Yolanta T. Kruszynska

The insulin-sensitizing effects of thiazolidinediones are thought to be mediated through peroxisome proliferator-activated receptor-gamma, a nuclear receptor that is highly abundant in adipose tissue. It has been reported that adipocytes secrete a variety of proteins, including tumor necrosis factor-alpha, resistin, plasminogen activator inhibitor-1, and adiponectin. Adiponectin is a fat cell-secreted protein that has been reported to increase fat oxidation and improve insulin sensitivity. Our aim was to study the effects of troglitazone on adiponectin levels in lean, obese, and diabetic subjects. Ten diabetic and 17 nondiabetic subjects (8 lean, BMI <27 kg/m(2) and 9 obese, BMI >27 kg/m(2)) participated in the study. All subjects underwent an 80 mU. m(-2). min(-1) hyperinsulinemic-euglycemic glucose clamp before and after 3 months' treatment with the thiazolidinedione (TZD) troglitazone (600 mg/day). Fasting plasma glucose significantly decreased in the diabetic group after 12 weeks of treatment compared with baseline (9.1 +/- 0.9 vs. 11.1 +/- 0.9 mmol/l, P < 0.005) but was unchanged in the lean and obese subjects. Fasting insulin for the entire group was significantly lower than baseline (P = 0.02) after treatment. At baseline, glucose disposal rate (R(d)) was lower in the diabetic subjects (3.4 +/- 0.5 mg. kg(-1). min(-1)) than in the lean (12.3 +/- 0.4) or obese subjects (6.7 +/- 0.7) (P < 0.001 for both) and was significantly improved in the diabetic and obese groups (P < 0.05) after treatment, and it remained unchanged in the lean subjects. Baseline adiponectin levels were significantly lower in the diabetic than the lean subjects (9.0 +/- 1.7 vs. 16.7 +/- 2.7 micro g/ml, P = 0.03) and rose uniformly in all subjects (12.2 +/- 2.3 vs. 25.7 +/- 2.6 micro g/ml, P < 10(-4)) after treatment, with no significant difference detected among the three groups. During the glucose clamps, adiponectin levels were suppressed below basal levels in all groups (10.2 +/- 2.3 vs. 12.2 +/- 2.3 micro g/ml, P < 0.01). Adiponectin levels correlated with R(d) (r = 0.46, P = 0.016) and HDL cholesterol levels (r = 0.59, P < 0.001) and negatively correlated with fasting insulin (r = -0.39, P = 0.042) and plasma triglyceride (r = -0.61, P < 0.001). Our findings show that TZD treatment increased adiponectin levels in all subjects, including normal subjects in which no other effects of TZDs are observed. Insulin also appears to suppress adiponectin levels. We have confirmed these results in normal rats. These findings suggest that adiponectin can be regulated by obesity, diabetes, TZDs, and insulin, and it may play a physiologic role in enhancing insulin sensitivity.

Low plasma fibrinolytic activity in association with increased plasma plasminogen activator inhibitor 1 (PAI-1) levels has been linked to an increased risk of atherosclerosis in obesity and type 2 diabetes. We tested the hypothesis that troglitazone, which improves insulin sensitivity and lowers plasma insulin levels in insulin-resistant obese subjects and patients with type 2 diabetes, would also lower circulating PAI-1 antigen concentrations and activity. We assessed insulin sensitivity (5-h, 80 mU x m(-2) x min(-1) hyperinsulinemic-euglycemic clamp) and measured plasma PAI-1 antigen and activities and tissue plasminogen activator (tPA) in 14 patients with type 2 diabetes and 20 normal control subjects (10 lean, 10 obese) before and after 3 months of treatment with troglitazone (600 mg/day). At baseline, plasma PAI-1 antigen levels after an overnight fast were significantly higher in the obese (33.5 +/- 4.7 microg/l) and type 2 diabetic subjects (54.9 +/- 6.3 microg/l) than in the lean control subjects (16.3 +/- 3.2 microg/l; P < 0.01 and P < 0.001, respectively). Troglitazone decreased plasma PAI-1 antigen concentrations in the diabetic patients (36.8 +/- 5.0 microg/l; P < 0.001 vs. baseline), but the reduction in the obese subjects did not reach statistical significance (baseline, 33.5 +/- 4.7; after troglitazone, 25.6 +/- 5.2 microg/l). Changes in plasma PAI-1 activity paralleled those of PAI-1 antigen. The extent of the reduction in plasma PAI-1 antigen concentrations in the diabetic patients after troglitazone correlated with the reductions in fasting plasma insulin (r = 0.60, P < 0.05), nonesterified fatty acid (r = 0.63, P < 0.02), and glucose concentrations (r = 0.64, P < 0.02) but not with the improvement in glucose disposal rates during the glucose clamps. Three nonresponders to troglitazone with respect to effects on insulin sensitivity and fasting glucose and insulin levels also had no reduction in circulating PAI-1. In conclusion, troglitazone enhances fibrinolytic system activity in insulin-resistant type 2 diabetic patients. This effect appears to be intimately linked to its potential to lower plasma insulin levels and improve glycemic control through its peripheral tissue insulin-sensitizing effects.

