Differential aetiology and impact of phosphoinositide 3-kinase (PI3K) and Akt signalling in skeletal muscle on in vivo insulin action

Aims/hypothesis

Insulin resistance in skeletal muscle is a key factor in the development of type 2 diabetes and although some studies indicate that this could be partly attributed to reduced content and activity of various proximal and distal insulin signalling molecules, consensus is lacking. We therefore aimed to investigate the regulation of proximal insulin signalling in skeletal muscle and its effect on glucose metabolism in a large non-diabetic population.

Methods

We examined 184 non-diabetic twins with gold-standard techniques including the euglycaemic–hyperinsulinaemic clamp. Insulin signalling was evaluated at three key levels, i.e. the insulin receptor, IRS-1 and V-akt murine thymoma viral oncogene (Akt) levels, employing kinase assays and phospho-specific western blotting.

Results

Proximal insulin signalling was not associated with obesity, age or sex. However, birthweight was positively associated with IRS-1-associated phosphoinositide 3-kinase (PI3K; IRS-1-PI3K) activity (p?=?0.04); maximal aerobic capacity $ \left( {\mathop {V}\limits^\cdot {{\hbox{O}}_{\rm{2max}}}} \right) $ , paradoxically, was negatively associated with IRS-1-PI3K (p?=?0.02) and Akt2 activity (p?=?0.01). Additionally, we found low heritability estimates for most measures of insulin signalling activity. Glucose disposal was positively associated with Akt-308 phosphorylation (p?<?0.001) and Akt2 activity (p?=?0.05), but not with insulin receptor tyrosine kinase or IRS-1-PI3K activity.

Conclusions/interpretation

With the exception of birthweight, ‘classical’ modifiers of insulin action, including genetics, age, sex, obesity and $ \mathop {V}\limits^\cdot {{\hbox{O}}_{\rm{2max}}} $ , do not seem to mediate their most central effects on whole-body insulin sensitivity through modulation of proximal insulin signalling in skeletal muscle. We also demonstrated an association between Akt activity and in vivo insulin sensitivity, suggesting a role of Akt in control of in vivo insulin resistance and potentially in type 2 diabetes.     

Global IRS-1 phosphorylation analysis in insulin resistance

Aims/hypothesis

IRS-1 serine phosphorylation is often elevated in insulin resistance models, but confirmation in vivo in humans is lacking. We therefore analysed IRS-1 phosphorylation in human muscle in vivo.

Methods

We used HPLC-electrospray ionisation (ESI)-MS/MS to quantify IRS-1 phosphorylation basally and after insulin infusion in vastus lateralis muscle from lean healthy, obese non-diabetic and type 2 diabetic volunteers.

Results

Basal Ser323 phosphorylation was increased in type 2 diabetic patients (2.1?±?0.43, p????0.05, fold change vs lean controls). Thr495 phosphorylation was decreased in type 2 diabetic patients (p????0.05). Insulin increased IRS-1 phosphorylation at Ser527 (1.4?±?0.17, p????0.01, fold change, 60?min after insulin infusion vs basal) and Ser531 (1.3?±?0.16, p????0.01, fold change, 60?min after insulin infusion vs basal) in the lean controls and suppressed phosphorylation at Ser348 (0.56?±?0.11, p????0.01, fold change, 240?min after insulin infusion vs basal), Thr446 (0.64?±?0.16, p????0.05, fold change, 60?min after insulin infusion vs basal), Ser1100 (0.77?±?0.22, p????0.05, fold change, 240?min after insulin infusion vs basal) and Ser1142 (1.3?±?0.2, p????0.05, fold change, 60?min after insulin infusion vs basal).

Conclusions/interpretation

We conclude that, unlike some aspects of insulin signalling, the ability of insulin to increase or suppress certain IRS-1 phosphorylation sites is intact in insulin resistance. However, some IRS-1 phosphorylation sites do not respond to insulin, whereas other Ser/Thr phosphorylation sites are either increased or decreased in insulin resistance.     

