Hiroyoshi Horikoshi

Troglitazone increases the number of small adipocytes without the change of white adipose tissue mass in obese Zucker rats.

Troglitazone (CS-045) is one of the thiazolidinediones that activate the peroxisome proliferator-activated receptor gamma (PPARgamma), which is expressed primarily in adipose tissues. To elucidate the mechanism by which troglitazone relieves insulin resistance in vivo, we studied its effects on the white adipose tissues of an obese animal model (obese Zucker rat). Administration of troglitazone for 15 d normalized mild hyperglycemia and marked hyperinsulinemia in these rats. Plasma triglyceride level was decreased by troglitazone in both obese and lean rats. Troglitazone did not change the total weight of white adipose tissues but increased the number of small adipocytes (< 2,500 micron2) approximately fourfold in both retroperitoneal and subcutaneous adipose tissues of obese rats. It also decreased the number of large adipocytes (> 5,000 micron2) by approximately 50%. In fact, the percentage of apoptotic nuclei was approximately 2.5-fold higher in the troglitazone-treated retroperitoneal white adipose tissue than control. Concomitantly, troglitazone normalized the expression levels of TNF-alpha which were elevated by 2- and 1.4-fold in the retroperitoneal and mesenteric white adipose tissues of the obese rats, respectively. Troglitazone also caused a dramatic decrease in the expression levels of leptin, which were increased by 4-10-fold in the white adipose tissues of obese rats. These results suggest that the primary action of troglitazone may be to increase the number of small adipocytes in white adipose tissues, presumably via PPARgamma. The increased number of small adipocytes and the decreased number of large adipocytes in white adipose tissues of troglitazone-treated obese rats appear to be an important mechanism by which increased expression levels of TNF-alpha and higher levels of plasma lipids are normalized, leading to alleviation of insulin resistance.

Characterization of New Oral Antidiabetic Agent CS-045: Studies in KK and ob/ob Mice and Zucker Fatty Rats

CS-045 is a new oral antidiabetic agent that was effective in insulin-resistant diabetic animal models, including the KK mouse, the ob/ob mouse, and the Zucker fatty rat. CS-045 was not effective in the streptozocin-treated mouse, an insulin-deficient diabetic animal model. In fed KK mice, CS-045 lowered the plasma glucose levels in a dosedependent manner after a single oral administration, and the hypoglycemic effect lasted for at least 18 h. In normal rats, however, plasma glucose levels were not changed after administration of CS-045. CS-045 when given chronically (2 wk) to diabetic KK and ob/ob mice as a 0.2% food admixture dramatically improved hyperglycemia, hyperinsulinemia, and hypertriglyceridemia to near-normal values and decreased plasma lactate, free fatty acid, and ketone body levels without reducing food intake or body weight. In the obese Zucker fatty rat, oral administration of CS-045 had a similar effect in lowering plasma glucose, insulin, triglyceride, free fatty acid, lactate, and ketone body levels. The CS-045–treated Zucker fatty rats showed increased glucose tolerance and decreased insulin secretion in response to oral glucose. After 9 days of treatment, insulin binding to adipocyte plasma membranes from both CS-045-treated Zucker fatty rats and KK mice was increased. Furthermore, 2-deoxyglucose uptake in CS-045-treated adipocytes was increased and the insulin dose-response curve was shifted to the left. These findings suggest that CS-045 increases not only insulin sensitivity but also insulin responsiveness. Based on its pharmacological profile, CS-045 is a new orally effective antidiabetic agent that may reduce abnormalities of glucose and lipid metabolism in obese and non-insulin-dependent diabetes mellitus patients with insulin resistance.

