Counteracting neuronal nitric oxide synthase proteasomal degradation improves glucose transport in insulin-resistant skeletal muscle from Zucker fa/fa rats

Aims/hypothesis

Insulin-mediated glucose transport and utilisation are decreased in skeletal muscle from type 2 diabetic and glucose-intolerant individuals because of alterations in insulin receptor signalling, GLUT4 translocation to the plasma membrane and microvascular blood flow. Catalytic activity of the muscle-specific isoform of neuronal nitric oxide synthase (nNOS) also participates in the regulation of glucose transport and appears to be decreased in a relevant animal model of drastic insulin resistance, the obese Zucker fa/fa rat. Our objective was to determine the molecular mechanisms involved in this defect.

Methods

Isolated rat muscles and primary cultures of myocytes were used for western blot analysis of protein expression, immunohistochemistry, glucose uptake measurements and GLUT4 translocation assays.

Results

nNOS expression was reduced in skeletal muscle from fa/fa rats. This was caused by increased ubiquitination of the enzyme and subsequent degradation by the ubiquitin proteasome pathway. The degradation occurred through a greater interaction of nNOS with the chaperone heat-shock protein 70 and the co-chaperone, carboxyl terminus of Hsc70-interacting protein (CHIP). In addition, an alteration in nNOS sarcolemmal localisation was observed. We confirmed the implication of nNOS breakdown in defective insulin-induced glucose transport by demonstrating that blockade of proteasomal degradation or overexpression of nNOS improved basal and/or insulin-stimulated glucose uptake and GLUT4 translocation in primary cultures of insulin-resistant myocytes.

Conclusions/interpretation

Recovery of nNOS in insulin-resistant muscles should be considered a potential new approach to address insulin resistance.     

Evidence coupling increased hexosamine biosynthesis pathway activity to membrane cholesterol toxicity and cortical filamentous actin derangement contributing to cellular insulin resistance

Hyperinsulinemia is known to promote the progression/worsening of insulin resistance. Evidence reveals a hidden cost of hyperinsulinemia on plasma membrane (PM) phosphatidylinositol 4,5-bisphosphate (PIP(2))-regulated filamentous actin (F-actin) structure, components critical to the normal operation of the insulin-regulated glucose transport system. Here we delineated whether increased glucose flux through the hexosamine biosynthesis pathway (HBP) causes PIP(2)/F-actin dysregulation and subsequent insulin resistance. Increased glycosylation events were detected in 3T3-L1 adipocytes cultured under conditions closely resembling physiological hyperinsulinemia (5 nm insulin; 12 h) and in cells in which HBP activity was amplified by 2 mm glucosamine (GlcN). Both the physiological hyperinsulinemia and experimental GlcN challenge induced comparable losses of PIP(2) and F-actin. In addition to protecting against the insulin-induced membrane/cytoskeletal abnormality and insulin-resistant state, exogenous PIP(2) corrected the GlcN-induced insult on these parameters. Moreover, in accordance with HBP flux directly weakening PIP(2)/F-actin structure, pharmacological inhibition of the rate-limiting HBP enzyme [glutamine-fructose-6-phosphate amidotransferase (GFAT)] restored PIP(2)-regulated F-actin structure and insulin responsiveness. Conversely, overexpression of GFAT was associated with a loss of detectable PM PIP(2) and insulin sensitivity. Even less invasive challenges with glucose, in the absence of insulin, also led to PIP(2)/F-actin dysregulation. Mechanistically we found that increased HBP activity increased PM cholesterol, the removal of which normalized PIP(2)/F-actin levels. Accordingly, these data suggest that glucose transporter-4 functionality, dependent on PIP(2) and/or F-actin status, can be critically compromised by inappropriate HBP activity. Furthermore, these data are consistent with the PM cholesterol accrual/toxicity as a mechanistic basis of the HBP-induced defects in PIP(2)/F-actin structure and impaired glucose transporter-4 regulation.     