Doc2b enrichment enhances glucose homeostasis in mice via potentiation of insulin secretion and peripheral insulin sensitivity

Aims/hypothesis

Insulin secretion from pancreatic beta cells and insulin-stimulated glucose uptake into skeletal muscle are processes regulated by similar isoforms of the soluble N-ethylmaleimide-sensitive factor-attachment protein receptor (SNARE) and mammalian homologue of unc-18 (Munc18) protein families. Double C2 domain β (Doc2b), a SNARE- and Munc18-interacting protein, is implicated as a crucial effector of glycaemic control. However, whether Doc2b is naturally limiting for these processes, and whether Doc2b enrichment might exert a beneficial effect upon glycaemia in vivo, remains undetermined.

Methods

Tetracycline-repressible transgenic (Tg) mice engineered to overexpress Doc2b simultaneously in the pancreas, skeletal muscle and adipose tissues were compared with wild-type (Wt) littermate mice regarding glucose and insulin tolerance, islet function in vivo and ex vivo, and skeletal muscle GLUT4 accumulation in transverse tubule/sarcolemmal surface membranes. SNARE complex formation was further assessed using Doc2b overexpressing L6-GLUT4-myc myoblasts to derive mechanisms relatable to physiological in vivo analyses.

Results

Doc2b Tg mice cleared glucose substantially faster than Wt mice, correlated with enhancements in both phases of insulin secretion and peripheral insulin sensitivity. Heightened peripheral insulin sensitivity correlated with elevated insulin-stimulated GLUT4 vesicle accumulation in cell surface membranes of Doc2b Tg mouse skeletal muscle. Mechanistic studies demonstrated Doc2b enrichment to enhance syntaxin-4–SNARE complex formation in skeletal muscle cells.

Conclusions/interpretation

Doc2b is a limiting factor in SNARE exocytosis events pertinent to glycaemic regulation in vivo. Doc2b enrichment may provide a novel means to simultaneously boost islet and skeletal muscle function in vivo in the treatment and/or prevention of diabetes.     

Skeletal muscle-specific overexpression of SIRT1 does not enhance whole-body energy expenditure or insulin sensitivity in young mice

Aims/hypothesis

The NAD⁺-dependent protein deacetylase sirtuin (SIRT)1 is thought to be a key regulator of skeletal muscle metabolism. However, its precise role in the regulation of insulin sensitivity is unclear. Accordingly, we sought to determine the effect of skeletal muscle-specific overexpression of SIRT1 on skeletal muscle insulin sensitivity and whole-body energy metabolism.

Methods

At 10 weeks of age, mice with muscle-specific overexpression of SIRT1 and their wild-type littermates were fed a standard diet with free access to chow or an energy-restricted (60% of standard) diet for 20 days. Energy expenditure and body composition were measured by indirect calorimetry and magnetic resonance imaging, respectively. Skeletal muscle insulin-stimulated glucose uptake was measured ex vivo in soleus and extensor digitorum longus muscles using a 2-deoxyglucose uptake technique with a physiological insulin concentration of 360 pmol/l (60 μU/ml).

Results

Sirt1 mRNA and SIRT1 protein levels were increased by approximately 100- and 150-fold, respectively, in skeletal muscle of mice with SIRT1 overexpression compared with wild-type mice. Despite this large-scale overexpression of SIRT1, body composition, whole-body energy expenditure, substrate oxidation and voluntary activity were comparable between genotypes. Similarly, skeletal muscle basal and insulin-stimulated glucose uptake were unaltered with SIRT1 overexpression. Finally, while 20 days of energy restriction enhanced insulin-stimulated glucose uptake in skeletal muscles of wild-type mice, no additional effect of SIRT1 overexpression was observed.

Conclusions/interpretation

These results demonstrate that upregulation of SIRT1 activity in skeletal muscle does not affect whole-body energy expenditure or enhance skeletal muscle insulin sensitivity in young mice on a standard diet with free access to chow or in young mice on energy-restricted diets.     

