scholarly journals Intracellular ceramide synthesis and protein kinase Cζ activation play an essential role in palmitate-induced insulin resistance in rat L6 skeletal muscle cells

2004 ◽  
Vol 382 (2) ◽  
pp. 619-629 ◽  
Author(s):  
Darren J. POWELL ◽  
Sophie TURBAN ◽  
Alexander GRAY ◽  
Eric HAJDUCH ◽  
Harinder S. HUNDAL
Keyword(s):  
Insulin Resistance ◽  
Skeletal Muscle ◽  
Protein Kinase ◽  
Glucose Transport ◽  
Insulin Signalling ◽  
Muscle Cells ◽  
Inhibitory Effect ◽  
L6 Myotubes ◽  
Skeletal Muscle Insulin Resistance ◽  
Ceramide Synthesis

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.

AMP kinase activation ameliorates insulin resistance induced by free fatty acids in rat skeletal muscle

2002 ◽  
Vol 283 (5) ◽  
pp. E965-E970 ◽  
Author(s):  
Grith S. Olsen ◽  
Bo F. Hansen
Keyword(s):  
Insulin Resistance ◽  
Skeletal Muscle ◽  
Glucose Transport ◽  
Prolonged Exposure ◽  
Glycogen Synthase ◽  
Glycogen Synthesis ◽  
Inhibitory Effect ◽  
Rat Skeletal Muscle ◽  
Compensatory Mechanisms ◽  
Skeletal Muscle Insulin Resistance

We examined whether acute activation of 5′-AMP-activated protein kinase (AMPK) by 5′-aminoimidazole-4-carboxamide-1-β-d-ribonucleoside (AICAR) ameliorates insulin resistance in isolated rat skeletal muscle. Insulin resistance was induced in extensor digitorum longus (EDL) muscles by prolonged exposure to 1.6 mM palmitate, which inhibited insulin-stimulated glycogen synthesis to 51% of control after 5 h of incubation. Insulin-stimulated glucose transport was less affected (22% of control). The decrease in glycogen synthesis was accompanied by decreased glycogen synthase (GS) activity and increased GS phosphorylation. When including 2 mM AICAR in the last hour of the 5-h incubation with palmitate, the inhibitory effect of palmitate on insulin-stimulated glycogen synthesis and glucose transport was eliminated. This effect of AICAR was accompanied by activation of AMPK. Importantly, AMPK inhibition was able to prevent this effect. Neither treatment affected total glycogen content. However, glucose 6-phosphate was increased after inclusion of AICAR, indicating increased influx of glucose. No effect of AICAR on the inhibited insulin-stimulated GS activity or increased GS phosphorylation by palmitate could be detected. Thus the mechanism by which AMPK activation ameliorates the lipid-induced insulin resistance probably involves induction of compensatory mechanisms overriding the insulin resistance. Our results emphasize AMPK as a promising molecular target for treatment of insulin resistance.

Modulating serine palmitoyl transferase (SPT) expression and activity unveils a crucial role in lipid-induced insulin resistance in rat skeletal muscle cells

2009 ◽  
Vol 417 (3) ◽  
pp. 791-801 ◽  
Author(s):  
Maria L. Watson ◽  
Matthew Coghlan ◽  
Harinder S. Hundal
Keyword(s):  
Insulin Resistance ◽  
Protein Kinase ◽  
Crucial Role ◽  
Insulin Signalling ◽  
Muscle Cells ◽  
Serine Phosphorylation ◽  
Short Term ◽  
Short Term Treatment ◽  
Ceramide Synthesis ◽  
Expression Activity

