Effects on insulin secretion and insulin action of a 48-h reduction of plasma free fatty acids with acipimox in nondiabetic subjects genetically predisposed to type 2 diabetes

Elevated plasma FFA cause beta-cell lipotoxicity and impair insulin secretion in nondiabetic subjects predisposed to type 2 diabetes mellitus [T2DM; i.e., with a strong family history of T2DM (FH+)] but not in nondiabetic subjects without a family history of T2DM. To determine whether lowering plasma FFA with acipimox, an antilipolytic nicotinic acid derivative, may enhance insulin secretion, nine FH+ volunteers were admitted twice and received in random order either acipimox or placebo (double-blind) for 48 h. Plasma glucose/insulin/C-peptide concentrations were measured from 0800 to 2400. On day 3, insulin secretion rates (ISRs) were assessed during a +125 mg/dl hyperglycemic clamp. Acipimox reduced 48-h plasma FFA by 36% (P < 0.001) and increased the plasma C-peptide relative to the plasma glucose concentration or DeltaC-peptide/Deltaglucose AUC (+177%, P = 0.02), an index of improved beta-cell function. Acipimox improved insulin sensitivity (M/I) 26.1 +/- 5% (P < 0.04). First- (+19 +/- 6%, P = 0.1) and second-phase (+31 +/- 6%, P = 0.05) ISRs during the hyperglycemic clamp also improved. This was particularly evident when examined relative to the prevailing insulin resistance [1/(M/I)], as both first- and second-phase ISR markedly increased by 29 +/- 7 (P < 0.05) and 41 +/- 8% (P = 0.02). There was an inverse correlation between fasting FFA and first-phase ISR (r2 = 0.31, P < 0.02) and acute (2-4 min) glucose-induced insulin release after acipimox (r2 =0.52, P < 0.04). In this proof-of-concept study in FH+ individuals predisposed to T2DM, a 48-h reduction of plasma FFA improves day-long meal and glucose-stimulated insulin secretion. These results provide additional evidence for the important role that plasma FFA play regarding insulin secretion in FH+ subjects predisposed to T2DM.     

Thiazolidinediones improve beta-cell function in type 2 diabetic patients

Thiazolidinediones (TZDs) improve glycemic control and insulin sensitivity in patients with type 2 diabetes mellitus (T2DM). There is growing evidence from in vivo and in vitro studies that TZDs improve pancreatic beta-cell function. The aim of this study was to determine whether TZD-induced improvement in glycemic control is associated with improved beta-cell function. We studied 11 normal glucose-tolerant and 53 T2DM subjects [age 53+/-2 yr; BMI 29.4+/-0.8 kg/m2; fasting plasma glucose (FPG) 10.3+/-0.4 mM; Hb A1c 8.2+/-0.3%]. Diabetic patients were randomized to receive placebo or TZD for 4 mo. Subjects received 1) 2-h OGTT with determination of plasma glucose, insulin, and C-peptide concentrations and 2) two-step euglycemic insulin (40 and 160 mU.m-2.min-1) clamp with [3-(3)H]glucose. T2DM patients were then randomized to receive 4 mo of treatment with pioglitazone (45 mg/day), rosiglitazone (8 mg/day), or placebo. Pioglitazone and rosiglitazone similarly improved FPG, mean plasma glucose during OGTT, Hb A1c, and insulin-mediated total body glucose disposal (Rd) and decreased mean plasma FFA during OGTT (all P<0.01, ANOVA). The insulin secretion/insulin resistance (disposition) index [DeltaISR(AUC)/Deltaglucose(AUC)/IR] was significantly improved in all TZD-treated groups: +1.8+/-0.7 (PIO+drug-na?ve diabetics), +0.7+/-0.3 (PIO+sulfonylurea-treated diabetics), and 0.7+/-0.2 (ROSI+sulfonylurea-withdrawn diabetics) vs. -0.2+/-0.3 in the two placebo groups (P<0.01, all TZDs vs. placebo, ANOVA). Improved insulin secretion correlated positively with increased body weight, fat mass, and Rd and inversely with decreased plasma glucose and FFA during the OGTT. In T2DM patients, TZD treatment leads to improved beta-cell function, which correlates strongly with improved glycemic control.     

