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Patients with type 2 diabetes mellitus exhibit hyperglycemia and dyslipidemia as well as a markedly increased incidence of atherosclerotic cardiovascular disease. Here we report the characterization of a novel arylthiazolidinedione capable of lowering both glucose and lipid levels in animal models. This compound, designated TZD18, is a potent agonist with dual human peroxisome proliferator-activated receptor (PPAR)-alpha/gamma activities. In keeping with its PPARgamma activity, TZD18 caused complete normalization of the elevated glucose in db/db mice and Zucker diabetic fatty rats. TZD18 lowered both cholesterol and triglycerides in hamsters and dogs. TZD18 inhibited cholesterol biosynthesis at steps before mevalonate and reduced hepatic levels of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity. Moreover, TZD18 significantly suppressed gene expression of fatty acid synthesis and induced expression of genes for fatty acid degradation and triglyceride clearance. Studies on 17 additional PPARalpha or PPARalpha/gamma agonists showed that lipid lowering in hamsters correlated with the magnitude of hepatic gene expression changes. Importantly, the presence of PPARgamma agonism did not affect the relationship between hepatic gene expression and lipid lowering. Taken together, these data suggest that PPARalpha/gamma agonists, such as TZD18, affect lipid homeostasis, leading to an antiatherogenic plasma lipid profile. Agents with these properties may provide favorable means for treatment of type 2 diabetes and dyslipidemia and the prevention of atherosclerotic cardiovascular disease.

Thiazolidinedione derivatives are a novel class of insulin-sensitizing agents that have demonstrated effective antidiabetic activity in vivo. Here, the effects of the potent thiazolidinedione derivative, AD-5075, on the rate-limiting enzyme of glycogen synthesis, glycogen synthase, were investigated in cultured rat adipose tissue. Short term preincubation of adipose tissue with AD-5075 potentiated acute insulin stimulation of I-form glycogen synthase activity in a concentration-dependent (EC50 approximately, 61nM) and time-dependent (t1/2 approximately, 2.3 h) manner. The thiazolidinedione derivative increased the responsiveness of I-form glycogen synthase activity to insulin stimulation at both maximal and submaximal insulin concentrations. In contrast, it had no effect on total glycogen synthase activity. Isoproterenol inhibited acute insulin activation of I-form glycogen synthase activity in a dose-dependent manner; maximal inhibition was attained at a concentration of 3 nM. AD-5075 antagonized isoproterenol inhibition of insulin's action. The concentration of glycogenolytic agent required to attain maximal inhibition was increased an order of magnitude in tissue treated with the antidiabetic agent. Short term preincubation of adipose tissue under hyperglycemic conditions (15 or 25 mM glucose) decreased insulin-stimulated I-form glycogen synthase activity. Concurrent treatment of the tissue with AD-5075 abrogated this glucose toxicity-induced inhibition of insulin action. Wortmannin, an inhibitor of phosphatidylinositol 3-kinase, blocked insulin activation of glycogen synthase in a dose-dependent manner. Half-maximal inhibition was observed at approximately 0.3 microM, and maximal inhibition occurred at 1.0 microM. AD-5075 did not antagonize wortmannin's inhibitory action. These results indicate that thiazolidinediones can act directly on adipose tissues to augment an important metabolic effect of insulin and counteract the inhibitory effects of catecholamines or hyperglycemia. As insulin stimulation of glycogen synthase remains wortmannin inhibitable in the presence of AD-5075, the effects of thiazolidinediones on insulin signal transduction may be phosphatidylinositol 3-kinase-dependent.

