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Background Caveolin‐3 (Cav3) is the principal structural component of caveolae in skeletal muscle. Dominant pathogenic mutations in the Cav3 gene, such as the Limb Girdle Muscular Dystrophy‐1C (LGMD1C) P104L mutation, result in substantial loss of Cav3 and myopathic changes characterized by muscle weakness and wasting. We hypothesize such myopathy may also be associated with disturbances in mitochondrial biology. Herein, we report studies assessing the effects of Cav3 deficiency on mitochondrial form and function in skeletal muscle cells. Methods L6 myoblasts were stably transfected with Cav3P104L or expression of native Cav3 repressed by shRNA or CRISPR/Cas9 genome editing prior to performing fixed/live cell imaging of mitochondrial morphology, subcellular fractionation and immunoblotting, or analysis of real time mitochondrial respiration. Skeletal muscle from wild‐type and Cav3−/− mice was processed for analysis of mitochondrial proteins by immunoblotting. Results Caveolin‐3 was detected in mitochondrial‐enriched membranes isolated from mouse gastrocnemius muscle and L6 myoblasts. Expression of Cav3P104L in L6 myoblasts led to its targeting to the Golgi and loss of native Cav3 (>95%), including that associated with mitochondrial membranes. Cav3P104L reduced mitochondrial mass and induced fragmentation of the mitochondrial network that was associated with significant loss of proteins involved in mitochondrial biogenesis, respiration, morphology, and redox function [i.e. PGC1α, succinate dehyrdogenase (SDHA), ANT1, MFN2, OPA1, and MnSOD). Furthermore, Cav3P104L myoblasts exhibited increased mitochondrial cholesterol and loss of cardiolipin. Consistent with these changes, Cav3P104L expression reduced mitochondrial respiratory capacity and increased myocellular superoxide production. These morphological, biochemical, and functional mitochondrial changes were phenocopied in myoblasts in which Cav3 had been silenced/knocked‐out using shRNA or CRISPR. Reduced mitochondrial mass, PGC1α, SDHA, ANT1, and MnSOD were also demonstrable in Cav3−/− mouse gastrocnemius. Strikingly, Cav3 re‐expression in Cav3KO myoblasts restored its mitochondrial association and facilitated reformation of a tubular mitochondrial network. Significantly, re‐expression also mitigated changes in mitochondrial superoxide, cholesterol, and cardiolipin content and recovered cellular respiratory capacity. Conclusions Our results identify Cav3 as an important regulator of mitochondrial homeostasis and reveal that Cav3 deficiency in muscle cells associated with the Cav3P104L mutation invokes major disturbances in mitochondrial respiration and energy status that may contribute to the pathology of LGMD1C.

Sustained nutrient (fuel) excess, as occurs during obesity and diabetes, has been linked to increased inflammation, impaired mitochondrial homeostasis, lipotoxicity, and insulin resistance in skeletal muscle. Precisely how mitochondrial dysfunction is initiated and whether it contributes to insulin resistance in this tissue remains a poorly resolved issue. Herein, we examine the contribution that an increase in proinflammatory NFkB signalling makes towards regulation of mitochondrial bioenergetics, morphology, and dynamics and its impact upon insulin action in skeletal muscle cells subject to chronic fuel (glucose and palmitate) overloading. We show sustained nutrient excess of L6 myotubes promotes activation of the IKKβ-NFkB pathway (as judged by a six-fold increase in IL-6 mRNA expression; an NFkB target gene) and that this was associated with a marked reduction in mitochondrial respiratory capacity (>50%), a three-fold increase in mitochondrial fragmentation and 2.5-fold increase in mitophagy. Under these circumstances, we also noted a reduction in the mRNA and protein abundance of PGC1α and that of key mitochondrial components (SDHA, ANT-1, UCP3, and MFN2) as well as an increase in cellular ROS and impaired insulin action in myotubes. Strikingly, pharmacological or genetic repression of NFkB activity ameliorated disturbances in mitochondrial respiratory function/morphology, attenuated loss of SDHA, ANT-1, UCP3, and MFN2 and mitigated the increase in ROS and the associated reduction in myotube insulin sensitivity. Our findings indicate that sustained oversupply of metabolic fuel to skeletal muscle cells induces heightened NFkB signalling and that this serves as a critical driver for disturbances in mitochondrial function and morphology, redox status, and insulin signalling. Electronic supplementary material The online version of this article (10.1007/s00018-019-03148-8) contains supplementary material, which is available to authorized users.

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.

AMT-L is supported by the School of Biology, University of St Andrews. AMT-L, PAR and FJGM were funded by the Anonymous Trust, University of St Andrews. PAR is supported by the Melville Trust for the Care and Cure of Cancer. The mass spectrometry work was supported by the Wellcome Trust [grant number 094476/Z/10/Z], which funded the purchase of the TripleTOF 5600 mass spectrometer at the BSRC Mass Spectrometry and Proteomics Facility, University of St Andrews. The clinical study was supported by the Department of Pathology, Albert Einstein College of Medicine/ Montefiore Medical Center. Crumbs3 (CRB3) is a component of epithelial junctions that has been implicated in apical-basal polarity, apical identity, apical stability, cell adhesion and cell growth. CRB3 undergoes alternative splicing to yield two variants: CRB3a and CRB3b. Here, we describe novel data demonstrating that as with previous studies on CRB3a, CRB3b also promotes the formation of tight junctions. However, significantly we demonstrate that the 4.1-ezrin-radixin-moesin (FERM) binding motif (FBM) of CRB3b is required for CRB3b functionality and that ezrin binds to the FBM of CRB3b. Furthermore, we show that ezrin contributes to CRB3b functionality and the correct distribution of tight junction proteins. We demonstrate that both CRB3 isoforms are required for the production of functionally mature tight junctions and also the localization of ezrin to the plasma membrane. Finally, we demonstrate that reduced CRB3b expression in head and neck squamous cell carcinoma (HNSCC) correlates with cytoplasmic ezrin, a biomarker for aggressive disease, and show evidence that whilst CRB3a expression has no effect, low CRB3b and high cytoplasmic ezrin expression combined may be prognostic for HNSCC. Postprint

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.