Ras-related C3 botulinum toxin substrate 1 (RAC1) regulates glucose-stimulated insulin secretion via modulation of F-actin

Aims/hypothesis

The small G-protein ras-related C3 botulinum toxin substrate 1 (RAC1) plays various roles in mammalian cells, such as in the regulation of cytoskeletal organisation, cell adhesion, migration and morphological changes. The present study examines the effects of RAC1 ablation on pancreatic beta cell function.

Methods

Isolated islets from pancreatic beta cell-specific Rac1-knockout (betaRac1 ^?/?) mice and RAC1 knockdown INS-1 insulinoma cells treated with small interfering RNA were used to investigate insulin secretion and cytoskeletal organisation in pancreatic beta cells.

Results

BetaRac1 ^?/? mice showed decreased glucose-stimulated insulin secretion, while there were no apparent differences in islet morphology. Isolated islets from the mice had blunted insulin secretion in response to high glucose levels. In RAC1 knockdown INS-1 cells, insulin secretion was also decreased in response to high glucose levels, consistent with the phenotype of betaRac1 ^?/? mice. Even under high glucose levels, RAC1 knockdown INS-1 cells remained intact with F-actin, which inhibits the recruitment of the insulin granules, resulting in an inhibition of insulin secretion.

Conclusions/interpretation

In RAC1-deficient pancreatic beta cells, F-actin acts as a barrier for insulin granules and reduces glucose-stimulated insulin secretion.     

Risk for obstructive sleep apnea in obese, nondiabetic adults varies with insulin resistance status

Purpose

Obstructive sleep apnea (OSA) is an increasingly common sleep disorder, especially among obese adults. Early identification of adults at risk for OSA would be of substantial benefit; however, the magnitude of the obesity epidemic requires that screening be performed judiciously. The study’s aim was to utilize questionnaires that assess OSA risk and symptoms to test the hypothesis that the most insulin-resistant subset of obese individuals is at highest risk for OSA.

Methods

Nondiabetic, overweight to obese volunteers underwent direct quantification of insulin sensitivity by measuring steady-state plasma glucose concentrations during the insulin suppression test. Insulin-sensitive and insulin-resistant individuals were administered the Berlin and STOP questionnaires to determine OSA risk status, and Epworth Sleepiness Scale (ESS) to evaluate daytime sleepiness. Fasting insulin and lipid/lipoprotein measurements were performed.

Results

Insulin-mediated glucose disposal differed threefold (p?<?0.001) between equally obese, insulin-resistant (n?=?22) and insulin-sensitive (n?=?14) individuals, associated with higher fasting insulin and triglyceride and lower high-density lipoprotein cholesterol (HDL-C) concentrations in insulin-resistant individuals. Fourteen (64 %) insulin-resistant as compared with 2 (14 %) insulin-sensitive individuals were found to be at high risk for OSA by both questionnaires (p?<?0.01). Whereas half of insulin-resistant individuals met the ESS criteria for excessive daytime sleepiness, only one insulin-sensitive individual did (p?=?0.011).

Conclusions

High risk for OSA and excessive daytime sleepiness is prevalent among the insulin-resistant subgroup of obese individuals. Surrogate estimates of insulin resistance based on fasting insulin, triglycerides, and/or HDL-C can be used to help identify those obese adults who would benefit most from OSA screening and referral for polysomnography.     

