Regulation of insulin receptor function.

Resistance to the biological actions of insulin contributes to the development of type 2 diabetes and risk of cardiovascular disease. A reduced biological response to insulin by tissues results from an impairment in the cascade of phosphorylation events within cells that regulate the activity of enzymes comprising the insulin signaling pathway. In most models of insulin resistance, there is evidence that this decrement in insulin signaling begins with either the activation or substrate kinase activity of the insulin receptor (IR), which is the only component of the pathway that is unique to insulin action. Activation of the IR can be impaired by post-translational modifications of the protein involving serine phosphorylation, or by binding to inhibiting proteins such as PC-1 or members of the SOCS or Grb protein families. The impact of these processes on the conformational changes and phosphorylation events required for full signaling activity, as well as the role of these mechanisms in human disease, is reviewed in this article.

Muscle insulin receptor concentrations in obese patients post bariatric surgery: relationship to hyperinsulinemia.

OBJECTIVE: Obesity results in insulin resistance. Bariatric surgery for obese individuals induces weight loss, improves insulin sensitivity, and lowers insulin levels. We investigated the mechanisms of this improvement. DESIGN: Insulin receptor (IR) content, IR signaling, and adiponectin levels were measured in nine morbidly obese subjects before and after bariatric surgery. SUBJECTS: Seven female and two male, average age 44+/-2y, BMI >40 kg/m(2) and/or at least 100 lbs over ideal body weight, undergoing elective bariatric surgery. MEASUREMENTS: Before surgery BMI, fasting plasma glucose, adiponectin, and insulin levels were measured. A fasting muscle biopsy was obtained from the vastus lateralis for IR concentration and autophosphorylation activity measurements. These procedures were repeated 1 y after surgery. RESULTS: At 1 y after surgery, the subjects had lost an average of 48.3+/-5.6 kg (P<0.001), insulin sensitivity had significantly increased as determined by the minimal model (SI 0.72+/-0.18 vs 3.86+/-1.43, P<0.05), and IR content had increased two-fold in muscle (2.1+/-0.4 vs 4.3+/-0.7 ng/mg protein, P<0.01). The increase in IR content was related to fasting insulin levels. In the subjects with the lowest IR function, there was also an increase in IR function. Plasma adiponectin increased by 40% following weight loss (7.4+/-1.6 pre vs 10.3+/-1.3 mg/ml post, P<0.05). There was no significant change in muscle content of the IR inhibitor, PC-1. CONCLUSION: Increased IR content, most likely regulated by insulin levels, may be one contributor to the increased insulin sensitivity that occurs when morbidly obese patients undergo bariatric surgery.

Impaired insulin-receptor autophosphorylation is an early defect in fat-fed, insulin-resistant rats.

High-fat feeding results in impaired insulin signaling in skeletal muscle, but the role of the insulin receptor (IR) remains controversial. In the present study, female Fischer 344 rats were fed diets either low in fat [low fat, complex carbohydrate (LFCC)] or high in fat and sucrose (HFS). Insulin-stimulated skeletal muscle glucose transport, measured in purified sarcolemmal vesicles, was lower in rats consuming the HFS diet for 2 and 8 wk compared with LFCC controls (72.9 +/- 3.5, 67.6 +/- 3.5, and 86.1 +/- 3.5 pmol x mg(-1) x 15 s(-1), respectively; P < 0.05). Muscle IR content was unchanged in 2-wk HFS animals but was 50% lower in the 8-wk HFS group (P < 0.001). However, compared with LFCC, insulin-stimulated IR autophosphorylation was 26% lower in 2-wk HFS and 40% lower in 8-wk HFS animals (P < 0.005). Total muscle content of the proposed IR inhibitors cytokine tumor necrosis factor-alpha and membrane glycoprotein PC-1 was not significantly changed in HFS animals at either 2 or 8 wk. These results demonstrate that high-fat feeding induces insulin resistance in muscle concomitant with a diminished IR signaling capacity, although the mechanism remains unknown.

Small molecule insulin receptor activators potentiate insulin action in insulin-resistant cells.