Glycogen synthase kinase-3 (GSK3) mediates phosphorylation of raptor on Ser859, which crucially supports activation of mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) signalling in response to amino acid availability. GSK3 inhibition is associated with reduced mTORC1 signalling that impacts negatively on cell growth, protein synthesis and promotes cellular autophagy., The mammalian or mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) is a ubiquitously expressed multimeric protein kinase complex that integrates nutrient and growth factor signals for the co-ordinated regulation of cellular metabolism and cell growth. Herein, we demonstrate that suppressing the cellular activity of glycogen synthase kinase-3 (GSK3), by use of pharmacological inhibitors or shRNA-mediated gene silencing, results in substantial reduction in amino acid (AA)-regulated mTORC1-directed signalling, as assessed by phosphorylation of multiple downstream mTORC1 targets. We show that GSK3 regulates mTORC1 activity through its ability to phosphorylate the mTOR-associated scaffold protein raptor (regulatory-associated protein of mTOR) on Ser859. We further demonstrate that either GSK3 inhibition or expression of a S859A mutated raptor leads to reduced interaction between mTOR and raptor and under these circumstances, irrespective of AA availability, there is a consequential loss in phosphorylation of mTOR substrates, such as p70S6K1 (ribosomal S6 kinase 1) and uncoordinated-51-like kinase (ULK1), which results in increased autophagic flux and reduced cellular proliferation.

A hyperglycemic and hyperinsulinemic environment characteristic of type 2 diabetes causes insulin resistance. In adipocytes, defects in both insulin sensitivity and maximum response of glucose transport have been demonstrated. To investigate the molecular mechanisms, freshly isolated rat adipocytes were incubated in control (5.6 mM glucose, no insulin) and high glucose (20 mM)/high insulin (100 nM) (HG/HI) for 18 hours to induce insulin resistance. Insulin-resistant adipocytes manifested decreased sensitivity of glucose uptake associated with defects in insulin receptor substrate (IRS)-1 Tyr phosphorylation, association of p85 subunit of phosphatidylinositol-3-kinase, Akt Ser473 and Thr308 phosphorylation, accompanied by impaired glucose transporter 4 translocation. In contrast, protein kinase C (PKC)-ζ activity was augmented by chronic HG/HI. Inhibition of PKC-ζ with a specific cell-permeable peptide reversed the signaling defects and insulin sensitivity of glucose uptake. Transfection of dominant-negative, kinase-inactive PKC-ζ blocked insulin resistance, whereas constitutively active PKC-ζ recapitulated the defects. The HG/HI incubation was associated with stimulation of IRS-1 Ser318 and Akt Thr34 phosphorylation, targets of PKC-ζ. Transfection of IRS-1 S318A and Akt T34A each partially corrected insulin signaling, whereas combined transfection of both completely normalized insulin signaling. In vivo hyperglycemia/hyperinsulinemia in rats for 48 hours similarly resulted in activation of PKC-ζ and increased phosphorylation of IRS-1 Ser318 and Akt Thr34. These data indicate that impairment of insulin signaling by chronic HG/HI is mediated by dual defects at IRS-1 and Akt mediated by PKC-ζ.

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.

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.

Background: The role of AMPK and PKCs as effectors of metformin action on glucose uptake (GU) in skeletal muscle cells was investigated. Results: Genetic loss/silencing of AMPK led to only a small repression in metformin-stimulated GU. Novel/conventional, but not atypical, PKCs support metformin-induced stimulation of GU. Conclusion: Metformin enhances GU by a mechanism largely independent of AMPK. Significance: Metformin can act via non-AMPK pathways to promote GU., The importance of AMP-activated protein kinase (AMPK) and protein kinase C (PKC) as effectors of metformin (Met) action on glucose uptake (GU) in skeletal muscle cells was investigated. GU in L6 myotubes was stimulated 2-fold following 16 h of Met treatment and acutely enhanced by insulin in an additive fashion. Insulin-stimulated GU was sensitive to PI3K inhibition, whereas that induced by Met was not. Met and its related biguanide, phenformin, stimulated AMPK activation/phosphorylation to a level comparable with that induced by the AMPK activator, 5-amino-1-β-d-ribofuranosyl-imidazole-4-carboxamide (AICAR). However, the increase in GU elicited by AICAR was significantly lower than that induced by either biguanide. Expression of a constitutively active AMPK mimicked the effects of AICAR on GU, whereas a dominant interfering AMPK or shRNA silencing of AMPK prevented AICAR-stimulated GU and Met-induced AMPK signaling but only repressed biguanide-stimulated GU by ∼20%. Consistent with this, analysis of GU in muscle cells from α1−/−/α2−/− AMPK-deficient mice revealed a significant retention of Met-stimulated GU, being reduced by ∼35% compared with that of wild type cells. Atypical PKCs (aPKCs) have been implicated in Met-stimulated GU, and in line with this, Met and phenformin induced activation/phosphorylation of aPKC in L6 myotubes. However, although cellular depletion of aPKC (>90%) led to loss in biguanide-induced aPKC phosphorylation, it had no effect on Met-stimulated GU, whereas inhibitors targeting novel/conventional PKCs caused a significant reduction in biguanide-induced GU. Our findings indicate that although Met activates AMPK, a significant component of Met-stimulated GU in muscle cells is mediated via an AMPK-independent mechanism that involves novel/conventional PKCs.