Thiazolidinedione (TZD) insulin sensitizers are specific agonists of peroxisome proliferator activated receptor (PPAR)gamma. However, their mechanism of action and the in vivo target tissue(s) that mediate insulin sensitization remain poorly defined. Although PPARgamma messenger RNA expression has been reported in skeletal muscle, the expression of PPARgamma within myocytes in intact muscle tissue has not been examined. An antipeptide PPARgamma antibody was generated; immunohistochemistry was then used to demonstrate that PPARgamma is present within nuclei of myocytes [in both skeletal (white and red fibers) and cardiac tissue (rodent and human)]. The effect of insulin sensitizer treatment on muscle insulin action was studied using ob/ob mice after 4 days dosing with a potent (6 nM PPARgamma Kd) TZD (10 mg/kg x day). 2-deoxyglucose (2-DOG) uptake was then assessed in freshly isolated soleus muscles from lean vs. ob/ob vs. TZD-treated ob/ob mice. In lean mouse muscles, 2-DOG uptake was stimulated by 82%, 95%, 165% (with 25, 100, 2000 microU/ml insulin); muscles from ob/ob were severely insulin resistant (80% stimulation with 2000 microU/ml insulin). Muscles from TZD-treated ob/ob displayed a normal insulin response with 100 (71%) or 2000 (158%) microU/ml insulin. Additional studies were performed using ZDF rats treated with/without TZD for 7 days. In vivo 2-DOG glucose uptake into soleus, gastrocnemius, and diaphragm muscles was measured during euglycemic-hyperinsulinemic clamp. Compared with lean rats, muscle 2-DOG uptake in ZDF was reduced by 52% (soleus) or 71% (diaphragm). Partial (40-60%) normalization of the reduced 2-DOG uptake was evident in TZD-treated ZDF rats. In contrast to the effect of in vivo treatment on muscle insulin action, preincubation of isolated soleus muscles from naive lean or ob/ob mice for 5 h with 100 nM TZD did not affect insulin-stimulated 2-DOG uptake. We conclude: 1) PPARgamma is expressed in myocytes within skeletal and cardiac muscle. 2) In vivo activation of PPARgamma by treatment of insulin-resistant mice/rats with a potent TZD corrects impaired muscle insulin action. 3) The lack of a direct effect on muscle after 5 h in vitro TZD incubation suggests that changes in insulin action may require a longer duration of PPARgamma activation or that improved muscle insulin sensitivity may result from an indirect in vivo effect of PPARgamma activation (e.g. changes in systemic lipid metabolism).

A role for peroxisome proliferator-activated receptors, PPAR gamma and PPAR alpha, as regulators of energy homeostasis and lipid metabolism, has been suggested. Recently, three distinct uncoupling protein isoforms, UCP-1, UCP-2, and UCP-3, have also been identified and implicated as mediators of thermogenesis. Here, we examined whether in vivo PPAR gamma or PPAR alpha activation regulates the expression of all three UCP isoforms. Rats or lean and db/db mice were treated with PPAR gamma [thiazolidinedione (TZD)] or PPAR alpha (WY-14643) agonists, followed by measurement of messenger RNAs (mRNAs) for UCP-1, UCP-2, and UCP-3 in selected tissues where they are expressed. TZD treatment (AD 5075 at 5 mg/kg x day) of rats (14 days) increased brown adipose tissue (BAT) depot size and induced the expression of each UCP mRNA (3x control levels for UCP-1 and UCP-2, 2.5x control for UCP-3). In contrast, UCP-2 and UCP-3 mRNA levels were not affected in white adipose tissue or skeletal muscle. Chronic (30 days) low-dose (0.3 mg/kg x day) TZD treatment induced UCP-1 mRNA and protein in BAT (2.5x control). In contrast, chronic TZD treatment (30 mg/kg x day) suppressed UCP-1 mRNA (>80%) and protein (50%) expression in BAT. This was associated with further induction of UCP-2 expression (>10-fold) and an increase in the size of lipid vacuoles, a decrease in the number of lipid vacuoles in each adipocyte, and an increase in the size of the adipocytes. TZD treatment of db/db mice (BRL 49653 at 10 mg/kg x day for 10 days) also induced UCP-1 and UCP-3 (but not UCP-2) expression in BAT. PPAR alpha is present in BAT, as well as liver. Treatment of rats or db/db mice with WY-14643 did not affect expression of UCP-1, -2, or -3 in BAT. Hepatic UCP-2 mRNA was increased (4x control level) in db/db and lean mice, although this effect was not observed in rats. Thus, in vivo PPAR gamma activation can induce expression of UCP-1, -2, and -3 in BAT; whereas chronic-intense PPAR gamma activation may cause BAT to assume white adipose tissue-like phenotype with increased UCP-2 levels. PPAR alpha activation in mice is sufficient to induce liver UCP-2 expression.