Albert Y. Chang

Ciglitazone, a New Hypoglycemic Agent: I. Studies in ob/ob and db/db Mice, Diabetic Chinese Hamsters, and Normal and Streptozotocin-Diabetic Rats

Ciglitazone, 5-[4-(1-methylcyclohexylmethoxy) benzyl]-thiazolidine-2,4-dione, is a new hypoglycemic agent orally active in the obese-hyperglycemic animal models. In C57BL 6J-ob ob mice, treatment with 100 mg /kg ciglitazone for 2 days elicited a drastic fall in blood glucose. It also decreased plasma insulin, triglycerides, and free fatty acids and food intake without affecting the body weight. Its hypoglycemic activity was independent of its effect on food intake. Regranulation of islet β-cells and increased pancreatic insulin content were observed in ob/ob mice treated for 41–44 days with 100 mg/kg/day ciglitazone. Ciglitazone showed no effect on food intake, blood glucose, or pancreatic islet β-cells in a group of lean C57BL/6J-+ /? mice concomitantly treated at a dose of 150 mg/kg/ day. In C57BL/KsJ-db/db mice, ciglitazone decreased blood glucose and food intake. The untreated db/db mice lost weight despite hyperphagia, whereas the cig-litazone-treated db/db mice gained weight. In the spontaneously diabetic Chinese hamsters, ciglitazone showed no significant effect on food intake, body weight, blood glucose, or insulin content in either plasma or pancreas, but it lowered plasma lipids. In normal rats, ciglitazone failed to affect fasting blood glucose but improved glucose tolerance without increasing insulin secretory response to glucose. In streptozotocin-diabetic rats, it showed no effect on blood glucose or glycemie response to exogenous insulin. The hypoglycemic activity of ciglitazone was specific for obese-hyperglycemic and insulin-resistant animals.

Ciglitazone, a New Hypoglycemic Agent

Kidneys of treated and control C57BL/KsJ-db/db mice were analyzed by semiquantitative light microscopy to determine the effects of ciglitazone on the deposition of fluorescein-conjugated IgM and IgG a

Ciglitazone, a New Hypoglycemic Agent: II. Effect on Glucose and Lipid Metabolisms and Insulin Binding in the Adipose Tissue of C57BL/6J-ob/ob and − + / ? Mice

The fat pads isolated from control and ciglitazone-treated C57BL/6J-ob/ob mice and their lean littermates (− + / ?) were incubated in vitro with glucose-1-14C/-5-3H in the presence of insulin. The formation of 14CO2 and 3H2O and the levels of free fatty acids and glycerol in the medium and the incorporation of 14C and 3H into esterified lipids and free fatty acids in the fat pads were measured. The basal rates of glucose-1-14C/-5-3H metabolism per unit weight were increased in the fat pads of ciglitazone-treated mice, more pronouncedly in the treated lean than in the treated obese. The insulin-dependent effects were enhanced in the treated ob/ob mice. To see the dose-response of insulin, a second experiment was carried out in which portions of the fat pads were incubated in vitro with glucose-1-14C in the presence of 0–40 ng/ml insulin and isolated adipo-cytes were used to estimate for 125l-insulin binding. The basal rates of 14CO2 and 14C-lipids formation per unit weight of fat pads were increased in both treated obese and treated lean groups but insulin-dependent elevation was seen only in the treated obese group. Ciglitazone significantly increased insulin binding capacity at the low-affinity sites in the adipocytes of obese mice but not in those of lean mice. The data showed that ciglitazone increased the basal rate of glucose metabolism, lipogenesis, insulin receptor numbers, and post-receptor responses in the C57BL/ 6J-ob/ob mice; it increased the basal rate of glucose metabolism and lipogenesis but not insulin sensitivity in the lean mice.

