Insulin resistance after a 72-h fast is associated with impaired AS160 phosphorylation and accumulation of lipid and glycogen in human skeletal muscle

During fasting, human skeletal muscle depends on lipid oxidation for its energy substrate metabolism. This is associated with the development of insulin resistance and a subsequent reduction of insulin-stimulated glucose uptake. The underlying mechanisms controlling insulin action on skeletal muscle under these conditions are unresolved. In a randomized design, we investigated eight healthy subjects after a 72-h fast compared with a 10-h overnight fast. Insulin action on skeletal muscle was assessed by a hyperinsulinemic euglycemic clamp and by determining insulin signaling to glucose transport. In addition, substrate oxidation, skeletal muscle lipid content, regulation of glycogen synthesis, and AMPK signaling were assessed. Skeletal muscle insulin sensitivity was reduced profoundly in response to a 72-h fast and substrate oxidation shifted to predominantly lipid oxidation. This was associated with accumulation of both lipid and glycogen in skeletal muscle. Intracellular insulin signaling to glucose transport was impaired by regulation of phosphorylation at specific sites on AS160 but not TBC1D1, both key regulators of glucose uptake. In contrast, fasting did not impact phosphorylation of AMPK or insulin regulation of Akt, both of which are established upstream kinases of AS160. These findings show that insulin resistance in muscles from healthy individuals is associated with suppression of site-specific phosphorylation of AS160, without Akt or AMPK being affected. This impairment of AS160 phosphorylation, in combination with glycogen accumulation and increased intramuscular lipid content, may provide the underlying mechanisms for resistance to insulin in skeletal muscle after a prolonged fast.

Download Full-text

Does impaired mitochondrial function affect insulin signaling and action in cultured human skeletal muscle cells?

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00267.2007 ◽

2008 ◽

Vol 294 (1) ◽

pp. E97-E102 ◽

Cited By ~ 8

Author(s):

Audrey E. Brown ◽

Matthias Elstner ◽

Stephen J. Yeaman ◽

Douglass M. Turnbull ◽

Mark Walker

Keyword(s):

Skeletal Muscle ◽

Glucose Uptake ◽

Insulin Signaling ◽

Mitochondrial Respiration ◽

Respiratory Function ◽

Insulin Action ◽

Human Skeletal Muscle ◽

Human Skeletal Muscle Cells ◽

Basal Glucose Uptake

Insulin-resistant type 2 diabetic patients have been reported to have impaired skeletal muscle mitochondrial respiratory function. A key question is whether decreased mitochondrial respiration contributes directly to the decreased insulin action. To address this, a model of impaired cellular respiratory function was established by incubating human skeletal muscle cell cultures with the mitochondrial inhibitor sodium azide and examining the effects on insulin action. Incubation of human skeletal muscle cells with 50 and 75 μM azide resulted in 48 ± 3% and 56 ± 1% decreases, respectively, in respiration compared with untreated cells mimicking the level of impairment seen in type 2 diabetes. Under conditions of decreased respiratory chain function, insulin-independent (basal) glucose uptake was significantly increased. Basal glucose uptake was 325 ± 39 pmol/min/mg (mean ± SE) in untreated cells. This increased to 669 ± 69 and 823 ± 83 pmol/min/mg in cells treated with 50 and 75 μM azide, respectively (vs. untreated, both P < 0.0001). Azide treatment was also accompanied by an increase in basal glycogen synthesis and phosphorylation of AMP-activated protein kinase. However, there was no decrease in glucose uptake following insulin exposure, and insulin-stimulated phosphorylation of Akt was normal under these conditions. GLUT1 mRNA expression remained unchanged, whereas GLUT4 mRNA expression increased following azide treatment. In conclusion, under conditions of impaired mitochondrial respiration there was no evidence of impaired insulin signaling or glucose uptake following insulin exposure in this model system.

