To determine whether the impaired insulin-stimulated glucose uptake in obese individuals is associated with altered insulin receptor signaling, we measured both glucose uptake and early steps in the insulin action pathway in intact strips of human skeletal muscle. Biopsies of rectus abdominus muscle were taken from eight obese and eight control subjects undergoing elective surgery (body mass index 52.9±3.6 vs 25.7±0.9). Insulin-stimulated 2-deoxyglucose uptake was 53% lower in muscle strips from obese subjects. Additional muscle strips were incubated in the basal state or with i0-7 M insulin for 2, 15, or 30 min. In the lean subjects, tyrosine phosphorylation of the insulin receptor and insulin receptor substrate-i (IRS-1), measured by immunoblotting with anti-phosphotyrosine antibodies, was significantly increased by insulin at all time points. In the skeletal muscle from the obese subjects, insulin was less effective in stimulating tyrosine phosphorylation (maximum receptor and IRS-1 phosphorylation decreased by 35 and 38%, respectively). Insulin stimulation of IRS-1 immunoprecipitable phosphatidylinositol 3-kinase (PI 3-kinase) activity also was markedly lower in obese subjects compared with controls (10-vs 35-fold above basal, respectively). In addition, the obese subjects had a lower abundance of the insulin receptor, IRS-1, and the p85 subunit of PI 3-kinase (55, 54, and 64% of nonobese, respectively). We conclude that impaired insulinstimulated glucose uptake in skeletal muscle from severely obese subjects is accompanied by a deficiency in insulin receptor signaling, which may contribute to decreased insulin action. (J. Clin. Invest. 1995.95:2195-2204 Key words:

The purpose of this study was to determine if a relationship exists among skeletal muscle fiber composition, adiposity, and in vitro muscle glucose transport rate in humans. Rectus abdominus muscle was obtained during elective abdominal surgery from nonobese control (n = 12), obese (n = 12), and obese non-insulin-dependent diabetes mellitus (NIDDM) patients (n = 10). The obese NIDDM group had a significantly lower percentage of type I muscle fibers (32.2 +/- 1.9%) than the obese group (40.4 +/- 2.7%), and both obese groups were significantly lower than the control group (50.0 +/- 2.6%). Insulin-stimulated glucose transport, determined on 28 subjects, was significantly lower in both the obese (3.83 +/- 0.48 nmol.min-1.mg-1) and NIDDM (3.93 +/- 1.0 nmol.min-1.mg-1) groups vs. the control group (7.35 +/- 1.50 nmol.min-1.mg-1). Body mass index (BMI) was inversely correlated to percent type I fibers (r = -0.50, P < 0.01) and to the insulin-stimulated glucose transport rate (r = -0.53, P < 0.01). The percentage of type I muscle fibers was related to the insulin-stimulated glucose transport rate (r = 0.57, P < 0.01), although this relationship was not significant after adjusting for BMI. Although these data do not support an independent relationship between fiber type and insulin action in obesity, a reduced skeletal muscle type I fiber population may be one component of a multifactorial process involved in the development of insulin resistance.

Insulin and muscle contraction stimulate glucose transport into muscle cells by separate signaling pathways, and hypoxia has been shown to operate via the contraction signaling pathway. To elucidate the mechanism of insulin resistance in human skeletal muscle, strips of rectus abdominis muscle from lean (body mass index [BMI] < 25), obese (BMI > 30), and obese non-insulin-dependent diabetes mellitus (NIDDM) (BMI > 30) patients were incubated under basal and insulin-, hypoxia-, and hypoxia + insulin-stimulated conditions. Insulin significantly stimulated 2-deoxyglucose transport approximately twofold in muscle from lean (P < 0.05) patients, but not in muscle from obese or obese NIDDM patients. Furthermore, maximally insulin-stimulated transport rates in muscle from obese and diabetic patients were significantly lower than rates in muscle from lean patients (P < 0.05). Hypoxia significantly stimulated glucose transport in muscle from lean and obese patients. There were no significant differences in hypoxia-stimulated glucose transport rates among lean, obese, and obese NIDDM groups. Hypoxia + insulin significantly stimulated glucose transport in lean, obese, and diabetic muscle. The results of the present study suggest that the glucose transport effector system is intact in diabetic human muscle when stimulated by hypoxia.

In response to insulin, several proteins are phosphorylated on tyrosine and on serine/threonine residues. Decreased phosphorylation of signaling peptides by a defective insulin receptor kinase may be a cause of insulin resistance. Accordingly, inhibition of the appropriate phosphatases might increase the phosphorylation state of these signaling peptides and thereby elicit increased glucose transport. The purpose of this study was to examine the effect of the serine/threonine phosphatase inhibitor okadaic acid and the tyrosine phosphatase inhibitors phenylarsine oxide and vanadate on 2-deoxyglucose transport in insulin-resistant human skeletal muscle. All three phosphatase inhibitors stimulated 2-deoxyglucose transport in insulin-resistant skeletal muscle. These data suggest that these compounds have bypassed a defect in at least one of the signaling pathways leading to glucose transport. Furthermore, maximal transport rates induced by the simultaneous presence of insulin and phosphatase inhibitor in insulin-resistant muscle were equal to insulin-stimulated rates in lean control subjects. However, both vanadate alone and vanadate plus insulin stimulated 2-deoxyglucose transport significantly more in insulin-sensitive tissue than in insulin-resistant tissue. These results demonstrate that although vanadate is able to stimulate glucose transport in insulin-resistant muscle, it is not able to normalize transport to the same rate achieved in insulin-sensitive muscle.

PU.1, a member of the ets transcription factor family, has been previously shown to be necessary for tetradecanoylphorbol-13 acetate (TPA)-induced U937 leukemic cell maturation. We examined the effects of TPA on PU.1 content and PU.1 DNA binding activity in U937 cells. Unstimulated cells expressed PU.1 mRNA transcripts and TPA did not increase these levels. However, TPA treatment induced phosphorylation of PU.1. Gel-shift analysis using a labeled PU.1 oligomer showed that TPA induced a unique PU.1 binding activity. This binding activity was phosphorylation-dependent, as indicated by the ability of phosphatase treatment to abolish its detection. The PU.1 binding activity was generated at TPA-13 concentrations stimulating growth arrest and was blocked by the PKC inhibitor GF109203X, which antagonized TPA-induced growth inhibition. Bryostatin 1, another protein kinase C activator, induced only a modest degree of U937 growth inhibition and antagonized TPA-stimulated growth arrest. Bryostatin 1 was unable to induce this TPA-generated PU.1 binding activity. High bryostatin 1 concentrations inhibited generation of this TPA-induced band shift. These data suggest that TPA-induced growth inhibition is associated with phosphorylation of PU.1 and generation of a unique PU.1 binding activity.