Insulin-stimulated Conditions Research Articles

Coffee Improves Insulin-Stimulated Akt Phosphorylation in Liver and Skeletal Muscle in Diabetic KK-Ay Mice

Coffee has an anti-diabetic effect, specifically the amelioration of both hyperglycemia and insulin resistance, in KK-A(y) mice, a type 2 diabetes animal model. To investigate coffee's effect on insulin signaling in liver, skeletal muscle, and adipose tissue (epididymal fat), we assayed the tyrosine phosphorylation of insulin receptor (IR) and serine phosphorylation of Akt. In Expt. 1, we assayed insulin signaling under nonfasting conditions in KK-A(y) mice that ingested water or coffee for 4 wk. Coffee ingestion ameliorated the development of hyperglycemia but did not affect insulin signaling in liver or skeletal muscle under such conditions. In Expt. 2, we assayed insulin signaling under basal and insulin-stimulated conditions in KK-A(y) mice that ingested water or coffee for 3 wk. The levels of tyrosine phosphorylation of insulin receptor in response to insulin injection in insulin-sensitive tissues were not different between mice that drank water and those that drank coffee. Coffee ingestion significantly increased the insulin-induced serine phosphorylation of Akt in liver and skeletal muscle, but not in epididymal fat, of KK-A(y) mice. Our results also indicated that coffee ingestion may contribute to the improvement of insulin resistance and hyperglycemia in KK-A(y) mice via the activation of Akt in insulin signaling in liver and skeletal muscle.

Improved Insulin Sensitivity by GLUT12 Overexpression in Mice

OBJECTIVEEvidence suggests that insulin-sensitive glucose transporters (GLUTs) other than GLUT4 may exist. To investigate whether GLUT12 may represent another insulin-sensitive GLUT, transgenic (TG) mice that overexpress GLUT12 were characterized.RESEARCH DESIGN AND METHODSTG mice that overexpressed GLUT12 under a β-actin promoter were generated. Glucose metabolism in TG and wild-type control mice was compared using glucose and insulin tolerance tests and hyperinsulinemic-euglycemic clamps. In addition, basal and insulin-stimulated glucose clearance rates into insulin-sensitive peripheral tissues were measured using [3H]-2-deoxy-d-glucose.RESULTSGLUT12 was overexpressed by 40–75% in TG compared with wild-type mice in insulin-sensitive tissues with no change in GLUT4 content. Body weight and fasting blood glucose did not differ between wild-type and TG mice; however, insulin concentrations were reduced in TG mice. Enhanced oral glucose tolerance was noted in TG mice by a reduced blood glucose excursion compared with wild-type mice (P < 0.05). Enhanced insulin sensitivity was noted by a greater decrease in blood glucose in TG mice during insulin tolerance testing. Hyperinsulinemic-euglycemic clamps confirmed enhanced insulin sensitivity in GLUT12-overexpressing mice (P < 0.01). Tissues of TG mice exhibited normal basal glucose clearance rates; however, under insulin-stimulated conditions, glucose clearance was significantly increased (P < 0.01) in tissues of TG mice.CONCLUSIONSIncreased expression of GLUT12 results in improved whole-body insulin sensitivity mediated by an increased glucose clearance rate in insulin-responsive tissues under insulin-stimulated, but not basal, conditions. These findings provide evidence that GLUT12 represents a novel, second insulin-sensitive GLUT.

New Insights into Cytosolic Glucose Levels during Differentiation of 3T3-L1 Fibroblasts into Adipocytes