Download Full-text

Protein kinase C modulates insulin action in human skeletal muscle

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.2000.278.3.e553 ◽

2000 ◽

Vol 278 (3) ◽

pp. E553-E562 ◽

Cited By ~ 67

Author(s):

Ronald N. Cortright ◽

John L. Azevedo ◽

Qian Zhou ◽

Madhur Sinha ◽

Walter J. Pories ◽

...

Keyword(s):

Insulin Resistance ◽

Skeletal Muscle ◽

Protein Kinase C ◽

Protein Kinase ◽

Glucose Uptake ◽

Glucose Transport ◽

Insulin Action ◽

Human Skeletal Muscle ◽

Kinase C ◽

Pkc Activation

There is good evidence from cell lines and rodents that elevated protein kinase C (PKC) overexpression/activity causes insulin resistance. Therefore, the present study determined the effects of PKC activation/inhibition on insulin-mediated glucose transport in incubated human skeletal muscle and primary adipocytes to discern a potential role for PKC in insulin action. Rectus abdominus muscle strips or adipocytes from obese, insulin-resistant, and insulin-sensitive patients were incubated in vitro under basal and insulin (100 nM)-stimulated conditions in the presence of GF 109203X (GF), a PKC inhibitor, or 12-deoxyphorbol 13-phenylacetate 20-acetate (dPPA), a PKC activator. PKC inhibition had no effect on basal glucose transport. GF increased ( P < 0.05) insulin-stimulated 2-deoxyglucose (2-DOG) transport approximately twofold above basal. GF plus insulin also increased ( P < 0.05) insulin receptor tyrosine phosphorylation 48% and phosphatidylinositol 3-kinase (PI 3-kinase) activity ∼50% ( P< 0.05) vs. insulin treatment alone. Similar results for GF on glucose uptake were observed in human primary adipocytes. Further support for the hypothesis that elevated PKC activity is related to insulin resistance comes from the finding that PKC activation by dPPA was associated with a 40% decrease ( P < 0.05) in insulin-stimulated 2-DOG transport. Incubation of insulin-sensitive muscles with GF also resulted in enhanced insulin action (∼3-fold above basal). These data demonstrate that certain PKC inhibitors augment insulin-mediated glucose uptake and suggest that PKC may modulate insulin action in human skeletal muscle.

Download Full-text

Effect of exercise on insulin action in human skeletal muscle

Journal of Applied Physiology ◽

10.1152/jappl.1989.66.2.876 ◽

1989 ◽

Vol 66 (2) ◽

pp. 876-885 ◽

Cited By ~ 228

Author(s):

E. A. Richter ◽

K. J. Mikines ◽

H. Galbo ◽

B. Kiens

Keyword(s):

Skeletal Muscle ◽

Glucose Uptake ◽

Insulin Action ◽

Human Skeletal Muscle ◽

Vastus Lateralis ◽

Femoral Vein ◽

Glycogen Synthesis ◽

Vastus Lateralis Muscle ◽

Glycogen Concentration ◽

Local Contraction

The effect of 1 h of dynamic one-legged exercise on insulin action in human muscle was studied in 6 healthy young men. Four hours after one-legged knee extensions, a three-step sequential euglycemic hyperinsulinemic clamp combined with arterial and bilateral femoral vein catheterization was performed. Increased insulin action on glucose uptake was found in the exercised compared with the rested thigh at mean plasma insulin concentrations of 23, 40, and 410 microU/ml. Furthermore, prior contractions directed glucose uptake toward glycogen synthesis and increased insulin effects on thigh O2 consumption and at some insulin concentrations on potassium exchange. In contrast, no change in insulin effects on limb exchange of free fatty acids, glycerol, alanine or tyrosine were found after exercise. Glycogen concentration in rested vastus lateralis muscle did not increase measurably during the clamp even though indirect estimates indicated net glycogen synthesis. In contrast, in exercised muscle estimated and biopsy-verified increases in muscle glycogen concentration agreed. Local contraction-induced increases in insulin sensitivity and responsiveness play an important role in postexercise recovery of human skeletal muscle.