固醇调节元件结合蛋白1c对骨骼肌细胞胰岛素受体底物1表达调控的影响

目的 探讨固醇调节元件结合蛋白1c（SREBP-1c）对大鼠骨骼肌细胞胰岛素受体底物1（IRS-1）表达调控的影响.方法 采用酶联合消化法取2～3 d SPF级雄性SD大鼠原代骨骼肌细胞,将原代细胞分为对照组（C）、对照＋胰岛素组（C＋I）、高脂组（PA）及高脂＋胰岛素组（PA＋I）.将表达SREBP-1c腺病毒转染L6细胞,根据感染复数（MOI）分为含绿色荧光蛋白阴性载体（GFP）组、MOI值为5、50、100、200组.将靶基因为SREBP-1c的干扰RNA （siRNA）转染L6细胞,并分为空白对照组、阴性siRNA组及SREBP-1c siRNA组.Western blotting和实时定量聚合酶链反应（RT-PCR）检测SREBP-1c、IRS-1、蛋白激酶B（Akt）基因及蛋白表达,油红O染色法检测细胞内脂质沉积情况.多组资料比较采用方差分析,两两比较采用最小显著差异法.结果 与C组相比,PA组SREBP-1c基因和蛋白水平升高（分别为2.72±0.08比1.00±0.18,3.02 ±0.19比1.00±0.05,t=15.240、18.289,均P＜0.05）,IRS-1基因和蛋白水平降低（分别为0.71 ±0.04比1.00 ±0.05,0.82 ±0.04比1.00±0.04,t=-7.960、-6.052,均P＜0.05）,丝氨酸磷酸化IRS-1蛋白表达升高,丝氨酸磷酸化Akt（p-Akt）蛋白表达下降（t=20.987、-5.869,均P＜0.05）.与GFP组相比,MOI值为50、100和200组的SREBP-1c基因和蛋白表达呈剂量依赖性上升（均P＜0.05）,IRS-1基因和蛋白表达水平呈剂量依赖性下降（均P＜0.05）.与空白对照组和阴性siRNA组相比,SREBP-1c siRNA组SREBP-1c基因和蛋白水平降低,IRS-1蛋白表达升高（均P＜0.05）.结论 SREBP-1c可抑制骨骼肌IRS-1胰岛素信号通路,参与肌细胞胰岛素抵抗的发生.     

AMPK phosphorylation of ACC2 is required for skeletal muscle fatty acid oxidation and insulin sensitivity in mice

Aims/hypothesis

Obesity is characterised by lipid accumulation in skeletal muscle, which increases the risk of developing insulin resistance and type 2 diabetes. AMP-activated protein kinase (AMPK) is a sensor of cellular energy status and is activated in skeletal muscle by exercise, hormones (leptin, adiponectin, IL-6) and pharmacological agents (5-amino-4-imidazolecarboxamide ribonucleoside [AICAR] and metformin). Phosphorylation of acetyl-CoA carboxylase 2 (ACC2) at S221 (S212 in mice) by AMPK reduces ACC activity and malonyl-CoA content but the importance of the AMPK–ACC2–malonyl-CoA pathway in controlling fatty acid metabolism and insulin sensitivity is not understood; therefore, we characterised Acc2 S212A knock-in (ACC2 KI) mice.

Methods

Whole-body and skeletal muscle fatty acid oxidation and insulin sensitivity were assessed in ACC2 KI mice and wild-type littermates.

Results

ACC2 KI mice were resistant to increases in skeletal muscle fatty acid oxidation elicited by AICAR. These mice had normal adiposity and liver lipids but elevated contents of triacylglycerol and ceramide in skeletal muscle, which were associated with hyperinsulinaemia, glucose intolerance and skeletal muscle insulin resistance.

Conclusions/interpretation

These findings indicate that the phosphorylation of ACC2 S212 is required for the maintenance of skeletal muscle lipid and glucose homeostasis.     

Pank1 deletion in leptin-deficient mice reduces hyperglycaemia and hyperinsulinaemia and modifies global metabolism without affecting insulin resistance

Aims/hypothesis

Pantothenate kinase (PANK) is the first enzyme in CoA biosynthesis. Pank1-deficient mice have 40% lower liver CoA and fasting hypoglycaemia, which results from reduced gluconeogenesis. Single-nucleotide polymorphisms in the human PANK1 gene are associated with insulin levels, suggesting a link between CoA and insulin homeostasis. We determined whether Pank1 deficiency (1) modified insulin levels, (2) ameliorated hyperglycaemia and hyperinsulinaemia, and (3) improved acute glucose and insulin tolerance of leptin (Lep)-deficient mice.

Methods

Serum insulin and responses to glucose and insulin tolerance tests were determined in Pank1-deficient mice. Levels of CoA and regulating enzymes were measured in liver and skeletal muscle of Lep-deficient mice. Double Pank1/Lep-deficient mice were analysed for the diabetes-related phenotype and global metabolism.

