Insulin Regulation Of Glucose Metabolism Research Articles

Leveraging continuous glucose monitoring for personalized modeling of insulin-regulated glucose metabolism

Continuous glucose monitoring (CGM) is a promising, minimally invasive alternative to plasma glucose measurements for calibrating physiology-based mathematical models of insulin-regulated glucose metabolism, reducing the reliance on in-clinic measurements. However, the use of CGM glucose, particularly in combination with insulin measurements, to develop personalized models of glucose regulation remains unexplored. Here, we simultaneously measured interstitial glucose concentrations using CGM as well as plasma glucose and insulin concentrations during an oral glucose tolerance test (OGTT) in individuals with overweight or obesity to calibrate personalized models of glucose-insulin dynamics. We compared the use of interstitial glucose with plasma glucose in model calibration, and evaluated the effects on model fit, identifiability, and model parameters’ association with clinically relevant metabolic indicators. Models calibrated on both plasma and interstitial glucose resulted in good model fit, and the parameter estimates associated with metabolic indicators such as insulin sensitivity measures in both cases. Moreover, practical identifiability of model parameters was improved in models estimated on CGM glucose compared to plasma glucose. Together these results suggest that CGM glucose may be considered as a minimally invasive alternative to plasma glucose measurements in model calibration to quantify the dynamics of glucose regulation.

A Synopsis of the Associations of Oxidative Stress, ROS, and Antioxidants with Diabetes Mellitus.

The Greek physician, Aretaios, coined the term “diabetes” in the 1st Century A.D. “Mellitus” arose from the observation that the urine exhibits a sweetness due to its elevated glucose levels. Diabetes mellitus (DM) accounted for 6.7 million deaths globally in 2021 with expenditures of USD 966 billion. Mortality is predicted to rise nearly 10-fold by 2030. Oxidative stress, an imbalance between the generation and removal of reactive oxygen species (ROS), is implicated in the pathophysiology of diabetes. Whereas ROS are generated in euglycemic, natural insulin-regulated glucose metabolism, levels are regulated by factors that regulate cellular respiration, e.g., the availability of NAD-linked substrates, succinate, and oxygen; and antioxidant enzymes that maintain the cellular redox balance. Only about 1–2% of total oxygen consumption results in the formation of superoxide anion and hydrogen peroxide under normal reduced conditions. However, under hyperglycemic conditions, about 10% of the respiratory oxygen consumed may be lost as free radicals. Under hyperglycemic conditions, the two-reaction polyol pathway is activated. Nearly 30% of blood glucose can flux through this pathway—a major path contributing to NADH/NAD+ redox imbalance. Under these conditions, protein glycation and lipid peroxidation increase, and inflammatory cytokines are formed, leading to the further formation of ROS. As mitochondria are the major site of intracellular ROS, these organelles are subject to the deleterious effects of ROS themselves and eventually become dysfunctional—a milestone in Metabolic Syndrome (MetS) of which insulin resistance and diabetes predispose to cardiovascular disease.

Prognostic criteria to assess the efficacy of the combined use of a low-frequency electrostatic field and transcranial magnetic therapy

The paper analyzes the dynamics of indicators characterizing the state of various functional body systems in patients with metabolic syndrome with the combined use of transcranial magnetic therapy and low-frequency electrostatic field in order to identify the main predictors of effectiveness. It was found that with such exposure, against the background of a significant increase in clinical efficacy, the degree of variation of dynamic changes increases, which is due to the difference in the initial condition of patients. It was revealed that the most pronounced therapeutic effect is observed in patients with preserved reserve and adaptive capabilities in the system of hemostasis, lipid peroxidation, micro-circulatory system and insulin regulation of glucose metabolism.