Cytosolic glucose concentration reflects the balance between glucose entry across the plasma membrane and cytosolic glucose utilization. In adipocytes, glucose utilization is considered very rapid, meaning that every glucose molecule entering the cytoplasm is quickly phosphorylated. Thus, the cytosolic free glucose concentration is considered to be negligible; however, it was never measured directly. In the present study, we monitored cytosolic glucose dynamics in 3T3-L1 fibroblasts and adipocytes by expressing a fluorescence resonance energy transfer (FRET)-based glucose nanosensor: fluorescent indicator protein FLIPglu-600μ. Specifically, we monitored cytosolic glucose responses by varying transmembrane glucose concentration gradient. The changes in cytosolic glucose concentration were detected in only 56% of 3T3-L1 fibroblasts and in 14% of 3T3-L1 adipocytes. In adipocytes, the resting cytosolic glucose concentration was reduced in comparison with the one recorded in fibroblasts. Membrane permeabilization increased cytosolic glucose concentration in adipocytes, and glycolytic inhibitor iodoacetate failed to increase cytosolic glucose concentration, indicating low adipocyte permeability for glucose at rest. We also examined the effects of insulin and adrenaline. Insulin significantly increased cytosolic glucose concentration in adipocytes by a factor of 3.6; however, we recorded no effect on delta ratio (ΔR) in fibroblasts. Adrenaline increased cytosolic glucose concentration in fibroblasts but not in adipocytes. However, in adipocytes in insulin-stimulated conditions, glucose clearance was significantly faster following adrenaline addition in comparison with controls (p < 0.001). Together, these results demonstrate that during differentiation, adipocytes develop more efficient mechanisms for maintaining low cytosolic glucose concentration, predominantly with reduced membrane permeability for glucose.

Stimulation of Human Whole-Body Energy Expenditure by Salsalate Is Fueled by Higher Lipid Oxidation under Fasting Conditions and by Higher Oxidative Glucose Disposal under Insulin-Stimulated Conditions

Nonsteroidal antiinflammatory drugs appear to improve insulin sensitivity and are currently tested in clinical trials. Salsalate, however, may blunt mitochondrial function, an unwarranted side effect for type 2 diabetics. We examined the effect of salsalate on ex vivo mitochondrial function and lipid-induced insulin resistance. In a crossover design, nine volunteers underwent a hyperinsulinemic-euglycemic clamp with simultaneous infusion of glycerol (control), Intralipid, or Intralipid preceded by 4 d of salsalate (4000 mg Disalsid). Oxidative glucose disposal and nonoxidative glucose disposal (NOGD), metabolic flexibility, energy expenditure (EE), and ex vivo muscle mitochondrial function were measured. Lipid infusion reduced insulin-stimulated glucose disposal by approximately 40%, glucose oxidation (CHOox) by approximately 50%, and NOGD by approximately 35%. Lipid-induced whole-body insulin resistance and decreased NOGD were not ameliorated by salsalate. However, salsalate repressed lipid-induced reduction in CHOox and reduced insulin clearance, resulting in higher insulin levels under basal as well as under clamp conditions (∼25 and ∼39%, respectively). Intriguingly, EE was higher after administration of salsalate (∼18 and ∼16% under basal and clamp conditions, respectively) and was fueled by increased fat oxidation in the basal state and increased CHOox upon insulin stimulation. Salsalate did not affect mitochondrial function and coupling. We conclude that salsalate failed to improve whole-body insulin sensitivity but increased basal fat oxidation and insulin-stimulated CHOox, indicating improved metabolic flexibility. The beneficial effects of salsalate on CHOox can be attributed to elevated insulin levels. Mitochondrial respirometry revealed no indications that the changes in substrate selection and EE could be attributed to changes in skeletal muscle mitochondrial capacity or mitochondrial coupling.

Impairment of cardiac insulin signaling in fructose-fed ovariectomized female Wistar rats

Fructose consumption produces deleterious metabolic effects in animal models. The sites of fructose-induced insulin resistance are documented to be the liver, skeletal muscle, and adipose tissue, but effects of fructose-rich diet on cardiac insulin signaling and action were not investigated. In order to study the potential fructose effects on development of cardiac insulin resistance, we analyzed biochemical parameters relevant for insulin action and phosphorylation of insulin signaling molecules, plasma membrane glucose transporter type 4 (GLUT4) content, and phosphorylation of endothelial nitric oxide synthase (eNOS), in ovariectomized female rats on fructose-enriched diet, in basal and insulin-stimulated conditions. Fructose-fed rats (FFR) had increased content of visceral adipose tissue, but not body weight. Food intake was decreased, while fluid and caloric intake were increased in FFR. Additionally, fructose diet increased plasma insulin, blood triglycerides level, and HOMA index. Stimulation of protein kinase B (Akt) signaling pathway by insulin was reduced in rats on fructose-enriched diet, but effect of fructose on extracellular signal-regulated kinase (Erk 1/2) phosphorylation was not observed. Furthermore, insulin-induced GLUT4 presence in plasma membranes of cardiac cells was decreased by fructose diet, as well as insulin stimulation of eNOS phosphorylation at Ser(1177). In summary, these results strongly support our hypothesis that fructose diet-induced changes of plasma lipid profile and insulin sensitivity are accompanied with decrease in cardiac insulin action in ovariectomized female rats.

