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Emerging evidence suggests that G protein coupled receptor 55 (GPR55) may influence adrenoceptor function/activity in the cardiovascular system. Whether this reflects direct interaction (dimerization) between receptors or signalling crosstalk has not been investigated. This study explored the interaction between GPR55 and the alpha 1A-adrenoceptor (α

Amino acids are essential for cellular metabolism, and it is important to understand how nutrient supply is coordinated with changing energy requirements during embryogenesis. Here, we show that the amino acid transporter Slc7a5/Lat1 is highly expressed in tissues undergoing morphogenesis and that Slc7a5‐null mouse embryos have profound neural and limb bud outgrowth defects. Slc7a5‐null neural tissue exhibited aberrant mTORC1 activity and cell proliferation; transcriptomics, protein phosphorylation and apoptosis analyses further indicated induction of the integrated stress response as a potential cause of observed defects. The pattern of stress response gene expression induced in Slc7a5‐null embryos was also detected at low level in wild‐type embryos and identified stress vulnerability specifically in tissues undergoing morphogenesis. The Slc7a5‐null phenotype is reminiscent of Wnt pathway mutants, and we show that Wnt/β‐catenin loss inhibits Slc7a5 expression and induces this stress response. Wnt signalling therefore normally supports the metabolic demands of morphogenesis and constrains cellular stress. Moreover, operation in the embryo of the integrated stress response, which is triggered by pathogen‐mediated as well as metabolic stress, may provide a mechanistic explanation for a range of developmental defects., The amino acid transporter Slc7a5 sustains the metabolic demands of tissues undergoing morphogenesis during mouse embryogenesis and so constrains the activation of the integrated stress response.

The endogenous cannabinoid (or endocannabinoid) system (ECS) is part of a central neuromodulatory system thought to play a key role in the regulation of feeding behavior and energy balance. However, increasing evidence suggests that modulation of the ECS may also act to regulate peripheral mechanisms involved in these processes, including lipogenesis in adipose tissue and liver, insulin release from pancreatic beta-cells, and glucose uptake into skeletal muscle. It was recently shown that cannabinoid receptor type 1 (CB1) and type 2 (CB2), both key components of the ECS, are expressed in human and rodent skeletal muscle. However, their role in modulating insulin sensitivity in this metabolically active tissue has yet to be determined. Our aim was to establish the role, if any, of these receptors in modulating insulin sensitivity in skeletal muscle cells.Cultured skeletal muscle cells were exposed to CB1 and/or CB2 pharmacological agonists/antagonists/inverse agonists, and the resulting effects on insulin-regulated phosphatidylinositol 3 kinase (PI 3-kinase)-protein kinase B (PKB) and extracellular signal-related kinases 1/2 (ERK1/2)-directed signaling were determined.Here, we report that modulating the activity of the ECS in skeletal muscle regulates both insulin-dependent mitogen-activated protein (MAP) kinase (ERK1/2) and the canonical PI 3-kinase/PKB signaling pathways. We show that pharmacological activation or inhibition of CB1 receptor activity exerts a differential effect with regard to MAP kinase- and PKB-directed signaling.Our study provides evidence that signaling via cannabinoid receptors can significantly modulate mitogenic and metabolic signaling in skeletal muscle with important implications for muscle growth and differentiation as well as the regulation of glucose and lipid metabolism.

Emerging evidence suggests that G protein coupled receptor 55 (GPR55) may influence adrenoceptor function/activity in the cardiovascular system. Whether this reflects direct interaction (dimerization) between receptors or signalling crosstalk has not been investigated. This study explored the interaction between GPR55 and the alpha 1A-adrenoceptor (α1A-AR) in the cardiovascular system and the potential to influence function/signalling activities. GPR55 and α1A-AR mediated changes in both cardiac and vascular function was assessed in male wild-type (WT) and GPR55 homozygous knockout (GPR55-/-) mice by pressure volume loop analysis and isolated vessel myography, respectively. Dimerization of GPR55 with the α1A-AR was examined in transfected Chinese hamster ovary-K1 (CHO-K1) cells via Bioluminescence Resonance Energy Transfer (BRET). GPR55 and α1A-AR mediated signalling (extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation) was investigated in neonatal rat ventricular cardiomyocytes using AlphaScreen proximity assays. GPR55-/- mice exhibited both enhanced pressor and inotropic responses to A61603 (α1A-AR agonist), while in isolated vessels, A61603 induced vasoconstriction was attenuated by a GPR55-dependent mechanism. Conversely, GPR55-mediated vasorelaxation was not altered by pharmacological blockade of α1A-ARs with tamsulosin. While cellular studies demonstrated that GPR55 and α1A-AR failed to dimerize, pharmacological blockade of GPR55 altered α1A-AR mediated signalling and reduced ERK1/2 phosphorylation. Taken together, this study provides evidence that GPR55 and α1A-AR do not dimerize to form heteromers, but do interact at the signalling level to modulate the function of α1A-AR in the cardiovascular system.

