Leptin inhibits glycogen synthesis in the isolated soleus muscle of obese (ob/ob) mice

The ob gene product, leptin, causes significant and dose-dependent inhibition of basal and insulin-stimulated glycogen synthesis in isolated soleus muscle from ob/ob mice, and a smaller, non-significant inhibition in muscle from wild-type mice. Leptin had no inhibitory effect on glycogen synthesis in soleus muscle from the diabetic (db/db) mice, which lack the functional leptin receptor. The full-length leptin receptor (Ob-Rb), is expressed in soleus muscle of both ob/ob and wild-type mice, however with no detectable differences in expression level. These results suggest that hyperleptinaemia may attenuate insulin action on glucose storage in skeletal muscle.     

Acute and chronic effects of leptin on glucose utilization in lean mice

Experiments described here show that in vivo glucose uptake is impaired in mice given 30 micrograms leptin by intraperitoneal injection 2 hours before an oral glucose tolerance test (GTT). When mice were infused for 7 days with 10 micrograms/day leptin, the 4-fold increase in circulating leptin caused a transient hypophagia, a sustained weight loss and significantly inhibited insulin release in response to an oral GTT. Adipocytes from these mice were not insulin responsive whereas insulin-stimulated muscle and liver glycogen synthesis were increased. In contrast, leptin added to 2 hour in vitro incubations had an insulin-like effect on muscle glucose utilization and augmented insulin stimulation of adipocyte lipid synthesis. Thus, normal mice treated chronically with leptin develop tissue specific changes in insulin sensitivity and compensate for inhibition of glucose-stimulated insulin release. The contrasting response to acute leptin exposure suggests these changes are not a direct effect of the protein.     

Islet amyloid polypeptide decreases the effects of insulin-like growth factor-I on glucose transport and glycogen synthesis in skeletal muscle

Previous studies have shown that islet amyloid polypeptide (IAPP) is co-secreted with insulin from the beta-cell. IAPP reduces insulin-stimulated rates of glycogen synthesis in skeletal muscle but the mechanisms are unclear. Insulin-like growth factor I (IGF-I) is an important regulator of glucose metabolism in skeletal muscle and acts through its own receptor, which has many structural and functional similarities with the insulin receptor. Despite this, the effects of IGF-I on glucose utilization are not identical to those of insulin. The aim of the study was to determine the effects of IAPP on IGF-I-stimulated rates of glucose transport and metabolism (measured by 3-O-methyl[3H]glucose and [U-14C]glucose, respectively) in rat soleus muscle, and compare them with those simulated by insulin. IAPP (10 nM) decreased the sensitivity of 3-O-methylglucose transport, the flux of glucose to hexosemonophosphate and the sensitivity of glycogen synthesis to IGF-I. In contrast, IAPP had no effect on IGF-I-stimulated rates of lactate formation (i.e., glycolysis). IAPP decreased the sensitivity of 3-O-methylglucose transport and glycogen synthesis to insulin. It is concluded that IAPP blunts the stimulation of glucose uptake and deposition by IGF-I or insulin in skeletal muscle. These observations expand those made initially for IAPP and insulin and suggest that IAPP affects IGF-I- or insulin-stimulated glucose metabolism in muscle by a mechanism which is common for both hormones. These experiments may serve as a framework for future studies in order to clarify the mechanisms by which IAPP affects glucose metabolism in skeletal muscle.     

The effects of insulin on transport and metabolism of glucose in skeletal muscle from hyperthyroid and hypothyroid rats

The effects of insulin on the rates of glucose disposal were studied in soleus muscles isolated from hyper- or hypothyroid rats. Treatment with triiodothyronine for 5 or 10 days decreased the sensitivity of glycogen synthesis but increased the sensitivity of lactate formation to insulin. The sensitivity of 3-O methylglucose to insulin was increased only after 10 days of treatment and was accompanied by an increase in the sensitivity of 2-deoxyglucose phosphorylation; however, 2-deoxyglucose and glucose 6-phosphate in response to insulin remained unaltered. In hypothyroidism, insulin-stimulated rates of 3-O-methylglucose transport and 2-deoxyglucose phosphorylation were decreased; however, at basal levels of insulin, 3-O-methylglucose transport was increased, while 2-deoxyglucose phosphorylation was normal. In these muscles, the sensitivity of lactate formation to insulin was decreased; this defect was improved after incubation of the muscles with prostaglandin E2. The results suggest: (a) in hyperthyroidism, insulin-stimulated rates of glucose utilization in muscle to form lactate are increased mainly because of a decrease in glycogen synthesis; when hyperthyroidism progresses in severity, increases in the sensitivity of glucose transport to insulin and in the activity of hexokinase may also be involved; (b) in hypothyroidism, the decrease in insulin-stimulated rates of glucose utilization is caused by decreased rates of glycolysis; (c) prostaglandins may be involved in the changes in sensitivity of glucose utilization to insulin observed in muscle in altered thyroid states.     