Results

Pank1-deficient mice had lower serum insulin and improved glucose tolerance and insulin sensitivity compared with wild-type mice. Hepatic and muscle CoA was abnormally high in Lep-deficient mice. Pank1 deletion reduced hepatic CoA but not muscle CoA, reduced serum glucose and insulin, but did not normalise body weight or improve acute glucose tolerance or protein kinase B phosphorylation in Lep-deficient animals. Pank1/Lep double-deficient mice exhibited reduced whole-body metabolism of fatty acids and amino acids and had a greater reliance on carbohydrate use for energy production.

Conclusions/interpretation

The results indicate that Pank1 deficiency drives a whole-body metabolic adaptation that improves aspects of the diabetic phenotype and uncouples hyperglycaemia and hyperinsulinaemia from obesity in leptin-deficient mice.     

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.     

Expression of anti-inflammatory macrophage genes within skeletal muscle correlates with insulin sensitivity in human obesity and type 2 diabetes

Aims/hypothesis

Low-grade systemic inflammation and adipose tissue inflammatory macrophages are frequently detected in patients with obesity and type 2 diabetes. Whether inflammatory macrophages also increase in skeletal muscle of individuals with metabolic disorders remains controversial. Here, we assess whether macrophage polarisation markers in skeletal muscle of humans correlate with insulin sensitivity in obesity and type 2 diabetes.

Methods

Skeletal muscle biopsies were obtained from individuals of normal weight and with normal glucose tolerance (NGT), and overweight/obese individuals with or without type 2 diabetes. Insulin sensitivity was determined by euglycaemic–hyperinsulinaemic clamps. Expression of macrophage genes was analysed by quantitative RT-PCR.

Results

Gene expression of the inflammatory macrophage phenotype marker cluster of differentiation (CD)11c was higher in muscle of type 2 diabetes patients (p?=?0.0069), and correlated with HbA_1c (p?=?0.0139, ρ?=?0.48) and fasting plasma glucose (p?=?0.0284, ρ?=?0.43), but not after correction for age. Expression of TGFB1, encoding the anti-inflammatory marker TGF-β1, correlated inversely with HbA_1c (p?=?0.0095, ρ?=??0.50; p?=?0.0484, ρ?=??0.50) and fasting plasma glucose (p?=?0.0471, ρ?=??0.39; p?=?0.0374, ρ?=??0.52) in two cohorts, as did HbA_1c with gene expression of macrophage galactose-binding lectin (MGL) (p?=?0.0425, ρ?=??0.51). TGFB1 expression was higher in NGT individuals than in individuals with type 2 diabetes (p?=?0.0303), and correlated with low fasting plasma insulin (p?=?0.0310, ρ?=??0.42). In exercised overweight/obese individuals, expression of genes for three anti-inflammatory macrophage markers, MGL (p?=?0.0031, ρ?=?0.71), CD163 (p?=?0.0268, ρ?=?0.57) and mannose receptor (p?=?0.0125, ρ?=?0.63), correlated with high glucose-disposal rate.

Conclusions/interpretation

Muscle expression of macrophage genes reveals a link between inflammatory macrophage markers, age and high glycaemia, whereas anti-inflammatory markers correlate with low glycaemia and high glucose-disposal rate.     

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.     

HLA-DRB1 reduces the risk of type 2 diabetes mellitus by increased insulin secretion

Aims/hypothesis

We sought to identify the physiological implications of genetic variation at the HLA-DRB1 region in full-heritage Pima Indians in Arizona.

Methods

Single-nucleotide polymorphisms from the HLA region on chromosome 6p were tested for association with skeletal muscle mRNA expression of HLA-DRB1 and HLA-DRA, and with type 2 diabetes mellitus and prediabetic traits.

Results

The A allele at rs9268852, which tags HLA-DRB1*02(1602), was associated both with higher HLA-DRB1 mRNA expression (n?=?133, p?=?4.27?×?10^?14) and decreased risk of type 2 diabetes (n?=?3,265, OR 0.723, p?=?0.002). Among persons with normal glucose tolerance (n?=?266) this allele was associated with a higher mean acute insulin response during an intravenous glucose tolerance test (p?=?0.005), higher mean 30?min insulin concentration during an oral glucose tolerance test (p?=?0.017) and higher body fat percentage (p?=?0.010). The polymorphism was not associated with HLA-DRA mRNA expression or insulin sensitivity.

