Insulin Function, Insulin Resistance, and Food Intake Control of Secretion

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Introduction to Insulin Activities

Insulin and Metabolism

Insulin is a major metabolism regulating hormone secreted by β-cells of the islets of Langerhans of the pancreas. The major function of insulin is to counter the concerted actions of a number of hyperglycemia-generating hormones and to maintain low blood glucose levels. In addition to its role in regulating glucose metabolism, insulin stimulates lipogenesis, diminishes lipolysis, and increases amino acid transport into cells. Because there are numerous hyperglycemic hormones, untreated disorders associated with insulin generally lead to severe hyperglycemia and shortened life span.












Insulin as Growth Factor

Insulin also exerts activities typically associated with growth factors. Insulin is a member of a family of structurally and functionally similar molecules that includes the insulin-like growth factors (IGF-1 and IGF-2), and relaxin. The tertiary structure of all four molecules is similar, and all have growth-promoting activities. Insulin modulates transcription and stimulates protein translocation, cell growth, DNA synthesis, and cell replication, effects that it holds in common with the insulin-like growth factors and relaxin.

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Insulin Secretion

Insulin is synthesized, from the INS gene, as a preprohormone in the β-cells of the islets of Langerhans. The INS gene is located on chromosome 11p15.5 and is composed of 3 exons that generate four alternatively spliced mRNAs, all of which encode the same 110 amino acid preproprotein. The signal peptide of preproinsulin is removed in the cisternae of the endoplasmic reticulum. The insulin proprotein is packaged into secretory vesicles in the Golgi, folded into its native structure, and locked in this conformation by the formation of two disulfide bonds. Specific protease activity cleaves the center third of the molecule, which dissociates as C peptide, leaving the amino terminal B peptide disulfide bonded to the carboxy terminal A peptide.

Insulin secretion from β-cells is principally regulated by plasma glucose levels. Increased uptake of glucose by pancreatic β-cells leads to a concomitant increase in metabolism. The increase in metabolism leads to an elevation in the ATP/ADP ratio. This in turn leads to the inhibition of an ATP-sensitive potassium channel (KATP channel). The net result is a depolarization of the cell leading to Ca2+ influx and insulin secretion.

Although glucose is the major fuel whose oxidation leads to energy-coupled insulin secretion there are other means for stimulated insulin secretion. One important process is referred to as the pyruvate cycle and involves coupling of amino acid metabolism to insulin secretion. The insulin secreting β-cells, in contrast to the liver, do not express the gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PEPCK) but do express robust levels of the gluconeogenic enzyme pyruvate carboxylase (PC). Coupled with the activity of PC is the activity of malic enzyme which together, is the only means for pyruvate cycling in β-cells. Cytoplasmic malic enzyme plays an important role in acetyl-CoA transport from the mitochondria to the cytosol for its use in lipid biosynthesis. Human cells express both cytoplasmic and mitochondrial versions of malic enzyme. The role of the mitochondrial malic enzyme is principally to provide the cell with an alternate source of pyruvate under conditions where glycolytic flux in reduced. In these circumstances, the pyruvate generated by the actions of mitochondrial malic enzyme comes from fumarate precursors such as glutamine. When glutamine is de-aminated by glutaminase the resulting glutamate can also be de-aminated by glutamate dehydrogenase yielding 2-oxoglutarate (α-ketoglutarate) which can then be shunted to malate synthesis in the TCA cycle. The malate can then be decarboxylated to pyruvate via mitochondrial malic enzyme. The pyruvate can then be decarboxylated by the PDHc and the resulting acetyl-CoA can enter the TCA cycle ultimately allowing for glutamine carbons to be oxidized for ATP synthesis. Within β-cells of the pancreas, this process, driven by mitochondrial malic enzyme serves as an important means for the use of amino acid carbon oxidation for the stimulated secretion of insulin. Indeed, this process is energetically equal to glucose-stimulated insulin secretion (GSIS).

The KATP channel is a complex of 8 polypeptides comprising four copies of the protein encoded by the ABCC8 (ATP-binding cassette, sub-family C, member 8) gene and four copies of the protein encoded by the KCNJ11 (potassium inwardly-rectifying channel, subfamily J, member 11) gene. The ABCC8 encoded protein is also known as the sulfonylurea receptor (SUR). The KCNJ11 encoded protein forms the core of the KATP channel and is called Kir6.2. As might be expected, the role of KATP channels in insulin secretion presents a viable therapeutic target for treating hyperglycemia due to insulin insufficiency as is typical in type 2 diabetes.

Chronic increases in numerous other hormones, such as growth hormone, placental lactogen, estrogens, and progestins, up-regulate insulin secretion, probably by increasing the preproinsulin mRNA and enzymes involved in processing the increased preprohormone.

Glucose-stimulated insulin secretion (GSIS) from pancreas

Mechanism of glucose-stimulated insulin secretion, GSIS. Increased blood glucose results in uptake by pancreatic β-cells through GLUT2. The glucose is oxidized to pyruvate via glycolysis. The pyruvate is oxidized by the PDHc and the resulting acetyl-CoA is oxidized in the TCA cycle. The resulting NADH and FADH2 are oxidized via the oxidative phosphorylation machinery resulting in increased ATP levels. The increased ATP inhibits the KATP channel resulting in membrane depolarization leading to an influx of Ca2+ ions triggering migration of insulin-containing vesicle to the plasma membrane releasing the insulin to the blood.

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Insulin Receptors

Structure of the insulin receptor with bound insulin

Insulin exerts all of its biological activities, both as a hormone and as a growth factor, by binding to a cell surface receptor complex. The insulin receptor is a member of the membrane-spanning receptor family that harbors intrinsic tyrosine kinase activity. However, the insulin receptor is unique in that it is a heterotetrameric complex composed of two completely extracellular α-peptides that are disulfide bonded to the two transmembrane-spanning β-peptides. Both the α- and β- subunits of the receptor complex are derived from a single gene (symbol: INSR). This processing of the receptor is reminiscent of the processing of the preproinsulin protein leading to two peptides (A and B) disulfide bonded together to form bioactive insulin.

The INSR gene is located on chromosome 19p13.3-p13.2 and is composed of 22 exons. Two alternative splicing variants of the insulin receptor preproprotein are generated from the INSR precursor mRNA. One form contains exon 11 sequences (termed the IR-B form or the Long preproprotein isoform) while the IR-A form (Short preproprotein isoform) does not. The result of the alternative splicing is that the α-subunit from the IR-B form has a 12-amino acid extension at its C-terminus. This form of the α-subunit is referred to as αCT. The insulin receptor can also bind the related growth factors mentioned above, IGF-1 and IGF-2. When insulin binds to the receptor it activates the intrinsic tyrosine kinase activity of the β-subunits resulting in autophosphorylation of the receptor. These autophosphorylations occur on between 6 and 13 tryosine residues with the most frequently observed being tyrosines at amino acid position 1316, 1322, 1146, 1150, and 1151 in the intracellular portions of the β-subunits.