AMPK enhances insulin-stimulated GLUT4 regulation via lowering membrane cholesterol

AMP-activated protein kinase (AMPK) enhances glucose transporter GLUT4 regulation. AMPK also suppresses energy-consuming pathways such as cholesterol synthesis. Interestingly, recent in vitro and in vivo data suggest that excess membrane cholesterol impairs GLUT4 regulation. Therefore, this study tested whether a beneficial, GLUT4-regulatory aspect of AMPK stimulation involved cholesterol lowering. Using L6 myotubes stably expressing an exofacial myc-epitope-tagged-GLUT4, AMPK stimulation by 5-aminoimidazole-4-carboxamide-1-β-d-ribonucleoside (AICAR; 45 min, 1 mm) or 2,4-dinitrophenol (DNP; 30 min, 200 μm) increased cell surface GLUT4myc labeling by approximately ≈ 25% (P < 0.05). Insulin (20 min, 100 nm) also increased GLUT4myc labeling by about 50% (P < 0.05), which was further enhanced (≈ 25%, P < 0.05) by AICAR or DNP. Consistent with AMPK-mediated suppression of cholesterol synthesis, AICAR and DNP decreased membrane cholesterol by 20-25% (P < 0.05). Whereas AMPK knockdown prevented the enhanced basal and insulin-stimulated GLUT4myc labeling by AICAR and DNP, cholesterol replenishment only blocked the AMPK-associated enhancement in insulin action. Cells cultured in a hyperinsulinemic milieu, resembling conditions in vivo that promote the progression/worsening of insulin resistance, displayed an increase in membrane cholesterol. This occurred concomitantly with a loss of cortical filamentous actin (F-actin) and defects in GLUT4 regulation by insulin. These derangements were prevented by AMPK stimulation. Examination of skeletal muscle from insulin-resistant Zucker rats revealed a similar elevation in membrane cholesterol and loss of F-actin. Lowering cholesterol to control levels restored F-actin structure and insulin sensitivity. In conclusion, these data suggest a novel aspect of GLUT4 regulation by AMPK involves membrane cholesterol lowering. Moreover, this AMPK-mediated process protected against hyperinsulinemia-induced insulin resistance.     

Altered amyloid precursor protein processing regulates glucose uptake and oxidation in cultured rodent myotubes

Aims/hypothesis

Impaired glucose uptake in skeletal muscle is an important contributor to glucose intolerance in type 2 diabetes. The aspartate protease, beta-site APP-cleaving enzyme 1 (BACE1), a critical regulator of amyloid precursor protein (APP) processing, modulates in vivo glucose disposal and insulin sensitivity in mice. Insulin-independent pathways to stimulate glucose uptake and GLUT4 translocation may offer alternative therapeutic avenues for the treatment of diabetes. We therefore addressed whether BACE1 activity, via APP processing, in skeletal muscle modifies glucose uptake and oxidation independently of insulin.

Methods

Skeletal muscle cell lines were used to investigate the effects of BACE1 and α-secretase inhibition and BACE1 and APP overexpression on glucose uptake, GLUT4 cell surface translocation, glucose oxidation and cellular respiration.

Results

In the absence of insulin, reduction of BACE1 activity increased glucose uptake and oxidation, GLUT4myc cell surface translocation, and basal rate of oxygen consumption. In contrast, overexpressing BACE1 in C₂C₁₂ myotubes decreased glucose uptake, glucose oxidation and oxygen consumption rate. APP overexpression increased and α-secretase inhibition decreased glucose uptake in C₂C₁₂ myotubes. The increase in glucose uptake elicited by BACE1 inhibition is dependent on phosphoinositide 3-kinase (PI3K) and mimicked by soluble APPα (sAPPα).

Conclusions/interpretation

Inhibition of muscle BACE1 activity increases insulin-independent, PI3K-dependent glucose uptake and cell surface translocation of GLUT4. As APP overexpression raises basal glucose uptake, and direct application of sAPPα increases PI3K–protein kinase B signalling and glucose uptake in myotubes, we suggest that α-secretase-dependent shedding of sAPPα regulates insulin-independent glucose uptake in skeletal muscle.     