Atypical protein kinase C (aPKC) isoforms (lambda and zeta) have been implicated in the control of insulin-stimulated glucose uptake in adipose and skeletal muscle, but their precise role in this process remains unclear, especially in light of accumulating evidence showing that, in response to numerous stimuli, including insulin and lipids such as ceramide, activation of aPKCs acts to negatively regulate key insulin-signaling molecules, such as insulin receptor substrate-1 (IRS-1) and protein kinase B (PKB)/cAMP-dependent PKC (Akt). In this study, we have depleted PKClambda in L6 skeletal muscle cells using RNA interference and assessed the effect this has upon insulin action. Muscle cells did not express detectable amounts of PKCzeta. Depletion of PKClambda (95%) had no significant effect on the expression of proteins participating in insulin signaling [i.e., insulin receptor, IRS-1, phosphatidylinositol 3-kinase (PI 3-kinase), PKB, or phosphate and tensin homolog deleted on chromosome 10] or those involved in glucose transport [Akt substrate of 160 kDa, glucose transporter (GLUT)1, or GLUT4]. However, PKClambda-depleted muscle cells exhibited greater activation of PKB/Akt and phosphorylation of its downstream target glycogen synthase kinase 3, in the basal state and displayed greater responsiveness to submaximal doses of insulin with respect to p85-PI 3-kinase/IRS-1 association and PKB activation. The increase in basal and insulin-induced signaling resulted in an associated enhancement of basal and insulin-stimulated glucose transport, both of which were inhibited by the PI 3-kinase inhibitor wortmannin. Additionally, like RNAi-mediated depletion of PKClambda, overexpression of a dominant-negative mutant of PKCzeta induced a similar insulin-sensitizing effect on PKB activation. Our findings indicate that aPKCs are likely to play an important role in restraining proximal insulin signaling events but appear dispensable with respect to insulin-stimulated glucose uptake in cultured L6 muscle cells.

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.

ABC50 is an ATP-binding cassette (ABC) protein, which, unlike most ABC proteins, does not possess membrane-spanning domains. ABC50 interacts with eukaryotic initiation factor 2 (eIF2), which plays a key role in translation initiation and its control. ABC50 binds to ribosomes, and this interaction requires both the N-terminal domain and at least one ABC domain. Knockdown of ABC50 by RNA interference impaired translation of both cap-dependent and -independent reporters, consistent with a positive role for ABC50 in the function of eIF2, which is required for both types of translation initiation. Mutation of the Walker box A or B motifs in both ABC regions of ABC50 yielded a mutant protein that exerted a dominant-interfering phenotype with respect to protein synthesis and translation initiation. Importantly, although dominant-interfering mutants of ABC50 impaired cap-dependent translation, translation driven by certain internal ribosome entry segments was not inhibited. ABC50 is located in the cytoplasm and nucleoplasm but not in the nucleolus. Thus, ABC50 is not likely to be directly involved in early ribosomal biogenesis, unlike some other ABC proteins. Taken together, the present data show that ABC50 plays a key role in translation initiation and has functions that are distinct from those of other non-membrane ABC proteins.

Amino acid transporters at the surface of cells are in an ideal location to relay nutritional information, as well as nutrients themselves, to the cell interior. These transporters are able to modulate signaling downstream of intracellular amino acid receptors by regulating intracellular amino acid concentrations through processes of coupled transport. The concept of dual-function amino acid transporter/receptor (or “transceptor”) proteins is well established in primitive eukaryotes such as yeast, where detection of extracellular amino acid deficiency leads to upregulation of proteins involved in biosynthesis and transport of the deficient amino acid(s). The evolution of the “extracellular milieu” and nutrient-regulated endocrine controls in higher eukaryotes, alongside their frequent inability to synthesize all proteinaceous amino acids (and, hence, the requirement for indispensable amino acids in their diet), appears to have lessened the priority of extracellular amino acid sensing as a stimulus for metabolic signals. Nevertheless, recent studies of amino acid transporters in flies and mammalian cell lines have revealed perhaps unanticipated “echoes” of these transceptor functions, which are revealed by cellular stresses (notably starvation) or gene modification/silencing. APC-transporter superfamily members, including slimfast, path, and SNAT2 all appear capable of sensing and signaling amino acid availability to the target of rapamycin (TOR) pathway, possibly through PI 3-kinase-dependent mechanisms. We hypothesize (by extrapolation from knowledge of the yeast Ssy1 transceptor) that, at least for SNAT2, the transceptor discriminates between extracellular and intracellular amino acid stimuli when evoking a signal.

Background: Cellular amino acid withdrawal/hypertonicity induces an adaptive increase in the expression, stability, and function of the SNAT2 amino acid transporter. Results: Linoleic acid, a polyunsaturated fatty acid, suppresses the adaptive increase by targeting SNAT2 for proteolysis. Conclusion: Linoleic acid promotes SNAT2 degradation via the ubiquitin/proteasome pathway. Significance: The study provides the first evidence that SNAT2 stability is modulated by fatty acid availability., Expression and activity of the System A/SNAT2 (SLC38A2) amino acid transporter is up-regulated by amino acid starvation and hypertonicity by a mechanism dependent on both ATF4-mediated transcription of the SLC38A2 gene and enhanced stabilization of SNAT2 itself, which forms part of an integrated cellular stress response to nutrient deprivation and osmotic stress. Here we demonstrate that this adaptive increase in System A function is restrained in cells subjected to prior incubation with linoleic acid (LOA, an unsaturated C18:2 fatty acid) for 24 h. While fatty acid treatment had no detectable effect upon stress-induced SNAT2 or ATF4 gene transcription, the associated increase in SNAT2 protein/membrane transport activity were strongly suppressed in L6 myotubes or HeLa cells preincubated with LOA. Cellular ubiquitination of many proteins was increased by LOA and although the fatty acid-induced loss of SNAT2 could be attenuated by proteasomal inhibition, the functional increase in System A transport activity associated with amino acid starvation/hypertonicity that depends upon processing/maturation and delivery of SNAT2 to the cell surface could not be rescued. LOA up-regulated cellular expression of Nedd4.2, an E3-ligase implicated in SNAT2 ubiquitination, but shRNA-directed Nedd4.2 gene silencing could not curb fatty acid-induced loss of SNAT2 adaptation. However, expression of SNAT2 in which seven putative lysyl-ubiquitination sites in the cytoplasmic N-terminal domain were mutated to alanine protected SNAT2 against LOA-induced proteasomal degradation. Collectively, our findings indicate that increased availability of unsaturated fatty acids can compromise the stress-induced induction/adaptation in SNAT2 expression and function by promoting its degradation via the ubiquitin-proteasome system.