The role of glucose 6-phosphate in the control of glycogen synthase

Elevated blood glucose concentrations result in increased intracellular levels of glucose 6-phosphate in liver, skeletal muscle, and adipose tissue. In liver, blood glucose concentrations are the main factor in control of the synthesis of glycogen; insulin has only a potentiating effect. In skeletal muscle and adipocytes, glucose alone has little effect on the activity of glycogen synthase, the limiting enzyme in glycogen synthesis. However, insulin released as a result of elevated blood glucose stimulates the translocation of specific glucose transporters to the cell membrane, increases the uptake of glucose, and causes the covalent, dephosphorylation-mediated activation of glycogen synthase. We present evidence that elevated intracellular contents of glucose 6-phosphate provoke the activation of glycogen synthase in liver, muscle, and adipose tissue. In addition, glucose 6-phosphate may inhibit the phosphorylation of glycogen synthase by cyclic AMP-stimulated protein kinase. We show that the stimulated glucose uptake and phosphorylation appear to play a major role in the control by insulin of the enzymes involved in glycogen synthesis.     

Acute stimulation of glucose metabolism in mice by leptin treatment

Leptin is an adipocyte hormone that functions as an afferent signal in a negative feedback loop regulating body weight, and acts by interacting with a receptor in the hypothalamus and other tissues. Leptin treatment has potent effects on lipid metabolism, and leads to a large, specific reduction of adipose tissue mass after several days. Here we show that leptin also acts acutely to increase glucose metabolism, although studies of leptin's effect on glucose metabolism have typically been confounded by the weight-reducing actions of leptin treatment, which by itself could affect glucose homoeostasis. We have demonstrated acute in vivo effects of intravenous and intracerebroventricular administrations of leptin on glucose metabolism. A five-hour intravenous infusion of leptin into wild-type mice increased glucose turnover and glucose uptake, but decreased hepatic glycogen content. The plasma levels of insulin and glucose did not change. Similar effects were observed after both intravenous and intracerebroventricular infusion of leptin, suggesting that effects of leptin on glucose metabolism are mediated by the central nervous system (CNS). These data indicate that leptin induces a complex metabolic response with effects on glucose as well as lipid metabolism. This response is unique to leptin, which suggests that new efferent signals emanate from the CNS after leptin treatment.     

Diverse effects of Glut 4 ablation on glucose uptake and glycogen synthesis in red and white skeletal muscle

The ability of muscles from Glut 4-null mice to take up and metabolize glucose has been studied in the isolated white EDL and red soleus muscles. In EDL muscles from male or female Glut 4-null mice, basal deoxyglucose uptake was lower than in control muscles and was not stimulated by insulin. In parallel, glycogen synthesis and content were decreased. Soleus muscles from male Glut 4-null mice took up twice more deoxyglucose in the absence of insulin than control muscles, but did not respond to insulin. In females, soleus deoxyglucose uptake measured in the absence of hormone was similar in Glut 4-null mice and in control mice. This uptake was stimulated twofold in Glut 4-null mice and threefold in control mice. Basal glycogen synthesis was increased by 4- and 2.2-fold in male and female null mice, respectively, compared to controls, and insulin had no or small (20% stimulation over basal) effect. These results indicate that while EDL muscles behaved as expected, soleus muscles were able to take up a large amount of glucose in the absence (males) or the presence of insulin (females). Whether this is due to a change in Glut 1 intrinsic activity or targeting and/or to the appearance of another glucose transporter remains to be determined.     