Loss of skeletal muscle mass is a characteristic feature of various pathologies including cancer, diabetes, and obesity, as well as being a general feature of ageing. However, the processes underlying its pathogenesis are not fully understood and may involve multiple factors. Importantly, there is growing evidence which supports a role for fatty acids and their derived lipid intermediates in the regulation of skeletal muscle mass and function. In this review, we discuss evidence pertaining to those pathways which are involved in the reduction, increase and/or preservation of skeletal muscle mass by such lipids under various pathological conditions, and highlight studies investigating how these processes may be influenced by dietary supplementation as well as genetic and/or pharmacological intervention.

Summary The endocannabinoid system can modulate energy homeostasis by regulating feeding behaviour as well as peripheral energy storage and utilization. Importantly, many of its metabolic actions are mediated through the cannabinoid type 1 receptor (CB1R), whose hyperactivation is associated with obesity and impaired metabolic function. Herein, we explored the effects of administering rimonabant, a selective CB1R inverse agonist, upon key metabolic parameters in young (4 month old) and aged (17 month old) adult male C57BL/6 mice. Daily treatment with rimonabant for 14 days transiently reduced food intake in young and aged mice; however, the anorectic response was more profound in aged animals, coinciding with a substantive loss in body fat mass. Notably, reduced insulin sensitivity in aged skeletal muscle and liver concurred with increased CB1R mRNA abundance. Strikingly, rimonabant was shown to improve glucose tolerance and enhance skeletal muscle and liver insulin sensitivity in aged, but not young, adult mice. Moreover, rimonabant‐mediated insulin sensitization in aged adipose tissue coincided with amelioration of low‐grade inflammation and repressed lipogenic gene expression. Collectively, our findings indicate a key role for CB1R in aging‐related insulin resistance and metabolic dysfunction and highlight CB1R blockade as a potential strategy for combating metabolic disorders associated with aging.

Iron is an indispensable micronutrient that regulates many aspects of cell function, including growth and proliferation. These processes are critically dependent upon signalling via the mammalian or mechanistic target of rapamycin complex 1 (mTORC1). Herein, we test whether iron depletion induced by cell incubation with the iron chelator, deferoxamine (DFO), mediates its effects on cell growth through mTORC1-directed signalling and protein synthesis. We have used Caco-2 cells, a well-established in vitro model of human intestinal epithelia. Iron depletion increased expression of iron-regulated proteins (TfR, transferrin receptor and DMT1, divalent metal transporter, as predicted, but it also promoted a marked reduction in growth and proliferation of Caco-2 cells. This was strongly associated with suppressed mTORC1 signalling, as judged by reduced phosphorylation of mTOR substrates, S6K1 and 4E-BP1, and diminished protein synthesis. The reduction in mTORC1 signalling was tightly coupled with increased expression and accumulation of REDD1 (regulated in DNA damage and development 1) and reduced phosphorylation of Akt and TSC2. The increase in REDD1 abundance was rapidly reversed upon iron repletion of cells but was also attenuated by inhibitors of gene transcription, protein phosphatase 2A (PP2A) and by REDD1 siRNA — strategies that also antagonised the loss in mTORC1 signalling associated with iron depletion. Our findings implicate REDD1 and PP2A as crucial regulators of mTORC1 activity in iron-depleted cells and indicate that their modulation may help mitigate atrophy of the intestinal mucosa that may occur in response to iron deficiency., Highlights • Cellular iron (Fe) depletion dramatically reduces growth of intestinal Caco-2 cells. • mTORC1-directed signalling and protein synthesis are reduced in Fe-depleted cells. • Fe deficiency induces expression and gain of REDD1 in a PP2A-dependent manner. • PP2A inhibition blocks REDD1 gain and restores mTORC1 activity in Fe-depleted cells.

Regulated in development and DNA damage response 1 (REDD1) has been functionally linked to the control of diverse cellular processes due, at least in part, to its ability to repress mammalian or mechanistic Target of Rapamycin (mTOR) Complex-1 (mTORC1), a key protein complex controlled by hormonal and nutrient cues. Notably, emerging evidence suggests that REDD1 also regulates several pathways involved in modulating energy balance and metabolism. Herein, we discuss evidence implicating REDD1 as a key modulator of insulin action and metabolic function, including its potential contribution to mitochondrial biology and pancreatic islet function. Collectively, the available evidence suggests that REDD1 has a more prominent role in energy homeostasis than was previously thought, and implicates REDD1 as a potential therapeutic target for treatment of metabolic disorders., Trends REDD1 has been implicated in a range of cellular processes due, at least in part, to its ability to interact with proteins that impact protein homeostasis and metabolic function Several obesity-related factors, including hyperinsulinaemia, hyperlipidaemia, and hypoxia, have been shown to upregulate REDD1, which may facilitate the development of insulin resistance through impaired PKB/Akt-directed signalling. Genetic inhibition of REDD1 has been reported to impair insulin sensitivity through dysregulation of the mTORC1/S6K pathway. Emerging evidence suggests that REDD1 acts as a key regulator of mitochondrial oxidative capacity and lipogenesis, as well as modulating insulin production and secretion. Understanding the role of REDD1 in insulin action and energy homeostasis may provide a roadmap toward designing strategies to counteract obesity-related metabolic disorders.