Elevation of plasma nonesterified fatty acid (NEFA) levels has been shown in various studies to induce peripheral tissue insulin resistance and impair the suppression of endogenous glucose production (EGP). These studies have been conducted predominantly in men. We compared the effects of elevated plasma NEFA levels on basal and insulin-stimulated glucose metabolism in 8 normal women (age 42 +/- 8 years [mean +/- SD], BMI 25 +/- 3 kg/m(2)) and 10 normal men (35 +/- 6 years, 24 +/- 3 kg/m(2)). Each subject underwent two 5-h 80 mU. m(-2). min(-1) hyperinsulinemic-euglycemic clamps with measurement of glucose kinetics (intravenous [3-(3)H]glucose) and substrate oxidation. Plasma NEFA levels were elevated in one study for 3 h before and during the clamp ( approximately 1 mmol/l in both groups) by infusion of 20% Intralipid (60 ml/h) and heparin (900 U/h). In the control studies, the men and women had similar insulin-stimulated glucose disposal rates (R(d)) and substrate oxidation rates. In the men, elevated NEFA levels decreased insulin-stimulated glucose R(d) during the final 40 min of the clamp by 23% (P < 0.001). By contrast, no significant change in glucose R(d) was found in the women (control 10.4 +/- 1.1, lipid study 9.9 +/- 1.3 mg. kg(-1). min(-1)). Glucose R(d) was also unchanged in six women studied at a lower insulin dose (40 mU. m(-2). min(-1)). During the last 40 min of the high-insulin dose clamps with elevated NEFA, glucose oxidation was decreased by 33% in the men (P < 0.001) and by 23% in the women (P < 0.02). Nonoxidative glucose R(d) at this time was decreased by 15% in the men (P = 0.02) but was not significantly affected in women. Basal EGP was unaffected by elevation of plasma NEFA levels in both groups. Suppression of EGP during the glucose clamps, however, was impaired. At the insulin infusion rate used, the magnitude of this defect was comparable in men and women. In summary, our findings suggest that although the effects on EGP appear comparable, the inhibitory effects of NEFA on peripheral tissue insulin sensitivity are observed in men but cannot be demonstrated in women.

The mechanisms by which elevated plasma nonesterified fatty acid (NEFA) levels induce skeletal muscle insulin resistance remain unclear. A NEFA-induced defect in the activation of PI3K, which plays a key role in insulin's stimulation of glucose transport, has been invoked. We sought to examine the effects of elevated plasma NEFA (approximately 1 mmol/liter) on muscle PI3K activity, insulin receptor substrate (IRS)-1 (important for activation of PI3K), and Akt, which is downstream of PI3K and activated by phosphorylation on serine and threonine in a PI3K-dependent manner. Ten normal men [age, 37 +/- 9 yr (mean +/- SD); body mass index, 25.2 +/- 3.8 kg/m(2)] underwent two 5-h hyperinsulinemic (80 mU/m(2) x min) euglycemic clamps with basal and end of clamp biopsies of the vastus lateralis muscle. Plasma NEFAs were increased in one study by infusion of 20% Intralipid (1 ml/min) and heparin (900 U/h) throughout and for 2.5 h beforehand. Skeletal muscle protein levels were quantified by Western blotting. Elevated plasma NEFA reduced whole-body insulin-stimulated glucose disposal by 24% (42.1 +/- 4.0 vs. 54.8 +/- 3.6 micromol/kg x min; P < 0.001). Basal muscle IRS-1 was the same in the two studies. IRS-1 levels decreased by 40% in the control glucose clamps (P < 0.005), but did not change during the Intralipid study. Total tyrosine phosphorylated IRS-1 increased by 29% during the control clamps (P < 0.05), but by only 18% (NS) during the Intralipid studies. Total levels of p85alpha subunit of PI3K and Akt were not influenced by plasma NEFA levels either in the basal state or during the glucose clamps. The insulin-induced increase in IRS-1-associated PI3K activity was impaired by elevated NEFA, so that activity at the end of the clamps with Intralipid was 35% lower than in the control clamps (P < 0.05). The percentage reduction in PI3K activation correlated with the reduction in insulin-stimulated glucose disappearance rate that was induced by elevated NEFA (r = 0.70; P < 0.05). Basal P-ser- and P-thr-Akt levels were very low and unaffected by NEFA levels. The glucose clamps resulted in a marked increase in P-ser and P-thr Akt levels. Despite the decrease in PI3K in the Intralipid study, no defect in Akt phosphorylation was found. In summary, NEFA-induced insulin resistance is associated with an impairment of IRS-1 tyrosine phosphorylation and IRS-1-associated PI3K activation. Down-regulation of IRS-1 levels is also impaired. The NEFA-induced defect in muscle glucose uptake appears to be a consequence of a defect in the insulin-signaling pathway leading to impaired PI3K activation. This in turn may lead to impaired glucose transport through an Akt-independent pathway because Akt phosphorylation was unaffected by elevated NEFA levels.