Increased Levels of Plasma Acylcarnitines in Obesity and Type 2 Diabetes and Identification of a Marker of Glucolipotoxicity

Dysregulation of fatty acid oxidation (FAO) is recognized as important in the pathophysiology of obesity and insulin resistance (IR). However, demonstrating FAO defects in vivo in humans has entailed complex and invasive methodologies. Recently, the identification of genetic blocks in FAO has been vastly simplified by using tandem mass spectrometry (MS/MS) of dried bloodspots to specify acylcarnitine (AcylCN) alterations characteristic for each disorder. This technology has recently been applied to examine FAO alterations in human and animal models of obesity and type 2 diabetes mellitus (T2DM). This study focused on characterizing AcylCN profiles in human plasma from individuals with obesity and T2DM during fasting and insulin-stimulated conditions. Following an overnight fast, plasma was obtained from lean (n = 12), obese nondiabetic (n = 14), and T2DM (n = 10) participants and analyzed for AcylCN using MS/MS. Plasma samples were also obtained at the end of a 4-h insulin-stimulated euglycemic clamp. In obesity and T2DM, long-chain AcylCNs were similarly significantly increased in the fasted state; free-CN levels were also elevated. Additionally, T2DM subjects of comparable BMI had increased short- and medium-chain AcylCNs, both saturated and hydroxy, as well as increased C(4)-dicarboxylcarnitine (C(4)DC-CN) that correlated with an index of poor glycemic control (HbA(1c); r = 0.74; P < 0.0001). Insulin infusion reduced all species of plasma AcylCN but this reduction was blunted in T2DM. Plasma long-chain AcylCN species are increased in obesity and T2DM, suggesting that more fatty acids can enter mitochondria. In T2DM, many shorter species accumulate, suggesting that they have a generalized complex oxidation defect.

AMP-activated Protein Kinase α2 Subunit Is Required for the Preservation of Hepatic Insulin Sensitivity by n-3 Polyunsaturated Fatty Acids

OBJECTIVEThe induction of obesity, dyslipidemia, and insulin resistance by high-fat diet in rodents can be prevented by n-3 long-chain polyunsaturated fatty acids (LC-PUFAs). We tested a hypothesis whether AMP-activated protein kinase (AMPK) has a role in the beneficial effects of n-3 LC-PUFAs.RESEARCH DESIGN AND METHODSMice with a whole-body deletion of the α2 catalytic subunit of AMPK (AMPKα2−/−) and their wild-type littermates were fed on either a low-fat chow, or a corn oil-based high-fat diet (cHF), or a cHF diet with 15% lipids replaced by n-3 LC-PUFA concentrate (cHF+F).RESULTSFeeding a cHF diet induced obesity, dyslipidemia, hepatic steatosis, and whole-body insulin resistance in mice of both genotypes. Although cHF+F feeding increased hepatic AMPKα2 activity, the body weight gain, dyslipidemia, and the accumulation of hepatic triglycerides were prevented by the cHF+F diet to a similar degree in both AMPKα2−/− and wild-type mice in ad libitum-fed state. However, preservation of hepatic insulin sensitivity by n-3 LC-PUFAs required functional AMPKα2 and correlated with the induction of adiponectin and reduction in liver diacylglycerol content. Under hyperinsulinemic-euglycemic conditions, AMPKα2 was essential for preserving low levels of both hepatic and plasma triglycerides, as well as plasma free fatty acids, in response to the n-3 LC-PUFA treatment.CONCLUSIONSOur results show that n-3 LC-PUFAs prevent hepatic insulin resistance in an AMPKα2-dependent manner and support the role of adiponectin and hepatic diacylglycerols in the regulation of insulin sensitivity. AMPKα2 is also essential for hypolipidemic and antisteatotic effects of n-3 LC-PUFA under insulin-stimulated conditions.