Cholecalciferol ameliorates insulin signalling and insulin regulation of enzymes involved in glucose metabolism in the rat heart

Context The evidence on potential cross-talk of vitamin D and insulin in the regulation of cardiac metabolism is very scanty. Objective Cholecalciferol was administered to male Wistar rats for six weeks to study its effects on cardiac glucose metabolism regulation. Materials and methods An expression, phosphorylation and/or subcellular localisation of insulin signalling molecules, glucose transport and metabolism key proteins were studied. Results Circulating non-esterified fatty acids (NEFA) level was lower after cholecalciferol administration. Cholecalciferol decreased cardiac insulin receptor substrate 1 Ser307 phosphorylation, while insulin-stimulated Akt Thr308 phosphorylation was increased. Cardiac 6-phosphofructo-2-kinase protein, hexokinase 2 mRNA level and insulin-stimulated glycogen synthase kinase 3β Ser9 phosphorylation were also increased. Finally, FOXO1 transcription factor cytosolic level was reduced. Conclusion Vitamin D-related improvement of insulin signalling and insulin regulation of glucose metabolism in the rat heart is accompanied by the decrease of blood NEFA level and dysregulation of cardiac FOXO1 signalling.

Enhancement of D-chiro-inositol transport across intestinal cells by alpha-Lactalbumin peptides.

This study aims to characterize in vitro D-chiro-inositol intestinal absorption and identify factors able to improve its bioavailability. D-chiro-inositol, one of the natural occurring stereoisomer of myo-inositol, acts as a second messenger in insulin-regulated glucose metabolism in complementary mode with myo-inositol. Because of their insulin-mimetic activities and safety, both myo-inositol and D-chiro-inositol are often employed as supplements in insulin-resistance treatment. Trans-epithelial passage of D-chiro-inositol was evaluated in the human intestinal Caco-2 cell line differentiated on filter, a widely established in vitro model to study intestinal absorption. D-chiro-inositol transport was assayed in a concentration range corresponding to an estimated in vivo concentration following oral supplementation. α-Lactalbumin peptides, obtained by in vitro simulated gastrointestinal digestion, were tested as possible modulators of the intestinal permeability of D-chiro-inositol. The absorption of this stereoisomer was relatively low and presumably due to passive diffusion, while it was greatly enhanced by the presence of α-Lactalbumin digest. α-Lactalbumin peptides induced an increase in paracellular permeability that was completely reversible, indicating lack of cytotoxicity. This effect involved temporary rearrangement of F-actin apical cytoskeleton and of the tight junction protein ZO-1. Although further studies are required to identify and characterize the most effective peptides, the ability of α-Lactalbumin digest to act as absorption enhancers may have very interesting and promising applications in the fields of nutritional supplements and pharmacology.

Kinetic Trans-omic Analysis Reveals Key Regulatory Mechanisms for Insulin-Regulated Glucose Metabolism in Adipocytes.

SummaryInsulin regulates glucose metabolism through thousands of regulatory mechanisms; however, which regulatory mechanisms are keys to control glucose metabolism remains unknown. Here, we performed kinetic trans-omic analysis by integrating isotope-tracing glucose flux and phosphoproteomic data from insulin-stimulated adipocytes and built a kinetic mathematical model to identify key allosteric regulatory and phosphorylation events for enzymes. We identified nine reactions regulated by allosteric effectors and one by enzyme phosphorylation and determined the regulatory mechanisms for three of these reactions. Insulin stimulated glycolysis by promoting Glut4 activity by enhancing phosphorylation of AS160 at S595, stimulated fatty acid synthesis by promoting Acly activity through allosteric activation by glucose 6-phosphate or fructose 6-phosphate, and stimulated glutamate synthesis by alleviating allosteric inhibition of Gls by glutamate. Most of glycolytic reactions were regulated by amounts of substrates and products. Thus, phosphorylation or allosteric modulator-based regulation of only a few key enzymes was sufficient to change insulin-induced metabolism.

AKT1 Regulates Endoplasmic Reticulum Stress and Mediates the Adaptive Response of Pancreatic β Cells.