Angptl3 regulates lipid metabolism in mice

The KK obese mouse is moderately obese and has abnormally high levels of plasma insulin (hyperinsulinemia), glucose (hyperglycemia) and lipids (hyperlipidemia). In one strain (KK/San), we observed abnormally low plasma lipid levels (hypolipidemia). This mutant phenotype is inherited recessively as a mendelian trait. Here we report the mapping of the hypolipidemia (hypl) locus to the middle of chromosome 4 and positional cloning of the autosomal recessive mutation responsible for the hypolipidemia. The hypl locus encodes a unique angiopoietin-like lipoprotein modulator, which we named Allm1. It is identical to angiopoietin-like protein 3, encoded by Angptl3, and has a highly conserved counterpart in humans. Overexpression of Angptl3 or intravenous injection of the purified protein in KK/San mice elicited an increase in circulating plasma lipid levels. This increase was also observed in C57BL/6J normal mice. Taken together, these data suggest that Angptl3 regulates lipid metabolism in animals.

Antihypertensive Effects of CS-045 Treatment in Obese Zucker Rats

The association of hypertension with obesity has been well recognized, but the etiology remains poorly understood. Obesity is characterized by hyperinsulinemia, which reflects peripheral insulin resistance. Insulin resistance may participate in the development of hypertension with obesity. CS-045 [(I)-5-[4-(5-hydroxy-2,5,7,8-tetramethylchroman-2-yl-methoxy)be nzy l]-2,4- thiazolidiendion] is a new orally effective antidiabetic agent that potentiates insulin action and reduces insulin resistance in obese Zucker rats and other obese diabetic animals. In this study, we examined the antihypertensive effect of CS-045 in obese male and female Zucker rats as a model of hypertension associated with obesity. CS-045 was administered as a food admixture (approximately 16 and approximately 70 mg/kg/d) for 4 weeks (female) and (approximately 15 and approximately 67 mg/kg/d) 8 weeks (male) in obese Zucker rats at 5 to 7 months of age. CS-045 slightly but significantly decreased plasma glucose levels. Plasma insulin levels were significantly decreased in obese male rats, but were not significantly decreased in obese female rats. Drug administration led to significant decreases in plasma triglyceride and cholesterol levels and systolic blood pressure (SBP) in a dose-dependent manner from 1 week after administration in obese Zucker rats. CS-045 increased urinary sodium excretion, sodium/potassium ratio, and creatine clearance in a dose-dependent manner, and also led to a remarkable decrease in urinary protein excretion. However, CS-045 did not reduce urinary catecholamine excretion. These data indicate that CS-045 may promote renal sodium excretion and improve decreased glomerular filtration rates, which may reflect the amelioration of insulin resistance.(ABSTRACT TRUNCATED AT 250 WORDS)

Suppression of Hepatic Gluconeogenesis in Long-Term Troglitazone Treated Diabetic KK and C57BL/KsJ-db/db Mice

The orally effective antidiabetic agent Troglitazone (CS-045) exerts hypoglycemic effects in various insulin-resistant obese and/or diabetic animals. Since increased hepatic gluconeogenesis is a major cause of hyperglycemia in these diabetic animals, we evaluated the effect of long-term Troglitazone treatment on hepatic gluconeogenesis. Troglitazone was administered for 7 days to normal ddY mice, diabetic KK mice, diabetic C57BL/KsJ-db/db mice, and its heterozygote, db/+ mice, as a 0.1% or 0.2% food admixture. Troglitazone significantly decreased plasma glucose in diabetic KK and db/db mice, but not in normal ddY and db/+ mice. 14C incorporation into blood glucose from NaH14CO3 was measured to assess hepatic gluconeogenesis in diabetic KK and normal ddY mice. Hepatic gluconeogenesis was significantly increased in diabetic KK mice (P < .01) as compared with normal mice, and was significantly suppressed (P < .05) after 7 days of Troglitazone treatment (approximately 200 mg/kg/d). Glucose-6-phosphate (G6P) and fructose-6-phosphate (F6P) were significantly decreased but fructose-1,6-bisphosphate (FBP) was not significantly increased in the liver of diabetic db/db mice treated with Troglitazone for 7 days (approximately 80 mg/kg/d) as compared with control db/db mice. These changes in G6P, F6P, and FBP corresponded with the activity of fructose-1,6-bisphosphatase (Fru-1,6P2ase) and 6-phosphofructo-1-kinase (6-PF-1K), which determined the content of F6P and FBP. Namely, Fru-1,6P2ase was significantly decreased in Troglitazone-treated db/db mice as compared with control mice, whereas 6-PF-1K activity was not affected by Troglitazone treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute Hyperglycemia Provides an Insulin-Independent Inducer for GLUT4 Translocation in C2C12 Myotubes and Rat Skeletal Muscle