Saturated fatty acids, such as palmitate, promote accumulation of ceramide, which impairs activation and signalling of PKB (protein kinase B; also known as Akt) to important end points such as glucose transport. SPT (serine palmitoyl transferase) is a key enzyme regulating ceramide synthesis from palmitate and represents a potential molecular target in curbing lipid-induced insulin resistance. In the present study we explore the effects of palmitate upon insulin action in L6 muscle cells in which SPT expression/activity has been decreased by shRNA (small-hairpin RNA) or sustained incubation with myriocin, an SPT inhibitor. Incubation of L6 myotubes with palmitate (for 16 h) increases intramyocellular ceramide and reduces insulin-stimulated PKB activation and glucose uptake. PKB inhibition was not associated with impaired IRS (insulin receptor substrate) signalling and was ameliorated by short-term treatment with myriocin. Silencing SPT expression (∼90%) by shRNA or chronic cell incubation with myriocin (for 7 days) markedly suppressed SPT activity and palmitate-driven ceramide synthesis; however, challenging these muscle cells with palmitate still inhibited the hormonal activation of PKB. This inhibition was associated with reduced IRS1/p85-PI3K (phosphoinositide 3-kinase) coupling that arises from diverting palmitate towards greater DAG (diacylglycerol) synthesis, which elevates IRS1 serine phosphorylation via activation of DAG-sensitive PKCs (protein kinase Cs). Treatment of SPT-shRNA cells or those treated chronically with myriocin with PKC inhibitors antagonized palmitate-induced loss in insulin signalling. The findings of the present study indicate that SPT plays a crucial role in desensitizing muscle cells to insulin in response to incubation with palmitate. While short-term inhibition of SPT ameliorates palmitate/ceramide-induced insulin resistance, sustained loss/reduction in SPT expression/activity promotes greater partitioning of palmitate towards DAG synthesis, which impacts negatively upon IRS1-directed insulin signalling.

Reversal of chronic alterations of skeletal muscle protein kinase C from fat-fed rats by BRL-49653

1997 ◽  
Vol 273 (5) ◽  
pp. E915-E921 ◽  
Author(s):  
Carsten Schmitz-Peiffer ◽  
Nicholas D. Oakes ◽  
Carol L. Browne ◽  
Edward W. Kraegen ◽  
Trevor J. Biden
Keyword(s):  
Insulin Resistance ◽  
Skeletal Muscle ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Muscle Protein ◽  
Skeletal Muscle Protein ◽  
Insulin Sensitizer ◽  
Muscle Insulin Resistance ◽  
Skeletal Muscle Insulin Resistance ◽  
Kinase C

We have recently shown that the reduction in insulin sensitivity of rats fed a high-fat diet is associated with the translocation of the novel protein kinase Cε(nPKCε) from cytosolic to particulate fractions in red skeletal muscle and also the downregulation of cytosolic nPKCθ. Here we have further investigated the link between insulin resistance and PKC by assessing the effects of the thiazolidinedione insulin-sensitizer BRL-49653 on PKC isoenzymes in muscle. BRL-49653 increased the recovery of nPKC isoenzymes in cytosolic fractions of red muscle from fat-fed rats, reducing their apparent activation and/or downregulation, whereas PKC in control rats was unaffected. Because BRL-49653 also improves insulin-stimulated glucose uptake in fat-fed rats and reduces muscle lipid storage, especially diglyceride content, these results strengthen the association between lipid availability, nPKC activation, and skeletal muscle insulin resistance and support the hypothesis that chronic activation of nPKC isoenzymes is involved in the generation of muscle insulin resistance in fat-fed rats.

Restoring AS160 phosphorylation rescues skeletal muscle insulin resistance and fatty acid oxidation while not reducing intramuscular lipids

2009 ◽  
Vol 297 (5) ◽  
pp. E1056-E1066 ◽  
Author(s):  
Hakam Alkhateeb ◽  
Adrian Chabowski ◽  
Jan F. C. Glatz ◽  
Brendon Gurd ◽  
Joost J. F. P. Luiken ◽  
...  
Keyword(s):  
Insulin Resistance ◽  
Skeletal Muscle ◽  
Glucose Transport ◽  
Acid Oxidation ◽  
Glut4 Translocation ◽  
Muscle Insulin Resistance ◽  
Palmitate Oxidation ◽  
Skeletal Muscle Insulin Resistance ◽  
Ampk Phosphorylation ◽  
Intramuscular Lipids