Oleate prevents saturated-fatty-acid-induced ER stress, inflammation and insulin resistance in skeletal muscle cells through an AMPK-dependent mechanism

Aims/hypothesis

Although the substitution of saturated fatty acids with oleate has been recommended in the management of type 2 diabetes mellitus, the mechanisms by which oleate improves insulin resistance in skeletal muscle cells are not completely known. Here, we examined whether oleate, through activation of AMP-activated protein kinase (AMPK), prevented palmitate-induced endoplasmic reticulum (ER) stress, which is involved in the link between lipid-induced inflammation and insulin resistance.

Methods

Studies were conducted in mouse C2C12 myotubes and in the human myogenic cell line LHCN-M2. To analyse the involvement of AMPK, activators and inhibitors of this kinase and overexpression of a dominant negative AMPK construct (K45R) were used.

Results

Palmitate increased the levels of ER stress markers, whereas oleate did not. In palmitate-exposed cells incubated with a lower concentration of oleate, the effects of palmitate were prevented. The induction of ER stress markers by palmitate was prevented by the presence of the AMPK activators AICAR and A-769662. Moreover, the ability of oleate to prevent palmitate-induced ER stress and inflammation (nuclear factor-kappa B [NF-κB] DNA-binding activity and expression and secretion of IL6) as well as insulin-stimulated Akt phosphorylation and 2-deoxyglucose uptake was reversed in the presence of the AMPK inhibitor compound C or by overexpression of a dominant negative AMPK construct. Finally, palmitate reduced phospho-AMPK levels, whereas this was not observed in oleate-exposed cells or in palmitate-exposed cells supplemented with oleate.

Conclusions/interpretation

Overall, these findings indicate that oleate prevents ER stress, inflammation and insulin resistance in palmitate-exposed skeletal muscle cells by activating AMPK.     

DOC2 isoforms play dual roles in insulin secretion and insulin-stimulated glucose uptake

Aims/hypothesis

Glucose-stimulated insulin secretion (GSIS) and insulin-stimulated glucose uptake are processes that rely on regulated intracellular vesicle transport and vesicle fusion with the plasma membrane. DOC2A and DOC2B are calcium-sensitive proteins that were identified as key components of vesicle exocytosis in neurons. Our aim was to investigate the role of DOC2 isoforms in glucose homeostasis, insulin secretion and insulin action.

Methods

DOC2 expression was measured by RT-PCR and western blotting. Body weight, glucose tolerance, insulin action and GSIS were assessed in wild-type (WT), Doc2a ^?/? (Doc2aKO), Doc2b ^?/? (Doc2bKO) and Doc2a ^?/?/Doc2b ^?/? (Doc2a/Doc2bKO) mice in vivo. In vitro GSIS and glucose uptake were assessed in isolated tissues, and exocytotic proteins measured by western blotting. GLUT4 translocation was assessed by epifluorescence microscopy.

Results

Doc2b mRNA was detected in all tissues tested, whereas Doc2a was only detected in islets and the brain. Doc2aKO and Doc2bKO mice had minor glucose intolerance, while Doc2a/Doc2bKO mice showed pronounced glucose intolerance. GSIS was markedly impaired in Doc2a/Doc2bKO mice in vivo, and in isolated Doc2a/Doc2bKO islets in vitro. In contrast, Doc2bKO mice had only subtle defects in insulin secretion in vivo. Insulin action was impaired to a similar degree in both Doc2bKO and Doc2a/Doc2bKO mice. In vitro insulin-stimulated glucose transport and GLUT4 vesicle fusion were defective in adipocytes derived from Doc2bKO mice. Surprisingly, insulin action was not altered in muscle isolated from DOC2-null mice.

Conclusions/interpretation

Our study identifies a critical role for DOC2B in insulin-stimulated glucose uptake in adipocytes, and for the synergistic regulation of GSIS by DOC2A and DOC2B in beta cells.     