In type 2 diabetes, impaired insulin signaling leads to hyperglycemia and other metabolic abnormalities. To study a new class of antidiabetic agents, we compared two small, nonpeptide molecules that activate insulin receptor (IR) beta-subunit tyrosine kinase activity: Merck L7, a direct IR agonist, and Telik's TLK16998, an IR sensitizer. In rat hepatoma cells (HTCs) that overexpress the IR (HTC-IR), IR autophosphorylation was directly activated by L7 in the absence of insulin. TLK16998 did not directly activate IR autophosphorylation, but it enhanced IR autophosphorylation in the presence of insulin. Tyrosine phosphorylation of an endogenous 185-kDa IR substrate was also significantly enhanced by both Merck L7 alone and TLK16998 plus insulin. Adding TLK16998 to L7 produced synergistic effects, further indicating that these two compounds act on the IR through separate mechanisms. We next studied HTC-IR(Delta485-599) cells, which overexpress a mutant IR with a deletion in the alpha-subunit connecting domain that does not undergo autophosphorylation in response to insulin binding. L7 was able to directly activate autophosphorylation of the deletion mutant IR in these cells, whereas TLK16998 had no effect. Compounds were then tested in three other cell models of impaired IR function. Both TLK16998 and Merck L7 improved IR autophosphorylation in cells with diminished IR signaling due to either treatment with tumor necrosis factor-alpha or overexpression of membrane glycoprotein PC-1. However, in TPA (tetradecanoylphorbol acetate)-treated cells, TLK16998 but not Merck L7 was able to significantly reverse the impaired insulin-stimulated IR autophosphorylation. In summary, these two classes of IR activators selectively increased IR function in a variety of insulin-resistant cell lines.

Enhanced muscle insulin receptor autophosphorylation with short-term aerobic exercise training.

Exercise training improves insulin action in skeletal muscle, but the mechanisms of this effect are not completely understood. In particular, the role of the insulin receptor (IR) is unclear. We examined the IR and an enzyme indicative of oxidative capacity in muscle in relation to improved insulin action in 20 previously sedentary individuals before and after a 7-day program of moderate-intensity cycle ergometry. After training, insulin sensitivity increased 33% (6.20 +/- 0.91 vs. 8.22 +/- 1.12 min. microU(-1). ml(-1) mean +/- SE, pre- vs. posttraining, respectively, P < 0.05). The mitochondrial marker enzyme cytochrome c oxidase (COX) increased in vastus lateralis biopsies by 21% (P < 0.05). After training, IR autophosphorylation, determined by ELISA, was significantly increased by approximately 40% at insulin concentrations from 1 to 100 nM (P < 0.05). The training-induced improvements in IR autophosphorylation were significantly correlated with changes in muscle COX content (r = 0.65, P < 0.05). These studies indicate that, in this model of increased physical activity, improvements in IR function are an early adaptation to exercise in humans, are correlated with increases in muscle oxidative capacity, and likely contribute to the beneficial effects of exercise training on insulin action.

Insulin receptor autophosphorylation in cultured myoblasts correlates to glucose disposal in Pima Indians.

In a previous study [Youngren, J. F., I. D. Goldfire, and R. E. Pratley. Am. J. Physiol. 273 (Endocrinol. Metab. 36): E276-E283, 1997] of skeletal muscle biopsies from insulin-resistant, nondiabetic Pima Indians, we demonstrated that diminished insulin receptor (IR) autophosphorylation correlated with in vivo insulin resistance. In the present study, to determine whether decreased IR function is a primary trait of muscle, and not secondary to an altered in vivo environment, we cultured myoblasts from 17 nondiabetic Pima Indians in whom insulin-stimulated glucose disposal (M) was measured during hyperinsulinemic-euglycemic glucose clamps. Myoblast IR autophosphorylation was determined by a highly sensitive ELISA. IR autophosphorylation directly correlated with M (r = 0.56, P = 0.02) and inversely correlated with the fasting plasma insulin (r = -0.58, P < 0.05). The relationship between M and IR autophosphorylation remained significant after M was adjusted for the effects of percent body fat (partial r = 0.53, P < 0.04). The relationship between insulin resistance and the capacity for myoblast IR autophosphorylation in nondiabetic Pima Indians suggests that variations in IR-signaling capacity may be intrinsic characteristics of muscle that contribute to the genetic component determining insulin action in this population.