Acute, same-day effects of antecedent exercise on counterregulatory responses to subsequent hypoglycemia in type 1 diabetes mellitus

Exercise-induced hypoglycemia can occur within hours after exercise in type 1 diabetes mellitus (T1DM) patients. This study tested the hypothesis that an acute exercise bout causes (within hours) blunted autonomic and metabolic responses to subsequent hypoglycemia in patients with T1DM. Twelve T1DM patients (3 W/9 M) were studied during a single-step, 2-h hyperinsulinemic (572 +/- 4 pmol/l) hypoglycemic (2.8 +/- 0.1 mmol/l) clamp 2 h after either a hyperinsulinemic euglycemic (AM EUG) or hypoglycemic clamp (AM HYPO) or after sitting in a chair with basal insulin infusion (AM CON) or 90 min of moderate-intensity exercise (50% Vo(2 max), AM EX). Both AM HYPO and AM EX significantly blunted epinephrine responses and muscle sympathetic nerve activity responses to subsequent hypoglycemia compared with both control groups. Endogenous glucose production was significantly lower and the exogenous glucose infusion rate needed to maintain the hypoglycemic level was significantly greater during subsequent hypoglycemia in AM EX vs. CON. Rate of glucose disposal (Rd) was significantly reduced following AM HYPO. In summary, within 2.5 h, both moderate-intensity AM EX and AM HYPO blunted key autonomic counterregulatory responses. Despite this, glucose Rd was reduced during afternoon hypoglycemia following morning hypoglycemia, indicating posthypoglycemic insulin resistance. After morning exercise, endogenous glucose production was blunted, but glucose Rd was maintained during afternoon hypoglycemia, thereby indicating reduced metabolic defenses against hypoglycemia. These data suggest that exercise-induced counterregulatory failure can occur very rapidly, increasing the risk for hypoglycemia in T1DM within hours.     

Lactate induces insulin resistance in skeletal muscle by suppressing glycolysis and impairing insulin signaling

Elevation of plasma lactate levels induces peripheral insulin resistance, but the underlying mechanisms are unclear. We examined whether lactate infusion in rats suppresses glycolysis preceding insulin resistance and whether lactate-induced insulin resistance is accompanied by altered insulin signaling and/or insulin-stimulated glucose transport in skeletal muscle. Hyperinsulinemic euglycemic clamps were conducted for 6 h in conscious, overnight-fasted rats with or without lactate infusion (120 micromol x kg(-1) x min(-1)) during the final 3.5 h. Lactate infusion increased plasma lactate levels about fourfold. The elevation of plasma lactate had rapid effects to suppress insulin-stimulated glycolysis, which clearly preceded its effect to decrease insulin-stimulated glucose uptake. Both submaximal and maximal insulin-stimulated glucose transport decreased 25-30% (P < 0.05) in soleus but not in epitrochlearis muscles of lactate-infused rats. Lactate infusion did not alter insulin's ability to phosphorylate the insulin receptor, the insulin receptor substrate (IRS)-1, or IRS-2 but decreased insulin's ability to stimulate IRS-1- and IRS-2-associated phosphatidylinositol 3-kinase activities and Akt/protein kinase B activity by 47, 75, and 55%, respectively (P < 0.05 for all). In conclusion, elevation of plasma lactate suppressed glycolysis before its effect on insulin-stimulated glucose uptake, consistent with the hypothesis that suppression of glucose metabolism could precede and cause insulin resistance. In addition, lactate-induced insulin resistance was associated with impaired insulin signaling and decreased insulin-stimulated glucose transport in skeletal muscle.     

Time-dependent effects of fatty acids on skeletal muscle metabolism

Increased plasma levels of free fatty acids (FFA) occur in states of insulin resistance such as type 2 diabetes mellitus, obesity, and metabolic syndrome. These high levels of plasma FFA seem to play an important role for the development of insulin resistance but the mechanisms involved are not known. We demonstrated that acute exposure to FFA (1 h) in rat incubated skeletal muscle leads to an increase in the insulin-stimulated glycogen synthesis and glucose oxidation. In conditions of prolonged exposure to FFA, however, the insulin-stimulated glucose uptake and metabolism is impaired in skeletal muscle. In this review, we discuss the differences between the effects of acute and prolonged exposure to FFA on skeletal muscle glucose metabolism and the possible mechanisms involved in the FFA-induced insulin resistance.     

