • AngII-mediated insulin resistance.

Finally, we discuss potential therapeutic approaches leading to modulation of the

  • ACE2 (angiotensin-converting enzyme 2)/Ang-(1–7)/Mas receptor axis

as a very attractive strategy in the therapy of the metabolic syndrome and diabetes-associated diseases.

Increased Skeletal Muscle Capillarization After Aerobic Exercise Training and Weight Loss Improves Insulin Sensitivity in Adults With IGT

Prior, JB. Blumenthal, LI. Katzel, AP. Goldberg, AS. Ryan. Diabetes Care 2014;37:1469–1475
http://dx.doi.org:/10.2337/dc13-2358

Transcapillary transport of insulin is one determinant of glucose uptake by skeletal muscle; thus,

  • a reduction in capillary density (CD) may worsen insulin sensitivity.

Skeletal muscle CD is lower in older adults with impaired glucose tolerance (IGT) compared with those with normal glucose tolerance and

  • may be modifiable through aerobic exercise training and weight loss (AEX+WL).

Insulin sensitivity (M) and 120-min postprandial glucose (G120) correlated with CD at baseline (r = 0.58 and r = 20.60, respectively, P < 0.05).

AEX+WL increased maximal oxygen consumption (VO2max) 18%(P = 0.02) and reduced weight and fat mass 8% (P < 0.02).

Regression analyses showed that the AEX+WL-induced increase in CD

  • independently predicted the increase in M (r = 0.74, P < 0.01)
  • as well as the decrease in G120 (r = 20.55, P < 0.05).

AEX+WL increases skeletal muscle CD in older adults with IGT. This represents one mechanism by which AEX+WL improves insulin sensitivity in older adults with IGT.

Glycaemic durability with dipeptidyl peptidase-4 inhibitors in type 2 diabetes: a systematic review and meta-analysis of long-term randomised controlled trials.

K Esposito, P Chiodini, MI Maiorino, G Bellastella, A Capuano, D Giugliano. BMJ Open 2014;4:e005442.
http://dx.doi.org:/10.1136/bmjopen-2014-005442

A systematic review and meta-analysis of longterm randomised trials of DPP-4 inhibitors (sitagliptin, vildagliptin, saxagliptin, linagliptin and alogliptin). on haemoglobin A1c (HbA1c) was conducted. The difference between final and intermediate HbA1c assessment was the primary outcome. All trials were of 76 weeks duration at least. The difference in HbA1c changes between final and intermediate points averaged 0.22% (95% CI 0.15% to 0.29%), with high heterogeneity (I2=91%, p<0.0001). Estimates
of differences were not affected by the analysis of six extension trials (0.24%, 0.02 to 0.46), or five trials in which a DPP-4 inhibitor was added to metformin (0.24%, 0.16 to 0.32).

  • The effect of DPP-4 inhibitors on HbA1c in type 2 diabetes significantly declines during the second year of treatment.

Overcoming Diabetes Mellitus & Borderline Diabetes
By Max Stanley Chartrand, Ph.D. (Behavioral Medicine)

The over-arching biomarker that has more to do with the ability to restore normal metabolic processes is in achieving a cellular pH 7.45 (via the Kreb’s Cycle). To say the least, getting one’s cellular pH to 7.45 and A1C score below 6.0 can be a daunting task!

SIRCLE®: Naturally Achieved Targets

 Cellular pH 7.35-7.45

 Oxygen 99-100% @55-65 bpm

 Resting Blood Pressure: 110-135/ 65-80

mmHg (differs male vs female)

 Fasting blood sugar consistently <70-99

mg/dL or 3.5-5.5 mmol/L

 HgA1C score: .04-5.8

 HDL: 40-60 mg/dL; LDL: 100 -140 mg/dL;

triglycerides: <85 mg/dL

 C-Reactive Protein (CRP) Score <.5

 Galectin-3 Assay <17.8 ng/mL

Antidiabetic Activity of Hydroalcoholic Extracts of Nardostachys jatamansi in Alloxan-induced Diabetic Rats