We examined whether AICAR or leptin rapidly rescued skeletal muscle insulin resistance via increased palmitate oxidation, reductions in intramuscular lipids, and/or restoration of insulin-stimulated AS60 phosphorylation. Incubation with palmitate (2 mM, 0–18 h) induced insulin resistance in soleus muscle. From 12–18 h, palmitate was removed or AICAR or leptin was provided while 2 mM palmitate was maintained. Palmitate oxidation, intramuscular triacylglycerol, diacylglycerol, ceramide, AMPK phosphorylation, basal and insulin-stimulated glucose transport, plasmalemmal GLUT4, and Akt and AS160 phosphorylation were examined at 0, 6, 12, and 18 h. Palmitate treatment (12 h) increased intramuscular lipids (triacylglycerol +54%, diacylglycerol +11%, total ceramide +18%, C16:0 ceramide +60%) and AMPK phosphorylation (+118%), whereas it reduced fatty acid oxidation (−60%) and insulin-stimulated glucose transport (−70%), GLUT4 translocation (−50%), and AS160 phosphorylation (−40%). Palmitate removal did not rescue insulin resistance or associated parameters. The AICAR and leptin treatments did not consistently reduce intramuscular lipids, but they did rescue palmitate oxidation and insulin-stimulated glucose transport, GLUT4 translocation, and AS160 phosphorylation. Increased AMPK phosphorylation was associated with these improvements only when AICAR and leptin were present. Hence, across all experiments, AMPK phosphorylation did not correlate with any parameters. In contrast, palmitate oxidation and insulin-stimulated AS160 phosphorylation were highly correlated ( r = 0.83). We speculate that AICAR and leptin activate both of these processes concomitantly, involving activation of unknown kinases in addition to AMPK. In conclusion, despite the maintenance of high concentrations of palmitate (2 mM), as well as increased concentrations of intramuscular lipids (triacylglycerol, diacylglycerol, and ceramide), the rapid AICAR- and leptin-mediated rescue of palmitate-induced insulin resistance is attributable to the restoration of insulin-stimulated AS160 phosphorylation and GLUT4 translocation.

scholarly journals Ketoisocaproic acid, a metabolite of leucine, suppresses insulin-stimulated glucose transport in skeletal muscle cells in a BCAT2-dependent manner

2016 ◽  
Vol 311 (3) ◽  
pp. C518-C527 ◽  
Author(s):  
Mahshid Moghei ◽  
Pegah Tavajohi-Fini ◽  
Brendan Beatty ◽  
Olasunkanmi A. J. Adegoke
Keyword(s):  
Insulin Resistance ◽  
Skeletal Muscle ◽  
Amino Acids ◽  
Glucose Transport ◽  
Direct Effect ◽  
Inhibitory Effect ◽  
Dependent Manner ◽  
Positive Effects ◽  
Mtorc1 Signaling ◽  
Branched Chain

Although leucine has many positive effects on metabolism in multiple tissues, elevated levels of this amino acid and the other branched-chain amino acids (BCAAs) and their metabolites are implicated in obesity and insulin resistance. While some controversies exist about the direct effect of leucine on insulin action in skeletal muscle, little is known about the direct effect of BCAA metabolites. Here, we first showed that the inhibitory effect of leucine on insulin-stimulated glucose transport in L6 myotubes was dampened when other amino acids were present, due in part to a 140% stimulation of basal glucose transport ( P < 0.05). Importantly, we also showed that α-ketoisocaproic acid (KIC), an obligatory metabolite of leucine, stimulated mTORC1 signaling but suppressed insulin-stimulated glucose transport (−34%, P < 0.05) in an mTORC1-dependent manner. The effect of KIC on insulin-stimulated glucose transport was abrogated in cells depleted of branched-chain aminotransferase 2 (BCAT2), the enzyme that catalyzes the reversible transamination of KIC to leucine. We conclude that although KIC can modulate muscle glucose metabolism, this effect is likely a result of its transamination back to leucine. Therefore, limiting the availability of leucine, rather than those of its metabolites, to skeletal muscle may be more critical in the management of insulin resistance and its sequelae.