Gangliosides constitute a large family of sialic acid-containing glycosphingolipids which play a key regulatory role in a diverse array of cellular processes, including receptor-associated signalling. Accordingly, the aberrant production of the ganglioside GM3 has been linked to pathophysiological changes associated with obesity, which in turn can lead to metabolic disorders such as insulin resistance and type 2 diabetes mellitus. This review examines the role of GM3 in mediating obesity-induced perturbations in metabolic function, including impaired insulin action. By doing so, we highlight the potential use of therapies targeting GM3 biosynthesis in order to counteract obesity-related metabolic disorders.

The plasma membrane-associated enzyme NEU3 sialidase functions to cleave sialic acid residues from the ganglioside GM3 thereby promoting its degradation, and has been implicated in the modulation of insulin action. Herein, we report for the first time that impaired insulin sensitivity in skeletal muscle and liver of obese Zucker fatty rats and aged C57BL/6 mice coincides with reduced NEU3 protein abundance. In addition, high fat feeding was found to significantly reduce NEU3 protein in white adipose tissue of rats. Notably, we also demonstrate the ability of the fatty acids palmitate and oleate to repress and induce NEU3 protein in L6 myotubes, concomitant with their insulin desensitising and enhancing effects, respectively. Moreover, we show that the palmitate-driven loss in NEU3 protein is mediated, at least in part, by intracellular ceramide synthesis but does not involve the proteasomal pathway. Strikingly, we further reveal that protein kinase B (PKB/Akt) acts as a key positive modulator of NEU3 protein abundance. Together, our findings implicate NEU3 as a potential biomarker of insulin sensitivity, and provide novel mechanistic insight into the regulation of NEU3 expression.

Carnosic acid (CA) is a major constituent of the labiate herbal plant Rosemary (Rosmarinus officinalis), which has been shown to exhibit a number of beneficial health properties. In particular, recently there has been growing interest into the anti-obesity effects conveyed by CA, including its ability to counteract obesity-associated hyperglycaemia and insulin resistance. However, the mechanisms underlying its anti-diabetic responses are not fully understood. In this study, we hypothesized that CA may act to improve glycaemic status through enhancing peripheral glucose clearance. Herein, we demonstrate that CA acts to mimic the metabolic actions of insulin by directly stimulating glucose uptake in rat skeletal L6 myotubes, concomitant with increased translocation of the GLUT4 glucose transporter to the plasma membrane. Mechanistically, CA-induced glucose transport was found to be dependent on protein kinase B (PKB/Akt) but not AMPK, despite both kinases being activated by CA. Crucially, in accordance with its ability to activate PKB and stimulate glucose uptake, we show that CA conveys these effects through a pathway involving PME-1 (protein phosphatase methylesterase-1), a key negative regulator of the serine/threonine phosphatase PP2A (protein phosphatase 2A). Herein, we demonstrate that CA promotes PME-1 mediated demethylation of the PP2A catalytic subunit leading to its suppressed activity, and in doing so, alleviates the repressive action of PP2A towards PKB. Collectively, our findings provide new insight into how CA may improve glucose homeostasis through enhancing peripheral glucose clearance in tissues such as skeletal muscle through a PME-1/PP2A/PKB signalling axis, thereby mitigating pathological effects associated with the hyperglycaemic state.

The endocannabinoid system (ECS) regulates numerous cellular and physiological processes through the activation of receptors targeted by endogenously produced ligands called endocannabinoids. Importantly, this signaling system is known to play an important role in modulating energy balance and glucose homeostasis. For example, current evidence indicates that the ECS becomes overactive during obesity whereby its central and peripheral stimulation drives metabolic processes that mimic the metabolic syndrome. Herein, we examine the role of the ECS in modulating the function of mitochondria, which play a pivotal role in maintaining cellular and systemic energy homeostasis, in large part due to their ability to tightly coordinate glucose and lipid utilization. Because of this, mitochondrial dysfunction is often associated with peripheral insulin resistance and glucose intolerance as well as the manifestation of excess lipid accumulation in the obese state. This review aims to highlight the different ways through which the ECS may impact upon mitochondrial abundance and/or oxidative capacity and, where possible, relate these findings to obesity-induced perturbations in metabolic function. Furthermore, we explore the potential implications of these findings in terms of the pathogenesis of metabolic disorders and how these may be used to strategically develop therapies targeting the ECS.