Role of PC-1 in the etiology of insulin resistance.

Defects in insulin receptor tyrosine kinase activity have been demonstrated in tissues from insulin resistant subjects, but mutations in the insulin receptor gene are rare. Therefore, other molecules that are capable of modulating the insulin receptor most likely play a major role in insulin resistance. In cultured fibroblasts from an insulin resistant patient with Type 2 diabetes, we first identified membrane glycoprotein PC-1 as an inhibitor of the insulin receptor tyrosine kinase activity. PC-1 is overexpressed in fibroblasts from other insulin resistant subjects, both with and without Type 2 diabetes. PC-1 is a large class II exoprotein whose function is unknown. Studies in muscle and fat of insulin resistant subjects two primary tissues for insulin activation, reveal that elevated levels of PC-1 are inversely correlated with decreased insulin action both in vivo and in vitro. Transfection and expression of PC-1 in cultured cells demonstrate that overexpression of PC-1 produces impairments in insulin receptor tyrosine kinase activity, and the subsequent cellular responses to insulin. These studies indicate, therefore, that PC-1 is a major factor in the etiology of insulin resistance, and is a potential new therapeutic target for anti-diabetic therapy.

Membrane glycoprotein PC-1 and insulin resistance.

Peripheral resistance to insulin is a major component of non-insulin dependent diabetes mellitus. Defects in insulin receptor tyrosine kinase activity have been demonstrated in several tissues from insulin resistant subjects, but mutations in the insulin receptor gene occur in only a small fraction of cases. Therefore, other molecules that are capable of modulating the function of the insulin receptor are likely candidates in the search for the cellular mechanisms of insulin resistance. We have isolated an inhibitor of insulin receptor tyrosine kinase activity from cultured fibroblasts of an insulin resistant NIDDM patient and identified it as membrane glycoprotein PC-1. Subsequently we have demonstrated that expression of PC-1 is elevated in fibroblasts from other insulin resistant subjects, both with and without NIDDM. Studies in muscle, the primary site for insulin-mediated glucose disposal, have shown that the levels of PC-1 in this tissue are inversely correlated to insulin action both in vivo and in vitro. Transfection of PC-1 into cultured cells has confirmed that overexpression of PC-1 can produce impairments in insulin receptor tyrosine kinase activity and the subsequent cellular responses to insulin. Preliminary data suggests a direct interaction between PC-1 and the insulin receptor. However, the mechanisms whereby PC-1 inhibits insulin receptor signaling remain to be determined.

Contributions of the American Journal of Physiology to the discovery of insulin.

Since its inception in 1898 the American Journal of Physiology has been a leader in diabetes research and has published many key articles on the subject. The Journal first published studies of phlorhizin-induced diabetes in 1898, and after many other contributions went on to publish the first reports of Banting, Best, Macleod, and Collip in 1922 concerning the isolation and purification of insulin (5-8, 13). This review highlights some of these key contributions of the Journal.

Decreased muscle insulin receptor kinase correlates with insulin resistance in normoglycemic Pima Indians.

Defects in insulin receptor tyrosine kinase activity are present in insulin-resistant non-insulin-dependent diabetes mellitus patients and certain nondiabetic individuals, both lean and obese. However, the relationship between insulin receptor function, insulin action, and obesity is unclear. To address this issue, we have employed a new and highly sensitive enzyme-linked immunosorbent assay to measure in vitro insulin-stimulated autophosphorylation of immunocaptured muscle insulin receptors in a group of 25 normoglycemic Pima Indians. Insulin action, determined during two-step euglycemic insulin clamps, varied widely in these subjects. Maximal in vitro insulin stimulation of insulin receptor autophosphorylation strongly correlated with both low (Mlow)- and high (Mhigh)-dose insulin-stimulated glucose disposal (r = 0.62 and 0.51, P < 0.002 and 0.011, respectively). Insulin receptor autophosphorylation was inversely related to percent body fat (r = -0.52, P < 0.009). After control for percent body fat, receptor autophosphorylation remained correlated with Mlow (partial r = 0.49, P < 0.025). These data therefore suggest that defects in insulin receptor function are major contributors to insulin resistance in both lean and obese normoglycemic Pima Indians.