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Insulin Signal Transduction

All of the post-receptor responses initiated by insulin binding to its receptor are mediated as a consequence of the activation of several divergent and/or intersecting signal transduction pathways (see Figure below). These include association of insulin receptor substrates (of which there are three: IRS1, IRS2, and IRS4) with the receptor resulting in the activation of phosphatidylinositol-3-kinase (PI3K) and growth factor receptor binding protein 2 (GRB2). Activated PI3K phosphorylates membrane phospholipids, the major product being phosphatidylinositol-3,4,5-trisphosphate, (PIP3). PIP3 in turn activates the enzyme, PIP3-dependent kinase 1, (PDK1). PDK1 activates another kinase called protein kinase B, PKB (also called Akt). There are three members of the PKB/Akt family of serine/threonine kinases identified as Akt1, Akt2, and Akt3. It is Akt2 that is important in insulin-mediated glucose homeostasis. Insulin-mediated activation of Akt also results in inhibition of lipolysis and gluconeogenesis and activation of protein synthesis and glycogen synthesis. Another important metabolism regulating enzyme activated by insulin receptor signaling is small ribosomal subunit protein 6 (p70) kinase, (p70S6K). Acting as a growth factor, insulin signaling activates the MAP kinase (MAPK) pathway either through insulin receptor phosphorylation of SRC homology 2 containing protein (Shc) which then interacts with growth factor receptor binding protein-2 (GRB2) or via IRS1 activation.

Insulin-mediated glucose uptake involves activated PDK1 which phosphorylates some isoforms of protein kinase C, PKC. The PKC isoform, PKCλ/ζ, phosphorylates intracellular vesicles containing the glucose transporter, GLUT4, resulting in their migration to and fusion with, the plasma membrane. This results in increased glucose uptake and metabolism. The activation of GRB2 results in signal transduction via the monomeric G-protein, RAS. Activation of RAS ultimately leads to changes in the expression of numerous genes via activation of members of the extracellular signal-regulated kinases, ERK. In addition to its effects on enzyme activity, insulin exerts effects on the transcription of numerous genes, effects that are primarily mediated by regulated activity of sterol-regulated element binding protein, SREBP. These transcriptional effects include (but are not limited to) increases in glucokinase, liver pyruvate kinase (L-PK), lipoprotein lipase (LPL), fatty acid synthase (FAS) and acetyl-CoA carboxylase (ACC) gene expression, and decreases in glucose 6-phosphatase, fructose 1,6-bisphosphatase and phosphoenolpyruvate carboxykinase (PEPCK) gene expression.

Signal transduction cascades initiated by activation of the insulin receptor

Multiple roles of insulin. When insulin binds to its receptor it triggers receptor autophosphorylation that generates docking sites for insulin receptor substrate proteins (IRS-1–IRS4). IRS proteins in turn trigger the activation of a wide array of signal transducing proteins (highly simplified in this Figure). The end results of insulin receptor activation are varied and in many cases cell-type specific but includes alterations in metabolism, ion fluxes, protein translocation, transcription rates, and growth properties of responsive cells. Arrows represent positive, activating functions. T-lines represent inhibitory functions. Most abbreviations are described within the text below. PDE3B = phosphodiesterase 3B (also called adipocyte cAMP phosphodiesterase), GS = glycogen synthase, HSL = hormone sensitive lipase, ACC = acetyl-CoA carboxylase, ACL = ATP-citrate lyase.

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Insulin Regulation of Metabolism

Insulin, secreted by the β-cells of the pancreas, is directly infused via the portal vein to the liver, where it exerts profound metabolic effects. These effects are the response of the activation of the insulin receptor which belongs to the class of cell surface receptors that exhibit intrinsic tyrosine kinase activity (see Signal Transduction). The insulin receptor is a heterotetramer of 2 extracellular α-subunits disulfide bonded to 2 transmembrane β-subunits. With respect to hepatic glucose homeostasis, the effects of insulin receptor activation are specific phosphorylation events that lead to an increase in the storage of glucose with a concomitant decrease in hepatic glucose release to the circulation as diagrammed below (only those responses at the level of glycogen synthase and glycogen phosphorylase are represented).

Insulin-mediated regulation of glycogen metabolism

Insulin receptor interactions at the level of insulin receptor substrate-1 (IRS1) and activation of the kinase cascade leading to altered activities of glycogen phosphorylase and glycogen synthase. PI3K = posphatidylinositol-3-kinase; PIP2 = phosphatidylinositol-4,5-bisphosphate; PIP3 = phosphatidylinositol-3,4,5-bisphosphate; PDK1 = PIP3-dependent protein kinase; Tsc1 and Tsc2 = Tuberous sclerosis tumor suppressors 1 (hamartin) and 2 (tuberin); Rheb = Ras homolog enriched in brain; mTOR = mammalian target of rapamycin. PKB/Akt = protein kinase B/Akt2; GSK3 = glycogen synthase kinase-3; S6K = 70kDa ribosomal protein S6 kinase, also called p70S6K. The insulin-mediated activation of mTOR also leads to changes in protein synthesis (see below). Arrows denote either direction of flow or positive effects, T lines represent inhibitory effects.

In most nonhepatic tissues, particularly in adipose tissue and skeletal muscle, insulin increases glucose uptake by stimulating an increase in the number of plasma membrane glucose transporters: GLUTs. Glucose transporters are in a continuous state of turnover. Increases in the plasma membrane content of GLUTs stem from an increase in the rate of recruitment of the transporters into the plasma membrane, deriving from a special pool of preformed transporters localized in the cytoplasm. GLUT1 is present in most tissues, GLUT2 is found primarily in intestine, pancreatic β-cells, kidney and liver, GLUT3 is found primarily in neurons but also found in the intestine, GLUT4 is found in insulin-responsive tissues such as heart, adipose tissue and skeletal muscle and GLUT5 is expressed in intestine, kidney, testes, skeletal muscle, adipose tissue and brain.

In the liver, glucose uptake is dramatically increased because of increased activity of the enzymes glucokinase, phosphofructokinase-1 (PFK-1), and pyruvate kinase (PK), the key regulatory enzymes of glycolysis. The latter effects are induced by insulin-dependent activation of phosphodiesterase (PDE3B) which hydrolyzes cAMP to AMP. Insulin triggers activation of PDE3B via the insulin receptorsignaltransduciton cascade that activates the kinase, PKB. Active PKB in turn phosphorylates and activates PDE3B. The resultant reduction in cAMP leads to decreased PKA activity and diminished phosphorylation of pyruvate kinase and phosphofructokinase-2, PFK-2. Dephosphorylation of pyruvate kinase increases its' activity while dephosphorylation of PFK-2 renders it active as a kinase. The kinase activity of PFK-2 converts fructose-6-phosphate into fructose-2,6-bisphosphate (F2,6BP). F2,6BP is a potent allosteric activator of the rate limiting enzyme of glycolysis, PFK-1, and an inhibitor of the gluconeogenic enzyme, fructose-1,6-bisphosphatase. In addition, phosphatases specific for the phosphorylated forms of the glycolytic enzymes increase in activity under the influence of insulin. All these events lead to conversion of the glycolytic enzymes to their active forms and consequently a significant increase in glycolysis. In addition, glucose-6-phosphatase activity is down-regulated. The net effect is an increase in the content of hepatocyte glucose and its phosphorylated derivatives, with diminished blood glucose. In addition to the above described events, diminished cAMP and elevated protein phosphatase activity combine to convert glycogen phosphorylase to its inactive form and glycogen synthase to its active form, with the result that not only is glucose funneled to glycolytic products, but glycogen content is increased as well.