Hepatic Enzyme Activities in Streptozotocin-Diabetic Rats Before and After Insulin Treatment

Streptozotocin causes hyperglycemia and hypoinsulinemia in rats. Striking elevations in hepatic phosphoenolpyruvate carboxykinase, pyruvate carboxylase, hexosediphosphatase and glucose-6-phosphate, and dramatic decreases in glucokinase, pyruvate kinase and 6-phosphogluconate dehydrogenase are observed in these diabetic animals. Daily treatment of insulin in diabetic rats brings about increase in liver weight, glycogen and soluble protein contents, and in the levels of glucokinase, pyruvate kinase, 6-phosphogluconate dehydrogenase and lactate dehydrogenase. In terms of unit/gm. liver, the activities of PEP carboxykinase, glucose-6-phosphatase and hexosediphosphatase are depressed by insulin injection. However, a multiphasic pattern is observed in the changes of these gluconeogenic enzymes following insulin treatments when their activities are expressed as units/100 gm. body weight or unit/mg. protein. Insulin withdrawal causes immediate rise in gluconeogenic enzymes, particularly in the extramitochondrial activity of pyruvate carboxylase, and decreases in glucose-catabolizing enzymes which proceed at different rates.

Analysis of Glycosaminoglycan from Diabetic and Normal Chinese Hamster Cells

Diabetic and normal cell lines from Chinese hamster kidneys were cultured in media containing 35SO4 and 3H-glucosamine. Glycosaminoglycans (GAG) were extracted and analyzed from the media, trypsin, and cell pellet by enzymatic and electrophoretic procedures. Significant increases in the hyaluronic acid content were noted in all three fractions of diabetic GAGs when compared with normals. In addition, an increased heparan sulfate content and decreased chondroitin sulfate amounts were noted in diabetic cell lines. These data suggest that in vivo changes in GAG types and amounts in diabetic kidneys seen by others may also be seen in cultured cells.

On the mechanism for the depression of liver NAD by streptozotocin

Abstract 1. 1. A depression in liver NAD content was observed in mice at 1, 2 and 6 h after intravenous injection of streptozotocin at a dose of 200 mg per kg of body weight. Studies on the incorporation of [7 −14 C]nicotinic acid into liver NAD in vivo revealed that liver NAD synthesis was not inhibited at 1 and 6 h after treatment. 2 h after the injection of streptozotocin, drastic reduction in hepatic [ 14 C]NAD formation concomitant with decreased uptake of labelled precursors was observed. 2. 2. The effect of streptozotocin in the removal of liver NAD was studied as follow. 10 min after prelabelling NAD with [7 −14C ]nicotic acid, streotozotocin was given together with a 10-fold excess of unlabelled nicotinic acid. Under these conditions, an initial rise in liver NAD content was followed by a streptozotocin-dependent depression. Furthermore, streptozotocin caused (1) reduction in the amount of 14 C-labelled materials recovered in liver extracts, (2) reduction in the fraction of recovered 14 C-labelled materials present as NAD, (3) increased in the fraction present as nicotinamide, and (4) decrease in the specific activity of [ 14 C]NAD. 3. 3. The suggest that streptozotocin exerts its initial action on the depression of liver NAD by facilitating its removal. A further decrease in NAD formation ensues and, probably, results from a reduction in tissue uptake of the precursors.

Rate of gluconeogenesis and levels of gluconeogenic enzymes in liver and kidney of diabetic and normal Chinese hamsters.

Abstract 1. 1. The rate of incorporation of sodium [2- 14 C]pyruvate into glucose was studied in vitro with liver and kidney slices isolated from diabetic and normal Chinese hamsters at the age of 7 to 14 months. The amount of glucose produced in one h per mg tissue was significantly higher in both liver and kidney slices of the diabetics than in those of normal animals. 2. 2. Studies on tissue enzyme levels revealed that livers of the diabetics had an elevated level of phosphoenolpyruvate carboxykinase and normal activity of pyruvate carboxylase, glucose-6-phosphatase and hexosediphosphatase. The kidneys of the diabetic hamsters showed significantly higher activity in phosphoenolpyruvate carboxykinase and pyruvate carboxylase, but similar activity in glucose-6-phosphatase and hexosediphosphate as the tissues from normal animals. 3. 3. In both groups of hamsters, kidney slices produced glucose more rapidly and had higher activity of gluconeogenic enzymes on a weight basis than liver.