GLUT4 translocation and activation of glucose uptake in skeletal muscle can be induced by both physiological (i.e., insulin, nerve stimulation, or exercise) and pharmacological (i.e., phorbol ester) means. Recently, we demonstrated that high glucose levels may mimic the effects of phorbol esters on protein kinase C (PKC) and insulin receptor function (J Biol Chem 269:3381–3386, 1994). In this study, we tested whether the previously described effects of phorbol esters on translocation of GLUT4 in myotubes in culture and also in rat skeletal muscle might be mimicked by glucose. We found that stimulation of C2C12 myotubes with both insulin (10–7) mol/l, 5 min) and glucose (25 mmol/l, 10 min) induces a comparable increase of the GLUT4 content in the plasma membrane. To test whether this effect occurs in intact rat skeletal muscle as well, two different model systems were used. As an in vitro model, isolated rat hindlimbs were perfused for 80 min with medium containing 6 mmol/l glucose ± insulin (1.6 × 10–9 mmol/l, 40 min) or 25 mmol/l glucose. As an in vivo model, acute hyperglycemia (> 11 mmol/l glucose, 20 min) was induced in Wistar rats by intraperitoneal injection of glucose under simultaneous suppression of the endogenous insulin release by injection of somatostatin. In both models, subcellular fractions were prepared from hindlimb skeletal muscle, and plasma membranes were characterized by the enrichment of the marker enzyme α1 Na+ -K+ -ATPase. Acute hyperglaycemia in vivo (n = 5) and in vitro (n = 6) induced an increases of GLUT4 content in the α1 Na+ -K+ -ATPase–enriched fraction (in vivo, 2.45 ± 0.47-fold increase to basal [mean ±SE]; in vitro, 1.71 ± 0.14-fold increase to basal), which was Quantitatively similar to that obtained after insulin treatment (in vivi, 2.35 ± 0.62-fold increase to basal; in vitro, 1.91 ± 0.21-fold increase to basal). Glucose-in-duced GLUT4 translocation in myotubes was prevented by prior addition of the PKC inhibitor 1-(5-isoquinolinyl-by prior addition of the PKC inhibitor 1-(5-isoquinolinyl-sulfonyl)-2-methylpiperazine; in rat skeletal muscle,GLUT4 translocation was paralleled by a translocation of PKC β, while no effect on PKC α, δ, ∊, and ζ was observed. These results suggest that glucose-induced GLUT4 translocation might represenet an insulin-independent autoregulatory mechanism of the skeletal muscle to rapidly increase glucose uptake in acute hyperglycemia. Activation of PKC β might be involved in this mechanisum in skeletal muscle. GLUT4 translocation was paralleled by a translocation of PKC β, while no effect on PKC α, δ, μ, and ¶ was observed. These results suggest that glucose-induced GLUT4 translocation might represent an insulin-independent autoregulatory mechanism of the skeletal muscle to rapidly increase glucose uptake in acute hyperglycemia. Activation of PKC P might be involved in this mechanism in skeletal muscle.

Thiazolidinedione derivatives as novel therapeutic agents to prevent the development of chronic pancreatitis.