The phospholipid l‐α‐lysophosphatidylinositol (LPI), an endogenous ligand for GPR55, is elevated in patients with acute coronary syndrome, and a GPR55 antagonist cannabidiol (CBD) reduces experimental ischemia/reperfusion (I/R) injury. While LPI activates multiple signaling pathways, little is known about which ones are important in cardiomyocytes. In this study we explored whether activation of the Rho kinase/ROCK/p38 MAPK pathway is responsible for LPI‐induced extension of I/R injury. Using a high‐throughput screening method (dynamic mass redistribution; DMR), mouse‐ and human‐induced pluripotent stem cell (iPSC) cardiomyocytes exposed to LPI were shown to exhibit a rapid, sustained, and concentration‐dependent (1 nmol L−1‐30 μmol L−1) cellular response. Y‐27632 (ROCK inhibitor; 10 & 50 μmol L−1) and CBD (1 μmol L−1) both abolished the DMR response to LPI (10 μmol L−1). In murine iPSC cardiomyocytes, LPI‐induced ROCK and p38 MAPK phosphorylation, both of which were prevented by Y‐27632 and CBD, but did not induce JNK activation or cleavage of caspase‐3. In hearts isolated from wild type (WT) mice subjected to 30 minutes global I/R, LPI (10 μmol L−1) administered via the coronary circulation increased infarct size when applied prior to ischemia onset, but not when given at the time of reperfusion. The exacerbation of tissue injury by LPI was not seen in hearts from GPR55−/− mice or in the presence of Y‐27632, confirming that injury is mediated via the GPR55/ROCK/p38 MAPK pathway. These findings suggest that raised levels of LPI in the vicinity of a developing infarct may worsen the outcome of AMI.

In recent years, abnormal regulation of the endocannabinoid system (ECS) has been proposed as a key factor in the development of obesity-related metabolic disorders such as diabetes. Indeed, this signaling system which includes the cannabinoid type 1 and 2 receptors (CB1R and CB2R) and their endogenous lipid ligands, has been shown to influence feeding behavior, energy expenditure, as well as glucose and lipid metabolism. Importantly, blocking CB1R function has been demonstrated to counteract metabolic aberrations associated with obesity in various murine models and in humans. Here we provide an update on recent findings describing the role of the ECS in energy balance and metabolism, and explore how recent experimental and clinical studies have delivered new insights into the therapeutic potential of this physiological system as a means of treating obesity-induced metabolic disorders. WIREs Membr Transp Signal 2013, 2:49–63. doi: 10.1002/wmts.79 For further resources related to this article, please visit the WIREs website.

The endocannabinoid system (ECS) is a key cellular signalling system that has been implicated in the regulation of diverse cellular functions. Importantly, growing evidence suggests that the biological actions of the ECS may, in part, be mediated through its ability to regulate the production and/or release of nitric oxide, a ubiquitous bioactive molecule, which functions as a versatile signalling intermediate. Herein, we review and discuss evidence pertaining to ECS-mediated regulation of nitric oxide production, as well as the involvement of reactive nitrogen species in regulating ECS-induced signal transduction by highlighting emerging work supporting nitrergic modulation of ECS function. Importantly, the studies outlined reveal that interactions between the ECS and nitrergic signalling systems can be both stimulatory and inhibitory in nature, depending on cellular context. Moreover, such crosstalk may act to maintain proper cell function, whereas abnormalities in either system can undermine cellular homoeostasis and contribute to various pathologies associated with their dysregulation. Consequently, future studies targeting these signalling systems may provide new insights into the potential role of the ECS–nitric oxide signalling axis in disease development and/or lead to the identification of novel therapeutic targets for the treatment of nitrosative stress-related neurological, cardiovascular, and metabolic disorders.

Growing evidence suggests that pathological overactivation of the endocannabinoid system (ECS) is associated with dyslipidemia, obesity and diabetes. Indeed, this signalling system acting through cannabinoid receptors has been shown to function both centrally and peripherally to regulate feeding behaviour as well as energy expenditure and metabolism. Consequently, modulation of these receptors can promote significant alterations in body weight and associated metabolic profile. Importantly, blocking cannabinoid receptor type 1 function has been found to prevent obesity and metabolic dysfunction in various murine models and in humans. Here we provide a detailed account of the known physiological role of the ECS in energy balance, and explore how recent studies have delivered novel insights into the potential targeting of this system as a therapeutic means for treating obesity and related metabolic disorders.

OBJECTIVE The endogenous cannabinoid (or endocannabinoid) system (ECS) is part of a central neuromodulatory system thought to play a key role in the regulation of feeding behavior and energy balance. However, increasing evidence suggests that modulation of the ECS may also act to regulate peripheral mechanisms involved in these processes, including lipogenesis in adipose tissue and liver, insulin release from pancreatic β-cells, and glucose uptake into skeletal muscle. It was recently shown that cannabinoid receptor type 1 (CB1) and type 2 (CB2), both key components of the ECS, are expressed in human and rodent skeletal muscle. However, their role in modulating insulin sensitivity in this metabolically active tissue has yet to be determined. Our aim was to establish the role, if any, of these receptors in modulating insulin sensitivity in skeletal muscle cells. RESEARCH DESIGN AND METHODS Cultured skeletal muscle cells were exposed to CB1 and/or CB2 pharmacological agonists/antagonists/inverse agonists, and the resulting effects on insulin-regulated phosphatidylinositol 3 kinase (PI 3-kinase)–protein kinase B (PKB) and extracellular signal–related kinases 1/2 (ERK1/2)-directed signaling were determined. RESULTS Here, we report that modulating the activity of the ECS in skeletal muscle regulates both insulin-dependent mitogen-activated protein (MAP) kinase (ERK1/2) and the canonical PI 3-kinase/PKB signaling pathways. We show that pharmacological activation or inhibition of CB1 receptor activity exerts a differential effect with regard to MAP kinase– and PKB-directed signaling. CONCLUSIONS Our study provides evidence that signaling via cannabinoid receptors can significantly modulate mitogenic and metabolic signaling in skeletal muscle with important implications for muscle growth and differentiation as well as the regulation of glucose and lipid metabolism.