Effect of telmisartan on selected adipokines, insulin sensitivity, and substrate utilization during insulin-stimulated conditions in patients with metabolic syndrome and impaired fasting glucose

Telmisartan improves glucose and lipid metabolism in rodents. This study evaluated the effect of telmisartan on insulin sensitivity, substrate utilization, selected plasma adipokines and their expressions in subcutaneous adipose tissue (SAT) in metabolic syndrome. Twelve patients with impaired fasting glucose completed the double-blind, randomized, crossover trial. Patients received telmisartan (160 mg/day) or placebo for 3 weeks and vice versa with a 2-week washout period. At the end of each period, a hyperinsulinemic euglycemic clamp (HEC) combined with indirect calorimetry was performed. During HEC (0, 30, and 120 min), plasma levels of adipokines were measured and a needle biopsy (0 and 30 min) of SAT was performed. Fasting plasma glucose was lower after telmisartan compared with placebo (P<0.05). There were no differences in insulin sensitivity and substrate utilization. We found no differences in basal plasma adiponectin, resistin and tumour necrosis factor α (TNFα), but an increase was found in basal leptin, after telmisartan treatment. Insulin-stimulated plasma adiponectin (P<0.05), leptin and resistin (P<0.001) were increased, whereas TNFα was decreased (P<0.05) after telmisartan treatment. Expression of resistin, but not adiponectin, TNFα and leptin was increased after telmisartan treatment. Despite the decrease in fasting plasma glucose, telmisartan does not improve insulin sensitivity and substrate utilization. Telmisartan increases plasma leptin as well as insulin-stimulated plasma adiponectin, leptin and resistin, and decreases plasma TNFα during HEC. Changes in plasma adipokines cannot be explained by their expressions in SAT. The changes in plasma adipokines might be involved in the metabolic effects of telmisartan in metabolic syndrome.

Disruption of AMPKα1 Signaling Prevents AICAR-Induced Inhibition of AS160/TBC1D4 Phosphorylation and Glucose Uptake in Primary Rat Adipocytes

The aim of this study was to investigate the molecular mechanisms by which AMP-kinase (AMPK) activation inhibits basal and insulin-stimulated glucose uptake in primary adipocytes. Rat epididymal adipocytes were exposed to 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR) for 1 h. Subsequently, basal and insulin-stimulated glucose uptake and the phosphorylation of AMPK, acetyl-CoA carboxylase, Akt, and the Akt substrate of 160 kDa (AS160/TBC1D4) were determined. In order to investigate whether these effects of AICAR were mediated by AMPK activation, these parameters were also assessed in adipocytes either expressing LacZ (control) or a kinase-dead AMPKalpha1 mutant. AICAR increased AMPK activation without affecting basal and insulin-stimulated Akt1/2 phosphorylation on Thr(308) and Ser(473) residues. However, AMPK activation suppressed the phosphorylation of AS160/TBC1D4 and its interaction with the 14-3-3 signal transduction-regulatory protein, which was accompanied by significant reductions in plasma membrane glucose transporter 4 content and glucose uptake under basal and insulin-stimulated conditions. Phosphorylation of Akt substrates glycogen synthase kinase 3alpha and -beta were unaltered by AICAR, indicating that the AMPK-regulatory effects were specific to the AS160/TBC1D4 signaling pathway. Expression of the kinase-dead AMPKalpha1 mutant fully prevented the suppression of AS160/TBC1D4 phosphorylation, plasma membrane glucose transporter 4 content, and the inhibitory effect of AICAR-induced AMPK activation on basal and insulin-stimulated glucose uptake. This study is the first to provide evidence that disruption of AMPKalpha1 signaling prevents the suppressive effects of AMPK activation on AS160/TBC1D4 phosphorylation and glucose uptake, indicating that insulin-signaling steps that are common to white adipose tissue and skeletal muscle regulation of glucose uptake are distinctly affected by AMPK activation.