Introduction Thiazolidinedione derivatives are known to be novel insulin-sensitizing agents and ligands of a nuclear hormone receptor peroxisome proliferator-activated receptor &ggr; (PPAR&ggr;). Recently, ligands of PPAR&ggr; have been shown to modulate proinflammatory cytokine production and NF-&kgr;B activation. Aim To show that thiazolidinedione derivatives interfere with the development of chronic pancreatitis. Methodology Rat chow containing 0.2% troglitazone was administered from 1 month to 7 months of age in WBN/Kob rats with spontaneous chronic pancreatitis. Morphologic evaluation of the pancreas was performed at 4 months and 7 months of age. Pancreas weight, protein, amylase, and insulin contents also were determined. Changes of cytokine levels were detected by enzyme-linked immunosorbent assay or semiquantitative reverse transcription–polymerase chain reaction. Localization and expression of PPAR&ggr; in the pancreas and isolated peritoneal macrophages were examined by immunohistochemical study. Results Administration of troglitazone reduced the severity of morphologic pancreatic damage including inflammatory cell infiltration, and fibrosis markedly improved by the administration of troglitazone. Further, troglitazone was able to prevent the decrease in amylase content and pancreas atrophy that were observed in WBN/Kob rats. Serum IL-8 levels and TNF-&agr; mRNA levels in the pancreas were significantly elevated in WBN/Kob rats, and these were dramatically attenuated by troglitazone. Peritoneal macrophages isolated from normal rats expressed PPAR&ggr; at low levels, whereas those from WBN/Kob rat abundantly expressed PPAR&ggr;. Conclusion Troglitazone prevented the progression of pancreatic inflammatory process in an animal model of chronic pancreatitis. Macrophages may be one of the targets of the PPAR&ggr; ligand to attenuate the severity of chronic pancreatitis, partially mediated by the inhibition of proinflammatory cytokine gene expression.

Troglitazone, a new antidiabetic agent possessing radical scavenging ability, improved decreased skin blood flow in diabetic rats

Troglitazone is a new class of antidiabetic agent possessing radical scavenging ability similar to vitamin E. Because of this ability, it is expected to improve decreased nutritive capillary blood flow in diabetes. In the present study, we investigated the effects of troglitazone on skin blood flow(SBF) in normal and streptozotocin(STZ)-induced diabetic rats. Effects of troglitazone on vasodilation, PGI2 and PGE2 production were also assessed in perfused hindlimb, isolated rat aorta rings and 3T6 fibroblasts, respectively. SBF at the base of the tail was decreased in STZ diabetic rats (2.1+/-0.2 ml/min/100 g) compared with normal rats (3.8+/-0.2 ml/min/100 g). This decrease of SBF was significantly improved (2.9+/-0.2 ml/min/100 g) by troglitazone treatment (approximately 220 mg/kg/day) for 7 days in STZ diabetic rats without alleviating hyperglycemia. Similar troglitazone treatment (approximately 160 mg/kg/day for 7 days) tended to increase SBF (approximately 30%) even in normal rats. In normal rats, subcutaneous administration of troglitazone (60 mg/kg) acutely increased SBF and, this increase was suppressed by 70% with pretreatment (10 mg/kg s.c.) of indomethacin, cyclooxygenase inhibitor, suggesting that troglitazone increases skin blood flow predominantly by increasing PGI2 and PGE2 production. In hindlimb perfusion under fixed flow rate, troglitazone infusion (20 microM) significantly decreased perfusion pressure by 13%, which reflects vasodilation of blood vessels. This decrease of perfusion pressure was inhibited by concomitant infusion of indomethacin but not N-monomethyl-L-arginine, inhibitor of nitric oxide synthase. In vitro studies, using isolated rat aorta rings, revealed that troglitazone (4.5 to 45 microM) increases PGI2 production by 31 and 70%, respectively. In 3T6 fibroblast (a component of skin tissue), troglitazone at a low dose of 0.3 microM increased PGI2 and PGE2 by 200% and 25%, respectively. Overall all, these results suggest that troglitazone increases nutritive SBF probably by virtue of its radical scavenging thus the resulting in an increase in PGI2 and PGE2 production in blood vessels and fibroblast. Troglitazone may alleviate impaired microcirculation in diabetic patients through these effects.

Troglitazone can prevent development of type 1 diabetes induced by multiple low-dose streptozotocin in mice.