System A and N amino acid transporters are key effectors of movement of amino acids across the plasma membrane of mammalian cells. These Na+-dependent transporters of the SLC38 gene family are highly sensitive to changes in pH within the physiological range, with transport markedly depressed at pH 7.0. We have investigated the possible role of histidine residues in the transporter proteins in determining this pH-sensitivity. The histidine-modifying agent DEPC (diethyl pyrocarbonate) markedly reduces the pH-sensitivity of SNAT2 and SNAT5 transporters (representative isoforms of System A and N respectively, overexpressed in Xenopus oocytes) in a concentration-dependent manner but does not completely inactivate transport activity. These effects of DEPC were reversed by hydroxylamine and partially blocked in the presence of excess amino acid substrate. DEPC treatment also blocked a reduction in apparent affinity for Na+ (K0.5Na+) of the SNAT2 transporter at low external pH. Mutation of the highly conserved C-terminal histidine residue to alanine in either SNAT2 (H504A) or SNAT5 (H471A) produced a transport phenotype exhibiting reduced, DEPC-resistant pH-sensitivity with no change in K0.5Na+ at low external pH. We suggest that the pH-sensitivity of these structurally related transporters results at least partly from a common allosteric mechanism influencing Na+ binding, which involves an H+-modifier site associated with C-terminal histidine residues.

It has been proposed that p38 mitogen-activated protein kinase (MAPK) isoforms sensitive to the pyridinylimidazole compounds SB 203580 and SB 202190 may participate in the acute insulin-dependent activation of glucose transporters recruited to the plasma membrane of adipocytes and skeletal muscle. Here, we explore whether these kinases support the insulin stimulation of glucose uptake in these tissues by investigating the effects of a genetic loss in p38beta and that of the p38 MAPK inhibitor SB 203580. Glucose uptake in adipocytes and soleus muscle was stimulated by insulin by up to fourfold irrespective of whether tissues were isolated from wild-type or p38beta-null mice. Consistent with this finding, mice lacking p38beta exhibited normal glucose tolerance, insulinemia, and glycemia compared with their wild-type counterparts. Insulin-stimulated glucose uptake was not inhibited by SB 203580 when adipocytes were preincubated with the drug at a cytocrit of 50%, but intriguingly, uptake was suppressed (by 35%) when the cytocrit was reduced by one-half. Despite the activation of glucose uptake at the higher cytocrit, insulin failed to induce any detectable activation of p38 MAPK, whereas p38 signaling was robustly activated by anisomycin in a SB 203580-sensitive manner. Although insulin also failed to induce any detectable activation of p38 MAPK in muscle, insulin-dependent glucose uptake was reduced by SB 203580 (approximately 44%) in muscle of both wild-type and p38beta-null mice. Our results indicate that p38beta is not required for insulin-stimulated glucose uptake in adipocytes or muscle. Moreover, given that insulin fails to promote any significant activation of p38 MAPK in these tissues and the finding that sensitivity of glucose uptake, but not that of the kinase, to SB 203580 can be influenced by cytocrit, we suggest that p38 signaling is unlikely to participate in any putative activation of transporters recruited to the cell surface by insulin and that SB 203580 suppresses insulin-stimulated glucose transport by a mechanism unrelated to its inhibitory effect on p38 MAPK.

We have investigated the expression and regulation of the Na+–K+–2Cl− cotransporter (NKCC) by insulin and hyperosmotic stress in L6 rat skeletal muscle cells. NKCC was identified by immunoblotting as a 170 kDa protein in L6 myotubes and mediated 54% of K+ (86Rb+) influx based on the sensitivity of ion transport to bumetanide, a NKCC inhibitor. The residual 86Rb+ influx occurred via the Na+,K+-ATPase and other transporters not sensitive to bumetanide or ouabain. NKCC-mediated 86Rb+ influx was enhanced significantly (∼1.6-fold) by acute cell exposure to insulin, but was inhibited significantly by tyrosine kinase inhibitors, wortmannin and rapamycin, consistent with a role for the insulin receptor tyrosine kinase, phosphoinositide 3 (PI3)-kinase and mTOR, respectively, in cotransporter activation. In contrast, the hormonal activation of NKCC was unaffected by inhibition of the classical Erk-signalling pathway. Subjecting L6 myotubes to an acute hyperosmotic challenge (420 mosmol l−1) led to a 40% reduction in cell volume and was accompanied by a rapid stimulation of NKCC activity (∼2-fold). Intracellular volume recovered to normal levels within 60 min, but this regulatory volume increase (RVI) was prevented if bumetanide was present. Unlike insulin, activation of NKCC by hyperosmolarity did not involve PI3-kinase but was suppressed by inhibition of tyrosine kinases and the Erk pathway. While inhibition of tyrosine kinases, using genistein, led to a complete loss in NKCC activation in response to hyperosmotic stress, immunoprecipitation of NKCC revealed that the cotransporter was not regulated directly by tyrosine phosphorylation. Simultaneous exposure of L6 myotubes to insulin and hyperosmotic stress led to an additive increase in NKCC-mediated 86Rb+ influx, of which, only the insulin-stimulated component was wortmannin-sensitive. Our findings indicate that L6 myotubes express a functional NKCC that is rapidly activated in response to insulin and hyperosmotic shock by distinct intracellular signalling pathways. Furthermore, activation of NKCC in response to hyperosmotic-induced cell shrinkage represents a critical component of the RVI mechanism that allows L6 muscle cells to volume regulate.