Conclusions/interpretation

HLA-DRB1*02 is protective for type 2 diabetes, probably by enhancing self tolerance, thereby protecting against the autoimmune-mediated reduction of insulin secretion.     

Protection from non-alcoholic steatohepatitis and liver tumourigenesis in high fat-fed insulin receptor substrate-1-knockout mice despite insulin resistance

Aims/hypothesis

Epidemiological studies have revealed that obesity and diabetes mellitus are independent risk factors for the development of non-alcoholic steatohepatitis (NASH) and hepatocellular carcinoma. However, the debate continues on whether insulin resistance as such is directly associated with NASH and liver tumourigenesis. Here, we investigated the incidence of NASH and liver tumourigenesis in Irs1 ^?/? mice subjected to a long-term high-fat (HF) diet. Our hypothesis was that hepatic steatosis, rather than insulin resistance may be related to the pathophysiology of these conditions.

Methods

Mice (8 weeks old, C57Bl/6J) were given free access to standard chow (SC) or an HF diet. The development of NASH and liver tumourigenesis was evaluated after mice had been on the above-mentioned diets for 60 weeks. Similarly, Irs1 ^?/? mice were also subjected to an HF diet for 60 weeks.

Results

Long-term HF diet loading, which causes obesity and insulin resistance, was sufficient to induce NASH and liver tumourigenesis in the C57Bl/6J mice. Obesity and insulin resistance were reduced by switching mice from the HF diet to SC, which also protected these mice against the development of NASH and liver tumourigenesis. However, compared with wild-type mice fed the HF diet, Irs1 ^?/? mice fed the HF diet were dramatically protected against NASH and liver tumourigenesis despite the presence of severe insulin resistance and marked postprandial hyperglycaemia.

Conclusions/interpretation

IRS-1 inhibition might protect against HF diet-induced NASH and liver tumourigenesis, despite the presence of insulin resistance.     

Muscle-specific activation of Ca2+/calmodulin-dependent protein kinase IV increases whole-body insulin action in mice

Aims/hypothesis

Aerobic exercise increases muscle glucose and improves insulin action through numerous pathways, including activation of Ca²⁺/calmodulin-dependent protein kinases (CAMKs) and peroxisome proliferator γ coactivator 1α (PGC-1α). While overexpression of PGC-1α increases muscle mitochondrial content and oxidative type I fibres, it does not improve insulin action. Activation of CAMK4 also increases the content of type I muscle fibres, PGC-1α level and mitochondrial content. However, it remains unknown whether CAMK4 activation improves insulin action on glucose metabolism in vivo.

Methods

The effects of CAMK4 activation on skeletal muscle insulin action were quantified using transgenic mice with a truncated and constitutively active form of CAMK4 (CAMK4^●) in skeletal muscle. Tissue-specific insulin sensitivity was assessed in vivo using a hyperinsulinaemic–euglycaemic clamp and isotopic measurements of glucose metabolism.

Results

The rate of insulin-stimulated whole-body glucose uptake was increased by ～25% in CAMK4^● mice. This was largely attributed to an increase of ～60% in insulin-stimulated glucose uptake in the quadriceps, the largest hindlimb muscle. These changes were associated with improvements in insulin signalling, as reflected by increased phosphorylation of Akt and its substrates and an increase in the level of GLUT4 protein. In addition, there were extramuscular effects: CAMK4^● mice had improved hepatic and adipose insulin action. These pleiotropic effects were associated with increased levels of PGC-1α-related myokines in CAMK4^● skeletal muscle.

Conclusions/interpretation

Activation of CAMK4 enhances mitochondrial biogenesis in skeletal muscle while also coordinating improvements in whole-body insulin-mediated glucose.     

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.     

Distinct patterns of tissue-specific lipid accumulation during the induction of insulin resistance in mice by high-fat feeding

Aims/hypothesis

While it is well known that diet-induced obesity causes insulin resistance, the precise mechanisms underpinning the initiation of insulin resistance are unclear. To determine factors that may cause insulin resistance, we have performed a detailed time-course study in mice fed a high-fat diet (HFD).

Methods

C57Bl/6 mice were fed chow or an HFD from 3 days to 16 weeks and glucose tolerance and tissue-specific insulin action were determined. Tissue lipid profiles were analysed by mass spectrometry and inflammatory markers were measured in adipose tissue, liver and skeletal muscle.