Erratum to: Trans Fatty Acid‐Induced NF‐κB Activation Does Not Induce Insulin Resistance in Cultured Murine Skeletal Muscle Cells

In error, an incorrect version of Fig. 2a was published. They-axis of Fig. 2a was erroneously labeled, whereas the description in the text contained the correct data. The correct version of Fig. 2a is displayed below. These changes do not impact the interpretation of the data. NF-κB transcriptional activity and insulin sensitivity in skeletal muscle cells after incubation with geometric and positional C18:1 or C18:2 isomers. C2C12 myoblasts stably transfected with the 6κB-TK-luciferase construct were differentiated for 5 days and incubated with 400 μmol/l of the indicated FAs for 24 h (a). Lysates were prepared for assessment of luciferase activity. Values were corrected for protein content and expressed as fold-induction over control. Alternatively, 5 days differentiated L6-GLUT4myc myotubes were incubated with and without the indicated fatty acids (200 μmol/l, 16 h). Nuclear extracts were prepared and assessed for RelA DNA binding activity to a consensus NF-κB oligonucleotide by EMSA (b). Relative DNA binding activity was determined by phosphor-imager analysis and expressed as fold induction. Furthermore, 2-deoxyglucose uptake (c) and GLUT4 translocation (d) were measured after stimulation with 25 nmol/l insulin for 15 min in 5 days differentiated L6-GLUT4myc myotubes. Data are expressed as percentage of insulin-stimulated glucose uptake or GLUT4 translocation in absence of FA. Basal and insulin-stimulated conditions are represented by the white and black bars, respectively. *p < 0.05 versus control. #p < 0.05 versus insulin-stimulated control. Data shown are representative examples of three independent experiments

Effects of periodontitis on aortic insulin resistance in an obese rat model

The combination of obesity and its associated risk factors, such as insulin resistance and inflammation, results in the development of atherosclerosis. However, the effects of periodontitis on atherosclerosis in an obese body remain unclear. The aim of the study was to investigate the effects of ligature-induced periodontitis in Zucker fatty rats on initiation of atherosclerosis by evaluating aortic insulin resistance. Zucker fatty rats (n=24) were divided into two groups. In the periodontitis group, periodontitis was ligature-induced for 4 weeks, whereas the control group was left unligated. After the 4-week experimental period, descending aorta was used for measuring the levels of lipid deposits, immunohistochemical analysis, and evaluation of gene expression. Levels of serum C-reactive protein (CRP), tumor necrosis factor-α (TNF-α), and insulin were also measured. Rats in the periodontitis group had significantly enhanced lipid deposits in the aorta, but not in the control group. Expression of suppressor of cytokine signaling 3, vascular cell adhesion molecule 1, reactive oxygen species, nitrotyrosine, and endothelin-1 in the periodontitis group was more intense than that in the control group. Significantly decreased levels of phosphatidylinositol 3-kinase (Pi3k) catalytic β-polypeptide (Pi3kcb), Pi3kp85, and insulin receptor substrate 1 and 2 were observed in the periodontitis group. Levels of serum CRP and TNF-α were significantly increased in the periodontitis group. Under insulin-stimulated conditions, aorta in the periodontitis group altered the Akt phosphorylation. Periodontitis in obesity induced the initial stage of atherosclerosis and disturbed aortic insulin signaling.

The Purinergic P2Y1 Receptor Supports Leptin Secretion in Adipose Tissue

Extracellular nucleotides have been shown to trigger intracellular calcium release and influence leptin secretion in differentiated white and brown adipocytes through activation of various but not clearly identified P2 receptors. In the present study, we wished to assess whether or not the P2Y1 ADP receptor is functional in white adipocytes and whether it could affect the secretion of adipocyte-derived hormones. Stromal cells and mature adipocytes were isolated from epididymal adipose tissue from wild-type and P2Y1 knockout (KO) C57-black/six male mice. The expression of the P2Y1 receptor in adipocytes was confirmed by RT-PCR and intracellular calcium measurements with fura 2-AM. KO of P2Y1 receptors did not affect the cell size and lipid content of mature adipocytes or the differentiation of the stromal cell fraction, but the leptin production of mature adipocytes was decreased under basal and insulin-stimulated conditions. A selective P2Y1 antagonist, MRS2500, reduced leptin release in isolated adipocytes. The plasma and adipose tissue mRNA levels of leptin were also lower in P2Y1 KO mice as compared with wild-type animals. However, in mice fed a high-fat diet, the plasma leptin levels were greatly enhanced and the inhibitory effect of P2Y1 KO was not observed. These results show that the P2Y1 receptor supports leptin production in isolated white adipocytes through a transcriptional mechanism. This function of the receptor may regulate plasma leptin in lean mice but is overcome in obese animals.