M.A. Aleem, B.S. Asad, T Mohammed, R.A. Khan, M.F. Ahmed, A. Anjum, M. Ibrahim. Brit J Med & Medical Res 4(28): 4665-4673, 2014. http://www.sciencedomain.org/review-history.php?iid=579&id=12&aid=5024

The antidiabetic study was carried out to estimate the anti hyperglycemic potential of Nardostachys Jatamansi rhizome’s hydroalcoholic extracts in alloxan induced diabetic rats over a period of two weeks. The hydroalcoholic extract HAE1 at a dose (500mg/kg) exhibited significantly greater antihyperglycemic activity than extract HAE2 at a dose (500mg/kg) in diabetic rats. The hydroalcoholic extracts showed improvement in different parameters associated with diabetes, like body weight, lipid
profile and biochemical parameters. Extracts also showed improvement in

  • regeneration of β-cells of pancreas in diabetic rats.

Histopathological studies support the healing of pancreas by hydro alcoholic extracts (HAE1& HAE2) of Nardostachys Jatamansi, as a probable mechanism of their antidiabetic activity.

Antidiabetic and Antihyperlipidemic Effect of Parmelia Perlata. Ach. in Alloxan Induced Diabetic Rats.
Jothi G and Brindha P
Internat J of Pharmacy and Pharmaceut Sciences 2014; 6(suppl 1)

The aqueous extract of the selected plant was administered at dose levels of 200mg and 400mg/kg body weight for 60 days. After the experimental period the blood and tissue samples were collected and subjected to various biochemical and enzymic parameters. There were profound alteration in

  • fasting blood glucose,
  • serum insulin,
  • glycosylated hemoglobin (HbA1C) and
  • liver glycogen levels in alloxanized rats.
  1. Glucose-6-phosphatase,
  2. glucokinase, and
  3. fructose 1-6 bisphosphatase activity
  • were also altered in diabetic rats.

Administration of plant extract significantly (P<0.05)

  • reduced the fasting blood glucose and HbA1C level and increased the level of plasma insulin.

The activities of glucose metabolizing enzymes were also resumed to normal. There was a profound improvement in serum lipid profiles by

  • reducing serum triglyceride, cholesterol, LDL, VLDL, free fatty acids, phospholipids and increasing the HDL level in a dose dependent manner.

The effects of leaf extract were compared with standard drug glibenclamide (600μg/Kg bw). The results indicate that Parmelia perlata. Ach., Linn. could be a good natural source for developing an antidiabetic drug that can effectively maintained the blood glucose levels and lipid profile to near normal values.

Pathophysiological Insights
Diabetic glomerulosclerosis

Reviewers: Nikhil Sangle, M.D.
Revised: 21 February 2014,
Copyright: (c) 2003-2012, PathologyOutlines.com, Inc.

General

==================================================

  • Diffuse capillary basement membrane thickening, diffuse and nodular glomerulosclerosis
  • Causes glomerular disease, arteriolar sclerosis, pyelonephritis, papillary necrosis; similar between type I and II patients
  • Accounts for 30% of long term dialysis patients in US; causes 20% of deaths in patients with diabetes < age 40
  • Changes may be related to nephronectin, which functions in the assembly of extracellular matrix (Nephrol Dial Transplant 2012;27:1889)

Clinical features

==================================================

  • Proteinuria occurs in 50%, usually 12-22 years after onset of diabetes
  • End stage renal disease occurs in 30% of type I patients
  • Early increased GFR and microalbuminemia (30-300 mg/day) are predictive of future diabetic nephropathy
  • Renal disease reduced by tight diabetic control; may recur with renal allografts; ACE inhibitors may reduce progression

Micro description

==================================================

  • Basement membrane thickening and increased mesangial matrix in ALL patients
  • Diffuse glomerulosclerosis: increase in mesangial matrix associated with PAS+ basement membrane thickening, eventually obliterates mesangial cells
  • Nodular glomerulosclerosis: also called intercapillary glomerulosclerosis or Kimmelstiel-Wilson disease; ovoid, spherical, laminated hyaline masses in peripheral of glomerulus, PAS+, eventually obliterates glomerular tuft; specific for diabetes and membranoproliferative glomerulonephritis, light-chain disease and amyloidosis (Hum Pathol 1993;24:77 (pathogenesis of Kimmelstiel-Wilson nodule))
  • Profound hyalinization of afferent arterioles (insudative lesion-intramural): specific for diabetes in afferent arterioles, but non-specific if in periphery of glomerular loop, Bowman’s capsule or mesangium; insudative material composed of proteins, lipids and mucopolysaccharides
  • Organizing fibroepithelial crescents: associated with aggressive clinical course
  • Diffuse thickening of tubular basement membrane, tubular atrophy and interstitial fibrosis
  • Isolated thickened glomerular basement membrane and proteinuria may be an early predictor of diabetic disease (Mod Pathol 2004;17:1506)