Effects of beta-adrenergic receptor stimulation and blockade on substrate metabolism during submaximal exercise

We used beta-adrenergic receptor stimulation and blockade as a tool to study substrate metabolism during exercise. Eight moderately trained subjects cycled for 60 min at 45% of VO(2 peak) 1) during a control trial (CON); 2) while epinephrine was intravenously infused at 0.015 microg. kg(-1) x min(-1) (beta-STIM); 3) after ingesting 80 mg of propranolol (beta-BLOCK); and 4) combining beta-BLOCK with intravenous infusion of Intralipid-heparin to restore plasma fatty acid (FFA) levels (beta-BLOCK+LIPID). beta-BLOCK suppressed lipolysis (i.e., glycerol rate of appearance) and fat oxidation while elevating carbohydrate oxidation above CON (135 +/- 11 vs. 113 +/- 10 micromol x kg(-1) x min(-1); P < 0.05) primarily by increasing rate of disappearance (R(d)) of glucose (36 +/- 2 vs. 22 +/- 2 micromol x kg(-1) x min(-1); P < 0.05). Plasma FFA restoration (beta-BLOCK+LIPID) attenuated the increase in R(d) glucose by more than one-half (28 +/- 3 micromol x kg(-1) x min(-1); P < 0.05), suggesting that part of the compensatory increase in muscle glucose uptake is due to reduced energy from fatty acids. On the other hand, beta-STIM markedly increased glycogen oxidation and reduced glucose clearance and fat oxidation despite elevating plasma FFA. Therefore, reduced plasma FFA availability with beta-BLOCK increased R(d) glucose, whereas beta-STIM increased glycogen oxidation, which reduced fat oxidation and glucose clearance. In summary, compared with control exercise at 45% VO(2 peak) (CON), both beta-BLOCK and beta-STIM reduced fat and increased carbohydrate oxidation, albeit through different mechanisms.     

Evidence that hyperglycaemia per se does not inhibit hepatic glucose production in man

The effect of hyperglycaemia on hepatic glucose production (Ra) was investigated in nine healthy men using sequential clamp protocols during somatostatin infusion and euglycaemia (0-150 min), at plasma glucose levels of 165 mg x dl-1 (9.2 mM, 150-270 min) and during insulin infusion (1.0 mU x kg-1 x min-1, 270-360 min) in study 1 or during hypo-insulinaemia and plasma glucose levels of 220 mg x dl-1 (12.2 mM; 270-390 min) in study 2. Somatostatin decreased Ra and glucose disposal rate (Rd) but increased plasma free fatty acids (FFA) and lipid oxidation during euglycaemia. Increasing plasma glucose to 165 mg x dl-1 (9.2 mM) and hypo-insulinaemia increased Rd, but no suppressive effects on Ra, plasma FFA and lipid oxidation were observed. By contrast hyperinsulinaemia (study 1), as well as a further increase in plasma glucose (study 2), both decreased Ra. However, more pronounced hyperglycaemia increased insulin secretion despite somatostatin resulting in a fall in plasma FFA and lipid oxidation. Our data questions the accepted dogma that hyperglycaemia inhibits Ra independently of insulin action.     

Intravenous estrogens increase insulin clearance and action in postmenopausal women

To test the hypothesis that estrogens alter insulin action, we evaluated the effects of intravenous conjugated estrogens (CE) on insulin-stimulated steady-state glucose infusion rate (SSGIR) and suppression of plasma glycerol in postmenopausal women (mean +/- SD; 56 +/- 4 yr; n = 12) not using hormone replacement. SSGIR and glycerol were measured during a two-stage (8 and 40 mU. m-2. min-1) hyperinsulinemic euglycemic clamp on 2 days, with or without a 2.5-mg intravenous CE bolus. Serum estradiol concentrations were increased approximately 200% on the estrogen (EST) compared with the control (CON) days. Serum insulin was reduced (P < 0.01) during stage 2 of the clamp for EST (63.3 +/- 12.8 micro U/ml) vs. CON (78.2 +/- 15.8 micro U/ml). Mean SSGIR and plasma glycerol did not differ between CON and EST days. With adjustment for differences in insulin concentration between conditions, stage 2 glucose disposals were significantly higher (8.63 vs. 7.20 mg. kg-1. min-1) and plasma glycerol concentrations were significantly lower (29.4 vs. 35.0 micro mol/l) for EST vs. CON. Our findings suggest that acute CE administration increases insulin clearance and action in postmenopausal women.     