Ciglitazone, a new hypoglycaemic agent. 3. effect on glucose disposal and gluconeogenesis in vivo in C57BL/6J-Ob/Ob and — +/? Mice

SummaryCiglitazone is orally active in preventing and reversing the hyperglycaemic syndrome in C57BL/6J-ob/ob mice and it is only mildly and transiently hypoglycaemic in lean littermates (C57BL/6J- +/?). Its effect on glucose disposal in vivo was estimated by injecting glucose-6-3H/14C and following the specific activity of radiolabelled glucose at 15, 30, 45, and 60 min after injection. The rate constants of glucose turnover were calculated to be as follows in decreasing order: treated obese (0.046/min), treated lean (0.032/min), control lean (0.026/min), and control obese (0.022/min). The obese mice showed less futile Cori cycle activity than the lean mice and ciglitazone had negligible effect on glucose recycling. The control obese mice incorporated more radiolabels in hepatic lipids, glycogen, and proteins than the control lean mice and ciglitazone further enhanced the incorporations. Ciglitazone also increased hepatic accumulations of radiolabels in the glycogen and lipid fractions in the lean littermates. Using lactate-14C as precursor, gluconeogenesis in vivo was measured in control and treated obese and lean mice. Ciglitazone significantly lowered the rate of conversion of lactate-14C to glucose-14C in the obese mice but not in the lean littermates.

Chemically and Hormonally Induced Diabetes Mellitus

The purpose of this chapter is to provide a review of the literature pertaining to chemically induced diabetes mellitus since 1977, the year when the first edition of this book was published. A thorough historical account (prior to 1977) of this topic has been published.1 This chapter is divided into four sections. The first two parts discuss the two chemical agents most widely used to induce diabetes mellitus in animals: streptozotocin and alloxan. The third section encompases various agents that cause transient or permanent hyperglycemia in man or experimental animal models. These agents include β-cytotoxic chemicals (dithizone, benzothiazides, Vacor, etc.), L-asparaginase, and a compound damaging the ventromedial hypothalamus (monosodium glutamate). The fourth section discusses the insulin-counteracting hormones, i.e., glucagon, growth hormone, sex hormones, and catecholamines.

Ciglitazone, a new hypoglycaemic agent. 4. Effect on pancreatic islets of C57BL/6J-ob/ob and C57BL/KsJ-db/db mice

SummaryPancreases of treated and control male C57BL/6J-ob/ob and C57BL/KsJ-db/db mice were evaluated by qualitative and morphometric microscopic techniques to determine the effects of chronic ciglitazone treatment on the morphology of β cells and surface area and number of pancreatic islets. The β cells of treated ob/ob and db/db mice displayed moderate to heavy granulation whereas most β cells of untreated obese and diabetic mice were extensively degranulated. Although moderate proliferation of the rough endoplasmic reticulum and Golgi apparatus was evident in some β cells of treated db/db mice, both groups of treated ob/ob and db/db mice displayed an improved pattern of insulin synthesis and storage. In contrast, the β cells of untreated ob/ob and db/db mice were in a severe state of stress which was indicated by extensive hypertrophy of the rough endoplasmic reticulum, Golgi apparatus and mitochondria. Some β cells of untreated db/ db mice also displayed lysosome aggregates indicative of early stages of necrosis. Morphometric analysis revealed that the surface area of islets of treated ob/ob mice was significantly smaller in comparison with that of untreated ob/ob mice. Since the surface area of islets of treated C57BL/6J-+/? mice (lean littermates of ob/ob mice) was less than that of treated ob/ob mice, the progression of islet hypertrophy in the obese mice was probably arrested or attenuated but not to the level of the treated +/? mice. The number of pancreatic islets was significantly greater in treated than in untreated db/ db mice. A majority of the islets of untreated db/db mice were atrophie and consisted of acinar and endocrine cells whereas most of the islets of treated db/db mice appeared to be intact and unremarkable. The results of this study suggest that ciglitazone is an effective hypoglycaemic agent which may directly or indirectly promote β-cell regranulation and an improved pattern of insulin synthesis and storage in ob/ob and db/db mice. However, in treated db/db mice, there still was some evidence of stress in the β cells. Overall, the prolonged treatment with ciglitazone also seemed to inhibit the hypertrophy of islets in ob/ob mice and protect the structural integrity and viability of islets in db/db mice.