Increased adipose tissue PC-1 protein content, but not tumour necrosis factor-alpha gene expression, is associated with a reduction of both whole body insulin sensitivity and insulin receptor tyrosine-kinase activity.

In the present study we measured PC-1 content, tumour necrosis factor (TNF)-alpha gene expression, and insulin stimulation of insulin receptor tyrosine-kinase activity in adipose tissue from non-obese, non-diabetic subjects. These parameters were correlated with in vivo insulin action as measured by the intravenous insulin tolerance test (Kitt values). PC-1 content was negatively correlated with Kitt values (r = -0.5, p = 0.04) and positively with plasma insulin levels both fasting (r = 0.58, p = 0.009) and after 120 min during oral glucose tolerance test (OGTT) (r = 0.67, p = 0.002). Moreover, adipose tissue PC-1 content was higher in relatively insulin-resistant subjects (Kitt values lower than 6) than in relatively insulin-sensitive subjects (Kitt values higher than 6) (525 +/- 49 ng/mg protein vs 336 +/- 45, respectively, p = 0.012). Adipose tissue insulin receptor tyrosine-kinase activity in response to insulin was significantly lower at all insulin concentrations tested (p = 0.017, by two-way analysis of variance test) in insulin-resistant than in insulin-sensitive subjects (Kitt values lower or higher than 6, respectively). In contrast to PC-1, no significant correlation was observed between adipose tissue TNF-alpha mRNA content and Kitt values, and plasma insulin levels, both fasting and at after 120 min during OGTT. Also, no difference was observed in TNF-alpha mRNA content between subjects with Kitt values higher or lower than 6. These studies in adipose tissue, together with our previous studies in skeletal muscle raise the possibility that PC-1, by regulating insulin receptor function, may play a role in the degree of insulin sensitivity in non-obese, non-diabetic subjects.

The development of insulin resistance with high fat feeding in rats does not involve either decreased insulin receptor tyrosine kinase activity or membrane glycoprotein PC-1.

Recent studies have suggested that the insulin receptor tyrosine kinase inhibitor, membrane glycoprotein PC-1, may play a role in certain insulin resistant states. In the present study, we examined whether either insulin receptor function or PC-1 activity was altered during the development of insulin resistance that occurs with high fat feeding in normal rats. Over the course of 14 days of high fat feeding, both maximal and submaximal (physiological) insulin-stimulated skeletal muscle glucose uptake decreased gradually; after 14 days of high fat feeding, submaximal and maximal insulin-stimulated glucose uptake decreased by approximately 40 and approximately 50%, respectively. In contrast, in the same muscles (tibialis anterior) of these animals, neither insulin receptor content nor insulin-stimulated insulin receptor autophosphorylation was altered after 14 days of high fat feeding. PC-1 has both nucleotide pyrophosphatase (EC 3.6.1.9) and alkaline phosphodiesterase I (EC 3.1.4.1) enzyme activities. These enzyme activities showed no changes during the course of 14 days of high fat feeding. Individual data revealed that there was no significant correlation between insulin-stimulated glucose uptake and alkaline phosphodiesterase or nucleotide pyrophosphatase activity (P > 0.05). Together, these data indicate that neither defects in insulin receptor function nor elevated PC-1 activities are involved in the development of insulin resistance in rats with high fat feeding, and the insulin resistance induced with high fat feeding is likely due to postreceptor defects in skeletal muscle.

Skeletal muscle content of membrane glycoprotein PC-1 in obesity. Relationship to muscle glucose transport.