Nodular glomerulosclerosis, Kidney

 Glomeruli:

  1.     Acellular, homogeneous, eosinophilic, globular nodules in the mesangial orintercapillary region of a glomerular tuft with capillary displaced to the periphery.
  2.     Diffuse intercapillary glomerulosclerosis: increasing eosinophilic mesangial matrix materials.
  3.     Capsular drop: eosinophilic small nodules on Bowman’s capsule.
  4.     Fibrin cap: eosinophilic, waxy, fatty structure within the lumen of one or more capillary loops of glomerular tufts.
nodular glomeruloschlerosis

nodular glomeruloschlerosis

http://www.kidneypathology.com/Imagenes/Diabetes/Imagen.Hial.jul.w.jpg

Islet amyloid polypeptide, islet amyloid, and diabetes mellitus.

Westermark P1, Andersson A, Westermark GT.
Physiol Rev. 2011 Jul;91(3):795-826.
http://dx.doi.org:/10.1152/physrev.00042.2009.

Islet amyloid polypeptide (IAPP), or amylin, was named for its tendency to

  • aggregate into insoluble amyloid fibrils, features typical of islets of most individuals with type 2 diabetes.

This pathological characteristic is most probably of

  • great importance for the development of the β-cell failure in this disease,
  • but the molecule also has regulatory properties in normal physiology.

In addition, it possibly contributes to the diabetic condition. This review deals with both these facets of IAPP.

Islet amyloid polypeptide (IAPP, or amylin) is one of the major secretory products of β-cells of the pancreatic islets of Langerhans. It is

  • a regulatory peptide with putative function
  • both locally in the islets, where it inhibits insulin and glucagon secretion, and at distant targets.

It has binding sites in the brain, possibly contributing also to satiety regulation and inhibits gastric emptying. Effects on several other organs have also been described.

IAPP was discovered through its ability to

  • aggregate into pancreatic islet amyloid deposits,

which are seen particularly in association with type 2 diabetes in humans and with diabetes in a few other mammalian species, especially monkeys and cats.

Aggregated IAPP has cytotoxic properties and is believed to be

  • of critical importance for the loss of β-cells in type 2 diabetes

and also in pancreatic islets transplanted into individuals with type 1 diabetes. This review deals both with physiological aspects of IAPP and with the

  • pathophysiological role of aggregated forms of IAPP,
  • including mechanisms whereby human IAPP forms toxic aggregates and amyloid fibrils.

Islet amyloid, initially named “islet hyalinization,” was described in 1901 by two researchers independently and for a long time was considered an enigma. It was found to occur in association with diabetes mellitus, particularly in elderly individuals, but its possible pathogenetic importance was often denied. The similarity of the hyaline substance to amyloid was noted at an early date, and some researchers reported staining reactions typical of amyloid. It had been shown in 1959 that

  • amyloid of several types has a characteristic ultrastructure,
  • and islet deposits were found to share this appearance.

When biochemical analyses of amyloid fibrils from systemic primary and secondary amyloidoses showed that

  • these consisted of distinctive proteins,
  • it was suspected that the islet deposits might also be a polymerized protein.

The chemical composition of islet amyloid did not attract much attention even after the characteristics of other amyloid fibrils had been elucidated. The finding that the amyloid in C cell-derived medullary thyroid carcinoma is of polypeptide hormonal origin was an important indication that amyloid in other endocrine tissues also comes from the local secretory products, and it was believed that

  • insulin, or proinsulin, or split products thereof constitute the islet amyloid fibrils.