Knockdown of PRAS40 inhibits insulin action via proteasome-mediated degradation of IRS1 in primary human skeletal muscle cells

Aims/hypothesis

The proline-rich Akt substrate of 40 kDa (PRAS40) is a component of the mammalian target of rapamycin complex 1 (mTORC1) and among the most prominent Akt substrates in skeletal muscle. Yet the cellular functions of PRAS40 are incompletely defined. This study assessed the function of PRAS40 in insulin action in primary human skeletal muscle cells (hSkMC).

Methods

Insulin action was examined in hSkMC following small interfering RNA-mediated silencing of PRAS40 (also known as AKT1S1) under normal conditions and following chemokine-induced insulin resistance.

Results

PRAS40 knockdown (PRAS40-KD) in hSkMC decreased insulin-mediated phosphorylation of Akt by 50% (p?<?0.05) as well as of the Akt substrates glycogen synthase kinase 3 (40%) and tuberous sclerosis complex 2 (32%) (both p?<?0.05). Furthermore, insulin-stimulated glucose uptake was reduced by 20% in PRAS40-KD myotubes (p?<?0.05). Exposing PRAS40-KD myotubes to chemokines caused no additional deterioration of insulin action. PRAS40-KD further reduced insulin-mediated phosphorylation of the mTORC1-regulated proteins p70S6 kinase (p70S6K) (47%), S6 (43%), and eukaryotic elongation 4E-binding protein 1 (100%), as well as protein levels of growth factor receptor bound protein 10 (35%) (all p?<?0.05). The inhibition of insulin action in PRAS40-KD myotubes was associated with a reduction in IRS1 protein levels (60%) (p?<?0.05), and was reversed by pharmacological proteasome inhibition. Accordingly, expression of the genes encoding E3-ligases F-box protein 32 (also known as atrogin-1) and muscle RING-finger protein-1 and activity of the proteasome was elevated in PRAS40-KD myotubes.

Conclusions/interpretation

Inhibition of insulin action in PRAS40-KD myotubes was found to associate with IRS1 degradation promoted by increased proteasome activity rather than hyperactivation of the p70S6K-negative-feedback loop. These findings identify PRAS40 as a modulator of insulin action.     

Increased neuronal nitric oxide synthase dimerisation is involved in rat and human pancreatic beta cell hyperactivity in obesity

Aims/hypothesis

Pancreatic beta cell hyperactivity is known to occur in obesity, particularly in insulin-resistant states. Our aim was to investigate whether changes in neuronal nitric oxide synthase (nNOS) function affect beta cell compensation in two relevant models: the Zucker fa/fa rats and pancreatic islets from obese humans.

Methods

Glucose-induced insulin response was evaluated in the isolated perfused rat pancreas and in human pancreatic islets from obese individuals. Expression of nNOS (also known as NOS1) and subcellular localisation of nNOS were studied by quantitative RT-PCR, immunoblotting, immunofluorescence and electron microscopy.

Results

Pancreatic beta cells from Zucker fa/fa rats and obese individuals were found to be hyper-responsive to glucose. Pharmacological blockade of nNOS was unable to modify beta cell response to glucose in fa/fa rats and in islets from obese individuals, suggesting an abnormal control of insulin secretion by the enzyme. In both cases, nNOS activity in islet cell extracts remained unchanged, despite a drastic increase in nNOS protein and an enhancement in the dimer/monomer ratio, pointing to the presence of high amounts of catalytically inactive enzyme. This relative decrease in activity could be mainly related to increases in islet asymmetric dimethyl-arginine content, an endogenous inhibitor of nNOS activity. In addition, mitochondrial nNOS level was decreased, which contrasts with a strongly increased association with insulin granules.

Conclusions/interpretation

Increased nNOS production and dimerisation, together with a relative decrease in catalytic activity and relocalisation, are involved in beta cell hyperactivity in insulin-resistant rats but also in human islets isolated from obese individuals.     