Improvement of insulin sensitivity by antagonism of the renin-angiotensin system

2007 ◽  
Vol 293 (3) ◽  
pp. R974-R980 ◽  
Author(s):  
Erik J. Henriksen
Keyword(s):  
Insulin Resistance ◽  
Skeletal Muscle ◽  
Glucose Transport ◽  
Renin Angiotensin System ◽  
Whole Body ◽  
Ang Ii ◽  
Angiotensin System ◽  
Skeletal Muscle Insulin Resistance

The reduced capacity of insulin to stimulate glucose transport into skeletal muscle, termed insulin resistance, is a primary defect leading to the development of prediabetes and overt type 2 diabetes. Although the etiology of this skeletal muscle insulin resistance is multifactorial, there is accumulating evidence that one contributor is overactivity of the renin-angiotensin system (RAS). Angiotensin II (ANG II) produced from this system can act on ANG II type 1 receptors both in the vascular endothelium and in myocytes, with an enhancement of the intracellular production of reactive oxygen species (ROS). Evidence from animal model and cultured skeletal muscle cell line studies indicates ANG II can induce insulin resistance. Chronic ANG II infusion into an insulin-sensitive rat produces a markedly insulin-resistant state that is associated with a negative impact of ROS on the skeletal muscle glucose transport system. ANG II treatment of L6 myocytes causes impaired insulin receptor substrate (IRS)-1-dependent insulin signaling that is accompanied by augmentation of NADPH oxidase-mediated ROS production. Further critical evidence has been obtained from the TG(mREN2)27 rat, a model of RAS overactivity and insulin resistance. The TG(mREN2)27 rat displays whole body and skeletal muscle insulin resistance that is associated with local oxidative stress and a significant reduction in the functionality of the insulin receptor (IR)/IRS-1-dependent insulin signaling. Treatment with a selective ANG II type 1 receptor antagonist leads to improvements in whole body insulin sensitivity, enhanced insulin-stimulated glucose transport in muscle, and reduced local oxidative stress. In addition, exercise training of TG(mREN2)27 rats enhances whole body and skeletal muscle insulin action. However, these metabolic improvements elicited by antagonism of ANG II action or exercise training are independent of upregulation of IR/IRS-1-dependent signaling. Collectively, these findings support targeting the RAS in the design of interventions to improve metabolic and cardiovascular function in conditions of insulin resistance associated with prediabetes and type 2 diabetes.

scholarly journals Involvement of protein kinase C in human skeletal muscle insulin resistance and obesity

Diabetes ◽  
2000 ◽  
Vol 49 (8) ◽  
pp. 1353-1358 ◽  
Author(s):  
S. I. Itani ◽  
Q. Zhou ◽  
W. J. Pories ◽  
K. G. MacDonald ◽  
G. L. Dohm
Keyword(s):  
Insulin Resistance ◽  
Skeletal Muscle ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Human Skeletal Muscle ◽  
Muscle Insulin Resistance ◽  
Skeletal Muscle Insulin Resistance ◽  
Kinase C

scholarly journals Dexamethasone impairs insulin signalling and glucose transport by depletion of insulin receptor substrate-1, phosphatidylinositol 3-kinase and protein kinase B in primary cultured rat adipocytes