Membrane glycoprotein PC-1, an inhibitor of insulin signaling, produces insulin resistance when overexpressed in cells transfected with PC-1 cDNA. In the present study, we determined whether PC-1 plays a role in the insulin resistance of skeletal muscle in obesity. Rectus abdominus muscle biopsies were taken from patients undergoing elective surgery. Subjects included both NIDDM patients (n = 14) and nondiabetic patients (n = 34) across a wide range of BMI values (19.5-90.1). Insulin-stimulated glucose transport was measured in incubated muscle strips, and PC-1 content, enzymatic activity, and insulin receptor content were measured in solubilized muscle extracts. Increasing BMI correlated with both an increase in the content of PC-1 in muscle (r = 0.55, P < 0.001) and a decrease in insulin stimulation of muscle glucose transport (r = -0.58, P = 0.008). NIDDM had no effect on either PC-1 content or glucose transport for any given level of obesity. Insulin stimulation of muscle glucose transport was negatively related to muscle PC-1 content (r = -0.68, P = 0.001) and positively related to insulin receptor content (r = 0.60, P = 0.005). Multivariate analysis indicated that both skeletal muscle PC-1 content and insulin receptor content, but not BMI, were independent predictors of insulin-stimulated glucose transport. Muscle PC-1 content accounted for 42% and insulin receptor content for 17% of the variance in glucose transport values. These studies raise the possibility that increased expression of PC-1 and a decreased insulin receptor content in skeletal muscle may be involved in the insulin resistance of obesity.

Effects of acute and chronic exercise on skeletal muscle glucose transport in aged rats.

The purpose of this study was to investigate the effects of acute and chronic exercise on skeletal muscle glucose transport in aged rats by using an isolated sarcolemmal membrane preparation. In 24-mo-old female Fischer 344 rats, a maximum dose of insulin increased glucose transport from 43 +/- 6 to 82 +/- 6 pmol.mg protein-1.15 s-1. A 45-min bout of exhaustive treadmill running increased glucose transport to the same maximum level (88 +/- 5 pmol.mg protein-1.15 s-1). Eight weeks of progressive exercise training resulted in a 65% increase in succinic dehydrogenase activity in hindlimb muscles and a 55% increase in total cellular GLUT-4 content. Despite these biochemical adaptations, there was no change in either basal or maximum insulin-stimulated glucose transport between control (43 +/- 6 and 82 +/- 6 pmol.mg protein-1.15 s-1, respectively) and trained (42 +/- 2 and 82 +/- 8 pmol.mg protein-1.15 s-1, respectively) animals. When hindlimb muscle succinate dehydrogenase activity and GLUT-4 content were compared for both the combined sedentary and trained groups, a significant correlation (r = 0.68) was obtained. This study demonstrates that the skeletal muscle glucose transport system of 24-mo-old rats is fully stimulated by acute exercise and that, although GLUT-4 levels are increased in aged animals after exercise training, this does not result in an enhancement of maximal insulin-stimulated glucose transport. Thus increases in GLUT-4 are not sufficient to improve muscle insulin responsiveness with training.

Diet, not aging, causes skeletal muscle insulin resistance.

The purpose of this study was to compare the effects of raising female Fischer rats on a low-fat, high-complex-carbohydrate diet (LFCC) versus a high-fat, sucrose diet (HFS) on serum glucose and insulin as well as skeletal muscle glucose transport. No significant differences were observed between 6- and 24-month-old rats raised on the LFCC diet for serum glucose (3.6 +/- 0.1 vs. 3.7 +/- 0.2 mM) and insulin (88 +/- 6 vs. 98 +/- 10 pM) or for basal (35 +/- 3 vs. 39 +/- 6 pmol/mg protein/15 s) or insulin-stimulated (74.2 +/- 7.6 vs. 69.4 +/- 3.8 pmol/mg protein/15 s) glucose transport. These data indicate that aging per se does not lead to insulin resistance. When the 24-month-old animals raised on the HFS diet were compared with those on the LFCC diet, major differences were observed. Fasting serum insulin was significantly higher in the HFS group (437 +/- 118 vs. 98 +/- 10 pM) and insulin-stimulated glucose transport was significantly reduced (52.5 +/- 3.7 vs. 69.4 +/- 3.8 pmol/mg protein/15 s). Fasting glucose (3.7 +/- 0.2 vs. 3.6 +/- 0.1 mM) and basal glucose transport (38 +/- 6 vs. 39 +/- 6 pmol/mg protein/15 s) were unchanged. These results indicate that diet and not aging per se caused insulin resistance.