Role of protein targeting to glycogen (PTG) in insulin-mediated regulation of glycogen metabolism

Insulin-mediated effects on glycogen homeostasis: Insulin activates the synthesis of glycogen, while smultaneously inhibiting glycogenolysis, through the concerted effects of several insulin receptor activated pathways. Shown in this Figure are the major insulin-regulated activities and how they can rapidly exert their effects since all the activities are closely associated through interactions with protein targeting to glycogen (PTG). PTG is actually a regulatory subunit of the heterotetrameric PP1. There is a muscle-specific PTG (PPP1R3A) and a liver-specific PTG (PPP1R3B). Also diagrammed is the response to insulin at the level of glucose transport into cells via GLUT4 translocation to the plasma membrane. PDK1: PIP3-dependent protein kinase 1. GS/GP kinase: glycogen synthase: gycogen phosphorylase kinase (PHK). PP1: protein phosphatase-1. PDE: phosphodiesterase. Arrows denote either direction of flow or positive effects, red T lines represent inhibitory effects.

Epinephrine, the fight-or-flight hormone, diminishes insulin secretion by a cAMP-coupled regulatory path. In addition, epinephrine counters the effect of insulin in liver and peripheral tissue, where it binds to β-adrenergic receptors, induces adenylate cycles activity, increases cAMP, and activates PKA similarly to that of glucagon. The latter events induce glycogenolysis and gluconeogenesis, both of which are hyperglycemic and which thus counter insulin's effect on blood glucose levels. In addition, epinephrine influences glucose homeostasis through interaction with α-adrenergic receptors.

Regulation of glycogen phosphorylase via activation of α-adrenergic receptors

Pathways involved in the regulation of glycogen phosphorylase by epinephrine activation of α1-adrenergic receptors. See Glycogen Metabolism for details of the epinephrine action in glycogen homeostasis. PLC-β is phospholipase C-β. The substrate for PLC-β is phosphatidylinositol-4,5-bisphosphate, (PIP2) and the products are inositol trisphosphate, IP3 and diacylglycerol, DAG. Gs-GP kinase is glycogen synthase-glycogen phosphorylase kinase. More commonly called phosphorylase kinase (PHK). Similar calmodulin-mediated activation of PHK phosphorylations lead to inhibition of glycogen synthase.

With respect to insulin responses and metabolism, activation of PKB and PKC-λ lead to translocation of GLUT4 molecules to the cell surface resulting in increased glucose uptake which is significant in skeletal muscle. Activation of PKB also leads to the phosphorylation and inhibition of glycogen synthase kinase-3 (GSK3), which is a major regulatory kinase of glycogen homeostasis. In addition, PKB phosphorylates and inhibits the activity of a transcription factor (FKHRL1), now called FoxO3a) that has pro-apoptotic activity. This results in reduced apoptosis in response to insulin action.

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Insulin Regulation of Protein Synthesis (Growth)

The role of insulin in the stimulation of protein synthesis occurs at the level of translational initiation and elongation and is exerted primarily via a cascade leading to the activation of mammalian target of rapamycin, mTOR, a protein with homology to a family of proteins first identified in yeast that bind to the immunosuppressant drug, rapamycin. Rapamycin gets its name from the fact that the compound was isolated from the bacterium Streptomyces hygroscopicus discovered on Easter Island (Rapa Nui). mTOR is a kinase whose catalytic domain shares significant homology with lipid kinases of the PI3K family.

mTOR is actually a component of two distinct multiprotein complexes termed mTORC1 and mTORC2 (mTOR complex 1 and mTOR complex 2). The activity of mTORC1 is sensitive to inhibition by by rapamycin whereas mTORC2 is not. Within the context of mTOR activity, mTORC1 is the central complex as it is responsible for integrating a diverse series of signal transduction cascades initiated by changes in both intra- and extracellular events. Activation and/or regulation of mTORC1 is involved in the control of cell proliferation, survival, metabolism and stress responses. These events can be triggered by nutrient availability, glucose, oxygen, and numerous different types of cell surface receptor activation, each of which eventually impinge on the activity of mTORC1. The components of mammalian mTORC1 include mTOR, Raptor (regulatory associated protein of TOR), Deptor (DEP domain containing mTOR-interacting protein), mLST8 (mammalian homolog of yeast LST8), and PRAS40 (proline-rich Akt/PKB substrate of 40kDa). Deptor and PRAS40 are inhibitors of mTOR activity within the complex. PRAS40 is a raptor-binding protein that is directly phosphorylated by mTOR, which then prevents PRAS40 inhibition of mTOR.

The components of mammalian mTORC2 include mTOR, Deptor, mLST8, Sin1, Poctor (protein observed with Rictor; also known as PRR5L for proline-rich 5-like protein), and Rictor (rapamycin-insensitive companion of mTOR). mTORC2 is involved in the control of the activity of serum- and glucocorticoid-induced kinase (SGK). Full activation of PKB/Akt requires the involvement of mTORC2.

Insulin-induced signaling cascade leading to regulation of translation

Insulin-mediated cascade leading to enhanced translation: (not intended to be a complete description of all of the targets of insulin action that affect translation rates). Also shown is the effect of an increase in the AMP to ATP ratio which activates AMP-activated kinase, AMPK. STK11-LKB1-PJS = serine-threonine kinase 11, Peutz-Jeghers syndrome gene. IRS1 = insulin receptor substrate-1; PI3K = phosphatidylinositol-3-kinase; PIP2 = phosphatidylinositol-4,5-bisphosphate; PTEN = phosphatase and tensin homolog deleted on chromosome 10; PDK1 = PIP3-dependent protein kinase; Tsc1 and Tsc2 = Tuberous sclerosis tumor suppressors 1 (hamartin) and 2 (tuberin); Rheb = Ras homolog enriched in brain; mTOR = mammalian target of rapamycin. PKB/Akt = protein kinase B; GSK3 = glycogen synthase kinase-3; 4EBP1 = eIF-4E binding protein; p70S6K = 70kDa ribosomal protein S6 kinase, also called S6K. Arrows denote either direction of flow or positive effects, red T-lines represent inhibitory effects.