Endoplasmic reticulum stress upregulates protein tyrosine phosphatase 1B and impairs glucose uptake in cultured myotubes

Aims/hypothesis

Endoplasmic reticulum (ER) stress has been recognised as a common pathway in the development of obesity-associated insulin resistance. Protein tyrosine phosphatase 1B (PTP1B) is a negative regulator of insulin signalling and is localised on the ER membrane. The aim of the study was to investigate the cross-talk between ER stress and PTP1B.

Methods

Leptin-deficient obese (ob/ob), Ptp1b (also known as Ptpn1) knockout and C57BL/6J mice were subjected to a high-fat or normal-chow diet for 20 weeks. ER stress was induced in cultured myotubes by treatment with tunicamycin. Immunohistochemistry and western blotting were used to assess proteins involved in the ER stress response. Myotube glucose uptake was determined by measuring 2-deoxy[³H]glucose incorporation.

Results

A high-fat diet induced ER stress and PTP1B expression in the muscle tissue of mice and these responses were attenuated by treatment with the ER chaperone tauroursodeoxycholic acid (TUDCA). Cultured myotubes exhibited increased levels of PTP1B in response to tunicamycin treatment. Silencing of Ptp1b with small interfering RNA (siRNA) or overexpression of Ptp1b with adenovirus construct failed to alter the levels of ER stress. Ptp1b knockout mice did not differ from the wild-type mice in the extent of tunicamycin-induced upregulation of glucose-regulated protein-78. However, tunicamycin-induced phosphorylation of eukaryotic initiation factor 2α and c-Jun NH₂-terminal kinase-2 were significantly attenuated in the Ptp1b knockout mice. Treatment with TUDCA or silencing of PTP1B reversed tunicamycin-induced blunted myotube glucose uptake.

Conclusions/interpretation

Our data suggest that PTP1B is activated by ER stress and is required for full-range activation of ER stress pathways in mediating insulin resistance in the skeletal muscle.     

Inducible liver-specific knockdown of protein tyrosine phosphatase 1B improves glucose and lipid homeostasis in adult mice

Aims/hypothesis

Protein tyrosine phosphatase 1B (PTP1B) is a key negative regulator of insulin signalling. Hepatic PTP1B deficiency, using the Alb-Cre promoter to drive Ptp1b deletion from birth in mice, improves glucose homeostasis, insulin sensitivity and lipid metabolism. The aim of this study was to investigate the therapeutic potential of decreasing liver PTP1B levels in obese and insulin-resistant adult mice.

Methods

Inducible Ptp1b liver-specific knockout mice were generated using SA-Cre-ER ^T2 mice crossed with Ptp1b floxed (Ptp1b ^fl/fl) mice. Mice were fed a high-fat diet (HFD) for 12 weeks to induce obesity and insulin resistance. Tamoxifen was administered in the HFD to induce liver-specific deletion of Ptp1b (SA-Ptp1b ^?/? mice). Body weight, glucose homeostasis, lipid homeostasis, serum adipokines, insulin signalling and endoplasmic reticulum (ER) stress were examined.

Results

Despite no significant change in body weight relative to HFD-fed Ptp1b ^fl/fl control mice, HFD-fed SA-Ptp1b ^?/? mice exhibited a reversal of glucose intolerance as determined by improved glucose and pyruvate tolerance tests, decreased fed and fasting blood glucose and insulin levels, lower HOMA of insulin resistance, circulating leptin, serum and liver triacylglycerols, serum NEFA and decreased HFD-induced ER stress. This was associated with decreased glycogen synthase, eukaryotic translation initiation factor-2α kinase 3, eukaryotic initiation factor 2α and c-Jun NH₂-terminal kinase 2 phosphorylation, and decreased expression of Pepck.

Conclusions/interpretation

Inducible liver-specific PTP1B knockdown reverses glucose intolerance and improves lipid homeostasis in HFD-fed obese and insulin-resistant adult mice. This suggests that knockdown of liver PTP1B in individuals who are already obese/insulin resistant may have relatively rapid, beneficial therapeutic effects.     