Insulin resistance is a recognized feature of PCOS (polycystic ovary syndrome). However, the molecular reason(s) underlying this reduced cellular insulin sensitivity is not clear. The present study compares the major insulin signalling pathways in skeletal muscle isolated from PCOS and controls. We measured whole-body insulin sensitivity and insulin signalling in skeletal muscle biopsies taken before and after acute exposure to hyperinsulinaemia in nine women diagnosed with PCOS and seven controls. We examined the expression, basal activity and response to in vivo insulin stimulation of three signalling molecules within these human muscle samples, namely IRS-1 (insulin receptor substrate-1), PKB (protein kinase B) and ERK (extracellular-signal-regulated kinase) 1/2. There was no significant difference in the expression, basal activity or activation of IRS-1 or PKB between PCOS and control subjects. However, there was a severe attenuation of insulin stimulation of the ERK pathway in muscle from all but two of the women with PCOS (the two most obese), and an accompanying trend towards higher basal phosphorylation of ERK1/2 in PCOS. These results are striking in that the metabolic actions of insulin are widely believed to require the IRS-1/PKB pathway rather than ERK, and the former has been reported as defective in some previous PCOS studies. Most importantly, the molecular defect identified was independent of adiposity. The altered response of ERK to insulin in PCOS was the most obvious signalling defect associated with insulin resistance in muscle from these patients.

PDK1 activates a group of kinases, including protein kinase B (PKB)/Akt, p70 ribosomal S6 kinase (S6K), and serum and glucocorticoid-induced protein kinase (SGK), that mediate many of the effects of insulin as well as other agonists. PDK1 interacts with phosphoinositides through a pleckstrin homology (PH) domain. To study the role of this interaction, we generated knock-in mice expressing a mutant of PDK1 incapable of binding phosphoinositides. The knock-in mice are significantly small, insulin resistant, and hyperinsulinemic. Activation of PKB is markedly reduced in knock-in mice as a result of lower phosphorylation of PKB at Thr308, the residue phosphorylated by PDK1. This results in the inhibition of the downstream mTOR complex 1 and S6K1 signaling pathways. In contrast, activation of SGK1 or p90 ribosomal S6 kinase or stimulation of S6K1 induced by feeding is unaffected by the PDK1 PH domain mutation. These observations establish the importance of the PDK1-phosphoinositide interaction in enabling PKB to be efficiently activated with an animal model. Our findings reveal how reduced activation of PKB isoforms impinges on downstream signaling pathways, causing diminution of size as well as insulin resistance.

Insulin regulation of hepatic gene transcription is a vital component of glucose homeostasis. Understanding the molecular regulationof thisprocess aids the searchfor the defect(s) that promotesinsulin-resistant states, such asdiabetesmellitus. We havepreviously shownthat the insulin regulationof hepatic IGF-binding protein-1 (IGFBP1) expression requiresthe signalling proteins phosphatidylinositol 3-kinase (PI 3-kinase) and mammalian target of rapamycin (mTOR). In this report, we demonstrate that activation of the mTOR pathway, without activation of its upstream regulator PI 3-kinase, reduces IGFBP1 expression. Therefore, mTOR activation is sufficient to mimic insulin regulation of this gene. However, longer exposure (>3 h) of cells to insulin reduces the importance of this pathway in insulin regulation of the gene, suggesting a temporal switch in signalling mechanisms linking insulin action to the IGFBP1 gene promoter. In contrast, the activation of PI 3-kinase is required for insulin regulation of IGFBP1 under all conditions tested. Therefore, an mTOR-independent, PI 3-kinase-dependent pathway becomes more important in IGFBP1 regulation after long exposure to insulin. This is a novel concept in insulin regulation of gene expression and demonstrates the importance of temporal analysis of signalling processes.

Reduced insulin-mediated glucose transport in skeletal muscle is a hallmark of the pathophysiology of T2DM (Type II diabetes mellitus). Impaired intracellular insulin signalling is implicated as a key underlying mechanism. Attention has focused on early signalling events such as defective tyrosine phosphorylation of IRS1 (insulin receptor substrate-1), a major target for the insulin receptor tyrosine kinase. This is required for normal induction of signalling pathways key to many of the metabolic actions of insulin. Conversely, increased serine/threonine phosphorylation of IRS1 following prolonged insulin exposure (or in obesity) reduces signalling capacity, partly by stimulating IRS1 degradation. We now show that IRS1 levels in human muscle are actually increased 3-fold following 1 h of hyperinsulinaemic euglycaemia. Similarly, transient induction of IRS1 (3-fold) in the liver or muscle of rodents occurs following feeding or insulin injection respectively. The induction by insulin is also observed in cell culture systems, although to a lesser degree, and is not due to reduced proteasomal targeting, increased protein synthesis or gene transcription. Elucidation of the mechanism by which insulin promotes IRS1 stability will permit characterization of the importance of this novel signalling event in insulin regulation of liver and muscle function. Impairment of this process would reduce IRS1 signalling capacity, thereby contributing to the development of hyperinsulinaemia/insulin resistance prior to the appearance of T2DM.