Insulin action leads to an increase in the activity of PI3K which in turn phosphorylates membrane phospholipids generating phosphatidylinositol-3,4,5-trisphophate (PIP3) from phosphatidylinositol-4,5-bisphosphate (PIP2). PIP3 then activates the kinase PDK1 which in turn phosphorylates and activates PKB/Akt. Activated PKB/Akt will phosphorylate TSC2 (tuberin) of the TSC1/TSC2 complex on two residues (S939 and T1462) resulting in altered activity of the complex. The TSC1/TSC2 complex functions as a GTPase-activating protein (GAP) which increases GTP hydrolyzing activity of Rheb. The GAP activity resides in the C-terminal portion of tuberin. The faster the GTPase action of Rheb the faster will be the reduction in Rheb activation of mTOR. When TSC1/TSC2 is phosphorylated by PKB it is less effective at stimulating the GTPase activity of Rheb and therefore Rheb activation of mTOR will remain high as is the case in response to insulin action.

AMPK phosphorylates TSC2 at two sites (T1271 and S1387) that are distinct from the sites that are the PKB/Akt targets for phosphorylation. Evidence indicates that the AMPK-mediated phosphorylation of TSC2 promotes the GTPase activity of Rheb resulting in inhibition of mTOR and thus a decrease in protein synthesis. Recent evidence has shown that PKB/Akt actually phosphorylates tuberin at 4 sites (S939, S1130, S1132, T1462) all of which result in inhibition of the Rheb-GAP activity of the TSC1/TSC2 complex.

The ultimate activation of mTOR leads to phosphorylation and activation of p70S6K which in turn leads to increased phosphorylation of eEF2 kinase. eEF2 kinase normally phosphorylates eEF2 leading to a decrease in its role in translation elongation. When phosphorylated by p70S6K, eEF2 kinase is less active at phosphorylating eEF2, thus eEF2 is much more active in response to insulin action. In addition, insulin action leads to a rapid dephosphorylation of eEF-2 via activation of protein phosphatase 2A (PP2A). Taken together, reduced eEF2K-mediated phosphorylation and increased eEF-2 dephosphorylation lead to increased protein synthesis.

Both mTOR and p70S6K have been shown to phosphorylate the regulator of translation initiation, eIF-4E binding protein, 4EBP1. Phosphorylation of 4EBP1 prevents it from binding to eIF-4E. Binding of 4EBP1 to eIF-4E prevents eIF-4E from interaction with the cap structure of mRNAs which is necessary for translational initiation. Thus, the consequences of 4EBP1:eIF-4E interaction is a reduction in translation initiation. As a consequence of the concerted actions of mTOR and p70S6K, insulin results in increased protein synthesis.

PKB activation will also lead to phosphorylation and inhibition of glycogen synthase kinase-3 (GSK3). One of the targets of GSK3, relative to translation, is eIF2B. Phosphorylation of eIF2B prevents it from performing its GTPase activating (GAP) function in association with eIF2 (see the Protein Synthesis page for more details) and as a consequence results in reduced translational initiation. However, when GSK3 is inhibited by PKB phosphorylation the GAP activity of eIF2B remains high and consequently the rate of translational initiation by eIF2 remains high so protein synthesis is favored.

Insulin also has profound effects on the transcription of numerous genes, effects that are primarily mediated by regulated function of sterol-regulated element binding protein, SREBP. These transcriptional effects include (but are not limited to) increases in glucokinase, pyruvate kinase, lipoprotein lipase (LPL), fatty acid synthase (FAS) and acetylCoA carboxylase (ACC) and decreases in glucose 6-phosphatase, fructose 1,6-bisphosphatase and phosphoenolpyruvate carboxykinase (PEPCK).

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Nutrient Intake and Hormonal Control of Insulin Action

Two of the many gastrointestinal hormones have significant effects on insulin secretion and glucose regulation. These hormones are the glucagon-like peptides (principally glucagon-like peptide-1, GLP-1) and glucose-dependent insulinotropic peptide (GIP). Both of these gut hormones constitute the class of molecules referred to as the incretins. Incretins are molecules associated with food intake-stimulation of insulin secretion from the pancreas.

Details of the actions of GLP-1 and GIP can be found in the Gut-Brain Interactions page. Briefly, GLP-1 is derived from the product of the proglucagon gene (gene symbol = GCG). This gene encodes a preproprotein that is differentially cleaved dependent upon the tissue in which it is synthesized. For example, in pancreatic α-cells prohormone convertase 2 action leads to the release of glucagon. In the gut prohormone convertase 1/3 action leads to release of several peptides including GLP-1. Upon nutrient ingestion GLP-1 is secreted from intestinal enteroendocrine L-cells that are found predominantly in the ileum and colon with some production from these cell types in the duodenum and jejunum. Bioactive GLP-1 consists of 2 forms; GLP-1(7-37) and GLP-1(7-36)amide, where the latter form constitutes the majority (80%) of the circulating hormone.

The primary physiological responses to GLP-1 are glucose-dependent insulin secretion, inhibition of glucagon secretion and inhibition of gastric acid secretion and gastric emptying. The latter effect will lead to increased satiety with reduced food intake along with a reduced desire to ingest food. The action of GLP-1 at the level of insulin and glucagon secretion results in significant reduction in circulating levels of glucose following nutrient intake. This activity has obvious significance in the context of diabetes, in particular the hyperglycemia associated with poorly controlled type 2 diabetes. The glucose lowering activity of GLP-1 is highly transient as the half-life of this hormone in the circulation is less than 2 minutes. Removal of bioactive GLP-1 is a consequence of N-terminal proteolysis catalyzed by dipeptidylpeptidase IV (DPP IV or DPP4). For more complete information on the activities of DPP4 go to the DPP4 page.

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Wnt Signaling, GLP-1, and Insulin Secretion

Although much of the research that has led to a detailed understanding of the signal transduction pathways initiated by Wnts was carried out in models of early development, evidence has been accumulating demonstrating a significant role for the Wnts in the control of metabolism. In particular Wnt action has been shown to be involved in metabolic control via its actions in both the gut and pancreas. In addition, Wnt signaling has been shown to interact with signaling pathways induced by insulin.

In the gut Wnt has been shown to be involved in regulated expression of the GCG gene. In intestinal enteroendocrine L cells the expression of the GCG gene results in the production of GLP-1. As indicated above, GLP-1 exerts its effects on the gut, the pancreas and in the brain. In the gut its effects lead to a reduced rate of gastric acid secretion and reduced gastric emptying. In the pancreas GLP-1 induced β-cell proliferation and inhibition of β-cell apoptosis. In the brain GLP-1 actins result in increased satiety leading to reduced desire for food intake.