Results

Glucose intolerance developed within 3 days of the HFD and did not deteriorate further in the period to 12 weeks. Whole-body insulin resistance, measured by hyperinsulinaemic–euglycaemic clamp, was detected after 1 week of HFD and was due to hepatic insulin resistance. Adipose tissue was insulin resistant after 1 week, while skeletal muscle displayed insulin resistance at 3 weeks, coinciding with a defect in glucose disposal. Interestingly, no further deterioration in insulin sensitivity was observed in any tissue after this initial defect. Diacylglycerol content was increased in liver and muscle when insulin resistance first developed, while the onset of insulin resistance in adipose tissue was associated with increases in ceramide and sphingomyelin. Adipose tissue inflammation was only detected at 16 weeks of HFD and did not correlate with the induction of insulin resistance.

Conclusions/interpretation

HFD-induced whole-body insulin resistance is initiated by impaired hepatic insulin action and exacerbated by skeletal muscle insulin resistance and is associated with the accumulation of specific bioactive lipid species.     

Exosomes participate in the alteration of muscle homeostasis during lipid-induced insulin resistance in mice

Aims/hypothesis

Exosomes released from cells can transfer both functional proteins and RNAs between cells. In this study we tested the hypothesis that muscle cells might transmit specific signals during lipid-induced insulin resistance through the exosomal route.

Methods

Exosomes were collected from quadriceps muscles of C57Bl/6 mice fed for 16 weeks with either a standard chow diet (SD) or an SD enriched with 20% palm oil (HP) and from C2C12 cells exposed to 0.5 mmol/l palmitate (EXO-Post Palm), oleate (EXO-Post Oleate) or BSA (EXO-Post BSA).

Results

HP-fed mice were obese and insulin resistant and had altered insulin-induced Akt phosphorylation in skeletal muscle (SkM). They also had reduced expression of Myod1 and Myog and increased levels of Ccnd1 mRNA, indicating that palm oil had a deep impact on SkM homeostasis in addition to insulin resistance. HP-fed mouse SkM secreted more exosomes than SD-fed mouse SkM. This was reproduced in-vitro using C2C12 cells pre-treated with palmitate, the most abundant saturated fatty acid of palm oil. Exosomes from HP-fed mice, EXO-Post Palm and EXO-Post Oleate induced myoblast proliferation and modified the expressions of genes involved in the cell cycle and muscle differentiation but did not alter insulin-induced Akt phosphorylation. Lipidomic analyses showed that exosomes from palmitate-treated cells were enriched in palmitate, indicating that exosomes likely transfer the deleterious effect of palm oil between muscle cells by transferring lipids. Muscle exosomes were incorporated into various tissues in vivo, including the pancreas and liver, suggesting that SkM could transfer specific signals through the exosomal route to key metabolic tissues.

Conclusions/interpretation

Exosomes act as ‘paracrine-like’ signals and modify muscle homeostasis during high-fat diets.     

Functional differences between aggregated and dispersed insulin-producing cells

Aims/hypothesis

Beta cells situated in the islet of Langerhans respond more vigorously to glucose than do dissociated beta cells. Mechanisms for this discrepancy were studied by comparing insulin-producing MIN6 cells aggregated into pseudoislets with MIN6 monolayer cells and mouse and human islets.

Methods

MIN6 monolayers, pseudoislets and mouse and human islets were exposed to glucose, α-ketoisocaproic acid (KIC), pyruvate, KIC plus glutamine and the phosphatidylinositol 3-kinase (PI3K) inhibitors LY294002 or wortmannin. Insulin secretion (ELISA), cytoplasmic Ca²⁺ concentration ([Ca²⁺]_c; microfluorometry), glucose oxidation (radiolabelling), the expression of genes involved in mitochondrial metabolism (PCR) and the phosphorylation of insulin receptor signalling proteins (western blotting) were measured.

Results

Insulin secretory responses to glucose, pyruvate, KIC and glutamine were higher in pseudoislets than monolayers and comparable to those of human islets. Glucose oxidation and genes for mitochondrial metabolism were upregulated in pseudoislets compared with single cells and monolayers, respectively. Phosphorylation at the inhibitory S636/639 site of IRS-1 was significantly higher in monolayers and dispersed human and mouse cells than pseudoislets and intact human and mouse islets. PI3K inhibition only slightly attenuated glucose-stimulated insulin secretion from monolayers, but substantially reduced that from pseudoislets and human and mouse islets without suppressing the glucose-induced [Ca²⁺]_c response.