Decreased basal, noninsulin-stimulated glucose uptake and metabolism by skeletal soleus muscle isolated from obese-hyperglycemic (ob/ob) mice

Insulin resistance of diaphragms of ob/ob mice has been repeatedly demonstrated previously both in vitro and in vivo. In the present study, transport and metabolism of glucose with and without insulin stimulation were compared in a skeletal muscle more likely than diaphragm or heart to be representative of the overall striated muscle mass, i.e. isolated soleus muscle. Compared with soleus muscle from lean controls, unstimulated lactate release in the presence of exogenous glucose was depressed from 16.2 to 12.3 nmol/60 min per mg wet wt in soleus from ob/ob mutants; glycolysis was decreased from 6.6 to 3.7 and [14C]glucose oxidation to 14CO2 from 0.90 to 0.33 nmol glucose/60 min per mg wet wt. Uptake of 2-deoxyglucose (2-DOG), both with and without insulin, was very much less for soleus from ob/ob than from lean mice, at 2-DOG concentrations ranging from 0.1 to 10 mM, and in mice of 6-15 wk. When 2-DOG concentration was 1 mM, its basal uptake was 0.53 nmol/30 min per mg wet wt for soleus of ob/ob as against 0.96 for soleus of lean mice. The absolute increment due to 1 mU/ml insulin was 0.49 in muscle of ob/ob as against 1.21 in that of lean mice. When the resistance to insulin action was decreased by pretreatment in vivo by either streptozotocin injection or fasting, the decreased basal 2-DOG uptake of subsequently isolated soleus muscle was not improved. Inhibition of endogenous oxidation of fatty acids by 2-bromostearate, while greatly increasing 14CO2 production from [14C]glucose, did not affect basal [5-3H]glucose metabolism or 2-DOG uptake. It is suggested that transport and/or phosphorylation of glucose under basal, unstimulated conditions are depressed in soleus muscle of ob/ob mice, whether or not resistance to insulin and hyperinsulinemia are also present. Although the origin of the decreased basal glucose uptake remains unknown it might be related to a similar decrease in basal glucose uptake by ventromedial hypothalamic cells, an event presumably resulting in a tendency to hyperphagia. Decreased basal glucose uptake by soleus muscle of ob/ob mice might explain the hyperglycemia, and hence partly the hyperinsulinemia and excessive fat deposition of those animals.     

Acute non-insulin-like stimulation of rat muscle glucose metabolism by troglitazone in vitro

1. The direct short-term effects of troglitazone on parameters of glucose metabolism were investigated in rat soleus muscle strips. 2. In muscle strips from Sprague-Dawley rats, troglitazone (3.25 micromol l(-1)) increased basal and insulin-stimulated glucose transport by 24% and 41%, respectively (P<0.01 each). 3. In the presence of 5 nmol l(-1) insulin, stimulation of glucose transport by 3.25 micromol l(-1) troglitazone was accompanied by a 36% decrease in glycogen synthesis, while glycolysis was increased (112% increase in lactate production) suggesting a catabolic response of intracellular glucose handling. 4. Whereas insulin retained its stimulant effect on [3H]-2-deoxy-glucose transport in hypoxia-stimulated muscle (by 44%; c.p.m. mg(-1) h(-1): 852+/-77 vs 1229+/-75, P<0.01), 3.25 micromol l(-1) troglitazone failed to increase glucose transport under hypoxic conditions (789+/-40 vs 815+/-28, NS) suggesting that hypoxia and troglitazone address a similar, non-insulin-like mechanism. 5. No differences between troglitazone and hypoxia were identified in respective interactions with insulin. 6. Troglitazone acutely stimulated muscle glucose metabolism in a hypoxia/contraction-like manner, but it remains to be elucidated whether this contributes to the long-term antidiabetic and insulin enhancing potential in vivo or is to be regarded as an independent pharmacological effect.     

Effect of soleus unweighting on stimulation of insulin-independent glucose transport activity

Unweighting of the rat soleus by tail-cast suspension results in increased insulin action on stimulation of glucose transport, which can be explained, at least in part, by increased insulin binding and enhanced glucose transporter protein levels. Glucose transport is also activated by an insulin-independent mechanism stimulated by in vitro muscle contractions or hypoxia. Therefore, the purpose of this study was to determine if soleus unweighting leads to an enhanced response of the insulin-independent pathway for stimulation of glucose transport. The hindlimbs of juvenile male Wistar rats were suspended by a tail-cast system for 3 or 6 days. Glucose transport activity in isolated soleus strips (approximately 18 mg) was then assessed by using 2-deoxy-[1,2-3H]glucose (2-DG) uptake. Insulin (2 mU/ml) had a progressively enhanced effect on 2-DG uptake after 3 and 6 days of unweighting (+44 and +72% vs. control, respectively; both P < 0.001). At these same times, there was no difference between groups for activation of 2-DG uptake by maximally effective treatments with contractions (10 tetanuses), hypoxia (60 min), or caffeine (5 mM). These results indicate that the enhanced capacity for stimulation of glucose transport after soleus unweighting is restricted to the insulin pathway, with no apparent enhancement of the insulin-independent pathway.     