Non-esterified fatty acids (NEFAs) have been implicated in the pathogenesis of skeletal muscle insulin resistance that may develop, in part, as a consequence of a direct inhibitory effect on early insulin signalling events. Here we report work investigating the mechanism by which palmitate (a saturated free fatty acid) inhibits insulin action in rat L6 myotubes. Palmitate suppressed the insulin-induced plasma membrane recruitment and phosphorylation of protein kinase B (PKB) and this was associated with a loss in insulin-stimulated glucose transport. The inhibition in PKB was not due to a loss in insulin receptor substrate (IRS)1 tyrosine phosphorylation, IRS-1/p85 (phosphoinositide 3-kinase) association or suppression in phosphatidyl 3,4,5 triphosphate synthesis, but was attributable to an elevated intracellular synthesis of ceramide (6-fold) from palmitate and a concomitant activation of protein kinase PKCζ (5-fold). Inhibitors of serine palmitoyl transferase suppressed the intracellular synthesis of ceramide from palmitate, prevented PKCζ activation, and antagonized the inhibition in PKB recruitment/phosphorylation and the loss in insulin-stimulated glucose transport elicited by the NEFA. Inhibiting the palmitate-induced activation of PKCζ with Ro 31.8220, also prevented the loss in the insulin-dependent phosphorylation of PKB caused by palmitate. These findings indicate that intracellular ceramide synthesis and PKCζ activation are important aspects of the mechanism by which palmitate desensitizes L6 muscle cells to insulin.

Amino acids exert modulatory effects on proteins involved in control of mRNA translation in animal cells through the target of rapamycin (TOR) signaling pathway. Here we use oocytes of Xenopus laevis to investigate mechanisms by which amino acids are "sensed" in animal cells. Small ( approximately 48%) but physiologically relevant increases in intracellular but not extracellular total amino acid concentration (or Leu or Trp but not Ala, Glu, or Gln alone) resulted in increased phosphorylation of p70(S6K) and its substrate ribosomal protein S6. This response was inhibited by rapamycin, demonstrating that the effects require the TOR pathway. Alcohols of active amino acids substituted for amino acids with lower efficiency. Oocytes were refractory to changes in external amino acid concentration unless surface permeability of the cell to amino acids was increased by overexpression of the System L amino acid transporter. Amino acid-induced, rapamycin-sensitive activation of p70(S6K) was conferred when System L-expressing oocytes were incubated in extracellular amino acids, supporting intracellular localization of the putative amino acid sensor. In contrast to lower eukaryotes such as yeast, which possess an extracellular amino acid sensor, our findings provide the first direct evidence for an intracellular location for the putative amino acid sensor in animal cells that signals increased amino acid availability to TOR/p70(S6K).

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.

Aims/hypothesis. Increased cellular production of ceramide has been implicated in the pathogenesis of insulin resistance and in the impaired utilisation of glucose. In this study we have used L6 muscle cells to investigate the mechanism by which the short-chain ceramide analogue, C2-ceramide, promotes a loss in insulin sensitivity leading to a reduction in insulin stimulated glucose transport and glycogen synthesis. Method. L6 muscle cells were pre-incubated with C2-ceramide and the effects of insulin on glucose transport, glycogen synthesis and the activities of key molecules involved in proximal insulin signalling determined. Results. Incubation of L6 muscle cells with ceramide (100 μmol/l) for 2 h led to a complete loss of insulin-stimulated glucose transport and glycogen synthesis. This inhibition was not due to impaired insulin receptor substrate 1 phosphorylation or a loss in phosphoinositide 3-kinase activation but was caused by a failure to activate protein kinase B. This defect could not be attributed to inhibition of 3-phosphoinositide-dependent kinase-1, or to impaired binding of phosphatidylinositol 3,4,5 triphosphate (PtdIns(3,4,5)P3) to the PH domain of protein kinase B, but results from the inability to recruit protein kinase B to the plasma membrane. Expression of a membrane-targetted protein kinase B led to its constitutive activation and an increase in glucose transport that was not inhibited by ceramide. Conclusions/interpretation. These findings suggest that a defect in protein kinase B recruitment underpins the ceramide-induced loss in insulin sensitivity of key cell responses such as glucose transport and glycogen synthesis in L6 cells. They also suggest that a stimulated rise in PtdIns(3,4,5)P3 is necessary but not sufficient for protein kinase B activation in this system. [Diabetologia (2001) 44: 173–183]