Retardant effect of hyperglycemia on the rise in plasma fatty acids following insulin withdrawal in man

The present study was undertaken to examine the influence of hyperglycemia in retarding the rise in circulating FFA noted after acute insulin withdrawal in man. The arterial FFA response to somatostatin administration was measured in the presence of (a) euglycemia and (b) hyperglycemia. In seven normal men who received somatostatin (0.9 mg/h) with euglycemia maintained by exogenous glucose infusion plasma insulin levels fell to levels 4 uU/ml and plasma FFA concentrations rose from 659 +/- 123 to 2057 +/- 268 uEq/l. When somatostatin was infused with hyperglycemia maintained at approximately 230 mg/dl, plasma insulin levels were again maintained at levels 4 uU/ml. Despite similar insulinopenia plasma FFA concentrations rose from 510 +/- 56 to only 1125 +/- 180 uEq/l, significantly less than in the previous protocol (p less than 0.01). These data indicate that hyperglycemia per se significantly attenuates the rise in circulating FFA caused by acute insulin withdrawal in man.     

PPARgamma2 Pro12Ala polymorphism in relation to free fatty acids concentration and composition in lean healthy Czech individuals with and without family history of diabetes type 2

Free fatty acids (FFAs) are natural ligands of the PPARgamma2 receptor. FFA plasma concentration and composition may represent one of the factors accounting for high heterogeneity of conclusions concerning the effect of the Pro12Ala on BMI, insulin sensitivity or diabetes type 2 (DM2) susceptibility. Our objective was to investigate the relation and possible interactions between the Pro12Ala polymorphism and FFA status, metabolic markers, and body composition in 324 lean nondiabetic subjects (M/F: 99/225; age 32+/-11 years; BMI 23.9+/-4.0 kg/m(2)) with and without family history of DM2. Family history of DM2 was associated with lower % PUFA and slightly higher % MUFA. The presence of Pro12Ala polymorphism was not associated with fasting plasma FFA concentration or composition, anthropometric or metabolic markers of glucose and lipid metabolism in tested population. However, the interaction of carriership status with FFA levels influenced the basal glucose levels, insulin sensitivity and disposition indices, triglycerides, HDL-cholesterol and leptin levels, especially in women. The metabolic effects of 12Ala carriership were influenced by FFA levels - the beneficial role of 12Ala was seen only in the presence of low concentration of plasma FFA. Surprisingly, a high PUFA/SFA ratio was associated with lower insulin sensitivity, the protective effect of 12Ala allele was apparent in subjects with family history of DM2. On the basis of our findings and published data we recommend the genotyping of diabetic patients for Pro12Ala polymorphism of the PPARgamma2 gene before treatment with thiazolidinediones and education of subjects regarding diet and physical activity, which modulate metabolic outcomes.     

Muscle glucose transport and phosphorylation in type 2 diabetic, obese nondiabetic, and genetically predisposed individuals