Isoforms of protein kinase B (also known as AKT) play important roles in mediating insulin and growth factor signals. Previous studies have suggested that the AKT2 isoform is critical for insulin-regulated glucose metabolism, while the role of the AKT1 isoform remains less clear. This study focuses on the effects of AKT1 on the adaptive response of pancreatic β cells. Using a mouse model with inducible β-cell-specific deletion of the Akt1 gene (βA1KO mice), we showed that AKT1 is involved in high-fat-diet (HFD)-induced growth and survival of β cells but is unnecessary for them to maintain a population in the absence of metabolic stress. When unchallenged, βA1KO mice presented the same metabolic profile and β-cell phenotype as the control mice with an intact Akt1 gene. When metabolic stress was induced by HFD, β cells in control mice with intact Akt1 proliferated as a compensatory mechanism for metabolic overload. Similar effects were not observed in βA1KO mice. We further demonstrated that AKT1 protein deficiency caused endoplasmic reticulum (ER) stress and potentiated β cells to undergo apoptosis. Our results revealed that AKT1 protein loss led to the induction of eukaryotic initiation factor 2 α subunit (eIF2α) signaling and ER stress markers under normal-chow-fed conditions, indicating chronic low-level ER stress. Together, these data established a role for AKT1 as a growth and survival factor for adaptive β-cell response and suggest that ER stress induction is responsible for this effect of AKT1.

Decreased secretion of tumor necrosis factor-α attenuates macrophages-induced insulin resistance in skeletal muscle

AimsMacrophages, as an important member of immune system, engulf and digest pathogens in innate immunity and help initiate adaptive immunity. However, macrophages also involve in occurrence and development of many diseases, such as obesity and type 2 diabetes. Here, we aimed to reveal how activated macrophages cause insulin resistance in skeletal muscle in vitro through simulating body environment. Main methodsWe established RAW264.7 macrophages and C2C12 myotubes co-incubation model in vitro using Transwell filter to simulate body environment and investigated effects of RAW264.7 cells on insulin-regulated glucose metabolism in C2C12 myotubes. Immunofluorescence, Immunoblot and glucose uptake tests were used to assess metabolic changes in C2C12 myotubes. ELISA test detected secretions of tumor necrosis factor α (TNF-α) and interleukin-6 (IL-6) from RAW264.7 cells. In addition, RNA interference and inhibitor treatment were used. Key findingsActivated RAW264.7 cells attenuated insulin response in C2C12 myotubes. Activated RAW264.7 cells secreted a lot of TNF-α and IL-6. We found that TNFα, but not IL-6, caused insulin resistance of skeletal muscle in a dose-dependent manner. The results further indicated that activation of TNF-α downstream proteins, inhibitor of nuclear factor κ-B kinase (IKK) and the jun-N-terminal kinase 1 (JNK1) led to phosphorylation of insulin receptor substrate 1 (IRS-1) at Ser residues and insulin resistance in C2C12 myotubes. SignificanceOur research provided further and direct demonstration on activated macrophage-induced insulin resistance in skeletal muscle, suggesting TNF-α might become a therapeutic target to ameliorate and treat type 2 diabetes.

SPARC is required for the maintenance of glucose homeostasis and insulin secretion in mice.

Obesity, metabolic syndrome, and type 2 diabetes, three strongly interrelated diseases, are associated to increased morbidity and mortality worldwide. The pathogenesis of obesity-associated disorders is still under study. Secreted protein acidic and rich in cysteine (SPARC) is a matricellular glycoprotein expressed in many cell types including adipocytes, parenchymal, and non-parenchymal hepatic cells and pancreatic cells. Studies have demonstrated that SPARC inhibits adipogenesis and promotes insulin resistance; in addition, circulating SPARC levels were positively correlated with body mass index in obese individuals. Therefore, SPARC is being proposed as a key factor in the pathogenesis of obesity-associated disorders. The aim of this study is to elucidate the role of SPARC in glucose homeostasis. We show here that SPARC null (SPARC-/-) mice displayed an abnormal insulin-regulated glucose metabolism. SPARC-/- mice presented an increased adipose tissue deposition and an impaired glucose homeostasis as animals aged. In addition, the absence of SPARC worsens high-fat diet-induced diabetes in mice. Interestingly, although SPARC-/- mice on high-fat diet were sensitive to insulin they showed an impaired insulin secretion capacity. Of note, the expression of glucose transporter 2 in islets of SPARC-/- mice was dramatically reduced. The present study provides the first evidence that deleted SPARC expression causes diabetes in mice. Thus, SPARC deficient mice constitute a valuable model for studies concerning obesity and its related metabolic complications, including diabetes.