High basal cell surface levels of fish GLUT4 are related to reduced sensitivity of insulin-induced translocation toward GGA and AS160 inhibition in adipocytes

Glucose entry into cells is mediated by a family of facilitative transporter proteins (GLUTs). In mammals, GLUT4 is expressed in insulin-sensitive tissues and is responsible for the postprandial uptake of glucose. In fish, GLUT4 also mediates insulin-regulated glucose entry into cells but differs from mammalian GLUT4 in its affinity for glucose and in protein motifs known to be important for the traffic of GLUT4. In this study, we have characterized the intracellular and plasma membrane (PM) traffic of two orthologs of GLUT4 in fish, trout (btGLUT4) and salmon (okGLUT4), that do not share the amino terminal FQQI targeting motif of mammalian GLUT4. btGLUT4 (FQHL) and, to a lesser extent, okGLUT4 (FQQL) showed higher basal PM levels, faster traffic to the PM after biosynthesis, and earlier acquisition of insulin responsiveness than rat GLUT4. Furthermore, btGLUT4 showed a similar profile of internalization than rat GLUT4. Expression of the dominant-interfering AS160-4P mutant caused a significant decrease in the insulin-induced PM levels of okGLUT4 and rat GLUT4 and, to a lesser extent, of btGLUT4, suggesting that btGLUT4 has reduced retention into the IRC. Contrary to rat GLUT4 and okGLUT4, the presence of btGLUT4 at the PM under insulin-stimulated conditions was not affected by coexpression of a dominant-interfering GGA mutant. These data suggest that fish GLUT4 follow a different trafficking pathway to the PM compared with rat GLUT4 that seems to be relatively independent of GGA. These results indicate that the regulated trafficking characteristics of GLUT4 have been modified during evolution from fish to mammals.

Insulin Induces REDD1 Expression through Hypoxia-inducible Factor 1 Activation in Adipocytes

REDD1 (regulated in development and DNA damage responses) is essential for the inhibition of mTORC1 (mammalian target of rapamycin complex) signaling pathway in response to hypoxia. REDD1 expression is regulated by many stresses such as hypoxia, oxidative stress, and energy depletion. However, the regulation of REDD1 expression in response to insulin remains unknown. In the present study, we demonstrate that in murine and in human adipocytes, insulin stimulates REDD1 expression. Insulin-induced REDD1 expression occurs through phosphoinositide 3-kinase/mTOR-dependent pathways. Moreover, using echinomycin, a hypoxia-inducible factor 1 (HIF-1) inhibitor, and HIF-1alpha small interfering RNA, we demonstrate that insulin stimulates REDD1 expression only through the transcription factor HIF-1. In conclusion, our study shows that insulin stimulates REDD1 expression in adipocytes.

Kinetic Evidence for Unique Regulation of GLUT4 Trafficking by Insulin and AMP-activated Protein Kinase Activators in L6 Myotubes

In L6 myotubes, redistribution of a hemagglutinin (HA) epitope-tagged GLUT4 (HA-GLUT4) to the cell surface occurs rapidly in response to insulin stimulation and AMP-activated protein kinase (AMPK) activation. We have examined whether these separate signaling pathways have a convergent mechanism that leads to GLUT4 mobilization and to changes in GLUT4 recycling. HA antibody uptake on GLUT4 in the basal steady state reached a final equilibrium level that was only 81% of the insulin-stimulated level. AMPK activators (5-aminoimidazole-4-carboxyamide ribonucleoside (AICAR) and A-769662) led to a similar level of antibody uptake to that found in insulin-stimulated cells. However, the combined responses to insulin stimulation and AMPK activation led to an antibody uptake level of ∼20% above the insulin level. Increases in antibody uptake due to insulin, but not AICAR or A-769662, treatment were reduced by both wortmannin and Akt inhibitor. The GLUT4 internalization rate constant in the basal steady state was very rapid (0.43 min−1) and was decreased during the steady-state responses to insulin (0.18 min−1), AICAR (0.16 min−1), and A-769662 (0.24 min−1). This study has revealed a nonconvergent mobilization of GLUT4 in response to activation of Akt and AMPK signaling. Furthermore, GLUT4 trafficking in L6 muscle cells is very reliant on regulated endocytosis for control of cell surface GLUT4 levels.