Immunological trials to characterize the amyloid yielded equivocal results. Only when concentrated formic acid was used on amyloid,

  • extracted from an amyloid-rich insulinoma, was it possible to purify the major fibril protein
  • and characterize it by NH2-terminal amino acid sequence analysis,

which very unexpectedly revealed a novel peptide,

  • not resembling any part of proinsulin
  • but with partial identity to the neuropeptide calcitonin gene-related peptide (CGRP).

Further characterization of the peptide purified from an insulinoma and from islet amyloid of human and feline origin proved it to be a 37-amino acid (aa) residue peptide. The peptide was initially named “insulinoma amyloid peptide” , later diabetes-associated peptide (DAP), and finally islet amyloid polypeptide (IAPP), or “amylin”.

IAPP is a 37-aa residue long peptide, but by the application of molecular biological methods it was quickly shown that IAPP is expressed initially as

  • part of an 89-aa residue preproprotein containing a 22-aa signal peptide and
  • two short flanking peptides, the latter cleaved off at double basic aa residues similar to proinsulin.

IAPP is expressed by one single-copy gene on the short arm of chromosome 12,

  • in contrast to insulin and the other members of the calcitonin family, including
  • CGRP,
  • adrenomedullin, and
  • calcitonin,

all of which are encoded by genes on the evolutionary related chromosome 11.

The preproIAPP gene contains three exons, of which

  • the last two encode the full prepromolecule.

The signal peptide is cleaved

  • off in the endoplasmic reticulum (ER), and
  • conversion of proIAPP to IAPP takes place in the secretory vesicles.

ProIAPP and proinsulin are both processed by the two endoproteases

  • prohormone convertase 2 (PC2) and
  • prohormone convertase 1/3 (PC1/3) and
  • by carboxypeptidase E (CPE) (Figure 1).
amylin

amylin

A: the amino acid sequence of human pro-islet amyloid polypeptide (proIAPP) with the cleavage site for PC2 at the NH2 terminus and the cleavage site for PC1/3 at the COOH terminus, indicated by arrows. The KR residues (blue) that remain at the COOH terminus after PC1/3 processing are removed by carboxypeptidase E. This event exposes the glycine residue that is used for COOH-terminal amidation.
Below is a cartoon of IAPP in blue with the intramolecular S-S bond between residues 2–7 and the amidated COOH terminus.

B: the amino acid sequence of human proinsulin with the basic residues at the B-chain/C-peptide junction and the A-chain/C-peptide/junction indicated in blue and the processing sites indicated by arrows. PC1/3 does almost exclusively process proinsulin at the B-chain/C-peptide junction while PC2 preferentially processes proinsulin at the A-chain/C-peptide junction. The basic residues (RR) (position 31, 32) that remain at the COOH terminus of the B-chain is removed by the carboxypeptidase CPE. Below is a cartoon of insulin A-chain and B-chain in red with intermolecular SS bonds between cystein residues 7 in the A and B chains, between cystein residues at position 19 in the B-chain and 20 in the A-chain and the intermolecular SS bond between cystein residues at position 6 and 11 of the A-chain.

http://physrev.physiology.org/content/physrev/91/3/795/F1.large.jpg

  1. IAPP and insulin genes contain similar promoter elements,
  2. and the transcription factor PDX1 regulates the effects of glucose on both genes.
  3. Glucose stimulated β-cells respond with a parallel expression pattern of IAPP and insulin in the rat.

However, this parallel secretion of IAPP and insulin is altered in experimental diabetes models in rodents. Perfused rat pancreas secreted relatively

  • more IAPP than insulin when exposed to dexamethasone, whereas
  • high doses of streptozotocin or alloxan reduced insulin secretion more than that of IAPP.

Oleat and palmitate increased the expression of IAPP but not of insulin in MIN6 cells. In mice fed a diet high in fat for 6 mo, plasma IAPP increased 4.5 times more than insulin compared with mice fed standard food containing 4% fat.

In human recipients who had become insulin-independent by intrahepatically transplanted islets, there was disproportionately

  • more IAPP than normal secreted during hyperglycemia.

These examples show that the strictly parallel expression of IAPP and insulin may be disturbed under certain conditions.

The crystalline structure of insulin in granules is well characterized.