TCPTP Regulates Insulin Signaling in AgRP Neurons to Coordinate Glucose Metabolism With Feeding.

Insulin regulates glucose metabolism by eliciting effects on peripheral tissues as well as the brain. Insulin receptor (IR) signaling inhibits AgRP-expressing neurons in the hypothalamus to contribute to the suppression of hepatic glucose production (HGP) by insulin, whereas AgRP neuronal activation attenuates brown adipose tissue (BAT) glucose uptake. The tyrosine phosphatase TCPTP suppresses IR signaling in AgRP neurons. Hypothalamic TCPTP is induced by fasting and degraded after feeding. Here we assessed the influence of TCPTP in AgRP neurons in the control of glucose metabolism. TCPTP deletion in AgRP neurons (Agrp-Cre;Ptpn2fl/fl ) enhanced insulin sensitivity, as assessed by the increased glucose infusion rates, and reduced HGP during hyperinsulinemic-euglycemic clamps, accompanied by increased [14C]-2-deoxy-d-glucose uptake in BAT and browned white adipose tissue. TCPTP deficiency in AgRP neurons promoted the intracerebroventricular insulin-induced repression of hepatic gluconeogenesis in otherwise unresponsive food-restricted mice, yet had no effect in fed/satiated mice where hypothalamic TCPTP levels are reduced. The improvement in glucose homeostasis in Agrp-Cre;Ptpn2fl/fl mice was corrected by IR heterozygosity (Agrp-Cre;Ptpn2fl/fl ;Insrfl/+ ), causally linking the effects on glucose metabolism with the IR signaling in AgRP neurons. Our findings demonstrate that TCPTP controls IR signaling in AgRP neurons to coordinate HGP and brown/beige adipocyte glucose uptake in response to feeding/fasting.

Phenethyl isothiocyanate protects against H2O2-induced insulin resistance in 3T3-L1 adipocytes.

Obesity is associated with systemic oxidative stress and leads to insulin resistance. Phenethyl isothiocyanate (PEITC), a natural dietary isothiocyanate, has been shown to have beneficial effects in improving cellular defense activities against oxidative stress through activation of nuclear factor erythroid-2 related factor 2 (Nrf2) pathway. However, little evidence exists if the antioxidative activity has beneficial effects on glucose metabolism. Here, we tested the preventive potential of PEITC for impaired insulin-induced glucose uptake by oxidative stress in 3T3-L1 adipocytes. Treatment with PEITC increased the expression of antioxidative enzymes regulated by Nrf2 such as γ-glutamylcysteine-synthetase, heme oxygenase 1, NAD(P)H:quinone oxidoreductase 1 and glutathione S-transferase, and reduced oxidative stress induced by H2O2. Furthermore, PEITC restored impaired insulin-stimulated glucose uptake, translocation of glucose transporter 4 and insulin signaling by H2O2. These results indicate that PEITC protected insulin-regulated glucose metabolism impaired by oxidative stress through the antioxidative activity in 3T3-L1 adipocytes.

Marine Peptides as Potential Agents for the Management of Type 2 Diabetes Mellitus-A Prospect.

An increasing prevalence of diabetes is known as a main risk for human health in the last future worldwide. There is limited evidence on the potential management of type 2 diabetes mellitus using bioactive peptides from marine organisms, besides from milk and beans. We summarized here recent advances in our understanding of the regulation of glucose metabolism using bioactive peptides from natural proteins, including regulation of insulin-regulated glucose metabolism, such as protection and reparation of pancreatic β-cells, enhancing glucose-stimulated insulin secretion and influencing the sensitivity of insulin and the signaling pathways, and inhibition of bioactive peptides to dipeptidyl peptidase IV, α-amylase and α-glucosidase activities. The present paper tried to understand the underlying mechanism involved and the structure characteristics of bioactive peptides responsible for its antidiabetic activities to prospect the utilization of rich marine organism proteins.