Erythrocyte Dematin Regulates Glucose Transport Via Akt Phosphorylation and 14-3-3zeta Association.

Abstract 1990Poster Board I-1012Erythrocyte dematin is a widely expressed actin-binding and bundling protein, and functions as a suppressor of RhoA signaling in fibroblasts (Mohseni and Chishti, Molecular Cell Biology 28: 4712-4718, 2008). Dematin is a substrate of multiple protein kinases, and its actin bundling activity is regulated by cAMP dependent protein kinase. Recently, we identified a novel interaction between dematin and glucose transporter-1 (GLUT1) that is critically important for erythrocyte shape and membrane mechanical properties (Khan et al., Journal of Biological Chemistry 283:14600-14609, 2008). Since homologues of dematin and GLUT1 exist in many non-erythroid cells, we proposed that a conserved mechanism might couple related sugar transporters, such as the insulin-responsive glucose transporter-4 (GLUT4), to the actin cytoskeleton via dematin. Immunocytochemistry established the presence of dematin in 3T3-L1 adipocytes, and a small pool of dematin and GLUT4-containing vesicles co-localized in 3T3-L1 cells under both basal and insulin-stimulated conditions. Plasma membrane sheet assays indicate that upon insulin stimulation, dematin translocates to the plasma membrane along with GLUT4, resulting in partial co-localization at the plasma membrane. Furthermore, dematin RNAi treated 3T3-L1 cells show reduced GLUT4 protein expression, suggesting that dematin may regulate a sub-population of GLUT4 via the lysosomal degradation pathway in adipocytes. Importantly, glucose transport was reduced by ∼28% in 3T3-L1 adipocytes depleted of dematin, and by ∼15% in the dematin headpiece knockout (HPKO) mouse primary adipocytes. Since a significant amount of dematin did not co-localize with GLUT4 in the cytosol and plasma membrane, biochemical interaction between dematin and GLUT4 could not be verified using immunoprecipitation and transfection assays. Although dematin does not bind directly to GLUT4 under these conditions, a possibility existed that this interaction may be transient and mediated through an adaptor protein. Interestingly, dematin contains seven 14-3-3 binding sites, and 14-3-3 adaptor has been shown to be functionally involved in GLUT4 trafficking. We demonstrate that phosphorylated dematin binds to 14-3-3 in 3T3-L1 adipocytes under both basal and insulin stimulated conditions. Mutagenesis studies identify serine-85 on dematin as the primary phospho-binding site for 14-3-3zeta. Furthermore, using pharmacological inhibitors, Akt is identified as the likely protein kinase that phosphorylates dematin to mediate the biochemical interactions between dematin and 14-3-3zeta. Together, our results identify erythrocyte dematin as a potential regulator of glucose transporter trafficking and degradation pathways in adipocytes with functional implications for glucose homeostasis, diabetes, and obesity. Disclosures:No relevant conflicts of interest to declare.

Insulin sensitivity, metabolic flexibility, and serum adiponectin concentration in women with anorexia nervosa

Anorexia nervosa (AN) is an eating disorder resulting in sustained low weight and marked decrease in fat mass. The lack of adipose tissue observed in lipodystrophies is accompanied by insulin resistance. It remains unclear if the same phenomenon would be present in AN. The objective of the study was to estimate insulin sensitivity, oxidative and nonoxidative glucose metabolism in insulin-stimulated conditions, metabolic flexibility, and serum adiponectin concentration in women with AN. We examined 21 women with AN and 24 healthy normal-weight female controls. Euglycemic hyperinsulinemic clamp, indirect calorimetry, and the measurement of serum adiponectin concentration were performed in all the subjects. We did not observe differences in insulin sensitivity, oxidative and nonoxidative glucose metabolism in insulin-stimulated conditions, and metabolic flexibility between AN and control subjects. Serum adiponectin was higher in AN women in comparison with control group ( P = .002). Women with AN have normal insulin sensitivity because of the preserved response of glucose oxidation, nonoxidative glucose metabolism in response to insulin, and normal metabolic flexibility. High adiponectin concentration and normal insulin sensitivity in anorectic women suggest that in AN the adipocytes are still capable of functioning at the level that is sufficient to prevent the metabolic consequences.

