The molecular bases underlying burn-or critical illness-induced insulin resistance still remain unclarified. Muscle protein catabolism is a ubiquitous feature of critical illness. Akt/PKB plays a central role in the metabolic actions of insulin and is a pivotal regulator of hypertrophy and atrophy of skeletal muscle. We therefore examined the effects of burn injury on insulin-stimulated Akt/PKB activation in skeletal muscle. Insulin-stimulated phosphorylation of Akt/PKB was significantly attenuated in burned compared with sham-burned rats. Insulin-stimulated Akt/PKB kinase activity, as judged by immune complex kinase assay and phosphorylation status of the endogenous substrate of Akt/PKB, glycogen synthase kinase-3␤ (GSK-3␤), was significantly impaired in burned rats. Furthermore, insulin consistently failed to increase the phosphorylation of p70 S6 kinase, another downstream effector of Akt/PKB, in rats with burn injury, whereas phosphorylation of p70 S6 kinase was increased by insulin in controls. The protein expression of Akt/PKB, GSK-3␤, and p70 S6 kinase was unaltered by burn injury. However, insulin-stimulated activation of ERK, a signaling pathway parallel to Akt/PKB, was not affected by burn injury. These results demonstrate that burn injury impairs insulin-stimulated Akt/PKB activation in skeletal muscle and suggest that attenuated Akt/PKB activation may be involved in deranged metabolism and muscle wasting observed after burn injury.protein kinase B; glycogen synthase kinase-3␤; insulin resistance FUNCTIONAL AND METABOLIC ABERRATIONS associated with critical illness such as burn injury include hypermetabolic response, increased protein catabolism, insulin resistance, and muscle wasting. Muscle wasting in critically ill patients leads to muscle weakness, resulting in hypoventilation, difficulties in weaning off respirators, decreased mobilization, prolonged rehabilitation and hospitalization, and even death (2, 3, 4, 6). Insulin resistance is a well-known phenomenon of critical illness and has long been considered to play a cardinal role in the derangements of metabolism and muscle wasting. Binding of insulin to its receptor results in activation of insulin receptor (IR) tyrosine kinase, which in turn phosphorylates the IR substrates (IRSs). Phosphorylation at the tyrosine residues of IRS-1 and IRS-2 transduces signal from IR to phosphatidylinositol 3-kinase (PI3K) (1, 4).A Ser/Thr protein kinase, Akt/PKB, is a major downstream effector of the IR-IRS-PI3K pathway. Akt/PKB is activated by phosphorylation of Thr 308 and Ser 473 residues of the kinase (8, 46, 52). The phosphorylation of Akt/PKB is dependent on phosphatidylinositol 3,4,5-triphosphate, a product of PI3K. Akt/PKB drives a major portion of the PI3K-mediated metabolic actions of insulin. Akt/PKB is required for insulinstimulated glucose uptake and glycogen synthesis (53). Akt/ PKB also promotes protein synthesis via activation of the mTOR-p70 S6 kinase pathway (19). Glycogen synthase kinase-3␤ (GSK-3␤), a negative regulator of glycogen synthase...

Chronic inflammation plays an important role in insulin resistance. Inducible nitric-oxide synthase (iNOS), a mediator of inflammation, has been implicated in many human diseases including insulin resistance. However, the molecular mechanisms by which iNOS mediates insulin resistance remain largely unknown. Here we demonstrate that exposure to NO donor or iNOS transfection reduced insulin receptor substrate (IRS)-1 protein expression without altering the mRNA level in cultured skeletal muscle cells. NO donor increased IRS-1 ubiquitination, and proteasome inhibitors blocked NO donor-induced reduction in IRS-1 expression in cultured skeletal muscle cells. The effect of NO donor on IRS-1 expression was cGMP-independent and accentuated by concomitant oxidative stress, suggesting an involvement of nitrosative stress. Inhibitors for phosphatidylinositol-3 kinase, mammalian target of rapamycin, and c-Jun amino-terminal kinase failed to block NO donor-induced IRS-1 reduction, whereas these inhibitors prevented insulin-stimulated IRS-1 decrease. Moreover iNOS expression was increased in skeletal muscle of diabetic (ob/ob) mice compared with lean wild-type mice. iNOS gene disruption or treatment with iNOS inhibitor ameliorated depressed IRS-1 expression in skeletal muscle of diabetic (ob/ob) mice. These findings indicate that iNOS reduces IRS-1 expression in skeletal muscle via proteasome-mediated degradation and thereby may contribute to obesity-related insulin resistance.

Chronic injection of an anti-c-KIT receptor tyrosine kinase monoclonal antibody (ACK2) results in the disruption of the normal motility patterns of young BALB/c mice intestine. This effect is accompanied by a drastic decrease in the number of intestinal c-kit-expressing (c-kit+) cells when studied immunohistochemically with the fluorescence-labelled antibody. In order to clarify the mechanism underlying the ACK2 action and the physiological roles of intestinal c-kit+ cells, we studied the excitability of intestinal c-kit+ cells in primary culture by use of the nystatin perforated-patch-clamp technique. Under voltage-clamp at -40 mV, the majority of c-kit+ cells tested (59/70) elicited rhythmic current waves with an amplitude and frequency of 263 +/- 24 pA and 2.30 +/- 0.25 cycles/min (mean +/- SEM), respectively. Intracellular perfusion of the c-kit+ cells with ethylenebis (okonitrilo) tetraacetate (EGTA) as well as a nominally Ca(2+)-free external solution or low holding voltage (< -60 mV) prevented the rhythmic current. The reversal potential of the rhythmic current was close to the equilibrium potential for Cl-(ECl). Moreover the rhythmic current was depressed by a Cl- channel blocker, 4-acetoamido-4-isothiocyanat-ostilbene-2,2'-disulphoni c acid (SITS). The smooth muscle cells freshly dissociated from the same intestinal specimen revealed a Ca(2+)-activated K+ current, as has been described in a variety of smooth muscle cells. Cultured smooth muscle cells from the ileum preparation lacked neither the Ca(2+)-activated K+ nor rhythmic Cl- currents. Smooth muscle cells freshly dissociated from the same ileum preparation and those in culture showed no immunoreactivity with the labelled ACK2, which was consistent with our previous in situ study. Results provided direct evidence that the intestinal c-kit+ cells, but not the smooth muscle cells, possess a rhythmic Cl- current oscillation, suggesting their participation in pacemaker activity for the peristaltic gut movement.