Modifications of glucose and lipid metabolism in cold-acclimated lean and genetically obese rats

Glucose turnover rate, 2-deoxy-D-[3H]glucose (2-DG) uptake, lipid synthesis in liver, white adipose tissue, and brown adipose tissue (BAT) were measured in lean FA/FA and genetically obese fa/fa rats either kept at 21 degrees C or acclimated to a cold environment (4 degrees C). After 10 days at 4 degrees C, lean rats increased their glucose turnover rate; 2-DG uptake as well as lipid synthesis in BAT were markedly stimulated. After cold acclimation, obese rats also increased glucose turnover; however, BAT glucose utilization was only slightly stimulated. Basal hyperinsulinemia and muscle insulin resistance of the obese group (as assessed by reduced 2-DG uptake in the soleus muscle) were present at room temperature and persisted at 4 degrees C. Total BAT lipid synthesis was increased to the same extent as in lean rats. Obese rat liver lipid synthesis, already much higher than normal at 21 degrees C, was further increased by cold exposure. We conclude that obese cold-acclimated fa/fa rats do not improve their muscle insulin resistance and barely improve BAT glucose utilization. We further suggest that an additional activation of hepatic lipid synthesis and oxidation thereof could participate in the heat production needed by the cold-acclimated obese rats.     

Leptin inhibits insulin-stimulated incorporation of glucose into lipids and stimulates glucose decarboxylation in isolated rat adipocytes

Leptin is an adipocyte hormone involved in the regulation of energy homeostasis. Generally accepted biological effects of leptin are inhibition of food intake and stimulation of metabolic rate in ob/ob mice, that are defective in the leptin gene. In contrast to these centrally mediated effects of leptin, we are reporting here on leptin effects on glucose incorporation into lipids and glucose decarboxylation in adipocytes isolated from male lean albino rats. Adipocytes previously cultivated (15 h) in the presence of leptin presented a 25% (P < 0.05) reduction of the insulin stimulated incorporation of glucose into lipids. Concurrently, the basal conversion of (U-14C)D-glucose into 14CO2 increased (110%) in the leptin cultivated adipocytes and reached values (1.54 nmol/10(5) cells) similar to the insulin stimulated group (not cultivated with leptin) (1.40 nmol/10(5) cells). In addition, in the presence of insulin, the leptin cultivated adipocytes elicited a 162% (P < 0.05) increase in 14CO2 production that was significantly higher than the increase observed for the not-leptin-cultivated insulin group (92%). We conclude that leptin: 1) directly inhibits the insulin stimulated glucose incorporation into lipids; 2) stimulates glucose decarboxylation, and also potentiates the effect of insulin on glucose decarboxylation in isolated adipocytes. Leptin per se does not alter glucose incorporation into lipids.     

Leptin constrains acetylcholine-induced insulin secretion from pancreatic islets of ob/ob mice

Hypersecretion of insulin from the pancreas is among the earliest detectable metabolic alterations in some genetically obese animals including the ob/ob mouse and in some obesity-prone humans. Since the primary cause of obesity in the ob/ob mouse is a lack of leptin due to a mutation in the ob gene, we tested the hypothesis that leptin targets a regulatory pathway in pancreatic islets to prevent hypersecretion of insulin. Insulin secretion is regulated by changes in blood glucose, as well as by peptides from the gastrointestinal tract and neurotransmitters that activate the pancreatic islet adenylyl cyclase (e.g., glucagon-like peptide-1) and phospholipase C (PLC) (e.g., acetylcholine) signaling pathways to further potentiate glucose-induced insulin secretion. Effects of leptin on each of these regulatory pathways were thus examined. Leptin did not influence glucose or glucagon-like peptide-1-induced insulin secretion from islets of either ob/ob or lean mice, consistent with earlier findings that these regulatory pathways do not contribute to the early-onset hypersecretion of insulin from islets of ob/ob mice. However, leptin did constrain the enhanced PLC- mediated insulin secretion characteristic of islets from ob/ob mice, without influencing release from islets of lean mice. A specific enhancement in PLC-mediated insulin secretion is the earliest reported developmental alteration in insulin secretion from islets of ob/ob mice, and thus a logical target for leptin action. This action of leptin on PLC-mediated insulin secretion was dose-dependent, rapid-onset (i.e., within 3 min), and reversible. Leptin was equally effective in constraining the enhanced insulin release from islets of ob/ob mice caused by protein kinase C (PKC) activation, a downstream mediator of the PLC signal pathway. One function of leptin in control of body composition is thus to target a PKC-regulated component of the PLC-PKC signaling system within islets to prevent hypersecretion of insulin.     