Download Full-text

Glucose infusion causes insulin resistance in skeletal muscle of rats without changes in Akt and AS160 phosphorylation

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00133.2007 ◽

2007 ◽

Vol 293 (5) ◽

pp. E1358-E1364 ◽

Cited By ~ 34

Author(s):

Andrew J. Hoy ◽

Clinton R. Bruce ◽

Anna Cederberg ◽

Nigel Turner ◽

David E. James ◽

...

Keyword(s):

Insulin Resistance ◽

Skeletal Muscle ◽

Glucose Uptake ◽

Insulin Signaling ◽

Glycogen Synthase ◽

Glycogen Synthesis ◽

Glucose Infusion ◽

Metabolic Effects ◽

Synthesis Rate ◽

Muscle Sample

Hyperglycemia is a defining feature of Type 1 and 2 diabetes. Hyperglycemia also causes insulin resistance, and our group (Kraegen EW, Saha AK, Preston E, Wilks D, Hoy AJ, Cooney GJ, Ruderman NB. Am J Physiol Endocrinol Metab Endocrinol Metab 290: E471–E479, 2006) has recently demonstrated that hyperglycemia generated by glucose infusion results in insulin resistance after 5 h but not after 3 h. The aim of this study was to investigate possible mechanism(s) by which glucose infusion causes insulin resistance in skeletal muscle and in particular to examine whether this was associated with changes in insulin signaling. Hyperglycemia (∼10 mM) was produced in cannulated male Wistar rats for up to 5 h. The glucose infusion rate required to maintain this hyperglycemia progressively lessened over 5 h (by 25%, P < 0.0001 at 5 h) without any alteration in plasma insulin levels consistent with the development of insulin resistance. Muscle glucose uptake in vivo (44%; P < 0.05) and glycogen synthesis rate (52%; P < 0.001) were reduced after 5 h compared with after 3 h of infusion. Despite these changes, there was no decrease in the phosphorylation state of multiple insulin signaling intermediates [insulin receptor, Akt, AS160 (Akt substrate of 160 kDa), glycogen synthase kinase-3β] over the same time course. In isolated soleus strips taken from control or 1- or 5-h glucose-infused animals, insulin-stimulated 2-deoxyglucose transport was similar, but glycogen synthesis was significantly reduced in the 5-h muscle sample (68% vs. 1-h sample; P < 0.001). These results suggest that the reduced muscle glucose uptake in rats after 5 h of acute hyperglycemia is due more to the metabolic effects of excess glycogen storage than to a defect in insulin signaling or glucose transport.

Download Full-text

NOD1 activation induces proinflammatory gene expression and insulin resistance in 3T3-L1 adipocytes

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00709.2010 ◽

2011 ◽

Vol 301 (4) ◽

pp. E587-E598 ◽

Cited By ~ 50

Author(s):

Ling Zhao ◽

Pan Hu ◽

Yijun Zhou ◽

Jaanki Purohit ◽

Daniel Hwang

Keyword(s):

