Loss of skeletal muscle mass and function is observed in many insulin-resistant disease states such as diabetes, cancer cachexia, renal failure and ageing although the mechanisms for this remain unclear. We hypothesised that impaired insulin signalling results in reduced muscle mass and function and that this decrease in muscle mass and function is due to both increased production of atrogenes and aberrant reactive oxygen species (ROS) generation. Maximum tetanic force of the extensor digitorum longus of muscle insulin receptor knockout (MIRKO) and lox/lox control mice was measured in situ. Muscles were removed for the measurement of mass, histological examination and ROS production. Activation of insulin signalling pathways, markers of muscle atrophy and indices of protein synthesis were determined in a separate group of MIRKO and lox/lox mice 15 min following treatment with insulin. Muscles from MIRKO mice had 36% lower maximum tetanic force generation compared with muscles of lox/lox mice. Muscle fibres of MIRKO mice were significantly smaller than those of lox/lox mice with no apparent structural abnormalities. Muscles from MIRKO mice demonstrated absent phosphorylation of AKT in response to exogenous insulin along with a failure to phosphorylate ribosomal S6 compared with lox/lox mice. Atrogin-1 and MuRF1 relative mRNA expression in muscles from MIRKO mice were decreased compared with muscles from lox/lox mice following insulin treatment. There were no differences in markers of reactive oxygen species damage between muscles from MIRKO mice and lox/lox mice. These data support the hypothesis that the absence of insulin signalling contributes to reduced muscle mass and function though decreased protein synthesis rather than proteasomal atrophic pathways.

Although many animal studies indicate insulin has cardioprotective effects, clinical studies suggest a link between insulin resistance (hyperinsulinemia) and heart failure (HF). Here we have demonstrated that excessive cardiac insulin signaling exacerbates systolic dysfunction induced by pressure overload in rodents. Chronic pressure overload induced hepatic insulin resistance and plasma insulin level elevation. In contrast, cardiac insulin signaling was upregulated by chronic pressure overload because of mechanical stretch-induced activation of cardiomyocyte insulin receptors and upregulation of insulin receptor and Irs1 expression. Chronic pressure overload increased the mismatch between cardiomyocyte size and vascularity, thereby inducing myocardial hypoxia and cardiomyocyte death. Inhibition of hyperinsulinemia substantially improved pressure overload-induced cardiac dysfunction, improving myocardial hypoxia and decreasing cardiomyocyte death. Likewise, the cardiomyocyte-specific reduction of insulin receptor expression prevented cardiac ischemia and hypertrophy and attenuated systolic dysfunction due to pressure overload. Conversely, treatment of type 1 diabetic mice with insulin improved hyperglycemia during pressure overload, but increased myocardial ischemia and cardiomyocyte death, thereby inducing HF. Promoting angiogenesis restored the cardiac dysfunction induced by insulin treatment. We therefore suggest that the use of insulin to control hyperglycemia could be harmful in the setting of pressure overload and that modulation of insulin signaling is crucial for the treatment of HF.

The class I(A) phosphatidylinsositol 3-kinases (PI3Ks) form a critical node in the insulin metabolic pathway; however, the precise roles of the different isoforms of this enzyme remain elusive. Using tissue-specific gene inactivation, we demonstrate that p110alpha catalytic subunit of PI3K is a key mediator of insulin metabolic actions in the liver. Thus, deletion of p110alpha in liver results in markedly blunted insulin signaling with decreased generation of PIP(3) and loss of insulin activation of Akt, defects that could not be rescued by overexpression of p110beta. As a result, mice with hepatic knockout of p110alpha display reduced insulin sensitivity, impaired glucose tolerance, and increased gluconeogenesis, hypolipidemia, and hyperleptinemia. The diabetic syndrome induced by loss of p110alpha in liver did not respond to metformin treatment. Together, these data indicate that the p110alpha isoform of PI3K plays a fundamental role in insulin signaling and control of hepatic glucose and lipid metabolism.