Regulation of glucose transport in skeletal muscle.

The entry of glucose into muscle cells is achieved primarily via a carrier-mediated system consisting of protein transport molecules. GLUT-1 transporter isoform is normally found in the sarcolemmal (SL) membrane and is thought to be involved in glucose transport under basal conditions. With insulin stimulation, glucose transport is accelerated by translocating GLUT-4 transporters from an intracellular pool out to the T-tubule and SL membranes. Activation of transporters to increase the turnover number may also be involved, but the evidence is far from conclusive. When insulin binds to its receptor, it autophosphorylates tyrosine and serine residues on the beta-subunit of the receptor. The tyrosine residues are thought to activate tyrosine kinases, which in turn phosphorylate/activate as yet unknown second messengers. Insulin receptor antibodies, however, have been reported to increase glucose transport without increasing kinase activity. Insulin resistance in skeletal muscle is a major characteristic of obesity and diabetes mellitus, especially NIDDM. A decrease in the number of insulin receptors and the ability of insulin to activate receptor tyrosine kinase has been documented in muscle from NIDDM patients. Most studies report no change in the intracellular pool of GLUT-4 transporters available for translocation to the SL. Both the quality and quantity of food consumed can regulate insulin sensitivity. A high-fat, refined sugar diet, similar to the typical U.S. diet, causes insulin resistance when compared with a low-fat, complex-carbohydrate diet. On the other hand, exercise increases insulin sensitivity. After an acute bout of exercise, glucose transport in muscle increases to the same level as with maximum insulin stimulation. Although the number of GLUT-4 transporters in the sarcolemma increases with exercise, neither insulin or its receptor is involved. After an initial acute phase, which may involve calcium as the activator, a secondary phase of increased insulin sensitivity can last for up to a day after exercise. The mechanism responsible for the increased insulin sensitivity with exercise is unknown. Regular exercise training also increases insulin sensitivity, which can be documented several days after the final bout of exercise, and again the mechanism is unknown. An increase in the muscle content of GLUT-4 transporters with training has recently been reported. Even though significant progress has been made in the past few years in understanding glucose transport in skeletal muscle, the mechanisms involved in regulating transport are far from being understood.

Effects of maturation and aging on the skeletal muscle glucose transport system.

Insulin resistance in old, compared with young, humans and animals has been well documented. The resistance is due primarily to defects in skeletal muscle. In the present study, skeletal muscle sarcolemmal membranes were purified from five age groups of female Fischer rats ranging from 2 to 24 mo. Basal specific D-glucose transport was not significantly different among any of the groups. Maximum insulin-stimulated transport was progressively decreased from 96.4 +/- 5.0 pmol.mg-1.15 s-1 in the 2-mo-old animals to 70.8 +/- 8.9 pmol.mg-1.15 s-1 in the 24-mo-old animals. Most of the decrease occurred during maturation, and in fact there was no significant difference in maximum transport among the 8-, 16-, and 24-mo-old rats. The decrease in insulin-stimulated transport in the 24-mo-old animals was due to a reduction in the number of glucose transporters translocated into the sarcolemma membrane (9.8 +/- 0.6 vs. 7.8 +/- 0.6 pmol/mg protein). The intracellular or microsomal pool of glucose transporters was not significantly different between the 2- and 24-mo-old animals (8.8 +/- 0.6 vs. 8.5 +/- 0.9/mg protein). Western blotting revealed no differences in the cellular GLUT-4 contents between the 2- and 24-mo-old rats. The number of insulin receptors (2.3 +/- 0.4 vs. 2.1 +/- 0.5 pmol/mg protein) was not significantly different. Tyrosine kinase activity of the insulin receptor was, however, significantly reduced in the 24-mo-old compared with the 2-mo-old animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of NIDDM on the glucose transport system in human skeletal muscle.