We have investigated the cellular mechanisms that participate in reducing insulin sensitivity in response to increased oxidant stress in skeletal muscle. Measurement of glucose transport and glycogen synthesis in L6 myotubes showed that insulin stimulated both processes, by 2- and 5-fold, respectively. Acute (30 min) exposure of muscle cells to hydrogen peroxide (H(2)O(2)) blocked the hormonal activation of both these processes. Immunoblot analyses of cell lysates prepared after an acute oxidant challenge using phospho-specific antibodies against c-Jun N-terminal kinase (JNK), p38, protein kinase B (PKB), and p42 and p44 mitogen-activated protein (MAP) kinases established that H(2)O(2) induced a dose-dependent activation of all five protein kinases. In vitro kinase analyses revealed that 1 mM H(2)O(2) stimulated the activity of JNK by approximately 8-fold, MAPKAP-K2 (the downstream target of p38 MAP kinase) by approximately 12-fold and that of PKB by up to 34-fold. PKB activation was associated with a concomitant inactivation of glycogen synthase kinase-3. Stimulation of the p38 pathway, but not that of JNK, was blocked by SB 202190 or SB203580, while that of p42/p44 MAP kinases and PKB was inhibited by PD 98059 and wortmannin respectively. However, of the kinases assayed, only p38 MAP kinase was activated at H(2)O(2) concentrations (50 microM) that caused an inhibition of insulin-stimulated glucose transport and glycogen synthesis. Strikingly, inhibiting the activation of p38 MAP kinase using either SB 202190 or SB 203580 prevented the loss in insulin-stimulated glucose transport, but not that of glycogen synthesis, by oxidative stress. Our data indicate that activation of the p38 MAP kinase pathway plays a central role in the oxidant-induced inhibition of insulin-regulated glucose transport, and unveils an important biochemical link between the classical stress-activated and insulin signaling pathways in skeletal muscle.

In this study we show that serotonin (5-hydroxytryptamine (5-HT)) causes a rapid stimulation in glucose uptake by approximately 50% in both L6 myotubes and isolated rat skeletal muscle. This activation is mediated via the 5-HT2A receptor, which is expressed in L6, rat, and human skeletal muscle. In L6 cells, expression of the 5-HT2A receptor is developmentally regulated based on the finding that receptor abundance increases by over 3-fold during differentiation from myoblasts to myotubes. Stimulation of the 5-HT2A receptor using methylserotonin (m-HT), a selective 5-HT2A agonist, increased muscle glucose uptake in a manner similar to that seen in response to 5-HT. The agonist-mediated stimulation in glucose uptake was attributable to an increase in the plasma membrane content of GLUT1, GLUT3, and GLUT4. The stimulatory effects of 5-HT and m-HT were suppressed in the presence of submicromolar concentrations of ketanserin (a selective 5-HT2A antagonist) providing further evidence that the increase in glucose uptake was specifically mediated via the 5-HT2A receptor. Treatment of L6 cells with insulin resulted in tyrosine phosphorylation of IRS1, increased cellular production of phosphatidylinositol 3,4,5-phosphate and a 41-fold activation in protein kinase B (PKB/Akt) activity. In contrast, m-HT did not modulate IRS1, phosphoinositide 3-kinase, or PKB activity. The present results indicate that rat and human skeletal muscle both express the 5-HT2A receptor and that 5-HT and specific 5-HT2A agonists can rapidly stimulate glucose uptake in skeletal muscle by a mechanism which does not depend upon components that participate in the insulin signaling pathway.

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.

We have investigated the subcellular distribution and association of cellubrevin, a low molecular weight protein implicated in the process of membrane fusion, with intracellular membranes containing the insulin-sensitive GLUT4 glucose transporter from rat adipocytes, rat skeletal muscle and human skeletal muscle. SDS-PAGE and immunoblot analyses of subcellular fractions of adipocytes and skeletal muscle indicated a positive correlation between the distribution of GLUT4 and cellubrevin in intracellular membrane fractions tested from all tissues. The identity of the polypeptide reacting with antiserum against cellubrevin was further confirmed on the basis of its susceptibility to proteolysis by tetanus toxin. Immunoisolation of GLUT4-containing vesicles from a microsomal fraction enriched with GLUT4 and cellubrevin revealed that cellubrevin could be coprecipitated with GLUT4 vesicles from adipocytes. In contrast, intracellular GLUT4 vesicles isolated from both rat and human skeletal muscle were devoid of any detectable immunoreactivity towards cellubrevin. The observation that cellubrevin does not colocalise with intracellular GLUT4 in skeletal muscle from two different species, rat and human, would strongly suggest that it is unlikely to participate in the insulininduced delivery of GLUT4 to the cell surface in skeletal muscle.

The glutamine and glutamate transporters in skeletal muscle and heart appear to play a role in control of the steady-state concentration of amino acids in the intracellular space and, in the case of skeletal muscle at least, in the rate of loss of glutamine to the plasma and to other organs and tissues. This article reviews what is currently known about transporter characteristics and mechanisms in skeletal muscle and heart, the alterations in transport activity in pathophysiological conditions and the implications for anabolic processes and cardiac function of altering the availability of glutamine. The possibilities that glutamine pool size is part of an osmotic signaling mechanism to regulate whole body protein metabolism is discussed and evidence is shown from work on cultured muscle cells. The possible uses of glutamine in maintaining cardiac function perioperatively and in promoting glycogen metabolism are discussed.

Glycogen synthase kinase-3 (GSK3) is inactivated in vitro by p70 S6 kinase or MAP kinase-activated protein kinase-1 beta (MAPKAP kinase-1 beta; also known as Rsk-2). Here we show that GSK3 isoforms are inhibited by 40% within minutes after stimulation of the rat skeletal-muscle cell line L6 with insulin-like growth factor-1 (IGF-1) or insulin. GSK3 was similarly inhibited in rabbit skeletal muscle after an intravenous injection of insulin. Inhibition resulted from increased phosphorylation of GSK3, probably at a serine/threonine residue(s), because it was reversed by incubation with protein phosphatase-2A. Rapamycin blocked the activation of p70 S6 kinase by IGF-1 in L6 cells, but had no effect on the inhibition of GSK3 or the activation of MAPKAP kinase-1 beta. In contrast, wortmannin, a potent inhibitor of PtdIns 3-kinase, prevented the inactivation of GSK3 and the activation of MAPKAP kinase-1 beta and p70 S6 kinase by IGF-1 or insulin. Wortmannin also blocked the activation of p74raf-1. MAP kinase kinase and p42 MAP kinase, but not the formation of GTP-Ras by IGF-1. The results suggest that the stimulation of glycogen synthase by insulin/IGF-1 in skeletal muscle involves the MAP-KAP kinase-1-catalysed inhibition of GSK3, as well as the previously described activation of the glycogen-associated form of protein phosphatase-1.