The GCG gene promoter region contains an enhancer that harbors a canonical Wnt response element that binds TCF factors, in particular the TCF7L2 protein. Genome wide screens for polymorphisms associated with type 2 diabetes demonstrated that two single nucleotide polymorphisms (SNPs) in the TCF7L2 gene were the most frequently occurring SNPs associated with this disease. The significance of Wnt in the control of GLP-1 production was demonstrated by the fact that reduction/loss of either β-catenin or TCF7L2 function completely blocks insulin-stimulated expression of the intestinal GCG gene. In addition, the effects of GLP-1 on the pancreas (i.e. proliferation and anti-apoptosis) are effected via the actions of β-catenin and TCF7L2. In the pancreas insulin inhibits expression from the GCG gene leading to reduced production of glucagon. This action has physiological significance because glucagon is the major counter-regulatory hormone to insulin action. The important role of TCF7L2 in pancreatic function can be demonstrated in experiments that lead to reduction in the levels of TCF7L2. In these types of experiments there is an increased rate of pancreatic β-cell apoptosis, reduced β-cell proliferation, and reduced glucose-dependent insulin secretion.

The demonstration of cross-talk between the Wnt and insulin signaling pathways is important as these observations may eventually lead to novel approaches to the treatment of type 2 diabetes.

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Insulin Resistance

Insulin resistance (IR) refers to the situation whereby insulin interaction with its receptor fails to elicit downstream signaling events such as those depicted in the Figures above. Metabolically and clinically the most detrimental effects of IR are due to disruption in insulin-mediated control of glucose and lipid homeostasis in the primary insulin-responsive tissues: liver, skeletal muscle, and adipose tissue. IR is a characteristic feature found associated with most cases of type 2 diabetes. In addition, IR is the hallmark feature of the metabolic syndrome (MetS). IR can occur for a number of reasons however, the most prevalent cause is the hyperlipidemic and pro-inflammatory states associated with obesity. How does an abnormal metabolism, as is associated with obesity, lead to the development of IR? The answer to this question can be found in the effects of excess free fatty acids (FFAs) on the insulin receptor-mediated signaling pathways in adipose tissue, liver, and skeletal muscle as well as the pro-inflammatory status induced by the toxic effects of excess FFAs principally in the liver and adipose tissues.

The precise mechanisms that underlie the promotion of a pro-inflammatory state in obese individuals in not completely established. However, both adipose tissue and liver are important mediators of systemic inflammation in obesity. One model proposes that the expansion of adipose tissue that occurs in obesity results in large adipocytes that have metabolic capacities that exceed the local oxygen supply. The resultant hypoxia leads to the activation of cellular stress response pathways causing cell autonomous inflammation and the release of pro-inflammatory cytokines. As a part of the chronic inflammation adipocytes secrete chemokines such as IL-8 and macrophage chemotactic protein-1 (MCP-1) that attract pro-inflammatory macrophages into the adipose tissue. These activated adipose tissue macrophages secrete cytokines that further exacerbate the pro-inflammatory state. In the liver inflammatory processes are also activated due to the excess accumulation of fatty acids and triglycerides which is the consequence of activated stress response pathways. Within the liver, Kupffer cells (resident liver macrophages) become activated by the generation of reactive oxygen species (ROS) and induction of stress responses. These activated Kupffer cells release locally acting cytokines that, like in adipose tissue, exacerbates the pro-inflammatory environment. Within the vasculature, saturated FFAs can directly activate pro-inflammatory pathways in endothelial cells and myeloid-derived cells resulting in the induction and propagation of a systemic pro-inflammatory state.

pathways to insulin resistance by fatty acids and inflammation

Model for how excess free fatty acids (FFAs) lead to insulin resistance and enhanced inflammatory responses in cells such as liver and adipose tissue. Only the major pathways regulated by insulin relative to glucose and lipid homeostasis are shown. Black arrows represent positive actions and red T-lines represent inhibitory actions. JNK = Jun N-terminal kinase. PKC = protein kinase C. IKKβ = inhibitor of nuclear factor kappa B kinase beta. ROS = reactive oxygen species. PI3K = phosphatidylinositol-3 kinase. DAG = diacylglycerol. TAG = triacylglycerols. LCA-CoA = long-chain acyl-CoAs. NFκB = nuclear factor kappa B. PKB (protein kinase B) is a serine/threonine kinase also known as Akt. The role of ceramides in the development of insulin resistance is discussed in the section below.

Hepatic IR is induced by the excess accumulation of FFAs. Within the hepatocyte, metabolites of the FFA re-esterification process, including long-chain acyl-CoAs and diacylglycerol (DAG), accumulate. Excess FFAs also participate in the relocation of several protein kinase C (PKC) isoforms, from the cytosol to the membrane compartment. These PKC isoforms include PKC-β2, PKC-δ, and PKC-theta (PKC-θ). DAG is a potent activator of these PKC isoforms and the membrane-associated PKCs will phosphorylate the intracellular portion of the insulin receptor on serine residues which results in impairment of insulin receptor interaction with downstream signaling proteins including insulin receptor substrate 1 (IRS1) and IRS2. Loss of IRS1 and IRS2 interaction with the receptor prevents interaction with phosphatidylinositol 3-kinase (PI3K) and its' subsequent activation. In addition to serine phosphorylation of the insulin receptor, various PKCs have been shown to phosphorylate IRS1 and IRS2 further impairing the ability of these insulin receptor substrates to associate with the insulin receptor and downstream effector proteins such as PI3K.

The FFA-induced down-regulation of insulin signaling pathways results in activation of several kinases involved in stress responses. These kinases include Jun N-terminal kinase (JNK), inhibitor of nuclear factor kappa B kinase beta (IKKβ), and suppressors of cytokine signaling-3 (SOCS-3). Like PKC, JNK activity is also regulated by FFAs and is an important regulator of IR. The target of JNK action is the Ser307 of IRS-1 and this phosphorylation plays an important role in the progression to hepatic IR. Activation of IKKβ (which is required for the activation of nuclear factor kappa B, NFκB) may have the most pronounced effect on inflammatory responses in the liver and adipose tissue. NFκB is the most important transcription factor activating the expression of numerous pro-inflammatory cytokine genes such as interleukin-1 (IL-1), IL-6, and tumor necrosis factor-alpha (TNF-α) each of which have been shown to be involved in promoting IR. NFκB-dependent inflammatory mediators produced in hepatocytes act to reduce insulin sensitivity and to promote liver injury.

Analysis of the effects of FFAs on macrophages in cell culture demonstrated that they can activate inflammatory signaling through the toll-like receptors (TLRs), specifically TLR4. The TLRs are a family of cell surface receptors involved in key events triggered via the innate immune system. The TLRs are pattern recognition receptors that recognize structurally conserved molecules from microbial pathogens. TLR4 is responsive to bacterially derived lipopolysaccharide (LPS) which is an endotoxin secreted by gram-negative bacteria. LPS stimulation of TLR4 results in activation of both the JNK and IKKβ signal transduction pathways leading to secretion of pro-inflammatory cytokines such as IL-1β, IL-6, MCP-1, and tumor necrosis factor alpha (TNFα). These cell culture experiments demonstrated that FFA addition to macrophages results in activation of NFκB and that this activation was deficient in macrophages from TLR4 knock-out mice. In the livers of TLR4 knock-out mice there is reduced inflammation even in the presence of hepatic steatosis suggesting that Kupffer cell TLR4 is important in hepatic inflammatory responses to excess FFA loading.