Non-insulin and non-exercise related increase of glucose utilization in rats and mice

The effects of high-energy phosphate contents in muscles on glucose tolerance and glucose uptake into tissues were studied in rats and mice. Enhanced glucose tolerance associated with depleted high-energy phosphates and elevated glycogen content in muscles and liver was observed in animals fed creatine analogue beta-guanidinopropionic acid (beta-GPA). Distribution of infused 2-[1-14C]deoxy-D-glucose in tissues especially in the soleus muscle, kidney, and brain was greater in mice fed beta-GPA than controls. The glucose uptake was decreased when the contents of ATP and glycogen were normalized following creatine supplementation. Plasma insulin in animals at rest was lower and its concentration after intraperitoneal glucose infusion tended to be less in animals fed beta-GPA than controls (p > 0.05), although the pattern of insulin response to glucose loading was similar to the control. The daily voluntary activity in beta-GPA fed mice was also less than controls. These results suggest that improved glucose tolerance is not related to elevated insulin concentration and/or decreased glycogen following exercise. Such improvement may be due to an increased mitochondrial energy metabolism caused by depletion of high-energy phosphates.     

Assignment of the gene encoding the limbic system-associated membrane protein (LAMP) to mouse chromosome 16B5 and human chromosome 3q13.2-q21

Both insulin and muscle contraction stimulate glucose transport activity. However, contraction stimulation does not involve the insulin signalling intermediate phosphatidylinositol 3-kinase (PI 3-kinase). Protein kinase B (PKB) has recently been identified as a direct downstream target of PI 3-kinase in the insulin signalling pathway. We have examined here whether the two stimuli share PKB as a convergent step in separate signalling pathways. Insulin stimulates both glucose transport, GLUT4 cell-surface content and PKB activity (by 4-6-fold above basal) in a wortmannin-sensitive manner in in vitro incubated rat soleus muscles. By contrast, muscle contraction, which stimulates glucose transport and the cell surface content of GLUT4 by 3-fold above basal levels, had no effect on PKB activity. These data demonstrate that PKB is not a mediator of contraction-induced glucose transport and GLUT4 translocation.     

Plasma leptin is not associated with insulin resistance and proinsulin in non-diabetic South Asian Indians

In an earlier study, we observed only a weak association between plasma insulin (non-specific assay) and leptin in South Asian Indians. This was in contrast to the observations in many other ethnic groups. With the availability of measurements of specific insulin (SI) and proinsulin (PI) in the same study group, we have reanalysed the data to look for possible correlation of leptin with proinsulin and with insulin resistance calculated from the fasting values of specific insulin and glucose using the HOMA model. Subjects with normoglycaemia (n = 117) and impaired glucose tolerance (n = 27, WHO criteria) were included in the analysis. Leptin values were higher in women. Multiple linear regression analysis showed that the variations in leptin concentrations in men were associated with BMI, WHR, and 2 h SI values (R2 = 56.2%) while fasting SI and proinsulin concentrations had no significant association. In women BMI and age showed a significant association with serum leptin values (R2 = 40.1%). Univariate and multivariate analyses using insulin resistance as the dependent variable showed that it had no association with leptin in both genders. Leptin had no correlation with proinsulin also. This study confirmed that in Asian Indians the association between plasma leptin and insulin concentrations is weak and that leptin has no influence on insulin resistance. Proinsulin and leptin are also not correlated in this population. Insulin resistance shows correlation with the beta-cell function both in men and women.     