Hindlimb skeletal muscles of the rat express two isoforms of the alpha (alpha 1 and alpha 2) and two isoforms of the beta (beta 1 and beta 2) subunits of the Na+,K(+)-ATPase. Because several muscles constitute the hindlimb, we investigated if specific isoforms are expressed in particular muscles. Northern blot analysis using isoform-specific cDNA probes demonstrated that soleus muscle expressed only the beta 1 transcript, whereas EDL or white gastrocnemius muscles expressed only the beta 2 transcript, and red gastrocnemius muscle expressed both mRNAs. All muscles tested expressed both alpha 1 and alpha 2 transcripts, albeit to various degrees: alpha 1 transcripts were present to about the same extent in all muscles but alpha 2 mRNA was 4-fold more abundant in soleus than in EDL for the same amount of total RNA. Beta subunit protein levels were investigated in purified plasma membrane fractions of pooled red (soleus + red gastrocnemius + red quadriceps) or white (white gastrocnemius + white quadriceps) muscles using isoform-specific antibodies. Red muscles expressed mostly the beta 1 protein while white muscles expressed mostly the beta 2 subunit. Both muscle groups had similar levels of alpha 1 or alpha 2 subunits, and crude membranes isolated from red muscles had 30% higher Na+,K(+)-ATPase activity than white muscle membranes. We conclude that oxidative muscles (slow and fast twitch) express beta 1 subunits, whereas glycolytic, fast twitch muscles express beta 2 subunits, and that both beta isoforms support the Na+,K(+)-ATPase activity of the alpha subunits.

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.

Intramuscular glutamine falls with injury and disease in circumstances associated with increases in blood corticosteroids. We have investigated the effects of corticosteroid administration (0.44 mg/kg dexamethasone daily for 8 days, 200 g female rats) on intramuscular glutamine and Na+, muscle glutamine metabolism and sarcolemmal glutamine transport in the perfused hindlimb. After dexamethasone treatment intramuscular glutamine fell by 45% and Na+ rose by 25% (the respective muscle/plasma distribution ratios changed from 8.6 to 4.5 and 0.12 to 0.15); glutamine synthetase and glutaminase activities were unchanged at 475 +/- 75 and 60 +/- 19 nmol/g muscle per min. Glutamine output by the hindlimb of anaesthetized rats was increased from 31 to 85 nmol/g per min. Sarcolemmal glutamine transport was studied by paired-tracer dilution in the perfused hindlimb: the maximal capacity (Vmax) for glutamine transport into muscle (by Na(+)-glutamine symport) fell from 1058 +/- 310 to 395 +/- 110 nmol/g muscle per min after dexamethasone treatment, accompanied by a decrease in the Km (from 8.1 +/- 1.9 to 2.1 +/- 0.4 mM glutamine). At physiological plasma glutamine concentration (0.75 mM) dexamethasone appeared to cause a proportional increase in sarcolemmal glutamine efflux over influx. Addition of dexamethasone (200 nM) to the perfusate of control rat hindlimbs caused acute changes in Vmax and Km of glutamine transport similar to those resulting from 8-day dexamethasone treatment. The reduction in muscle glutamine concentration after dexamethasone treatment may be primarily due to a reduction in the driving force for intramuscular glutamine accumulation, i.e., in the Na+ electrochemical gradient. The prolonged increase in muscle glutamine output after dexamethasone treatment (which occurs despite a reduction in the size of the intramuscular glutamine pool) appears to be due to a combination of (a) accelerated sarcolemmal glutamine efflux and (b) increased intramuscular synthesis of glutamine.

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.

Mammalian nutrient sensors are novel targets for therapeutic intervention in disease states such as insulin resistance and muscle wasting; however, the proteins responsible for this important task are largely uncharacterized. To address this issue we have dissected an amino acid (AA) sensor/effector regulon that controls the expression of the System A amino acid transporter SNAT2 in mammalian cells, a paradigm nutrient-responsive process, and found evidence for the convergence of at least two sensor/effector pathways. During AA withdrawal, JNK is activated and induces the expression of SNAT2 in L6 myotubes by stimulating an intronic nutrient-sensitive domain. A sensor for large neutral AA (e.g. Tyr, Gln) inhibits JNK activation and SNAT2 up-regulation. Additionally, shRNA and transporter chimeras demonstrate that SNAT2 provides a repressive signal for gene transcription during AA sufficiency, thus echoing AA sensing by transceptor (transporter-receptor) orthologues in yeast (Gap1/Ssy1) and Drosophila (PATH). Furthermore, the SNAT2 protein is stabilized during AA withdrawal.

An increase in circulating levels of specific NEFAs (non-esterified fatty acids) has been implicated in the pathogenesis of insulin resistance and impaired glucose disposal in skeletal muscle. In particular, elevation of SFAs (saturated fatty acids), such as palmitate, has been correlated with reduced insulin sensitivity, whereas an increase in certain MUFAs and PUFAs (mono- and poly-unsaturated fatty acids respectively) has been suggested to improve glycaemic control, although the underlying mechanisms remain unclear. In the present study, we compare the effects of palmitoleate (a MUFA) and palmitate (a SFA) on insulin action and glucose utilization in rat L6 skeletal muscle cells. Basal glucose uptake was enhanced approx. 2-fold following treatment of cells with palmitoleate. The MUFA-induced increase in glucose transport led to an associated rise in glucose oxidation and glycogen synthesis, which could not be attributed to activation of signalling proteins normally modulated by stimuli such as insulin, nutrients or cell stress. Moreover, although the MUFA-induced increase in glucose uptake was slow in onset, it was not dependent upon protein synthesis, but did, nevertheless, involve an increase in the plasma membrane abundance of GLUT1 and GLUT4. In contrast, palmitate caused a substantial reduction in insulin signalling and insulin-stimulated glucose transport, but was unable to antagonize the increase in transport elicited by palmitoleate. Our findings indicate that SFAs and MUFAs exert distinct effects upon insulin signalling and glucose uptake in L6 muscle cells and suggest that a diet enriched with MUFAs may facilitate uptake and utilization of glucose in normal and insulin-resistant skeletal muscle.

