Phosphatidylinositol 3-kinase (PI 3-kinase) has been implicated in the regulation of numerous cellular processes, including the insulin-induced regulation of glycogen synthase kinase 3 (GSK-3) and glucose transport. The hormonal-induced inactivation of GSK-3 is mediated by protein kinase B (PKB), a downstream target of PI 3-kinase, whose involvement in other insulin-stimulated responses remains poorly defined at present. In this study, we investigated whether the uptake of glucose, system A amino acid transport, and cellular protein synthesis are regulated by PKBalpha in L6 skeletal muscle cells. L6 cells stably overexpressing wild-type PKBalpha (wtPKBalpha) or a constitutively active membrane-targeted PKBalpha (mPKBalpha) showed a 3- and 15-fold increase in PKB activity, respectively. Both wtPKBalpha and mPKBalpha expression led to a significant increase in the basal uptake of glucose and methyl-aminoisobutyric acid (a substrate for the system A amino acid transporter), at least to a level seen in control cells treated with insulin. The stimulation in glucose transport was facilitated, in part, by the increased translocation of GLUT4 to the plasma membrane and also through an increase in the cellular synthesis of GLUT3. In the absence of insulin, only muscle cells expressing the constitutively active PKBalpha showed a significant increase in protein synthesis and an inhibition in GSK-3. Our results indicate that constitutive activation of PKBalpha in skeletal muscle stimulates the uptake of glucose, system A amino acids, and protein synthesis and promotes the inactivation of GSK-3. These observations imply that PKBalpha may have a role in the insulin-regulated control of these processes in skeletal muscle.

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 (PIP 3 ) 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 PIP 3 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)P 3 ] 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(3), allowing them to be phosphorylated by two upstream kinases. One of these, 3-phosphoinositide-dependent kinase-1 (PDK1), phosphorylates Thr 308 (4, 44), whereas the identity of the second kinase that phosphorylates Ser 473 (putatively termed PDK2) remains unknown, although a number of potential candidates have recently been proposed (for a review, see reference 15).The activation of PKB elicited by insulin and g...

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.

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 PKCzeta (5-fold). Inhibitors of serine palmitoyl transferase suppressed the intracellular synthesis of ceramide from palmitate, prevented PKCzeta 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 PKCzeta 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 PKCzeta activation are important aspects of the mechanism by which palmitate desensitizes L6 muscle cells to insulin.

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.

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.

The activation of phosphoinositide 3-kinase (PI3K) by insulin represents a key signalling event in the hormonal stimulation of diverse cellular responses including glucose transport and glycogen synthesis. The activation of PI3K increases the production of 4,5 trisphosphate [PtdIns(3,4,5)P 3 ] and phosphatidylinositol 3,4 bisphosphate [PtdIns(3,4)P 2 ]), which act as important signalling intermediates in the downstream activation of the serine/threonine kinase, Protein Kinase B (PKB/Akt). Activation of PKB depends upon its phosphorylation on two key amino acid residues, Thr 308 and Ser 473, with full activation requiring the phosphorylation of both [1]. The Nterminal domain of PKB contains a pleckstrin homology (PH) domain, which is thought to be critical in allowing the kinase to interact with 3-phospho- Diabetologia (2001) Abstract 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, C 2 -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 C 2 -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 mmol/l) for 2 h led to a complete loss of insulinstimulated 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-phosphoinositidedependent kinase-1, or to impaired binding of phosphatidylinositol 3,4,5 triphosphate (PtdIns(3,4,5)P 3 ) 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)P 3 is necessary but not sufficient for protein kinase B activation in this system. [Diabetologia (2001) 44: 173±183]

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.