  • Hexameric insulin, together with zinc, constitutes the core of the mature granules, while
  • IAPP, together with a large number of additional components, including the C peptide, is found in the halo region.

The highly fibrillogenic human IAPP has to be protected in some way from aggregation, which otherwise would take place spontaneously. The fact that very fibril-prone proteins can be kept in solution at high concentrations is known from studies of arthropod silk. The composition of the β-cell granule is extremely complex, and it has many components in addition to insulin and C peptide, in micromolar concentrations.

It is probable that IAPP is protected from aggregation by interaction with other components. Plausible candidates are

  • proinsulin, insulin, or their processing intermediates.

Insulin has been found to be

  • a strong inhibitor of IAPP fibril formation.

This finding has been verified in a number of subsequent studies, which have also shown the potency of the inhibition. The inhibition seems to depend

  • solely on the B-chain,
  • which binds specifically to a short segment of IAPP.

An insulin-to-IAPP ratio of between 1:5 and 1:100 had a strong inhibitory effect. The molar ratio between IAPP and insulin in the granule as a whole is ∼1–2:50.

Type 2 Diabetes, APOE Gene, and the Risk for Dementia and Related Pathologies. The Honolulu-Asia Aging Study

Rita Peila, Beatriz L. Rodriguez and Lenore J. Launer
Diabetes Apr 2002; 51(4): 1256-1262
http://dx.doi.org:/10.2337/diabetes.51.4.1256

Type 2 diabetes may be a risk factor for dementia, but the associated pathological mechanisms remains unclear. We evaluated the association of diabetes

  • alone or combined with the apolipoprotein E (APOE) gene
  • with incident dementia and neuropathological outcomes

in a population-based cohort of 2,574 Japanese-American men enrolled in the Honolulu-Asia Aging Study, including 216 subjects who underwent autopsy. Type 2 diabetes was ascertained by interview and direct glucose testing. Dementia was assessed in 1991 and 1994 by clinical examination and magnetic resonance imaging and was diagnosed according to international guidelines. Logistic regression was used to assess the RR of developing dementia, and log-linear regression was used to estimate the incident rate ratio (IRR) of neuropathological outcomes.

Diabetes was associated with

  1. total dementia (RR 1.5 [95% CI 1.01–2.2]),
  2. Alzheimer’s disease (AD; 1.8 [1.1–2.9]), and
  3. vascular dementia (VsD; 2.3 [1.1–5.0]).

Individuals with both type 2 diabetes and the APOE ε4 allele

  • had an RR of 5.5 (CI 2.2–13.7) for AD compared with those with neither risk factor.

Participants with type 2 diabetes and the ε4 allele had

  • a higher number of hippocampal neuritic plaques (IRR 3.0 [CI 1.2–7.3]) and
  • neurofibrillary tangles in the cortex (IRR 3.5 [1.6–7.5]) and hippocampus (IRR 2.5 [1.5–3.7]), and
  • they had a higher risk of cerebral amyloid angiopathy (RR 6.6, 1.5–29.6).

Type 2 diabetes is a risk factor for AD and VsD. The association between diabetes and AD is particularly strong among carriers of the APOE ε4 allele. The neuropathological data are consistent with the clinical results.

Role of insulin signaling impairment, adiponectin and dyslipidemia in peripheral and central neuropathy in mice

  1. Anderson, MR. King, L Delbruck, CG. Jolivalt
    Dis. Model. Mech. June 2014; 7(6): 625-633
    http://dx.doi.org:/10.1242/dmm.015750

One of the tissues or organs affected by diabetes is the nervous system,

  • predominantly the peripheral system (peripheral polyneuropathy and/or painful peripheral neuropathy)
  • but also the central system with impaired learning, memory and mental flexibility.

The aim of this study was to test the hypothesis that the pre-diabetic or diabetic condition caused by a high-fat diet (HFD) can damage both the peripheral and central nervous systems. Groups of C57BL6 and Swiss Webster mice were fed a diet containing 60% fat for 8 months and compared to control and streptozotocin (STZ)-induced diabetic groups that were fed a standard diet containing 10% fat. Aspects of peripheral nerve function (conduction velocity, thermal sensitivity) and central nervous system function (learning ability, memory) were measured at assorted times during the study. Both strains of mice on HFD developed impaired glucose tolerance, indicative of insulin resistance, but

  • only the C57BL6 mice showed statistically significant hyperglycemia.