Our objectives were to quantitate insulin-stimulated inward glucose transport and glucose phosphorylation in forearm muscle in lean and obese nondiabetic subjects, in lean and obese type 2 diabetic (T2DM) subjects, and in normal glucose-tolerant, insulin-resistant offspring of two T2DM parents. Subjects received a euglycemic insulin (40 mU.m(-2).min(-1)) clamp with brachial artery/deep forearm vein catheterization. After 120 min of hyperinsulinemia, a bolus of d-mannitol/3-O-methyl-d-[(14)C]glucose/d-[3-(3)H]glucose (triple-tracer technique) was given into brachial artery and deep vein samples obtained every 12-30 s for 15 min. Insulin-stimulated forearm glucose uptake (FGU) and whole body glucose metabolism (M) were reduced by 40-50% in obese nondiabetic, lean T2DM, and obese T2DM subjects (all P < 0.01); in offspring, the reduction in FGU and M was approximately 30% (P < 0.05). Inward glucose transport and glucose phosphorylation were decreased by approximately 40-50% (P < 0.01) in obese nondiabetic and T2DM groups and closely paralleled the decrease in FGU. The intracellular glucose concentration in the space accessible to glucose was significantly greater in obese nondiabetic, lean T2DM, obese T2DM, and offspring compared with lean controls. We conclude that 1) obese nondiabetic, lean T2DM, and offspring manifest moderate-to-severe muscle insulin resistance (FGU and M) and decreased insulin-stimulated glucose transport and glucose phosphorylation in forearm muscle; these defects in insulin action are not further reduced by the combination of obesity plus T2DM; and 2) the increase in intracelullar glucose concentration under hyperinsulinemic euglycemic conditions in obese and T2DM groups suggests that the defect in glucose phosphorylation exceeds the defect in glucose transport.     

Differing physiological effects of epinephrine in type 1 diabetes and nondiabetic humans

Acute increases of the key counterregulatory hormone epinephrine can be modified by a number of physiological and pathological conditions in type 1 diabetic patients (T1DM). However, it is undecided whether the physiological effects of epinephrine are also reduced in T1DM. Therefore, the aim of this study was to determine whether target organ (liver, muscle, adipose tissue, pancreas, cardiovascular) responses to epinephrine differ between healthy subjects and T1DM patients. Thirty-four age- and weight-matched T1DM (n = 17) and healthy subjects (n = 17) underwent two randomized, single-blind, 2-h hyperinsulinemic euglycemic clamp studies with (Epi) and without epinephrine infusion. Muscle biopsy was performed at the end of each study. Epinephrine levels during Epi were similar in all groups (4,039 +/- 384 pmol/l). Glucose (5.3 +/- 0.06 mmol/l) and insulin levels (462 +/- 18 pmol/l) were also similar in all groups during the glucose clamps. Glucagon responses to Epi were absent in T1DM and significantly reduced compared with healthy subjects. Endogenous glucose production during the final 30 min was significantly greater during Epi in healthy subjects compared with T1DM (8.4 +/- 1.3 vs. 4.4 +/- 0.6 micromol.kg(-1).min(-1), P = 0.041). Glucose uptake showed almost a twofold greater decrease with Epi in healthy subjects vs. T1DM (Delta31 +/- 2 vs. Delta17 +/- 2 nmol.kg(-1).min(-1), respectively, P = 0.026). Glycerol, beta-hydroxybutyrate, and nonesterified fatty acid (NEFA) all increased significantly more in T1DM compared with healthy subjects. Increases in systolic blood pressure were greater in healthy subjects, but reductions of diastolic blood pressure were greater in T1DM patients with Epi. Reduction of glycogen synthase was significantly greater during epinephrine infusion in T1DM vs. healthy subjects. In summary, despite equivalent epinephrine, insulin, and glucose levels, changes in glucose flux, glucagon, and cardiovascular responses were greater in healthy subjects compared with T1DM. However, T1DM patients had greater lipolytic responses (glycerol and NEFA) during Epi. Thus we conclude that there is a spectrum of significant in vivo physiological differences of epinephrine action at the liver, muscle, adipose tissue, pancreas, and cardiovascular system between T1DM and healthy subjects.     

Discordant effects of glucosamine on insulin-stimulated glucose metabolism and phosphatidylinositol 3-kinase activity.