The conserved amino-terminal domain of hSRP1 alpha is essential for nuclear protein import

Transgenic mice that overexpressed IGFBP-1 are hyperinsulinemic in the first week of life and gradually develop fasting hyperglycemia. In adult transgenic mice, the hypoglycemic response to IGF-I but not insulin or des (1-3) IGF-I was attenuated (P < 0.05) compared with wild-type mice. Furthermore, in isolated adipocytes from transgenic mice, the stimulatory effect of IGF-I but not insulin on 2-deoxy-[3H]-glucose uptake was reduced (P < 0.02). In contrast, in isolated soleus muscle, the effects of both IGF-I and insulin on 2-deoxy-3H-glucose uptake and on [3H]-glucose incorporation into glycogen were significantly reduced compared to wild-type mice. The decline in specific activity of the 2-deoxy-3H-glucose, a measure of glucose appearance in the circulation, was more marked in transgenic animals (P < 0.05). In addition, tissue uptake of glucose was significantly higher in diaphragm, heart, intestine, liver, soleus muscle, and adipose tissue from fasting transgenic mice. Plasma concentrations of alanine, lysine, and methionine were also elevated in transgenic mice. These data suggest that overexpression of IGFBP-1 attenuates the hypoglycemic effect of endogenous IGF-I, which is initially compensated for by enhanced pancreatic insulin production. However, in adult mice pancreatic insulin content is reduced, insulin resistance is demonstrable in skeletal muscle and fasting hyperglycemia develops.     

Regulation of exogenous and endogenous glucose metabolism by insulin and acetoacetate in the isolated working rat heart. A three tracer study of glycolysis, glycogen metabolism, and glucose oxidation

Myocardial glucose use is regulated by competing substrates and hormonal influences. However, the interactions of these effectors on the metabolism of exogenous glucose and glucose derived from endogenous glycogen are not completely understood. In order to determine changes in exogenous glucose uptake, glucose oxidation, and glycogen enrichment, hearts were perfused with glucose (5 mM) either alone, or glucose plus insulin (40 microU/ml), glucose plus acetoacetate (5 mM), or glucose plus insulin and acetoacetate, using a three tracer (3H, 14C, and 13C) technique. Insulin-stimulated glucose uptake and lactate production in the absence of acetoacetate, while acetoacetate inhibited the uptake of glucose and the oxidation of both exogenous glucose and endogenous carbohydrate. Depending on the metabolic conditions, the contribution of glycogen to carbohydrate metabolism varied from 20-60%. The addition of acetoacetate or insulin increased the incorporation of exogenous glucose into glycogen twofold, and the combination of the two had additive effects on the incorporation of glucose into glycogen. In contrast, the glycogen content was similar for the three groups. The increased incorporation of glucose in glycogen without a significant change in the glycogen content in hearts perfused with glucose, acetoacetate, and insulin suggests increased glycogen turnover. We conclude that insulin and acetoacetate regulate the incorporation of glucose into glycogen as well as the relative contributions of exogenous glucose and endogenous carbohydrate to myocardial energy metabolism by different mechanisms.     

Characterization of apolipoprotein E7 (Glu(244)-->Lys, Glu(245)--->Lys), a mutant apolipoprotein E associated with hyperlipidemia and atherosclerosis

To examine the mechanism by which free fatty acids (FFA) induce insulin resistance in human skeletal muscle, glycogen, glucose-6-phosphate, and intracellular glucose concentrations were measured using carbon-13 and phosphorous-31 nuclear magnetic resonance spectroscopy in seven healthy subjects before and after a hyperinsulinemic-euglycemic clamp following a five-hour infusion of either lipid/heparin or glycerol/heparin. IRS-1-associated phosphatidylinositol 3-kinase (PI 3-kinase) activity was also measured in muscle biopsy samples obtained from seven additional subjects before and after an identical protocol. Rates of insulin stimulated whole-body glucose uptake. Glucose oxidation and muscle glycogen synthesis were 50%-60% lower following the lipid infusion compared with the glycerol infusion and were associated with a approximately 90% decrease in the increment in intramuscular glucose-6-phosphate concentration, implying diminished glucose transport or phosphorylation activity. To distinguish between these two possibilities, intracellular glucose concentration was measured and found to be significantly lower in the lipid infusion studies, implying that glucose transport is the rate-controlling step. Insulin stimulation, during the glycerol infusion, resulted in a fourfold increase in PI 3-kinase activity over basal that was abolished during the lipid infusion. Taken together, these data suggest that increased concentrations of plasma FFA induce insulin resistance in humans through inhibition of glucose transport activity; this may be a consequence of decreased IRS-1-associated PI 3-kinase activity.