International audience; Aims/Hypothesis: Reduced skeletal muscle insulin sensitivity is a feature associated with sustained exposure to excess saturated fatty acids (SFA), whereas mono and polyunsaturated fatty acids (MUFA and PUFA) not only improve insulin sensitivity but blunt SFA-induced insulin resistance. The mechanisms by which MUFAs and PUFAs institute these favourable changes remain unclear, but may involve stimulating insulin signalling by counter-modulation/repression of protein phosphatase 2A (PP2A). This study investigated the effects of oleic acid (OA; a MUFA), linoleic acid (LOA; a PUFA) and palmitate (PA; a SFA) in cultured myotubes and determined whether changes in insulin signalling can be attributed to PP2A regulation. Principal Findings: We treated cultured skeletal myotubes with unsaturated and saturated fatty acids and evaluated insulin signalling, phosphorylation and methylation status of the catalytic subunit of PP2A. Unlike PA, sustained incubation of rat or human myotubes with OA or LOA significantly enhanced Akt-and ERK1/2-directed insulin signalling. This was not due to heightened upstream IRS1 or PI3K signalling nor to changes in expression of proteins involved in proximal insulin signalling, but was associated with reduced dephosphorylation/inactivation of Akt and ERK1/2. Consistent with this, PA reduced PP2Ac demethylation and tyrosine 307 phosphorylation-events associated with PP2A activation. In contrast, OA and LOA strongly opposed these PA-induced changes in PP2Ac thus exerting a repressive effect on PP2A.Conclusions/Interpretation: Beneficial gains in insulin sensitivity and the ability of unsaturated fatty acids to oppose palmitate-induced insulin resistance in muscle cells may partly be accounted for by counter-modulation of PP2A.

International audience; Ceramides are known to promote insulin resistance in a number of metabolically important tissues including skeletal muscle, the predominant site of insulin-stimulated glucose disposal. Depending on cell type, these lipid intermediates have been shown to inhibit protein kinase B (PKB/Akt), a key mediator of the metabolic actions of insulin, via two distinct pathways: one involving the action of atypical protein kinase C (aPKC) isoforms, and the second dependent on protein phosphatase-2A (PP2A). The main aim of this study was to explore the mechanisms by which ceramide inhibits PKB/Akt in three different skeletal muscle-derived cell culture models; rat L6 myotubes, mouse C2C12 myotubes and primary human skeletal muscle cells. Our findings indicate that the mechanism by which ceramide acts to repress PKB/Akt is related to the myocellular abundance of caveolin-enriched domains (CEM) present at the plasma membrane. Here, we show that ceramide-enriched-CEMs are markedly more abundant in L6 myotubes compared to C2C12 myotubes, consistent with their previously reported role in coordinating aPKC-directed repression of PKB/Akt in L6 muscle cells. In contrast, a PP2A-dependent pathway predominantly mediates ceramide-induced inhibition of PKB/Akt in C2C12 myotubes. In addition, we demonstrate for the first time that ceramide engages an aPKC-dependent pathway to suppress insulin-induced PKB/Akt activation in palmitate-treated cultured human muscle cells as well as in muscle cells from diabetic patients. Collectively, this work identifies key mechanistic differences, which may be linked to variations in plasma membrane composition, underlying the insulin-desensitising effects of ceramide in different skeletal muscle cell models that are extensively used in signal transduction and metabolic studies.

Insulin resistance (IR), an impaired cellular, tissue and whole body response to insulin, is a major pathophysiological defect of type 2 diabetes mellitus. Although IR is closely associated with obesity, the identity of the molecular defect(s) underlying obesity-induced IR in skeletal muscle remains controversial; reduced post-receptor signalling of the insulin receptor substrate 1 (IRS1) adaptor protein and downstream effectors such as protein kinase B (PKB) have previously been implicated. We examined expression and/or activation of a number of components of the insulin-signalling cascade in skeletal muscle of 22 healthy young men (with body mass index (BMI) range, 20-37 kg/m2). Whole body insulin sensitivity (M value) and body composition was determined by the hyperinsulinaemic (40 mU. min-1.m-2.), euglycaemic clamp and by dual energy X-ray absorptiometry (DEXA) respectively. Skeletal muscle (vastus lateralis) biopsies were taken before and after one hour of hyperinsulinaemia and the muscle insulin signalling proteins examined by western blot and immunoprecipitation assay. There was a strong inverse relationship between M-value and BMI. The most striking abnormality was significantly reduced insulin-induced activation of p42/44 MAP kinase, measured by specific assay, in the volunteers with poor insulin sensitivity. However, there was no relationship between individuals' BMI or M-value and protein expression/phosphorylation of IRS1, PKB, or p42/44 MAP kinase protein, under basal or hyperinsulinaemic conditions. In the few individuals with poor insulin sensitivity but preserved p42/44 MAP kinase activation, other signalling defects were evident. These findings implicate defective p42/44 MAP kinase signalling as a potential contributor to obesity-related IR in a non-diabetic population, although clearly multiple signalling defects underlie obesity associated IR.

