Jonathan S. Fisher

Skeletal Muscle Insulin Resistance: Roles of Fatty Acid Metabolism and Exercise

The purpose of this review is to provide information about the role of exercise in the prevention of skeletal muscle insulin resistance, that is, the inability of insulin to properly cause glucose uptake into skeletal muscle. Insulin resistance is associated with high levels of stored lipids in skeletal muscle cells. Aerobic exercise training decreases the amounts of these lipid products and increases the lipid oxidative capacity of muscle cells. Thus, aerobic exercise training may prevent insulin resistance by correcting a mismatch between fatty acid uptake and fatty acid oxidation in skeletal muscle. Additionally, a single session of aerobic exercise increases glucose uptake by muscle during exercise, increases the ability of insulin to promote glucose uptake, and increases glycogen accumulation after exercise, all of which are important to blood glucose control. There also is some indication that resistance exercise may be effective in preventing insulin resistance. The information provided is intended to help clinicians understand and explain the roles of exercise in reducing insulin resistance.

Ataxia telangiectasia mutated influences cytochrome c oxidase activity.

Cells lacking ataxia telangiectasia mutated (ATM) have impaired mitochondrial function. Furthermore, mammalian cells lacking ATM have increased levels of reactive oxygen species (ROS) as well as mitochondrial DNA (mtDNA) deletions in the region encoding for cytochrome c oxidase (COX). We hypothesized that ATM specifically influences COX activity in skeletal muscle. COX activity was ∼40% lower in tibialis anterior from ATM-deficient mice than for wild-type mice (P < 0.01, n = 9/group). However, there were no ATM-related differences in activity of succinate dehydrogenase, isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase, mitochondrial glycerol 3-phosphate dehydrogenase, or complex III. Incubation of wild-type extensor digitorum longus muscles for 1h with the ATM inhibitor KU55933 caused a ∼50% reduction (P<0.05, n = 5/group) in COX activity compared to muscles incubated with vehicle alone. Among the control muscles and muscles treated with the ATM inhibitor, COX activity was correlated (r = 0.61, P<0.05) with activity of glucose 6-phosphate dehydrogenase, a key determinant of antioxidant defense through production of NADPH. Overall, the findings suggest that ATM has a protective role for COX activity.

A role for AMPK in increased insulin action after serum starvation

Serum starvation is a common cell culture procedure for increasing cellular response to insulin, though the mechanism for the serum starvation effect is not understood. We hypothesized that factors known to potentiate insulin action [e.g., AMP-activated protein kinase (AMPK) and p38] or to be involved in insulin signaling leading to glucose transport [e.g., Akt, PKCζ, AS160, and ataxia telangiectasia mutated (ATM)] would be phosphorylated during serum starvation and would be responsible for increased insulin action after serum starvation. L6 myotubes were incubated in serum-containing or serum-free medium for 3 h. Levels of phosphorylated AMPK, Akt, and ATM were greater in serum-starved cells than in control cells. Serum starvation did not affect p38, PKCζ, or AS160 phosphorylation or insulin-stimulated Akt or AS160 phosphorylation. Insulin had no effect on glucose transport in control cells but caused an increase in glucose uptake for serum-starved cells that was preventable by compound C (an AMPK inhibitor), by expression of dominant negative AMPK (AMPK-DN), and by KU55933 (an ATM inhibitor). ATM protein levels increased during serum starvation, and this increase in ATM was prevented by compound C and AMPK-DN. Thus, it appears that AMPK is required for the serum starvation-related increase in insulin-stimulated glucose transport, with ATM as a possible downstream effector.

Role of GLUT1 in regulation of reactive oxygen species.