Conclusions/interpretation

We propose that islet architecture is critical for proper beta cell mitochondrial metabolism and IRS-1 signalling, and that PI3K regulates insulin secretion at a step distal to the elevation of [Ca²⁺]_c.     

A vascular mechanism for high-sodium-induced insulin resistance in rats

Aims/hypothesis

High sodium (HS) effects on hypertension are well established. Recent evidence implicates a relationship between HS intake and insulin resistance, even in the absence of hypertension. The aim of the current study was to determine whether loss of the vascular actions of insulin may be the driving factor linking HS intake to insulin resistance.

Methods

Sprague Dawley rats were fed a control (0.31% wt/wt NaCl) or HS (8.00% wt/wt NaCl) diet for 4 weeks and subjected to euglycaemic–hyperinsulinaemic clamp (10 mU min^?1 kg^?1) or constant-flow pump-perfused hindlimb studies following an overnight fast. A separate group of HS rats was given quinapril during the dietary intervention and subjected to euglycaemic–hyperinsulinaemic clamp as above.

Results

HS intake had no effect on body weight or fat mass or on fasting glucose, insulin, endothelin-1 or NEFA concentrations. However, HS impaired whole body and skeletal muscle glucose uptake, in addition to a loss of insulin-stimulated microvascular recruitment. These effects were present despite enhanced insulin signalling (Akt) in both liver and skeletal muscle. Constant-flow pump-perfused hindlimb experiments revealed normal insulin-stimulated myocyte glucose uptake in HS-fed rats. Quinapril treatment restored insulin-mediated microvascular recruitment and muscle glucose uptake in vivo.

Conclusions/interpretation

HS-induced insulin resistance is driven by impaired microvascular responsiveness to insulin, and is not due to metabolic or signalling defects within myocytes or liver. These results imply that reducing sodium intake may be important not only for management of hypertension but also for insulin resistance, and highlight the vasculature as a potential therapeutic target in the prevention of insulin resistance.     

Matrix metalloproteinase 9 opposes diet-induced muscle insulin resistance in mice

Aims/hypothesis

Increased extracellular matrix (ECM) collagen is a characteristic of muscle insulin resistance. Matrix metalloproteinase (MMP) 9 is a primary enzyme that degrades collagen IV (ColIV). As a component of the basement membrane, ColIV plays a key role in ECM remodelling. We tested the hypotheses that genetic deletion of MMP9 in mice increases muscle ColIV, induces insulin resistance in lean mice and worsens diet-induced muscle insulin resistance.

Methods

Wild-type (Mmp9 ^+/+) and Mmp9-null (Mmp9 ^?/?) mice were chow or high-fat (HF) fed for 16 weeks. Insulin action was measured by the hyperinsulinaemic–euglycaemic clamp in conscious weight-matched surgically catheterised mice.

Results

Mmp9 ^?/? and HF feeding independently increased muscle ColIV. ColIV in HF-fed Mmp9 ^?/? mice was further increased. Mmp9 ^?/? did not affect fasting insulin or glucose in chow- or HF-fed mice. The glucose infusion rate (GIR), endogenous glucose appearance (EndoRa) and glucose disappearance (Rd) rates, and a muscle glucose metabolic index (Rg), were the same in chow-fed Mmp9 ^+/+ and Mmp9 ^?/? mice. In contrast, HF-fed Mmp9 ^?/? mice had decreased GIR, insulin-stimulated increase in Rd and muscle Rg. Insulin-stimulated suppression of EndoRa, however, remained the same in HF-fed Mmp9 ^?/? and Mmp9 ^+/+ mice. Decreased muscle Rg in HF-fed Mmp9 ^?/? was associated with decreased muscle capillaries.

Conclusions/interpretation

Despite increased muscle ColIV, genetic deletion of MMP9 does not induce insulin resistance in lean mice. In contrast, this deletion results in a more profound state of insulin resistance, specifically in the skeletal muscle of HF-fed mice. These results highlight the importance of ECM remodelling in determining muscle insulin resistance in the presence of HF diet.