The relationship between glucose and lipid metabolism has been of significant interest in understanding the pathogenesis of obesity-induced insulin resistance. To gain insight into this metabolic paradigm, we explored the potential interplay between cellular glucose flux and lipid-induced metabolic dysfunction within skeletal muscle. Here, we show that palmitate (PA)-induced insulin resistance and proinflammation in muscle cells, which is associated with reduced mitochondrial integrity and oxidative capacity, can be attenuated under conditions of glucose withdrawal or glycolytic inhibition using 2-deoxyglucose (2DG). Importantly, these glucopenic-driven improvements coincide with the preservation of mitochondrial function and are dependent on PA oxidation, which becomes markedly enhanced in the absence of glucose. Intriguingly, despite its ability to upregulate mitochondrial PA oxidation, glucose withdrawal did not attenuate PA-induced increases in total intramyocellular diacylglycerol and ceramide. Furthermore, consistent with our findings in cultured muscle cells, we also report enhanced insulin sensitivity and reduced proinflammatory tone in soleus muscle from obese Zucker rats fed a 2DG-supplemented diet. Notably, this improved metabolic status after 2DG dietary intervention is associated with markedly reduced plasma free fatty acids. Collectively, our data highlight the key role that mitochondrial substrate availability plays in lipid-induced metabolic dysregulation both in vitro and in vivo.

Obesity is a major risk factor for a variety of chronic diseases, including diabetes mellitus, and comorbidities such as cardiovascular disorders. Despite recommended alterations in lifestyle, including physical activity and energy restriction, being the foundation of any anti-obesity therapy, this approach has so far proved to be of little success in tackling this major public health concern. Because of this, alternative means of tackling this problem are currently being investigated, including pharmacotherapeutic intervention. Consequently, much attention has been directed towards elucidating the molecular mechanisms underlying the development of insulin resistance. This review discusses some of these potential mechanisms, with particular focus on the involvement of the sphingolipid ceramide. Various factors associated with obesity, such as saturated fatty acids and inflammatory cytokines, promote the synthesis of ceramide and other intermediates. Furthermore, studies performed in cultured cells and in vivo associate these sphingolipids with impaired insulin action. In light of this, we provide an account of the research investigating how pharmacological inhibition or genetic manipulation of enzymes involved in regulating sphingolipid synthesis can attenuate the insulin-desensitising effects of these obesity-related factors. By doing so, we outline potential therapeutic targets that may prove useful in the treatment of metabolic disorders.

Myostatin deficiency leads to both an increased rate of protein synthesis and skeletal muscle hypertrophy. However, the mechanisms involved in mediating these effects are not yet fully understood. Here, we demonstrate that genetic loss of myostatin leads to enhanced muscle expression of both protein kinase B and mammalian target of rapamycin/S6K signalling components, consistent with their elevated activity. This is associated with a reduction in the expression of PGC1α and COX IV, proteins which play important roles in maintaining mitochondrial function. Furthermore, we show that these changes in signalling and protein expression are largely independent of alterations in intramuscular amino acid content. Our findings, therefore, reveal potential new mechanisms and further contribute to our understanding of myostatin-regulated skeletal muscle growth and function.

Elevated ceramide concentrations in adipocytes and skeletal muscle impair PKB (protein kinase B; also known as Akt)-directed insulin signalling to key hormonal end points. An important feature of this inhibition involves the ceramide-induced activation of atypical PKCζ (protein kinase C-ζ), which associates with and negatively regulates PKB. In the present study, we demonstrate that this inhibition is critically dependent on the targeting and subsequent retention of PKCζ–PKB within CEM (caveolin-enriched microdomains), which is facilitated by kinase interactions with caveolin. Ceramide also recruits PTEN (phosphatase and tensin homologue detected on chromosome 10), a 3′-phosphoinositide phosphatase, thereby creating a repressive membrane microenvironment from which PKB cannot signal. Disrupting the structural integrity of caveolae by cholesterol depletion prevented caveolar targeting of PKCζ and PKB and suppressed kinase–caveolin association, but, importantly, also ameliorated ceramide-induced inhibition of PKB. Consistent with this, adipocytes from caveolin-1−/− mice, which lack functional caveolae, exhibit greater resistance to ceramide compared with caveolin-1+/+ adipocytes. We conclude that the recruitment and retention of PKB within CEM contribute significantly to ceramide-induced inhibition of PKB-directed signalling.