In skeletal muscle cells, GLUT1 is responsible for a large portion of basal uptake of glucose and dehydroascorbic acid, both of which play roles in antioxidant defense. We hypothesized that conditions that would decrease GLUT1-mediated transport would cause increased reactive oxygen species (ROS) levels in L6 myoblasts, while conditions that would increase GLUT1-mediated transport would result in decreased ROS levels. We found that the GLUT1 inhibitors fasentin and phloretin increased the ROS levels induced by antimycin A and the superoxide generator pyrogallol. However, indinavir, which inhibits GLUT4 but not GLUT1, had no effect on ROS levels. Ataxia telangiectasia mutated (ATM) inhibitors and activators, previously shown to inhibit and augment GLUT1-mediated transport, increased and decreased ROS levels, respectively. Mutation of an ATM target site on GLUT1 (GLUT1-S490A) increased ROS levels and prevented the ROS-lowering effect of the ATM activator doxorubicin. In contrast, expression of GLUT1-S490D lowered ROS levels during challenge with pyrogallol, prevented an increase in ROS when ATM was inhibited, and prevented the pyrogallol-induced decrease in insulin signaling and insulin-stimulated glucose transport. Taken together, the data suggest that GLUT1 plays a role in regulation of ROS and could contribute to maintenance of insulin action in the presence of ROS.

Role of ataxia telangiectasia mutated in insulin signalling of muscle-derived cell lines and mouse soleus

Aim:  Ataxia telangiectasia mutated (ATM) reportedly plays a role in insulin‐stimulated activation of Akt in some cell types but not in others. The role of ATM in insulin signalling has not been firmly resolved for skeletal muscle cells, for which Akt phosphorylation is a pivotal step in stimulation of glucose transport. Accordingly, our aim was to determine the role of ATM in insulin effects for cell lines derived from skeletal muscle and for skeletal muscle.

Regional differences in interstitial glycerol concentration in subcutaneous adipose tissue of women.

The aims of this study were to 1) compare two methods of determining interstitial glycerol concentration in subcutaneous adipose tissue (AT) and 2) determine whether there are regional differences in interstitial glycerol concentration in subcutaneous AT of nonobese, premenopausal women. Microdialysis probes were inserted under local anesthesia into the abdominal (2 probes) and femoral (1 probe) subcutaneous AT in each subject ( n = 5) and perfused with a Ringer solution containing 2.5 mM glucose and glycerol in concentrations ranging from 0 to 900 μM. Microdialysis probe relative recoveries and interstitial glycerol concentrations were determined by the no-net-flux method (NNF) and the internal reference method (IR) with the use of [13C]glycerol. Microdialysis probe relative recoveries were 57.4 ± 3.6% by NNF and 61.2 ± 10.1% by IR in femoral AT [ P = not significant (NS)] and were 55.2 ± 6.0% by NNF and 66.6 ± 4.2% by IR in abdominal AT ( P = NS). The calculated interstitial glycerol concentrations determined by NNF and IR were 236.4 ± 42.7 and 241.1 ± 39.6 μM ( P = NS) in femoral AT and 151.4 ± 29.7 and 129.4 ± 18.7 μM in abdominal AT (NNF vs. IR, P = NS; femoral vs. abdominal, P < 0.05). It can be concluded that the interstitial glycerol concentration in the femoral AT of nonobese, premenopausal females is ∼240 μM and is higher than in abdominal AT (140 μM). Furthermore, the use of a stable isotope of glycerol as an internal reference is suitable for determining interstitial glycerol concentrations in subcutaneous adipose tissue in humans at rest.

ATM and GLUT1-S490 Phosphorylation Regulate GLUT1 Mediated Transport in Skeletal Muscle

Objective The glucose and dehydroascorbic acid (DHA) transporter GLUT1 contains a phosphorylation site, S490, for ataxia telangiectasia mutated (ATM). The objective of this study was to determine whether ATM and GLUT1-S490 regulate GLUT1. Research Design and Methods L6 myoblasts and mouse skeletal muscles were used to study the effects of ATM inhibition, ATM activation, and S490 mutation on GLUT1 localization, trafficking, and transport activity. Results In myoblasts, inhibition of ATM significantly diminished cell surface GLUT1, glucose and DHA transport, GLUT1 externalization, and association of GLUT1 with Gα-interacting protein-interacting protein, C-terminus (GIPC1), which has been implicated in recycling of endosomal proteins. In contrast, ATM activation by doxorubicin (DXR) increased DHA transport, cell surface GLUT1, and the GLUT1/GIPC1 association. S490A mutation decreased glucose and DHA transport, cell surface GLUT1, and interaction of GLUT1 with GIPC1, while S490D mutation increased transport, cell surface GLUT1, and the GLUT1/GIPC1 interaction. ATM dysfunction or ATM inhibition reduced DHA transport in extensor digitorum longus (EDL) muscles and decreased glucose transport in EDL and soleus. In contrast, DXR increased DHA transport in EDL. Conclusions These results provide evidence that ATM and GLUT1-S490 promote cell surface GLUT1 and GLUT1-mediated transport in skeletal muscle associated with upregulation of the GLUT1/GIPC1 interaction.