The impact of increased GlcN availability on insulin-stimulated p85/p110 phosphatidylinositol 3-kinase (PI3K) activity in skeletal muscle was examined in relation to GlcN-induced defects in peripheral insulin action. Primed continuous GlcN infusion (750 micromol/kg bolus; 30 micromol/kg.min) in conscious rats limited both maximal stimulation of muscle PI3K by acute insulin (I) (1 unit/kg) bolus (I + GlcN = 1.9-fold versus saline = 3.3-fold above fasting levels; p < 0.01) and chronic activation of PI3K following 3-h euglycemic, hyperinsulinemic (18 milliunits/kg.min) clamp studies (I + GlcN = 1.2-fold versus saline = 2.6-fold stimulation; p < 0.01). To determine the time course of GlcN-induced defects in insulin-stimulated PI3K activity and peripheral insulin action, GlcN was administered for 30, 60, 90, or 120 min during 2-h euglycemic, hyperinsulinemic clamp studies. Activation of muscle PI3K by insulin was attenuated following only 30 min of GlcN infusion (GlcN 30 min = 1.5-fold versus saline = 2.5-fold stimulation; p < 0.05). In contrast, the first impairment in insulin-mediated glucose uptake (Rd) developed following 110 min of GlcN infusion (110 min = 39.9 +/- 1.8 versus 30 min = 42.8 +/- 1.4 mg/kg.min, p < 0.05). However, the ability of insulin to stimulate phosphatidylinositol 3,4, 5-trisphosphate production and to activate glycogen synthase in skeletal muscle was preserved following up to 180 min of GlcN infusion. Thus, increased GlcN availability induced (a) profound and early inhibition of proximal insulin signaling at the level of PI3K and (b) delayed effects on insulin-mediated glucose uptake, yet (c) complete sparing of insulin-mediated glycogen synthase activation. The pattern and time sequence of GlcN-induced defects suggest that the etiology of peripheral insulin resistance may be distinct from the rapid and marked impairment in insulin signaling.     

Role of tumor suppressor PTEN in tumor necrosis factor alpha-induced inhibition of insulin signaling in murine skeletal muscle C2C12 cells.

In an attempt to clarify the role of phosphatase and tensin homologue deleted on chromosome 10 (PTEN) in muscle insulin resistance, we investigated the effect of PTEN on phosphoinositide 3 (PI3)-kinase/Akt related insulin signaling pathway in skeletal muscle-like C2C12 cells damaged by tumor necrosis factor-alpha (TNFalpha). C2C12 cells cultured with TNFalpha (10 ng/ml) for 1 h displayed a marked decrease of insulin-stimulated 2-[14C]-deoxy-D-glucose (2-DG) uptake in parallel with an elevation of PTEN mRNA and protein levels. However, pretreatment of PTEN antisense oligonucleotide (AS) (1 micromol/l for 3 days) for specific inhibition of PTEN expression in C2C12 cells abolished the TNFalpha-induced changes in 2-DG uptake. Similar pretreatment with PTEN AS, but not with sense oligonucleotide (1 micromol/l for 3 days), eliminated the ability of TNFalpha to impair insulin-stimulated signals including p85 regulatory subunit of PI3-kinase expression and the degree of Akt serine phosphorylation as well as protein expression in glucose transporter subtype 4. Data taken from cultured C2C12 cells emphasize the negative regulatory of muscle PI3-kinase/Akt signaling pathways as the major substrate of PTEN but also support the concept that PTEN contributes to the development of insulin resistance in skeletal muscle.     

FFA cause hepatic insulin resistance by inhibiting insulin suppression of glycogenolysis

Free fatty acids (FFA) have been shown to inhibit insulin suppression of endogenous glucose production (EGP). To determine whether this is the result of stimulation by FFA of gluconeogenesis (GNG) or glycogenolysis (GL) or a combination of both, we have determined rates of GNG and GL (with (2)H(2)O) and EGP in 16 healthy nondiabetic volunteers (11 males, 5 females) during euglycemic-hyperinsulinemic (~450 pM) clamping performed either with or without simultaneous intravenous infusion of lipid plus heparin. During insulin infusion, FFA decreased from 571 to 30 micromol/l (P < 0.001), EGP from 15.7 to 2.0 micromol x kg(-1) x min(-1) (P < 0.01), GNG from 8.2 to 3.7 micromol x kg(-1). min(-1) (P < 0.05), and GL from 7.4 to -1.7 micromol x kg(-1). min(-1) (P < 0.02). During insulin plus lipid/heparin infusion, FFA increased from 499 to 1,247 micromol/l (P < 0.001). EGP decreased 64% less than during insulin alone (-5.1 +/- 0.7 vs. -13.7 +/- 3.4 micromol x kg(-1). min(-1)). The decrease in GNG was not significantly different from the decrease of GNG during insulin alone (-2.6 vs. -4.5 micromol x kg(-1). min(-1), not significant). In contrast, GL decreased 66% less than during insulin alone (-3.1 vs. -9.2 micromol x kg(-1). min(-1), P < 0.05). We conclude that insulin suppressed EGP by inhibiting GL more than GNG and that elevated plasma FFA levels attenuated the suppression of EGP by interfering with insulin suppression of GL.     