MicroRNA-593-3p regulates insulin-promoted glucose consumption by targeting Slc38a1 and CLIP3.

Insulin plays an important role in the regulation of glucose metabolism. However, the molecular mechanisms involved are not entirely clarified. In this context, we found that miR-593-3p negatively regulates insulin-regulated glucose metabolism in hepatocellular carcinoma HepG2 and Bel7402 cells. We then identified Slc38a1 and CLIP3 as novel targets of miR-593-3p. Further studies demonstrated that Slc38a1 and CLIP3 mediate insulin-regulated glucose metabolism. Interestingly, we also demonstrated that miR-593-3p expression was negatively associated with Slc38a1 and CLIP3 expression in insulin-treated HepG2 cells, and insulin-induced Slc38a1 and CLIP3 expression via downregulation of miR-593-3p. Taken together, this study indicates that inhibition of miRNA-593-3p by insulin promotes glucose metabolism through the regulation of Slc38a1 and CLIP3 expression, and provides a new insight into the role and mechanism of insulin-induced glycolysis.

Modulation of cellular glucose metabolism in human HepG2 cells by combinations of structurally related flavonoids.

Insulin-regulated glucose metabolism in cells is critical for proper metabolic functioning, and insulin resistance leads to type 2 diabetes. We performed a human study to assess the availability of structurally related dietary flavonols and tested their ability to affect cellular glucose uptake, metabolism, and glucose transporter gene expression in a liver HepG2 cell model. Eight healthy volunteers consumed a meal containing galangin, kaempferol, quercetin, and myricetin. In plasma, myricetin was absent, but the others were present, mostly as conjugates. In HepG2 cells, a combination of galangin, kaempferol, and quercetin (5 μM each) for 12 h increased the acute uptake of [U-(14)C]-glucose and 2-[U-(14)C]-deoxyglucose by almost 100 and ∼10%, respectively. All of the combinations increased glucose metabolism, but the effect on transport was less pronounced and mixed. A mixture of all flavonols significantly increased mRNA expression of the main glucose transporter Glut1 in HepG2 cells. These results for the first time show the presence of galangin conjugates in human plasma, and allow direct comparison between absorption of flavonols. A combination of flavonols has the potential to modulate sugar metabolism, both uptake into cells as evident from effects on deoxyglucose, and also further cellular glucose metabolism.

Effects of inhibitors of key enzymes of sphingolipid metabolism on insulin-induced glucose uptake and glycogen synthesis in liver cells of old rats.

Sphingolipids play an important role in the development of insulin resistance. Ceramides are the most potent inhibitors of insulin signal transduction. Ceramides are generated in response to stress stimuli and in old age. In this work, we studied the possible contribution of different pathways of sphingolipid metabolism in age-dependent insulin resistance development in liver cells. Inhibition of key enzymes of sphingolipid synthesis (serine palmitoyl transferase, ceramide synthase) and degradation (neutral and acidic SMases) by means of specific inhibitors (myriocin, fumonisin B1, imipramine, and GW4869) was followed with the reduction of ceramide level and partly improved insulin regulation of glucose metabolism in "old" hepatocytes. Imipramine and GW4869 decreased significantly the acidic and neutral SMase activities, respectively. Treatment of "old" cells with myriocin or fumonisin B1 reduced the elevated in old age ceramide and SM synthesis. Ceramide and SM levels and glucose metabolism regulation by insulin could be improved with concerted action of all tested inhibitors of sphingolipid turnover on hepatocytes. The data demonstrate that not only newly synthesized ceramide and SM but also neutral and acidic SMase-dependent ceramide accumulation plays an important role in development of age-dependent insulin resistance.