STZ-diabetic C57BL6 mice

  • developed learning deficits in the Barnes maze after 8 weeks of diabetes, whereas
  • neither C57BL6 nor Swiss Webster mice fed a HFD showed signs of defects at that time point.

By 6 months on HFD, Swiss Webster mice developed

  • learning and memory deficits in the Barnes maze test,
  • whereas their peripheral nervous system remained normal.

In contrast, C57BL6 mice fed the HFD developed peripheral nerve dysfunction,

  • as indicated by nerve conduction slowing and thermal hyperalgesia,
  • but showed normal learning and memory functions.

Our data indicate that STZ-induced diabetes or a HFD can damage

  • both peripheral and central nervous systems,
  • but learning deficits develop more rapidly in insulin-deficient than in insulin-resistant conditions
  • and only in Swiss Webster mice.

In addition to insulin impairment, dyslipidemia or adiponectinemia might determine the neuropathy phenotype.

Neuroinflammation and neurologic deficits in diabetes linked to brain accumulation of amylin

S Srodulski, S Sharma, AB Bachstetter, JM Brelsfoard, et al.
Molecular Neurodegeneration  2014; 9(30):
http://dx.doi.org:/10.1186/1750-1326-9-30

Background: We recently found that brain tissue from patients with type-2 diabetes (T2D) and cognitive impairment

  • contains deposits of amylin, an amyloidogenic hormone synthesized and co-secreted with insulin by pancreatic β-cells.

Amylin deposition is promoted by

  • chronic hypersecretion of amylin (hyperamylinemia), which is common in humans with obesity or pre-diabetic insulin resistance.

Human amylin oligomerizes quickly when oversecreted, which is toxic,

  • induces inflammation in pancreatic islets and
  • contributes to the development of T2D.

Here, we tested the hypothesis that accumulation of oligomerized amylin affects brain function.

Methods: In contrast to amylin from humans,

  • rodent amylin is neither amyloidogenic nor cytotoxic.

We exploited this fact by comparing

  • rats overexpressing human amylin in the pancreas (HIP rats) with their littermate rats

which express only wild-type (WT) non-amyloidogenic rodent amylin. Cage activity, rotarod and novel object recognition tests were performed on animals nine months of age or older. Amylin deposition in the brain was documented by immunohistochemistry, and western blot. We also measured neuroinflammation by immunohistochemistry, quantitative real-time PCR and cytokine protein levels.

Results: Compared to WT rats, HIP rats show

i) reduced exploratory drive,
ii) impaired recognition memory and
iii) no ability to improve the performance on the rotarod.

The development of neurological deficits is

  • associated with amylin accumulation in the brain.

The level of oligomerized amylin in supernatant fractions and pellets from brain homogenates

  • is almost double in HIP rats compared with WT littermates (P < 0.05).

Large amylin deposits (>50 μm diameter) were also occasionally seen in HIP rat brains. Accumulation of oligomerized amylin

  • alters the brain structure at the molecular level.

Immunohistochemistry analysis with an ED1 antibody indicates possible activated microglia/macrophages which

  • are clustering in areas positive for amylin infiltration.

Multiple inflammatory markers are expressed in HIP rat brains as opposed to WT rats, confirming that

  • amylin deposition in the brain induces a neuroinflammatory response.

Conclusions:

  1. Hyperamylinemia promotes accumulation of oligomerized amylin in the brain
  2. leading to neurological deficits through an oligomerized amylin-mediated inflammatory response.

Additional studies are needed to determine

  • whether brain amylin accumulation may predispose to diabetic brain injury and cognitive decline.

Keywords: Diabetes, Alzheimer’s Disease, Amylin, Pre-diabetes, Insulin Resistance, Inflammation, Behavior


Some people who want to avoid prescribed pharmaceuticals for high blood pressure, anxiety, and other conditions are increasingly looking for alternative or natural sources of beta blockers.

Source: www.newsmax.com

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