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.

Ceramide is generated in response to numerous stress-inducing stimuli and has been implicated in the regulation of diverse cellular responses, including cell death, differentiation, and insulin sensitivity. Recent evidence indicates that ceramide may regulate these responses by inhibiting the stimulus-mediated activation of protein kinase B (PKB), a key determinant of cell fate and insulin action. Here we show that inhibition of this kinase involves atypical PKC, which physically interacts with PKB in unstimulated cells. Insulin reduces the PKB-PKC interaction and stimulates PKB. However, dissociation of the kinase complex and the attendant hormonal activation of PKB were prevented by ceramide. Under these circumstances, ceramide activated PKC, leading to phosphorylation of the PKB-PH domain on Thr 34 . This phosphorylation inhibited phosphatidylinositol 3,4,5-trisphosphate (PIP3) binding to PKB, thereby preventing activation of the kinase by insulin. In contrast, a PKB-PH domain with a T34A mutation retained the ability to bind PIP3 even in the presence of a ceramide-activated PKC and, as such, expression of PKB T34A mutant in L6 cells was resistant to inhibition by ceramide treatment. Inhibitors of PKC and a kinase-dead PKC both antagonized the inhibitory effect of ceramide on PKB. Since PKB confers a prosurvival signal and regulates numerous pathways in response to insulin, suppressing its activation by a PKC-dependent process may be one mechanism by which ceramide promotes cell death and induces insulin resistance. Protein kinase B (PKB), also known as c-Akt, is a serine/ threonine kinase that has been implicated in the control of diverse cellular functions, including glucose metabolism, gene transcription, cell proliferation, and apoptosis (16, 27, 34, 48). Three PKB isoforms (, , and ) have been identified, and these can be activated rapidly in response to insulin and growth factors in a phosphoinositide 3-kinase (PI3K)-dependent manner. PI3K activation results in the increased production of 3-phosphoinositides, e.g., phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3] and phosphatidylinositol 3,4-bisphosphate, which play a key role in the recruitment of PKB to the plasma membrane (5). The N-terminal domain of all three PKB isoforms contains a pleckstrin homology (PH) domain, which is considered critical in allowing the kinase to interact with 3-phosphoinositides and possibly other signaling proteins (13, 16, 19). Binding of 3-phosphoinositides to the PH domain of PKB is also thought to induce conformational changes in the kinase that expose two key regulatory sites, Thr 308 and Ser 473

Glycogen synthase kinase 3 (GSK3) is inactivated by insulin and lithium and, like insulin, Li also activates glycogen synthase (GS) via inhibition of GSK3. Li also mimics insulin's ability to stimulate glucose transport (GT), an observation that has led to the suggestion that GSK3 may coordinate hormonal increases in GT and glycogen synthesis. Here we have used Li and SB-415286, a selective GSK3 inhibitor, to establish the importance of GSK3 in the hormonal activation of GT in terms of its effect on GS in L6 myotubes and 3T3-L1 adipocytes. Insulin, Li and SB-415286 all induced a significant inhibition of GSK3, which was associated with a marked dephosphorylation and activation of GS. In L6 myotubes, SB-415286 induced a much greater activation of GS (6.8-fold) compared to that elicited by insulin (4.2-fold) or Li (4-fold). In adipocytes, insulin, Li and SB-415286 all caused a comparable activation of GS despite a substantial differentiation-linked reduction in GSK3 expression ( approximately 85%) indicating that GSK3 remains an important determinant of GS activation in fat cells. Whilst Li and SB-415286 both inhibit GSK3 in muscle and fat cells, only Li stimulated GT. This increase in GT was not sensitive to inhibitors of PI3-kinase, MAP kinase or mTOR, but was suppressed by the p38 MAP kinase inhibitor, SB-203580. Consistent with this, phosphorylation of p38 MAP kinase induced by Li correlated with its stimulatory effect on GT. Our findings support a crucial role for GSK3 in the regulation of GS, but based on the differential effects of Li and SB-415286, it is unlikely that acute inhibition of GSK3 contributes towards the rapid stimulation of GT by insulin in muscle and fat cells.

Amino acid availability regulates cellular physiology by modulating gene expression and signal transduction pathways. However, although the signalling intermediates between nutrient availability and altered gene expression have become increasingly well documented, how eukaryotic cells sense the presence of either a nutritionally rich or deprived medium is still uncertain. From recent studies it appears that the intracellular amino acid pool size is particularly important in regulating translational effectors, thus, regulated transport of amino acids across the plasma membrane represents a means by which the cellular response to amino acids could be controlled. Furthermore, evidence from studies with transportable amino acid analogues has demonstrated that flux through amino acid transporters may act as an initiator of nutritional signalling. This evidence, coupled with the substrate selectivity and sensitivity to nutrient availability classically associated with amino acid transporters, plus the recent discovery of transporter-associated signalling proteins, demonstrates a potential role for nutrient transporters as initiators of cellular nutrient signalling. Here, we review the evidence supporting the idea that distinct amino acid “receptors” function to detect and transmit certain nutrient stimuli in higher eukaryotes. In particular, we focus on the role that amino acid transporters may play in the sensing of amino acid levels, both directly as initiators of nutrient signalling and indirectly as regulators of external amino acid access to intracellular receptor/signalling mechanisms.