Insulin Increases Phosphorylation of Mitochondrial Proteins in Human Skeletal Musclein Vivo

There is increasing evidence that multiple proteins involved in key regulatory processes in mitochondria are phosphorylated in mammalian tissues. Insulin regulates glucose metabolism by phosphorylation-dependent signaling and has been shown to stimulate ATP synthesis in human skeletal muscle. Here, we investigated the effect of insulin on the phosphorylation of mitochondrial proteins in human skeletal muscle in vivo. Using a combination of TiO(2) phosphopeptide-enrichment, HILIC fractionation, and LC-MS/MS, we compared the phosphoproteomes of isolated mitochondria from skeletal muscle samples obtained from healthy individuals before and after 4 h of insulin infusion. In total, we identified 207 phosphorylation sites in 95 mitochondrial proteins. Of these phosphorylation sites, 45% were identified in both basal and insulin-stimulated samples. Insulin caused a 2-fold increase in the number of different mitochondrial phosphopeptides (87 ± 7 vs 40 ± 7, p = 0.015) and phosphoproteins (46 ± 2 vs 26 ± 3, p = 0.005) identified in each mitochondrial preparation. Almost half of the mitochondrial phosphorylation sites (n = 94) were exclusively identified in the insulin-stimulated state and included the majority of novel sites. Phosphorylation sites detected more often or exclusively in insulin-stimulated samples include multiple sites in mitochondrial proteins involved in oxidative phosphorylation, tricarboxylic acid cycle, and fatty acid metabolism, as well as several components of the newly defined mitochondrial inner membrane organizing system (MINOS). In conclusion, the present study demonstrates that insulin increases the phosphorylation of several mitochondrial proteins in human skeletal muscle in vivo and provides a first step in the understanding of how insulin potentially regulates mitochondrial processes by phosphorylation-dependent mechanisms.

Spatiotemporal Confinement of GLUT4 in Plasma Membrane Domains

Translocation of glucose transporter-4 (GLUT4) to the plasma membrane (PM) is the hallmark of insulin regulation of glucose metabolism. However, GLUT4 translocation to PM proceeds through two distinct exocytotic mechanisms: classical fusion with dispersal and fusion with retention in specialized GLUT4 domains (clusters). In this study we examined the properties of GLUT4 domains and investigated insulin effect on spatiotemporal organization of GLUT4 in PM of live adipose cells by direct microscopic observation of single monomers tagged with photoswitchable fluorescent protein.Using live cell fluorescent Photo-Activation Localization Microscopy (fPALM), we tracked diffusion of GLUT4-EOS in PM, and measured lifetime of GLUT4 molecules trapped inside the domains. While dispersed GLUT4 monomers followed Brownian motion with diffusion coefficient of ∼0.09 μm2/s, GLUT4 associated with domains exhibited constrained diffusion limited by the boundaries of the elongated domains (sized 60-240 nm). We next analyzed the rates of GLUT4 monomer association with and dissociation from the domains, and found that insulin stimulated the latter by ∼3-fold, but did not influence the former. Interestingly, outside the domains GLUT4 monomers appeared to frequently collide, but did not form new domains. Rather, GLUT4 domain formation was observed exclusively upon GLUT4 exocytosis. Consistent with previous findings, insulin increased the probability of fusion with dispersal (bypassing domain formation), and decreased the rate of GLUT4 internalization from domains, thus further augmenting the total amount of GLUT4 in PM and increasing the relative fraction of dispersed GLUT4.All together these three effects of insulin act synergistically to shift the distribution of PM GLUT4 from clustered to dispersed states and concomitantly increase the residency time of GLUT4 monomers at the cell surface. Thus, we propose that spatiotemporal confinement of GLUT4 in PM domains represents a novel kinetic mechanism for insulin regulation of glucose homeostasis.

Maternal protein restriction before pregnancy reduces offspring early body mass and affects glucose metabolism in C57BL/6JBom mice.