Recent investigations suggest that cytotoxic cytokines such as tumor necrosis factor (TNF)alpha and interleukin (IL)-1beta or free radicals play an essential role in destruction of pancreatic beta cells in Type 1 diabetes and that, therefore, anti-oxidant or anti-TNF alpha and IL-1beta therapy could prevent the development of Type I diabetes. Troglitazone belongs to a novel class of antidiabetic agent possessing the ability to enhance insulin action provably through activating PPAR gamma and to scavenge free radicals. In the present study, we examined whether troglitazone can prevent the development of Type 1 diabetes in multiple, low-dose streptozotocin (MLDSTZ)-injected mice. In addition, effects of troglitazone on cytokine-induced pancreatic beta cell damage were examined in vitro. Type 1 diabetes was induced by MLDSTZ injection to DBA/2 mice (40 mg/kg/day for 5 days). Troglitazone was administered as a 0.2% food admixture (240 mg/kg/day) for 4 weeks from the start of or immediately after STZ injection. MLDSTZ injection elevated plasma glucose to 615 +/- 8 mg/dl 4 weeks after final STZ injection and was accompanied by infiltration of leukocytes to pancreatic islets (insulitis). Troglitazone treatment with MLDSTZ injection prevented hyperglycemia (230 +/- 30 mg/dl) and, suppressed insulitis and TNF alpha production from intraperitoneal exudate cells. TNF alpha (10 pg/ml) and IL-1beta (1 pg/ml) addition to hamster insulinoma cell line HIT-T15 for 7 days in vitro decreased insulin secretion and cell viability. Simultaneous troglitazone addition (0.03 to approximately 3 microM) significantly improved cytokine-induced decrease in insulin secretion and in cell viability. These findings suggest that troglitazone prevents the development of Type 1 diabetes in the MLDSTZ model by suppressing insulitis associated with decreasing TNF alpha production from intraperitoneal exudate cells and the subsequent TNF alpha and IL-1beta-induced beta cell damage.

Acute effect of troglitazone on glucose metabolism in the absence or presence of insulin in perfused rat hindlimb.

Troglitazone (CS-045) is a new type of antidiabetic agent that decreases plasma glucose by enhancing insulin action in insulin-resistant diabetic animals and non-insulin-dependent diabetes mellitus (NIDDM) patients. To examine the direct effect of troglitazone on glucose metabolism and insulin action in skeletal muscle, we infused troglitazone solution into perfused rat hindlimbs in the presence of 6 mmol/L glucose and in the absence or presence of insulin. In the absence of insulin, even 50 mumol/L troglitazone did not elicit glucose uptake. Troglitazone did increase lactate and pyruvate release at concentrations of 20 mumol/L and higher; however, it decreased the ratio of lactate to pyruvate (L/P ratio) and increased oxygen consumption at concentrations higher than 5 and 20 mumol/L, respectively. In hindlimb muscle, 20 mumol/L troglitazone decreased glycogen content without changing fructose 2,6-bisphosphate (F2,6P2) content in the absence of insulin. Insulin infusion with 250 microU/mL obtained half-maximal effects, causing a 2.8-fold increase in glucose uptake and a 1.5-fold increase in lactate and pyruvate release. When 20 mumol/L troglitazone was infused for 30 minutes together with 250 microU/mL insulin, insulin-induced glucose uptake significantly increased 30 minutes after troglitazone infusion, and this increase was further augmented after withdrawal of troglitazone. In insulin plus troglitazone infusion at 30 minutes after troglitazone removal, glycogen content in hindlimb muscle was significantly decreased compared with that obtained with insulin infusion alone. In summary, in the absence of insulin, troglitazone does not elicit glucose uptake, but causes an increase in glycolysis accompanied by a decrease in muscle glycogen content and L/P ratio and an increase in oxygen consumption. In the presence of insulin, troglitazone increases insulin-induced glucose uptake, and this increase is further augmented after troglitazone removal. Addition of troglitazone to insulin infusion decreased the glycogen content in hindlimb muscle. This decrease in muscle glycogen content may trigger an enhancement of insulin-induced glucose uptake similar to that observed during muscle contraction or epinephrine treatment.