2002 ◽  
pp. 419-429 ◽  
Author(s):  
J Buren ◽  
HX Liu ◽  
J Jensen ◽  
JW Eriksson
Keyword(s):  
Insulin Resistance ◽  
Protein Kinase ◽  
Glucose Uptake ◽  
Glucose Transport ◽  
Insulin Signalling ◽  
Protein Kinase B ◽  
Insulin Binding ◽  
Insulin Receptor Substrate ◽  
Phosphatidylinositol 3 Kinase ◽  
Rat Adipocytes

OBJECTIVE: Glucocorticoid excess leads to insulin resistance. This study explores the effects of glucocorticoids on the glucose transport system and insulin signalling in rat adipocytes. The interaction between glucocorticoids and high levels of insulin and glucose is also addressed. DESIGN AND METHODS: Isolated rat adipocytes were cultured for 24 h at different glucose concentrations (5 and 15 mmol/l) with or without the glucocorticoid analogue dexamethasone (0.3 micromol/l) and insulin (10(4) microU/ml). After the culture period, the cells were washed and then basal and insulin-stimulated glucose uptake, insulin binding and lipolysis as well as cellular content of insulin signalling proteins (insulin receptor substrate-1 (IRS-1), IRS-2, phosphatidylinositol 3-kinase (PI3-K) and protein kinase B (PKB)) and glucose transporter isoform GLUT4 were measured. RESULTS: Dexamethasone in the medium markedly decreased both basal and insulin-stimulated glucose uptake at both 5 and 15 mmol/l glucose (by approximately 40-50%, P<0.001 and P<0.05 respectively). Combined long-term treatment with insulin and dexamethasone exerted additive effects in decreasing basal, and to a lesser extent insulin-stimulated, glucose uptake capacity (P<0.05) compared with dexamethasone alone, but this was seen only at high glucose (15 mmol/l). Insulin binding was decreased (by approximately 40%, P<0.05) in dexamethasone-treated cells independently of surrounding glucose concentration. Following dexamethasone treatment a approximately 75% decrease (P<0.001) in IRS-1 expression and an increase in IRS-2 (by approximately 150%, P<0.001) was shown. Dexamethasone also induced a subtle decrease in PI3-K (by approximately 20%, P<0.01) and a substantial decrease in PKB content (by approximately 45%, P<0.001). Insulin-stimulated PKB phosphorylation was decreased (by approximately 40%, P<0.01) in dexamethasone-treated cells. Dexamethasone did not alter the amount of total cellular membrane-associated GLUT4 protein. The effects of dexamethasone per se on glucose transport and insulin signalling proteins were mainly unaffected by the surrounding glucose and insulin levels. Dexamethasone increased the basal lipolytic rate (approximately 4-fold, P<0.05), but did not alter the antilipolytic effect of insulin. CONCLUSIONS: These results suggest that glucocorticoids, independently of the surrounding glucose and insulin concentration, impair glucose transport capacity in fat cells. This is not due to alterations in GLUT4 abundance. Instead dexamethasone-induced insulin resistance may be mediated via reduced cellular content of IRS-1 and PKB accompanied by a parallel reduction in insulin-stimulated activation of PKB.

Cellular depletion of atypical PKCλ is associated with enhanced insulin sensitivity and glucose uptake in L6 rat skeletal muscle cells

2010 ◽  
Vol 299 (3) ◽  
pp. E402-E412 ◽  
Author(s):  
Clare Stretton ◽  
Ashleigh Evans ◽  
Harinder S. Hundal
Keyword(s):  
Skeletal Muscle ◽  
Protein Kinase ◽  
Insulin Receptor ◽  
Glucose Uptake ◽  
Glucose Transport ◽  
Insulin Signaling ◽  
Glycogen Synthase ◽  
Muscle Cells ◽  
Skeletal Muscle Cells ◽  
Dominant Negative Mutant

Atypical protein kinase C (aPKC) isoforms (λ and ζ) 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 PKCλ 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 PKCζ. Depletion of PKCλ (>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, PKCλ-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 PKCλ, overexpression of a dominant-negative mutant of PKCζ 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.

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