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Ceramides and Insulin Resistance

Numerous lines of evidence over the past 10 years have shown that various inducers of cellular stress such as inflammatory activation, excess saturated fatty acid intake, and chemotherapeutics, result in increased rates of ceramide synthesis. In addition, there is ample evidence demonstrating that the accumulation of cellular ceramides is associated with the pathogenesis of diseases such as obesity, diabetes, atherosclerosis, and cardiomyopathy. For example, studies in mice have correlated endogenous ceramides and glucosylceramides with the antagonism of insulin-stimulated glucose uptake and synthesis. In animal models of obesity, evidence shows that genetic or pharmacological inhibition of ceramide or glucosylceramide biosynthesis leads to increased peripheral insulin sensitivity while at the same time reducing the severity of pathologies associated with insulin resistance including diabetes, atherosclerosis, hepatic steatosis, and/or cardiomyopathy. With respect to overall lipid homeostasis and the role of adipose tissue in disease pathology, studies have revealed roles for the adipokines leptin, adiponectin, and TNFα in the modulation of cellular ceramide levels.

An enhanced systemic inflammatory status as well as cellular stress have both been associated with insulin resistance. With respect to biological lipids, excess lipid intake, especially saturated fatty acids, leads to mitochondrial and endoplasmic reticulum (ER) stress. Increased fat oxidation in mitochondria leads to the production of reactive oxygen species (ROS) which are known to result in insulin resistance. Both mitochondrial and ER stress can result in apoptosis. Excess fatty acid intake also interferes with normal insulin receptor-mediated signal transduction resulting in insulin resistance. Excess saturated fatty acid intake, particularly palmitic acid, results in increased ceramide synthesis which has been shown to be both a cause and effector of pancreatic β-cell stress resulting in impaired insulin secretion. Obesity, which results in insulin resistance and development of type 2 diabetes, has long been associated with low-grade systemic inflammation. The correlation between obesity, ceramide synthesis and insulin resistance is discussed below.

The ability of ceramides to interfere with insulin receptor signaling is the result of blocking the receptors ability to activate the downstream effector kinase, PKB/Akt. Experiments in cell culture, involving both adipocytes and skeletal muscle cells, have shown that ceramides inhibit insulin-stimulated glucose uptake by blocking translocation of GLUT4 to the plasma membrane as well as interfering with glycogen synthesis. That blockade of PKB/Akt activation is central to the effects of ceramides can be demonstrated by constitutive overexpression of the kinase which negates the effects of ceramides. Thus, far the action of ceramides at blocking activation of PKB/Akt has been shown in all cell types tested.

Several lines of evidence have solidified the model of ceramides leading to insulin resistance as a consequence of blockade of PKB/Akt activation. Administration of ceramide to cells in culture blocks the translocation of PKB/Akt to the plasma membrane. This inhibition of translocation is the result of the phosphorylation of a regulatory site in the PH domain. The phosphorylation leads to reduced affinity of the kinase for phosphoinositides. The kinase responsible for the ceramide-induced phosphorylation of PKB/Akt is likely to be the atypical PKC isoform PKCζ since this kinase is activated by ceramides in vitro. Additional evidence pointing to a link between ceramides and activation of PKCζ is that mutation of a target serine in the kinase, S34, to alanine confers resistance to ceramide action. Also, ceramide addition has been shown to stabilize interactions between PKB/Akt and PKCζ via their recruitment membrane rafts or caveolae. Another mechanism by which ceramides impact the activity of PKB/Akt is by activating protein phosphatase 2A (PP2A) to dephosphorylate the kinase. Experiments that were designed to specifically inhibit PP2A were shown to prevent the effects of ceramide on PKB/Akt in a number of different cell types. In some cell types, both mechanisms are functional, while in other cell culture systems either PKCζ or PP2A is the central mediator of ceramide effects.

Palmitic acid (C16:0) is the most abundant saturated fatty aid in the circulation. The role of saturated fatty acids in increased levels of ceramides has been demonstrated by adding palmitate to cultured muscle cells. In this system the addition of palmitate results in increased ceramide accumulation while simultaneously inhibiting PKB/Akt. Ceramide synthesis was indeed required for the effect of palmitate addition on the activity of PKB/Akt since pharmacological inhibition of ceramide synthesis or siRNA-mediated knockdown of several enzymes required for ceramide biosynthesis (serine palmitoyltransferase, ceramide synthases, or dihydroceramide desaturase) completely blocks the effects of palmitate on insulin signaling.

An alternative means to examine the effects of ceramides on insulin sensitivity is to block the pathways of ceramide metabolism. Treatment of cells with acid ceramidase inhibitors results in increased endogenous ceramide levels while simultaneously blocking insulin-mediated activation of PKB/Akt. Under conditions of ceramidase inhibition there is an exagerated effect of palmitic acid addition on insulin resistance. Conversely, if one overexpresses acid ceramidase, the inhibition of insulin signaling induced by palmitate addition is completely blocked.

The cellular effects of glucosylceramide, although similar to ceramides themselves, does exhibit cell-type specificity. Glucosylceramide is the precursor for a complex family of gangliosides, for example the GM3 ganglioside. Adipocytes are highly sensitive to the insulin inhibitory effects of glucosylated sphingolipids, whereas muscle cells are unaffected. Addition of GM3 ganglioside to adipocytes inhibits insulin activation of the IRS-1. In addition, TNFα treatment induces GM3 accumulation in membrane lipid rafts allowing for association with the insulin receptor through caveolin-1 present in the rafts. The the antagonistic effects of the TNFα can be prevented by depleting cells of glucosylated ceramides. Obesity is associated with adipose tissue enrichment in the complpex gangliosides, GM2, GM1, and GD1a. The significance of the accumulation of these gangliosides has been demonstrated in mice lacking GM3 synthase which generates the major ganglioside precursor. These mice are protected from insulin resistance and glucose intolerance when fed a high-fat diet. Treatment of genetically obese or diet-induced obese mice with highly specific glucosylceramide synthase (GCS) inhibitors results in improved glucose tolerance and increased insulin sensitivity in muscle and liver. Collectively, these studies strongly implicate a role for glucosylated ceramides in increased adipose tissue inflammation, peripheral insulin resistance, and hepatic steatosis.