Ataxia telangiectasia mutated impacts insulin-like growth factor 1 signalling in skeletal muscle

•  What is the central question of this study? In some cultured cells, ataxia telangiectasia mutated (ATM) is required for activation of Akt by insulin. However, this is not the case in other cell or tissue types, including skeletal muscle. Furthermore, it is not known whether ATM plays a role in skeletal muscle insulin‐like growth factor 1 (IGF‐1) signalling. •  What is the main finding and its importance? We found that IGF‐1 caused autophosphorylation of ATM in skeletal muscle. However, IGF‐1‐stimulated phosphorylation of Akt, p70 S6 kinase and mammalian target of rapamycin (but not insulin receptor substrate 1) was impaired in C2C12 myotubes with reduced ATM expression and/or muscle from ATM‐haploinsufficient mice. These findings demonstrate activation of ATM by IGF‐1 and a role for ATM in IGF‐1 signalling downstream of insulin receptor substrate 1.

Possibility of Autocrine β-Adrenergic Signaling in C2C12 Myotubes

Levodopa reportedly inhibits insulin action in skeletal muscle. Here we show that C2C12 myotubes produce levodopa and that insulin-stimulated glucose transport is enhanced when endogenous levodopa is depleted. Exogenous levodopa prevented the stimulation of glucose transport by Insulin (P < 0.05) and Increased cAMP concentrations (P < 0.05). The decrease in Insulin-stimulated glucose transport caused by levodopa was attenuated by propranolol (a β-adrenergic antagonist) and Prevented by NSD-1015 (NSD), an inhibitor of DOPA decarboxylase (DDC; converts levodopa to dopamine). Propranolol and NSD both prevented levodopa-related increases in [cAMP]. However, the effects of levodopa were unlikely to be dependent on the conversion of levodopa to catecholamines because we could detect neither DDC in myotubes nor catecholamines in media after incubation of myotubes with levodopa. The data suggest the possibility of novel autocrine β-adrenergic action in C2C12 myotubes in which levodopa, produced by myotubes, could have hormone-like effects that impinge on glucose metabolism.

Impaired insulin-stimulated glucose transport in ATM-deficient mouse skeletal muscle.

There are reports that ataxia telangiectasia mutated (ATM) plays a role in insulin-stimulated Akt phosphorylation, although this is not the case in some cell types. Because Akt plays a key role in insulin signaling, which leads to glucose transport in skeletal muscle, the predominant tissue in insulin-stimulated glucose disposal, we examined whether insulin-stimulated Akt phosphorylation and (or) glucose transport would be decreased in skeletal muscle of mice lacking functional ATM, compared with muscle from wild-type mice. We found that in vitro insulin-stimulated Akt phosphorylation was normal in soleus muscle from mice with 1 nonfunctional allele of ATM (ATM+/-) and from mice with 2 nonfunctional alleles (ATM-/-). However, insulin did not stimulate glucose transport or the phosphorylation of AS160 in ATM-/- soleus. ATM protein level was markedly higher in wild-type extensor digitorum longus (EDL) than in wild-type soleus. In EDL from ATM-/- mice, insulin did not stimulate glucose transport. However, in contrast to findings for soleus, insulin-stimulated Akt phosphorylation was blunted in ATM-/- EDL, concomitant with a tendency for insulin-stimulated phosphatidylinositol 3-kinase activity to be decreased. Together, the findings suggest that ATM plays a role in insulin-stimulated glucose transport at the level of AS160 in muscle comprised of slow and fast oxidative-glycolytic fibers (soleus) and at the level of Akt in muscle containing fast glycolytic fibers (EDL).