Insulin resistance influences the association of adiponectin levels with diabetes incidence in two population-based cohorts: the Cooperative Health Research in the Region of Augsburg (KORA) S4/F4 study and the Framingham Offspring Study

Aims/hypothesis

Lower adiponectin levels are associated with higher risk of incident type 2 diabetes. Most analyses have been adjusted for confounding factors, but few have taken into account insulin resistance per se. We tested the hypothesis that the association of adiponectin levels with incident type 2 diabetes differs between insulin-resistant and insulin-sensitive individuals.

Methods

We studied two prospective cohorts: the Framingham Offspring Study (n?=?2,023) and the Cooperative Health Research in the Region of Augsburg (KORA) S4/F4 study (n?=?887) cohorts. Insulin resistance was estimated by HOMA-insulin resistance (HOMA-IR). We used logistic regression analysis to test the association between adiponectin and incident type 2 diabetes overall and in insulin-resistant vs insulin-sensitive individuals (defined by ?? vs <75th percentile of HOMA-IR).

Results

At baseline, Framingham??s participants were 60?±?9?years old and 56% were women; KORA??s participants were 63?±?5?years old and 49% were women. Type 2 diabetes incidence was 5.4% over 6.5?years (n?=?109) in Framingham and 10.5% over 8?years (n?=?93) in KORA. Lower adiponectin levels were associated with type 2 diabetes incidence in both cohorts. In insulin-resistant individuals, lower adiponectin levels were associated with higher risk of type 2 diabetes incidence (OR 1.60 [95% CI 1.10?C2.31] per SD decrease in Framingham, p?=?0.01; and OR 2.34 [95% CI 1.16?C4.73] in KORA, p?=?0.02); while this was not observed in insulin-sensitive individuals (OR 1.10 [95% CI 0.73?C1.67] in Framingham, p?=?0.64; and OR 1.34 [95%CI: 0.88?C2.03] in KORA, p?=?0.18).

Conclusions/interpretation

We conclude that lower adiponectin levels are associated with higher risk of type 2 diabetes in insulin-resistant but not in insulin-sensitive individuals. This suggests that some level of insulin resistance is needed to see deleterious effects of low adiponectin.     

Exendin-4 protects pancreatic beta cells from palmitate-induced apoptosis by interfering with GPR40 and the MKK4/7 stress kinase signalling pathway

Aims/hypothesis

The mechanisms of the protective effects of exendin-4 on NEFA-induced beta cell apoptosis were investigated.

Methods

The effects of exendin-4 and palmitate were evaluated in human and murine islets, rat insulin-secreting INS-1E cells and murine glucagon-secreting alpha-TC1-6 cells. mRNA and protein expression/phosphorylation were measured by real-time RT-PCR and immunoblotting or immunofluorescence, respectively. Small interfering (si)RNAs for Ib1 and Gpr40 were used. Cell apoptosis was quantified by two independent assays. Insulin release was assessed with an insulin ELISA.

Results

Exposure of human and murine primary islets and INS-1E cells, but not alpha-TC1-6 cells, to exendin-4 inhibited phosphorylation of the stress kinases, c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK), and prevented apoptosis in response to palmitate. Exendin-4 increased the protein content of islet-brain 1 (IB1), an endogenous JNK blocker; however, siRNA-mediated reduction of IB1 did not impair the ability of exendin-4 to inhibit JNK and prevent apoptosis. Exendin-4 reduced G-protein-coupled receptor 40 (GPR40) expression and inhibited palmitate-induced phosphorylation of mitogen-activated kinase kinase (MKK)4 and MKK7. The effects of exendin-4 were abrogated in the presence of the protein kinase A (PKA) inhibitors, H89 and KT5720. Knockdown of GPR40, as well as use of a specific GPR40 antagonist, resulted in diminished palmitate-induced JNK and p38 MAPK phosphorylation and apoptosis. Furthermore, inhibition of JNK and p38 MAPK activity prevented palmitate-induced apoptosis.