The most potent reagent used to study the effects of the manipulation of enzymes involved in sphingolipid biosynthesis is the compound myriocin [2-Amino-3,4-dihydroxy-2-(hydroxymethyl)-14-oxoicos-6-enoic acid]. Myriocin is a highly specific inhibitor of serine palmitoyltransferase (SPT), which is the first and rate-limiting enzyme in the de novo pathway of ceramide synthesis. See the Figure above showing sphingosine and ceramide synthesis. Myriocin (also known as antibiotic ISP-1 and thermozymocidin) was isolated from themophilic fungi such as Mycelia sterilia and Isaria sinclairii. Extracts from these fungi have been used in traditional Chinese medicine as a treatment for numerous conditions including diabetes. Myriocin can be administered chronically to rodents and it appears to be well tolerated. Addition of myriocin to animals that are models of obesity prevents insulin resistance and the development of diabetes, atherosclerosis, and cardiomyopathy. In addition, myriocin improves glucose tolerance, insulin sensitivity and ameliorates hypertension when administered to rodents.

Genetic manipulation of several enzymes in ceramide metabolism has also been shown to insulin sensitizing. In mice heterozygous for the SPT subunit SPTLC2 (serine palmitoyltransferase, long-chain base subunit 2) there is a reduction in peripheral ceramide levels and improved insulin sensitivity when these animals are fed a high-fat diet. Similar results are seen in mice heterozygous for dihydroceramide desaturase-1 (DES1). Both SPT and DES1 are required for ceramide biosynthesis. As described above, a large family of ceramide synthases (CerS) have been identified in mammals. CerS1 is the most abundant isoform expressed in skeletal muscle and is involved primarily in the synthesis of C18:0 ceramides. The level of expression of CerS1 was shown to be significantly elevated in mice fed a high-fat diet. This increase in CerS1 expression was associated with alterations in ceramide levels and reduced glucose tolerance.

Collectively these data demonstrate a complex interrelationship between sphingosine and ceramide metabolism and insulin resistance. As pointed out ceramides can be deacetylated by ceramidases to form sphingosine. As discussed below, sphingosine can be phosphorylated to S1P which is an important biologically active lipid. Ceramides can also be glucosylated (catalyzed GCS) forming glucosylceramides which then serve as the building blocks of complex glycosphingolipids; they can act as substrates for the sphingomyelin synthases yielding sphingomyelins; or they can be phosphorylated (by ceramide kinase) to yield ceramide-1-phosphate. Thus, it is clear that multiple products of the actions of SPT, CerS, and DES1 could all potentially contribute to the development of insulin resistance and diabetes.

As pointed out earleir, obesity is associated with a low-grade systemic inflammatory state. One of the mechanisms involved in this inflammatory status is the activation of toll-like receptors (TLRs). TLR activation leads to enhanced transcription of pro-inflammatory cytokines such as TNFα and interleukin-6 (IL6). Saturated fatty acids are known to activate TLR4 and this activation is requisite for lipid induction of TNFα and other cytokines. When TLRs are knocked-out in mice the animals are protected from lipid-induced insulin resistance. The signal transduction cascade initiated by TLR activation involves the downstream effectors IKKβ and NFκB. TLR4 activation has been shown to selectively and strongly increase the levels of sphingolipids within cells. Several studies have shown that ceramide is indeed an obligate intermediate linking TLR4 activation to the induction of insulin resistance.

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Hexosamine Biosynthesis and Insulin Resistance

The details of the hexosamine biosynthesis pathway and its role in metabolism and development can be found in the Glycoproteins page.

Numerous proteins involved in insulin signaling and the downstream targets of these signaling cascades have been shown to be O-GlcNAcylated. With respect to insulin receptor signaling proteins, IRS-1, PI3K, PKB/Akt, PDK1, and GSK3β are all known to be O-GlcNAcylated. These modifications have all been observed in adipocytes which are a major target for the actions of insulin. Insulin-stimulated glucose uptake into adipocytes occurs via insulin-mediated mobilization of GLUT4 to the plasma membrane. Increased glucose uptake, in response to insulin, can therefore, significantly modify the rate of flux through the HBP. Evidence linking the correlation between the HBP and insulin resistance in adipocytes was demonstrated at least 20 years ago. Using cultured rat adipocytes experiments demonstrated that chronic exposure to both insulin and glucose was required for the adipocytes to become insulin-resistant. This is now a common theme underlying insulin resistance in other insulin-sensitive tissues such as skeletal muscle. In these early experiments is was shown that the impairment in insulin-stimulated glucose uptake, under hyperglycemic and hyperinsulinemic conditions, was exclusively dependent on the presence of the amino acid glutamine. Remember that glutamine is required as a substrate for GFAT, the rate-limiting enzyme in the HBP. Inhibition of GFAT activity was observed in the hyperglycemic and hyperinsulinemic conditions likely due to feedback inhibition by UDP-GlcNAc as the HBP product was shown to accumulate in the treated cells. However, if GFAT was inhibited with the use of various amidotransferase inhibitors the hyperglycemia-induced insulin resistance was prevented. Additionally, if cells are treated with glucosamine, which enters the HBP after the GFAT catalyzed reaction, there was a greater reduction in insulin-mediated glucose uptake compared to the hyperglycemic condition. As expected, since GFAT is bypassed, the glucosamine-induced insulin resistance does not require glutamine. Although glucose and glutamine metabolism are key inducers of the flux through the HBP, free fatty acids (FFA) and uridine are also potent modulators of the HBP.

Utilizing experiments in whole animals, as opposed to cell culture, has provided additional direct evidence that excess flux through the HBP leads to modulation of insulin sensitivity in adipocytes. When GFAT is overexpressed in mice under the control of a GLUT4 promoter the animals develop classical insulin-resistant phenotype with hyperinsulinemia and reduction in whole-body glucose disposal rate. Because GLUT4 is highly expressed in adipose tissue and skeletal muscle, two major insulin-responsive tissues, it is not surprising that defective whole-body glucose disposal was observed. Elevation in serum leptin level was also observed in these GFAT overexpressing mice. Interestingly, muscle explants from GLUT4-GFAT mice showed normal insulin-stimulated glucose uptake. This latter observation is strong evidence that adipocytes play a major regulatory role in the HBP-mediated whole-body insulin resistance.

Another strain of mice has been utilized for studies on the role of HBP in insulin sensitivity that express GFAT specifically in adipose tissue by the use of an aP2 (adipocyte lipid binding protein) promoter driving its expression. Adipose tissue-restricted elevations in O-GlcNAc levels are detected in these mice and this is associated the development of whole-body insulin resistance. The results in these animals is characterized by a reduction in both glucose disposal rate and skeletal muscle glucose uptake. An increase in serum leptin and a decrease in serum adiponectin levels were also found in these mice.