Dietary protein restriction in pregnant females reduces offspring birth weight and increases the risk of developing obesity, type 2 diabetes and cardiovascular disease. Despite these grave consequences, few studies have addressed the effects of preconceptional maternal malnutrition. Here we investigate how a preconceptional low-protein (LP) diet affects offspring body mass and insulin-regulated glucose metabolism. Ten-week-old female mice (C57BL/6JBom) received either an LP or isocaloric control diet (8% and 22% crude protein, respectively) for 10 weeks before conception, but were thereafter fed standard laboratory chow (22.5% crude protein) during pregnancy, lactation and offspring growth. When the offspring were 10 weeks old, they were subjected to an intraperitoneal glucose tolerance test (GTT), and sacrificed after a 5-day recovery period to determine visceral organ mass. Body mass of LP male offspring was significantly lower at weaning compared with controls. A similar, nonsignificant, tendency was observed for LP female offspring. These differences in body mass disappeared within 1 week after weaning, a consequence of catch-up growth in LP offspring. GTTs of 10-week-old offspring revealed enhanced insulin sensitivity in LP offspring of both sexes. No differences were found in body mass, food intake or absolute size of visceral organs of adult offspring. Our results indicate that maternal protein restriction imposed before pregnancy produces effects similar to postconceptional malnutrition, namely, low birth weight, catch-up growth and enhanced insulin sensitivity at young adulthood. This could imply an increased risk of offspring developing lifestyle-acquired diseases during adulthood.

Regulation of Glucose Homeostasis by KSR1 and MARK2

Protein scaffolds control the intensity and duration of signaling and dictate the specificity of signaling through MAP kinase pathways. KSR1 is a molecular scaffold of the Raf/MEK/ERK MAP kinase cascade that regulates the intensity and duration of ERK activation. Relative to wild-type mice, ksr1-/- mice are modestly glucose intolerant, but show a normal response to exogenous insulin. However, ksr1-/- mice also demonstrate a three-fold increase in serum insulin levels in response to a glucose challenge, suggesting a role for KSR1 in insulin secretion. The kinase MARK2 is closely related to C-TAK1, a known regulator of KSR1. Mice lacking MARK2 have an increased rate of glucose disposal in response to exogenous insulin, increased glucose tolerance, and are resistant to diet-induced obesity. mark2-/-ksr1-/- (DKO) mice were compared to wild type, mark2-/-, and ksr1-/- mice for their ability to regulate glucose homeostasis. Here we show that disruption of KSR1 in mark2-/- mice reverses the increased sensitivity to exogenous insulin resulting from MARK2 deletion. DKO mice respond to exogenous insulin similarly to wild type and ksr1-/- mice. These data suggest a model whereby MARK2 negatively regulates insulin sensitivity in peripheral tissue through inhibition of KSR1. Consistent with this model, we found that MARK2 binds and phosphorylates KSR1 on Ser392. Phosphorylation of Ser392 is a critical regulator of KSR1 stability, subcellular location, and ERK activation. These data reveal an unexpected role for the molecular scaffold KSR1 in insulin-regulated glucose metabolism.

Insulin Controls the Spatial Distribution of GLUT4 on the Cell Surface through Regulation of Its Postfusion Dispersal

While the glucose transporter-4 (GLUT4) is fundamental to insulin-regulated glucose metabolism, its dynamic spatial organization in the plasma membrane (PM) is unclear. Here, using multicolor TIRF microscopy in transfected adipose cells, we demonstrate that insulin regulates not only the exocytosis of GLUT4 storage vesicles but also PM distribution of GLUT4 itself. In the basal state, domains (clusters) of GLUT4 molecules in PM are created by an exocytosis that retains GLUT4 at the fusion site. Surprisingly, when insulin induces a burst of GLUT4 exocytosis, it does not merely accelerate this basal exocytosis but rather stimulates approximately 60-fold another mode of exocytosis that disperses GLUT4 into PM. In contradistinction, internalization of most GLUT4, regardless of insulin, occurs from pre-existing clusters via the subsequent recruitment of clathrin. The data fit a new kinetic model that features multifunctional clusters as intermediates of exocytosis and endocytosis.