The purpose of this study was to investigate cellular changes in the glucose transport system in skeletal muscle of lean non-insulin-dependent diabetes mellitus (NIDDM) compared to lean nondiabetic control patients. NIDDM patients had significantly elevated fasting levels (means +/- SE) of serum glucose (10.1 +/- 1.3 vs. 5.4 +/- 0.4 mM, P less than 0.001) and serum insulin (110.8 +/- 31.1 vs. 35.9 +/- 3.6 pM, P less than 0.0025). Basal glucose transport (35.1 +/- 5.5 vs. 30.8 +/- 8.0 pM/mg protein) and cytochalasin-beta binding (3.5 +/- 1.2 vs 3.8 +/- 1.0 pM/mg protein) in isolated sarcolemmal vesicles were not significantly different between NIDDM and control groups. Insulin binding was reduced in NIDDM (0.82 +/- 0.03 vs. 1.63 +/- 0.18 pM/mg protein) as was the Kd (0.93 +/- 0.03 vs. 1.38 + 0.12 nM). Tyrosine kinase activity, as assessed from incorporation of [32P]ATP into Glu 4:Tyr 1, was significantly (P less than 0.005) reduced in NIDDM at insulin concentrations from 1-100 nM. Maximum kinase activity was depressed (1.88 +/- 0.04 vs. 2.97 +/- 0.07 fM 32P/fM insulin binding at 100 nM insulin). The number of glucose transporters in the low-density microsomes was not significantly different between NIDDM and control groups (7.01 +/- 1.40 vs. 7.65 +/- 0.90 pM cytochalasin-beta bound/mg protein). These results suggest that decreased insulin binding and diminished receptor tyrosine kinase activity play a substantial role in the development of skeletal muscle insulin resistance associated with NIDDM.

Effects of streptozotocin-induced diabetes on glucose transport in skeletal muscle.

Female Sprague-Dawley rats were injected with streptozotocin (45 mg/kg) to induce mild diabetes (glucose, greater than 13 mM). Half of the animals received daily insulin injections to reduce hyperglycemia. After 10 weeks, sarcolemmal membranes were isolated from hindlimb muscles to study glucose transport, and the number of glucose transporters was assessed by cytochalasin-beta binding. Both glucose transport (19.2 +/- 1.6 vs. 31.93 +/- 3.29 pmol/mg protein.15 sec) and cytochalasin-beta binding (3.06 +/- 0.28 vs. 6.14 +/- 0.59 pmol/mg protein) were significantly (P less than 0.05) reduced in the diabetic untreated rats compared to control values. Daily insulin injections restored both (P less than 0.05) basal transport (33.22 +/- 3.62 pmol/mg protein.15 sec) and cytochalasin-beta binding (5.52 +/- 0.66 pmol/mg protein) to control levels. Maximum insulin stimulation (1 U/kg, iv) significantly increased (P less than 0.05) both glucose transport (30.18 +/- 3.76 vs. 96.48 +/- 4.21 pmol/mg protein.15 sec) and cytochalasin-beta binding (4.38 +/- 0.29 vs. 9.40 +/- 0.42 pmol/mg protein) in the untreated diabetic and control rats. However, the stimulation in the untreated diabetic rats only reached basal control levels, which was significantly (P less than 0.05) below the insulin-stimulated value for the controls. In the rats receiving daily insulin injections, maximum insulin stimulation increased (P less than 0.05) both glucose transport (58.67 +/- 15.24 pmol/mg protein.15 sec) and cytochalasin-beta binding (6.4 +/- 0.7 pmol/mg protein), but both transport and binding were significantly (P less than 0.05) below insulin-stimulated values for the control rats. These data show that insulin deficiency adversely affected the glucose transport system in skeletal muscle. Both basal and maximum insulin-stimulated transport and the number of transport molecules were reduced. Daily insulin treatment corrected some of the defects, but maximum insulin stimulation was still significantly below values for control animals.