As pointed out above, numerous proteins downstream of the insulin receptor that are critical to insulin-mediated signal transduction are known to be O-GlcNAcylated. Therefore, it is not difficult to assume that HBP-mediated glucose desensitization will occur at multiple stages, in particular through insulin-mediated signal transduction. Under high glucose-induced insulin resistance, there is a reduction in insulin-stimulated phosphorylation of PKB/Akt. There has been some discrepancy in determining precisely how HBP flux affects PKB/Akt phosphorylation in response to insulin binding its receptor. Recent research has shown that when cells are exposed to chronically high glucose and insulin there is a concomitant reduction in PIP3 which is a product of activated PI3K, a target of the activated insulin receptor. This reduction in PIP3 levels is correlated with an increase in PTEN (phosphatase and tensin homolog deleted on chromosome 10) levels. PTEN is a known inhibitor of PI3K. In addition, it was shown that there is an increase in IRS-1 phosphorylation on Ser636 and Ser639. Since rapamycin treatment inhibits the alteration of PIP3 and PTEN levels under insulin-resistant conditions, it is believed that mammalian target of rapamycin complex 1 (mTORC1) is involved in negatively regulating the IRS-1/PI3K/Akt signaling cascade downstream of the insulin receptor. The sites on IRS-1 seen to be phosphorylated by chronic hyperglycemic and hypeinsulinemic conditions (S636/S639) are known to be substrates of mTORC1.

The regulation of insulin-stimulated GLUT4 translocation is also affected by changes in the flux rate through the HBP. Several cytoskeletal proteins involved in mobilization of GLUT4 to the plasma membrane are known to be O-GlcNAcylated. In addition, several of the proteins involved in the translocation process are targets of signaling proteins downstream of the insulin receptor. In cell culture models of both glucose- and glucosamine-induced insulin-resistance a reduction in the acute insulin-stimulated GLUT4 translocation is associated with a significant alteration in membrane redistribution of vesicle proteins such as t-(target membrane) SNARE, v-(vesicle membrane) SNARE and Munc18c (mammalian uncoordinated). SNARE stands for soluble-N-ethylmaleimide-sensitive factor attachment protein receptor. Munc18c is a negative regulator of both t- and v-SNAREs. Munc18c is known to be a target for O-GlcNAcylation. These results suggest a direct involvement of excess HBP flux in desensitizing the fusion between GLUT4-containing intracellular vesicles and the plasma membrane.

In addition to GLUT4 translocation, insulin-mediated PI3K and PKB/Akt activation also stimulates glycogen synthesis. The net effect is to balance the level of glucose metabolism in response to excess glucose influx. Insulin-dependent glycogen synthesis is mediated via the activation of of glycogen synthase (GS). Like other downstream targets of the insulin receptor, GS regulation involves a PKB/Akt-mediated inhibition of GSK3β which normally phosphorylates and inhibits GS. The insulin-stimulated increase in glycogen synthesis decreases the pool of G6P and subsequently F6P, thereby restricting flux through the HBP. PKB/Akt activation also leads to reduced dephosphorylation of GS via protein phosphatase 1 (PP1). Exposing cells to either high glucose or glucosamine results in a reduction in insulin-stimulated GS activity. Additionally, GS is a known O-GlcNAcylated protein and as might be expected it has been shown that GS becomes more resistant to dephosphorylation by PP1 under conditions of excess HBP flux.

While increased global O-GlcNAc levels are implicated in the development of insulin resistance, OGT is also regulated by insulin in adipocyte cell cultures. OGT is tyrosine-phosphorylated by the insulin receptor upon acute insulin stimulation and this phosphorylation increases the activity of the enzyme. In addition there is an observed shift in OGT localization from the nucleus to the cytosol in response to insulin stimulation. This OGT translocation to the plasma membrane is PI3K-dependant in response to acute insulin stimulation.

In summary, given that genetic and pharmacologic elevation in O-GlcNAc levels in cultured adipocytes and mouse models is associated with insulin-resistant phenotypes, it is likely that reducing O-GlcNAc levels in adipocytes should reverse the HBP-induced insulin resistance. A proof-of-concept experiment in transgenic mice (the insulin-resistant db/db mouse model which harbors a mutated leptin receptor) showed that overexpression of OGA, which reduces the level of O-GlcNAcylation, significantly improves whole-body glucose tolerance and insulin sensitivity. This result suggests that lowering O-GlcNAc levels in vivo should be of significant clinical beneficial.

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Insulin Action and Endothelial Functions

The metabolic functions of insulin are primarily reflective of its role in glucose and lipid homeostasis in skeletal muscle, adipose tissue, and liver. However, insulin also exerts important functions in other non-classical insulin target tissues such as the brain, pancreas, and the vascular endothelium. The ability of insulin to exert vasodilator action in the vascular endothelium as a result of increased nitric oxide (NO) production is an important component of the ability of this hormone to enhance glucose uptake by skeletal muscle. The insulin-mediated signaling pathway that triggers production of NO in vascular endothelium involves the same signaling proteins (PI3K, PKD, and PKB/Akt) that are components of metabolic regulatory pathways induced by insulin. Therefore, it is understandable why the same disruptions to insulin signaling that lead to IR (see above) caused by excess FFAs and hyperglycemia result in endothelial dysfunction.

The production of NO in endothelial cells is the result of the activation of endothelial nitric oxide synthase (eNOS). The production and actions of NO and the various NOSs involved are discussed in more detail in the Amino Acid Derivatives page. With respect to insulin action, the activation of endothelial PKB/Akt leads to phosphorylation and activation of eNOS and thus increased NO production. In addition to modulating vascular tone by activating signaling events in the underlying vascular smooth muscle cells, endothelial cell-derived NO reduces the production of pro-inflammatory cytokines, reduces leukocyte and monocyte recruitment and adhesion to the endothelium, inhibits the proliferation of vascular smooth muscle cells, inhibits apoptosis, and attenuates platelet aggregation. Inactivation of endothelial cell NO production, as occurs due to IR, results in endothelial dysfunction and promotes the development of atherosclerosis. As described above for the liver and adipose tissue, elevated levels of circulating FFAs lead to impaired insulin signaling via the PI3K-PDK-PKB/Akt pathway in vascular endothelial cells.

Insulin exerts its mitogenic, growth promoting, and differentiation effects via a signaling pathway that involves mitogen-activated protein kinase (MAPK) which is distinct from the PI3K-PDK-PKB/Akt pathway that is involved in metabolic regulation by insulin. The MAPK-induced pathway does not play a role in the production of NO by insulin. This MAPK-induced pathway plays a significant role in the development of atherosclerosis in the IR state. When insulin signaling via PI3K-PDK-PKB/Akt is impaired as described above for the IR state, the MAPK signaling pathway in endothelial cells is enhanced. In the endothelium MAPK activation by insulin results in increased expression of endothelin-1 (ET-1), plasminogen activator inhibitor type-1 (PAI-1), and the adhesion molecules intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin. ET-1 is a potent vasoconstrictor and contributes to endothelial cell dysfunction in the presence of IR. The increased expression of numerous cell adhesion molecules accelerates the adherence to the endothelium of pro-inflammatory leukocytes which in turn contributes to the development of atherosclerosis. Therefore, the molecules beneficial to vascular endothelial health that are induced by insulin (e.g. NO) are reduced in the IR state and those that are proatherogenic (e.g. ET-1, PAI-1) are increased.

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Last modified: September 15, 2015