Insulin and insulin-like growth factor-1 (IGF-1) act on highly homologous receptors, yet in vivo elicit distinct effects on metabolism and growth. To investigate how the insulin and IGF-1 receptors exert specificity in their biological responses, we assessed their role in the regulation of gene expression using three experimental paradigms: 1) preadipocytes before and after differentiation into adipocytes that express both receptors, but at different ratios; 2) insulin receptor (IR) or IGF1R knock-out preadipocytes that only express the complimentary receptor; and 3) IR/IGF1R double knock-out (DKO) cells reconstituted with the IR, IGF1R, or both. In wild-type preadipocytes, which express predominantly IGF1R, microarray analysis revealed approximately 500 IGF-1 regulated genes (p 0.05). The largest of these were confirmed by quantitative PCR, which also revealed that insulin produced a similar effect, but with a smaller magnitude of response. After differentiation, when IR levels increase and IGF1R decrease, insulin became the dominant regulator of each of these genes. Measurement of the 50 most highly regulated genes by quantitative PCR did not reveal a single gene regulated uniquely via the IR or IGF1R using cells expressing exclusively IGF-1 or insulin receptors. Insulin and IGF-1 dose responses from 1 to 100 nm in WT, IRKO, IGFRKO, and DKO cells re-expressing IR, IGF1R, or both showed that insulin and IGF-1 produced effects in proportion to the concentration of ligand and the specific receptor on which they act. Thus, IR and IGF1R act as identical portals to the regulation of gene expression, with differences between insulin and IGF-1 effects due to a modulation of the amplitude of the signal created by the specific ligand-receptor interaction.

Class Ia phosphoinositide 3-kinase (PI3K), an essential mediator of the metabolic actions of insulin, is composed of a catalytic (p110alpha or p110beta) and regulatory (p85alphaalpha, p85betaalpha or p55alpha) subunit. Here we show that p85alphaalpha interacts with X-box-binding protein-1 (XBP-1), a transcriptional mediator of the unfolded protein response (UPR), in an endoplasmic reticulum (ER) stress-dependent manner. Cell lines with knockout or knockdown of p85alphaalpha show marked alterations in the UPR, including reduced ER stress-dependent accumulation of nuclear XBP-1, decreased induction of UPR target genes and increased rates of apoptosis. This is associated with a decreased activation of inositol-requiring protein-1alpha (IRE1alpha) and activating transcription factor-6alphaalpha (ATF6alpha). Mice with deletion of p85alpha in liver (L-Pik3r1(-/-)) show a similar attenuated UPR after tunicamycin administration, leading to an increased inflammatory response. Thus, p85alphaalpha forms a previously unrecognized link between the PI3K pathway, which is central to insulin action, and the regulation of the cellular response to ER stress, a state that when unresolved leads to insulin resistance.

Sirtuins are NAD(+)-dependent protein deacetylases. They mediate adaptive responses to a variety of stresses, including calorie restriction and metabolic stress. Sirtuin 3 (SIRT3) is localized in the mitochondrial matrix, where it regulates the acetylation levels of metabolic enzymes, including acetyl coenzyme A synthetase 2 (refs 1, 2). Mice lacking both Sirt3 alleles appear phenotypically normal under basal conditions, but show marked hyperacetylation of several mitochondrial proteins. Here we report that SIRT3 expression is upregulated during fasting in liver and brown adipose tissues. During fasting, livers from mice lacking SIRT3 had higher levels of fatty-acid oxidation intermediate products and triglycerides, associated with decreased levels of fatty-acid oxidation, compared to livers from wild-type mice. Mass spectrometry of mitochondrial proteins shows that long-chain acyl coenzyme A dehydrogenase (LCAD) is hyperacetylated at lysine 42 in the absence of SIRT3. LCAD is deacetylated in wild-type mice under fasted conditions and by SIRT3 in vitro and in vivo; and hyperacetylation of LCAD reduces its enzymatic activity. Mice lacking SIRT3 exhibit hallmarks of fatty-acid oxidation disorders during fasting, including reduced ATP levels and intolerance to cold exposure. These findings identify acetylation as a novel regulatory mechanism for mitochondrial fatty-acid oxidation and demonstrate that SIRT3 modulates mitochondrial intermediary metabolism and fatty-acid use during fasting.