Conclusions/interpretation

Exendin-4 counteracts the proapoptotic effects of palmitate in beta cells by reducing GPR40 expression and inhibiting MKK7- and MKK4-dependent phosphorylation of the stress kinases, JNK and p38 MAPK, in a PKA-dependent manner.     

Chronic muscle stimulation improves insulin sensitivity while increasing subcellular lipid droplets and reducing selected diacylglycerol and ceramide species in obese Zucker rats

Aims/hypothesis

Although insulin resistance has been associated with accumulations of specific intramuscular fatty acids and altered subcellular localisation of lipid droplets, these concepts remain controversial. Therefore, we aimed to identify specific intramuscular fatty acids and subcellular lipid localisations associated with improved insulin sensitivity following chronic muscle contraction.

Methods

In lean and insulin-resistant obese Zucker rats the tibialis anterior muscle was stimulated (6 h/day for 6 days). Thereafter, muscles were examined for insulin sensitivity, intramuscular lipid droplet localisation and triacylglycerol (TAG), diacylglycerol (DAG) and ceramide fatty acid composition.

Results

In lean and obese animals, regardless of muscle type, chronic muscle contraction improved muscle insulin sensitivity and increased intramuscular levels of total and most C14–C22 TAG fatty acids (p?<?0.05). Therefore, accumulation in subcellular lipid droplet compartments reflected the oversupply of lipids within muscle. In contrast, improvements in insulin sensitivity induced by muscle contraction were associated with reductions in specific DAG and ceramide species that were not uniform in red and white muscle of obese rats. However, these reductions were insufficient to fully normalise insulin sensitivity, indicating that other mechanisms are involved.

Conclusions/interpretation

Reductions in 18 C length DAG and ceramide species were the most consistent in red and white muscle and therefore may represent therapeutic targets for improving insulin sensitivity.     

Ceramide accumulation in L6 skeletal muscle cells due to increased activity of ceramide synthase isoforms has opposing effects on insulin action to those caused by palmitate treatment

Aims/hypothesis

An accumulation of ceramides has been implicated in the generation of insulin resistance in skeletal muscle upon an oversupply of fatty acid. Different ceramide species are generated through the actions of ceramide synthases (CerSs), which incorporate specific acyl side chains. We tested whether particular CerS isoforms promoted insulin resistance through the generation of more inhibitory ceramide species, thus representing potential targets for intervention.

Methods

CerS isoforms CerS1, CerS2, CerS4, CerS5 and CerS6 were overexpressed in L6 myotubes using adenovirus, and cells were treated with palmitate and stimulated with insulin. Alternatively, CerS isoforms were knocked down using siRNAs. Sphingolipids were examined by mass spectrometry and tracer incorporation. Phosphorylation of IRS1 and Akt was measured by immunoblotting, while glucose disposal was assessed by measuring GLUT4 translocation and the incorporation of [¹⁴C]glucose into glycogen.

Results

Palmitate treatment increased the levels of several ceramides but reduced the levels of sphingomyelins, while insulin had no effect. The fatty acid also inhibited insulin-stimulated Akt phosphorylation and glycogen synthesis. Overexpression of CerS isoforms increased specific ceramides. Unexpectedly, the overexpression of CerS1 and CerS6 promoted insulin action, while no isoform had inhibitory effects. CerS6 knockdown had effects reciprocal to those of CerS6 overexpression.

Conclusions/interpretation

Palmitate may increase intracellular ceramide levels through sphingomyelin hydrolysis as well as de novo synthesis, but no particular species were implicated in the generation of insulin resistance. The modulation of ceramides through an alteration of CerS expression does not affect the action of insulin in the same way as ceramide generation by palmitate treatment. Conversely, certain isoforms promote insulin action, indicating the importance of ceramides in cell function.