Thioredoxin-independent Regulation of Metabolism by the α-Arrestin Proteins

Thioredoxin-interacting protein (Txnip), originally characterized as an inhibitor of thioredoxin, is now known to be a critical regulator of glucose metabolism in vivo. Txnip is a member of the alpha-arrestin protein family; the alpha-arrestins are related to the classical beta-arrestins and visual arrestins. Txnip is the only alpha-arrestin known to bind thioredoxin, and it is not known whether the metabolic effects of Txnip are related to its ability to bind thioredoxin or related to conserved alpha-arrestin function. Here we show that wild type Txnip and Txnip C247S, a Txnip mutant that does not bind thioredoxin in vitro, both inhibit glucose uptake in mature adipocytes and in primary skin fibroblasts. Furthermore, we show that Txnip C247S does not bind thioredoxin in cells, using thiol alkylation to trap the Txnip-thioredoxin complex. Because Txnip function was independent of thioredoxin binding, we tested whether inhibition of glucose uptake was conserved in the related alpha-arrestins Arrdc4 and Arrdc3. Both Txnip and Arrdc4 inhibited glucose uptake and lactate output, while Arrdc3 had no effect. Structure-function analysis indicated that Txnip and Arrdc4 inhibit glucose uptake independent of the C-terminal WW-domain binding motifs, recently identified as important in yeast alpha-arrestins. Instead, regulation of glucose uptake was intrinsic to the arrestin domains themselves. These data demonstrate that Txnip regulates cellular metabolism independent of its binding to thioredoxin and reveal the arrestin domains as crucial structural elements in metabolic functions of alpha-arrestin proteins.

Forkhead transcription factor FoxO1 inhibits insulin- and transforming growth factor-β-stimulated plasminogen activator inhibitor-1 expression

Elevated levels of plasminogen activator inhibitor-1 (PAI-1) are considered a risk factor for chronic liver disease in patients with hyperinsulinemia. Insulin increases the expression of PAI-1, and inactivates the forkhead box-containing protein FoxO1. We were interested in whether the inactivation of FoxO1 is involved in the activation of PAI-1 expression under conditions of insulin stimulation. Here, we examined whether adenoviral-mediated expression of a constitutively active form of FoxO1 (Ad-CA-FoxO1) inhibited insulin-stimulated PAI-1 expression in human HepG2 hepatocellular liver carcinoma cells and mouse AML12 hepatocytes. Treatment of cells with insulin increased PAI-1 gene expression, and this effect was abolished by Ad-CA-FoxO1. Insulin also increased the transforming growth factor (TGF)-β-induced expression of PAI-1 mRNA, and Ad-CA-FoxO1 inhibited this effect. Transient transfection assays using a reporter gene under the control of the PAI-1 promoter revealed that CA-FoxO1 inhibits Smad3-stimulated PAI-1 promoter activity. Taken together, our results indicate that FoxO1 inhibits PAI-1 expression through the inhibition of TGF-β/Smad-mediated signaling pathways. Our data also suggest that in the hyperinsulinemic state, FoxO1 is inactivated by increased levels of insulin, and does not function as an inhibitor of TGF-β-induced PAI-1 expression.

APPL1 Potentiates Insulin-Mediated Inhibition of Hepatic Glucose Production and Alleviates Diabetes via Akt Activation in Mice

Hepatic insulin resistance is the major contributor to fasting hyperglycemia in type 2 diabetes. Here we report that the endosomal adaptor protein APPL1 increases hepatic insulin sensitivity by potentiating insulin-mediated suppression of the gluconeogenic program. Insulin-stimulated activation of Akt and suppression of gluconeogenesis in hepatocytes are enhanced by APPL1 overexpression, but are attenuated by APPL1 knockdown. APPL1 interacts with Akt and blocks the association of Akt with its endogenous inhibitor tribble 3 (TRB3) through direct competition, thereby promoting Akt translocation to the plasma membrane and the endosomes for further activation. In db/db diabetic mice, the blockage of the augmented interaction between Akt and TRB3 by hepatic overexpression of APPL1 is accompanied by a marked attenuation of hyperglycemia and insulin resistance. These results suggest that the potentiating effects of APPL1 on insulin-stimulated suppression of hepatic glucose production are attributed to its ability in counteracting the inhibition of Akt activation by TRB3.