No in vitro effects of fatty acids on glucose uptake, lipolysis or insulin signaling in rat adipocytes.

Elevated plasma levels of free fatty acids (FFA) can produce insulin resistance in skeletal muscle tissue and liver and, together with alterations in beta-cell function, this has been referred to as lipotoxicity. This study explores the effects of FFAs on insulin action in rat adipocytes. Cells were incubated 4 or 24 h with or without an unsaturated FFA, oleate or a saturated FFA, palmitate (0.6 and 1.5 mM, respectively). After the culture period, cells were washed and insulin effects on glucose uptake and lipolysis as well as cellular content of insulin signaling proteins (IRS-1, PI3-kinase, PKB and phosphorylated PKB) and the insulin regulated glucose transporter GLUT4 were measured. No significant differences were found in basal or insulin-stimulated glucose uptake in FFA-treated cells compared to control cells, regardless of fatty acid concentration or incubation period. Moreover, there were no significant alterations in the expression of IRS-1, PI3-kinase, PKB and GLUT4 following FFA exposure. Insulin's ability to stimulate PKB phosphorylation was also left intact. Nor did we find any alterations following FFA exposure in basal or cAMP-stimulated lipolysis or in the ability of insulin to inhibit lipolysis. The results indicate that oleate or palmitate does not directly influence insulin action to stimulate glucose uptake and inhibit lipolysis in rat fat cells. Thus, lipotoxicity does not seem to occur in the fat tissue itself.     

Suppression of glucose production by GLP-1 independent of islet hormones: a novel extrapancreatic effect

Glucagon-like peptide-1 (GLP-1) is an intestinal hormone that stimulates insulin secretion and decreases glucagon release. It has been hypothesized that GLP-1 also reduces glycemia independent of its effect on islet hormones. Based on preliminary evidence that GLP-1 has independent actions on endogenous glucose production, we undertook a series of experiments that were optimized to address this question. The effect of GLP-1 on glucose appearance (Ra) and glucose disposal (Rd) was measured in eight men during a pancreatic clamp that was performed by infusing octreotide to suppress secretion of islet hormones, while insulin and glucagon were infused at rates adjusted to maintain blood glucose near fasting levels. After stabilization of plasma glucose and equilibration of [3H]glucose tracer, GLP-1 was given intravenously for 60 min. Concentrations of insulin, C-peptide, and glucagon were similar before and during the GLP-1 infusion (115 +/- 14 vs. 113 +/- 11 pM; 0.153 +/- 0.029 vs. 0.156 +/- 0.026 nM; and 64.7 +/- 11.5 vs. 65.8 +/- 13.8 ng/l, respectively). With the initiation of GLP-1, plasma glucose decreased in all eight subjects from steady-state levels of 4.8 +/- 0.2 to a nadir of 4.1 +/- 0.2 mM. This decrease in plasma glucose was accounted for by a significant 17% decrease in Ra, from 22.6 +/- 2.8 to 19.1 +/- 2.8 micromol. kg-1. min-1 (P < 0.04), with no significant change in Rd. These findings indicate that, under fasting conditions, GLP-1 decreases endogenous glucose production independent of its actions on islet hormone secretion.     

Free fatty acid-induced peripheral insulin resistance augments splanchnic glucose uptake in healthy humans