Background Glycogen Synthase Kinase-3 (GSK3) activity is repressed following insulin treatment of cells. Pharmacological inhibition of GSK3 mimics the effect of insulin on Phosphoeno lpyruvate Carboxykinase (PEPCK), Glucose-6 Phosphatase (G6Pase) and IGF binding protein-1 (IGFBP1) gene expression. CAAT/enhancer binding protein alpha (C/EBPα) regulates these gene promoters in liver and is phosphorylated on two residues (T222/T226) by GSK3, although the functional outcome of the phosphorylation has not been established. We aimed to establish whether CEBPα is a link between GSK3 and these gene promoters. Results C/EBPα represses the IGFBP1 thymine-rich insulin response element (TIRE), but mutation of T222 or T226 of C/EBPα to non-phosphorylatable alanines has no effect on C/EBPα activity in liver cells (towards the TIRE or a consensus C/EBP binding sequence). Phosphorylation of T222/T226 is decreased by GSK3 inhibition, suggesting GSK3 does phosphorylate T222/226 in intact cells. However, phosphorylation was not altered by treatment of liver cells with insulin. Meanwhile C/EBPα activity in 3T3 L1 preadipocytes was enhanced by mutation of T222/T226 and/or S230 to alanine residues. Finally, we demonstrate that C/EBPα is a very poor substrate for GSK3 in vitro and in cells. Conclusion The work demonstrates an important role for this domain in the regulation of C/EBPα activity in adipocytes but not hepatocytes, however GSK3 phosphorylation of these residues does not mediate regulation of this C/EBP activity. In short, we find no evidence that C/EBPα activity is regulated by direct phosphorylation by GSK3.

GSK3 (glycogen synthase kinase-3) regulation is proposed to play a key role in the hormonal control of many cellular processes. Inhibition of GSK3 in animal models of diabetes leads to normalization of blood glucose levels, while high GSK3 activity has been reported in Type II diabetes. Insulin inhibits GSK3 by promoting phosphorylation of a serine residue (Ser-21 in GSK3α, Ser-9 in GSK3β), thereby relieving GSK3 inhibition of glycogen synthesis in muscle. GSK3 inhibition in liver reduces expression of the gluconeogenic genes PEPCK (phosphoenolpyruvate carboxykinase), G6Pase (glucose-6-phosphatase), as well as IGFBP1 (insulin-like growth factor binding protein-1). Overexpression of GSK3 in cells antagonizes insulin regulation of these genes. In the present study we demonstrate that regulation of these three genes by feeding is normal in mice that express insulin-insensitive GSK3. Therefore inactivation of GSK3 is not a prerequisite for insulin repression of these genes, despite the previous finding that GSK3 activity is absolutely required for maintaining their expression. Interestingly, insulin injection of wild-type mice, which activates PKB (protein kinase B) and inhibits GSK3 to a greater degree than feeding (50% versus 25%), does not repress these genes. We suggest for the first time that although pharmacological inhibition of GSK3 reduces hepatic glucose production even in insulin-resistant states, feeding can repress the gluconeogenic genes without inhibiting GSK3.

The liver plays an important role in insulin-regulated glucose homoeostasis. To study the function of the PDK1 (3-phospho-inositide-dependent protein kinase-1) signalling pathway in mediating insulin's actions in the liver, we employed CRE recombinase/loxP technology to generate L(liver)-PDK1 - / - mice, which lack expression of PDK1 in hepatocytes and in which insulin failed to induce activation of PKB in liver. The L-PDK1 - / - mice were not insulin-intolerant, possessed normal levels of blood glucose and insulin under normal feeding conditions, but were markedly glucose-intolerant when injected with glucose. The L-PDK1 - / - mice also possessed 10-fold lower levels of hepatic glycogen compared with control littermates, and were unable to normalize their blood glucose levels within 2 h after injection of insulin. The glucose intolerance of the L-PDK1 - / - mice may be due to an inability of glucose to suppress hepatic glucose output through the gluconeogenic pathway, since the mRNA encoding hepatic PEPCK (phosphoenolpyruvate carboxykinase), G6Pase (glucose-6-phosphatase) and SREBP1 (sterol-regulatory-element-binding protein 1), which regulate gluconeogenesis, are no longer controlled by feeding. Furthermore, three other insulin-controlled genes, namely IGFBP1 (insulin-like-growth-factor-binding protein-1), IRS2 (insulin receptor substrate 2) and glucokinase, were regulated abnormally by feeding in the liver of PDK1-deficient mice. Finally, the L-PDK1 - / - mice died between 4-16 weeks of age due to liver failure. These results establish that the PDK 1 signalling pathway plays an important role in regulating glucose homoeostasis and controlling expression of insulin-regulated genes. They suggest that a deficiency of the PDK pathway in the liver could contribute to development of diabetes, as well as to liver failure.

Insulin rapidly and completely inhibits expression of the hepatic insulin-like growth factor binding protein-1 (IGFBP-1), phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) genes. This inhibition is mediated through a phosphatidyl inositol 3-kinase-dependent regulation of a DNA element, termed the thymine-rich insulin response element, found within the promoters of each of these genes. This has led to the conclusion that these three promoters are regulated by insulin using the same molecular mechanism. However, we recently found that the regulation of the IGFBP1 but not the PEPCK or G6Pase genes by insulin was sensitive to rapamycin, an inhibitor of mTOR. Here, we present further evidence that different regulatory pathways mediate the insulin regulation of these promoters. Importantly, we identify a protein phosphatase activity in the pathway connecting mTOR to the IGFBP-1 promoter. These data have major implications for the development of molecular therapeutics for the treatment of insulin-resistant states such as diabetes and hypertension.