We have previously shown that expression of the transcription factor ARNT/HIF1beta is reduced in islets of humans with type 2 diabetes. We have now found that ARNT is also reduced in livers of diabetics. To study the functional effect of its reduction, we created mice with liver-specific ablation (L-ARNT KO) using ARNT loxP mice and adenoviral-mediated delivery of Cre. L-ARNT KO mice had normal blood glucose but increased fed insulin levels. These mice also exhibited features of type 2 diabetes with increased hepatic gluconeogenesis, increased lipogenic gene expression, and low serum beta-hydroxybutyrate. These effects appear to be secondary to increased expression of CCAAT/enhancer-binding protein alpha (C/EBPalpha), farnesoid X receptor (FXR), and sterol response element-binding protein 1c (SREBP-1c) and a reduction in phosphorylation of AMPK without changes in the expression of enzymes in ketogenesis, fatty acid oxidation, or FGF21. These results demonstrate that a deficiency of ARNT action in the liver, coupled with that in beta cells, could contribute to the metabolic phenotype of human type 2 diabetes.

Insulin-like growth factor-1 (IGF-1) signaling has recently been implicated in the development of cardiac hypertrophy after long-term endurance training, via mechanisms that may involve energetic stress. Given the potential overlap of insulin and IGF-1 signaling we sought to determine if both signaling pathways could contribute to exercise-induced cardiac hypertrophy following shorter-term exercise training. Studies were performed in mice with cardiac-specific IGF-1 receptor (IGF1R) knockout (CIGFRKO), mice with cardiac-specific insulin receptor (IR) knockout (CIRKO), CIGFRKO mice that lacked one IR allele in cardiomyocytes (IGFR-/-IR+/-), and CIRKO mice that lacked one IGF1R allele in cardiomyocytes (IGFR+/-IR-/-). Intravenous administration of IGF-1 or 75 hours of swimming over 4 weeks increased IGF1R tyrosine phosphorylation in the heart in control and CIRKO mice but not in CIGFRKO mice. Intriguingly, IR tyrosine phosphorylation in the heart was also increased following IGF-1 administration or exercise training in control and CIGFRKO mice but not in CIRKO mice. The extent of cardiac hypertrophy following exercise training in CIGFRKO and CIRKO mice was comparable to that in control mice. In contrast, exercise-induced cardiac hypertrophy was significantly attenuated in IGFR-/-IR+/- and IGFR+/-IR-/- mice. Thus, IGF-1 and exercise activates both IGF1R and IR in the heart, and IGF1R- and IR-mediated signals may serve redundant roles in the hypertrophic responses of the heart to exercise training.

AIMS/HYPOTHESIS: Previous findings in rodents used as a model of diabetes suggest that insulin activation of atypical protein kinase C (aPKC) is impaired in muscle, but, unexpectedly, conserved in liver, despite impaired hepatic protein kinase B (PKB/Akt) activation. Moreover, aPKC at least partly regulates two major transactivators: (1) hepatic sterol receptor binding protein-1c (SREBP-1c), which controls lipid synthesis; and (2) nuclear factor kappa B (NFkappaB), which promotes inflammation and systemic insulin resistance. METHODS: In Goto-Kakizaki rats used as a model of type 2 diabetes, we examined: (1) whether differences in hepatic aPKC and PKB activation reflect differences in activation of IRS-1- and IRS-2-dependent phosphatidylinositol 3-kinase (PI3K); (2) whether hepatic SREBP-1c and NFkappaB are excessively activated by aPKC; and (3) metabolic consequences of excessive activation of hepatic aPKC, SREBP-1c and NFkappaB. RESULTS: In liver, as well as in muscle, IRS-2/PI3K activation by insulin was intact, whereas IRS-1/PI3K activation by insulin was impaired. Moreover, hepatic IRS-2 is known to control hepatic aPKC during insulin activation. Against this background, selective inhibition of hepatic aPKC by adenoviral-mediated expression of mRNA encoding kinase-inactive aPKC or short hairpin RNA targeting Irs2 mRNA and partially depleting hepatic IRS-2 diminished hepatic SREBP-1c production and NFkappaB activities, concomitantly improving serum lipids and insulin signalling in muscle and liver. Similar improvements in SREBP-1c, NFkappaB and insulin signalling were seen in ob/ob mice following inhibition of hepatic aPKC. CONCLUSIONS/INTERPRETATION: In diabetic rodent liver, diminished PKB activation may largely reflect impaired IRS-1/PI3K activation, while conserved aPKC activation reflects retained IRS-2/PI3K activity. Hepatic aPKC may also contribute importantly to excessive SREPB-1c and NFkappaB activities. Excessive hepatic aPKC-dependent activation of SREBP-1c and NFkappaB may contribute importantly to hyperlipidaemia and systemic insulin resistance.