To investigate the effect of elevated plasma free fatty acid (FFA) concentrations on splanchnic glucose uptake (SGU), we measured SGU in nine healthy subjects (age, 44 +/- 4 yr; body mass index, 27.4 +/- 1.2 kg/m(2); fasting plasma glucose, 5.2 +/- 0.1 mmol/l) during an Intralipid-heparin (LIP) infusion and during a saline (Sal) infusion. SGU was estimated by the oral glucose load (OGL)-insulin clamp method: subjects received a 7-h euglycemic insulin (100 mU x m(-2) x min(-1)) clamp, and a 75-g OGL was ingested 3 h after the insulin clamp was started. After glucose ingestion, the steady-state glucose infusion rate (GIR) during the insulin clamp was decreased to maintain euglycemia. SGU was calculated by subtracting the integrated decrease in GIR during the period after glucose ingestion from the ingested glucose load. [3-(3)H]glucose was infused during the initial 3 h of the insulin clamp to determine rates of endogenous glucose production (EGP) and glucose disappearance (R(d)). During the 3-h euglycemic insulin clamp before glucose ingestion, R(d) was decreased (8.8 +/- 0.5 vs. 7.6 +/- 0.5 mg x kg(-1) x min(-1), P < 0.01), and suppression of EGP was impaired (0.2 +/- 0.04 vs. 0.07 +/- 0.03 mg x kg(-1) x min(-1), P < 0.01). During the 4-h period after glucose ingestion, SGU was significantly increased during the LIP vs. Sal infusion study (30 +/- 2 vs. 20 +/- 2%, P < 0.005). In conclusion, an elevation in plasma FFA concentration impairs whole body glucose R(d) and insulin-mediated suppression of EGP in healthy subjects but augments SGU.     

Regulation of Myosin Light Chain Kinase during Insulin-Stimulated Glucose Uptake in 3T3-L1 Adipocytes

Myosin II (MyoII) is required for insulin-responsive glucose transporter 4 (GLUT4)-mediated glucose uptake in 3T3-L1 adipocytes. Our previous studies have shown that insulin signaling stimulates phosphorylation of the regulatory light chain (RLC) of MyoIIA via myosin light chain kinase (MLCK). The experiments described here delineate upstream regulators of MLCK during insulin-stimulated glucose uptake. Since 3T3-L1 adipocytes express two MyoII isoforms, we wanted to determine which isoform was required for insulin-stimulated glucose uptake. Using a siRNA approach, we demonstrate that a 60% decrease in MyoIIA protein expression resulted in a 40% inhibition of insulin-stimulated glucose uptake. We also show that insulin signaling stimulates the phosphorylation of MLCK. We further show that MLCK can be activated by calcium as well as signaling pathways. We demonstrate that adipocytes treated with the calcium chelating agent, 1,2-b (iso-aminophenoxy) ethane-N,N,N'',N''-tetra acetic acid, (BAPTA) (in the presence of insulin) impaired the insulin-induced phosphorylation of MLCK by 52% and the RLC of MyoIIA by 45% as well as impairing the recruitment of MyoIIA to the plasma membrane when compared to cells treated with insulin alone. We further show that the calcium ionophore, A23187 alone stimulated the phosphorylation of MLCK and the RLC associated with MyoIIA to the same extent as insulin. To identify signaling pathways that might regulate MLCK, we examined ERK and CaMKII. Inhibition of ERK2 impaired phosphorylation of MLCK and insulin-stimulated glucose uptake. In contrast, while inhibition of CaMKII did inhibit phosphorylation of the RLC associated with MyoIIA, inhibition of CAMKIIδ did not impair MLCK phosphorylation or translocation to the plasma membrane or glucose uptake. Collectively, our results are the first to delineate a role for calcium and ERK in the activation of MLCK and thus MyoIIA during insulin-stimulated glucose uptake in 3T3-L1 adipocytes.     

Interaction between altered insulin and lipid metabolism in CEACAM1-inactive transgenic mice

Inactivation of CEACAM1 in L-SACC1 mice by a dominant-negative transgene in liver impairs insulin clearance and increases serum free fatty acid (FFA) levels, resulting in insulin resistance. The contribution of elevated FFAs in the pathogenesis of insulin resistance is herein investigated. Treatment of L-SACC1 female mice with carnitine restored plasma FFA content. Concomitantly, it normalized insulin levels without directly regulating receptor-mediated insulin internalization and prevented glucose tolerance in these mice. Similarly, treatment with nicotinic acid, a lipolysis inhibitor, restored insulin-stimulated receptor uptake in L-SACC1 mice. Taken together, these data suggest that chronic elevation in plasma FFAs levels contributes to the regulation of insulin metabolism and action in L-SACC1 mice.