Insulin Resistance ◽

Glucose Uptake ◽

Insulin Signaling ◽

Serine Phosphorylation ◽

Dependent Manner ◽

Proinflammatory Response ◽

Downstream Target ◽

Synthetic Ligand ◽

Underlying Mechanisms ◽

Oligomerization Domain

Chronic inflammation is associated with obesity and insulin resistance; however, the underlying mechanisms are not fully understood. Pattern recognition receptors Toll-like receptors and nucleotide-oligomerization domain-containing proteins play critical roles in innate immune response. Here, we report that activation of nucleotide binding oligomerization domain-containing protein-1 (NOD1) in adipocytes induces proinflammatory response and impairs insulin signaling and insulin-induced glucose uptake. NOD1 and NOD2 mRNA are markedly increased in differentiated murine 3T3-L1 adipocytes and human primary adipocyte culture upon adipocyte conversion. Moreover, NOD1 mRNA is markedly increased only in the fat tissues in diet-induced obese mice, but not in genetically obese ob/ob mice. Stimulation of NOD1 with a synthetic ligand Tri-DAP induces proinflammatory chemokine MCP-1, RANTES, and cytokine TNF-α and MIP-2 (human IL-8 homolog) and IL-6 mRNA expression in 3T3-L1 adipocytes in a time- and dose-dependent manner. Similar proinflammatory profiles are observed in human primary adipocyte culture stimulated with Tri-DAP. Furthermore, NOD1 activation suppresses insulin signaling, as revealed by attenuated tyrosine phosphorylation and increased inhibitory serine phosphorylation, of IRS-1 and attenuated phosphorylation of Akt and downstream target GSK3α/3β, resulting in decreased insulin-induced glucose uptake in 3T3-L1 adipocytes. Together, our results suggest that NOD1 may play an important role in adipose inflammation and insulin resistance in diet-induced obesity.

Download Full-text

Abstract 1185: Role Of Sphingosine-1-Phosphate (S1P) In Insulin Signaling.

Circulation ◽

10.1161/circ.116.suppl_16.ii_239-d ◽

2007 ◽

Vol 116 (suppl_16) ◽

Author(s):

Suzanne M Nicholl ◽

Elisa Roztocil ◽

Mark G Davies

Keyword(s):

Insulin Resistance ◽

Skeletal Muscle ◽

Glucose Uptake ◽

Insulin Signaling ◽

Receptor Expression ◽

Serine Phosphorylation ◽

Glucose Disposal ◽

Sphingosine 1 Phosphate ◽

Low Glucose

A failure to increase glucose disposal into peripheral tissues in response to insulin leads to impaired insulin signaling and an inability to uptake glucose leading to the onset of insulin resistance, a major contributing factor to diabetes. We examined the role of sphingosine-1-phosphate (S1P) in insulin signaling and its ability to regulate glucose uptake in skeletal muscle cells. S1P, a sphingolipid found in abundance in the circulation, has been implicated in not only mediating crosstalk with other signaling pathways but has also been implicated in insulin resistance. We hypothesize that S1P interacts with post-receptor insulin signaling to increase glucose disposal in an in vitro model of insulin resistance using differentiated mouse skeletal C2C12 myotubes. Our data demonstrates that S1P (10μM) increases basal glucose levels similar to that observed in response to insulin (100nM) under conditions of low glucose (** p < 0.005: n = 3). Conversely, high glucose conditions completely inhibit both insulin and S1P stimulated glucose uptake (*p < 0.01:n = 3). Pre-incubation with S1P does not augment insulin-induced glucose uptake (***p < 0.001:n = 3), suggesting that S1P does not act via a separate signaling pathway. This is confirmed by our data demonstrating that S1P-induced glucose uptake is abrogated by Cytochalasin B (*p < 0.001:n = 3). In addition, the PI3-K inhibitors, LY294002 and Wortmannin, the Akt inhibitor, AKT2 and the p38MAPK inhibitor, SB203580 significantly inhibited glucose uptake in response to S1P, demonstrating their importance in S1P-induced glucose uptake (*p < 0.05:n = 3). S1P2 and S1P3 receptor expression were upregulated in response to insulin (~2-fold over basal) under low glucose conditions suggesting that insulin may regulate S1P signaling via one or both of these receptors. S1P increased serine phosphorylation of IRS1, both at serine 307 and serines 636/639 maximally after 15 minutes of stimulation. This data has important clinical implications in patients with metabolic syndrome who have impaired skeletal muscle glucose disposal due to insulin resistance and will help guide present and future therapy for patients who have this rapidly growing disease.

Download Full-text