Beatrice M. Wyse

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.

Studies on the ability of compounds to block the diabetogenic activity of streptozotocin.

Evidence is presented that the early hyperglycemia (two to four hours after injection) following streptozotocin administration is not a result of an interference with insulin action, since streptozotocin did not block insulin action on skeletal muscle in vitro. Compounds with known metabolic effects were tested for their ability to block streptozotocin-induced diabetes. Mannoheptulose, glucosamine, diazoxide, NAD, glucose, epinephrine, 3,5-dimethylpyrazole, 3-carboxy-5-methylpyrazole, glutamic acid, glycine, asparagine, cysteine, sodium tolbutamide, guanidoacetic acid, glutathione, p-aminobenzoic acid, and ethyl alcohol were ineffective as blocking agents. Pretreatment with pyrazinamid blocked streptozotocin diabetes,but was ineffective following streptozotocin treatment. 2-carboxypyrazine (the metabolite of pyrazinamide) did not block streptozotocin diabetogenic activity. Administration of 2-deoxyglucose blocked streptozotocin-induced diabetes; this is postulated to be the result of an interference with streptozotocin transport into the β cell. Pretreatment with nicotinamide, or treatment as long as two hours after streptozotocin, blocked the diabetogenic effect of streptozotocin. Nicotinic acid was ineffective. It is postulated that streptozotocin interferes with NAD formation in the β cell, and that nicotinamide treatment abolishes the streptozotocin effect by its ability to maintain ah adequate concentration of NAD in the β cell.

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.

The KKAy mouse: a model for the rapid development of glomerular capillary basement membrane thickening.

The renal tissue of 12-, 29-, 90- and 165-day-old genetically obese, hyperphagic, diabetic KKAy and age-matched nondiabetic C57BL/6 mice was morphometrically analyzed to characterize the development of peripheral glomerular capillary basement membrane thickening in the kidney of this animal model. Peripheral glomerular basement membrane (GBM) thickness was unremarkable in KKAy mice at 12 days of age (prior to onset of hyperinsulinemia) or at 29 days of age (after development of hyperinsulinemia). By 90 days of age, when the KKAy mice became severely hyperinsulinemic and hyperglycemic, the peripheral GBM thickness was greater (13%, p less than 0.002) in the diabetic compared with the nondiabetic mice. Furthermore, the peripheral GBM thickness was exacerbated (20%, p less than 0.001) by 165 days of age in the KKAy mice. The results of the present study suggest that peripheral glomerular capillary basement membrane thickening has an early onset and develops rapidly in the KKAy mouse in comparison with other diabetic animal models. Therefore, the KKAy mouse seems to be an appropriate model for further investigation of early structural and functional defects in the glomerular filtration barrier.

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.

Reversal of streptozotocin diabetes with nicotinamide.

Summary Animals given nicotinamide either as a pretreatment or as long as 2 hr (but not 4 hr) after streptozotocin were protected from diabetes. Nicotinic acid and NAD pretreatment were ineffective in preventing streptozotocin diabetes. It was postualted that streptozotocin may interfere with NAD formation in the beta cell and that treatment with nicotinamide reverses the streptozotocin effect by reversing its effect on levels of NAD within beta cells.

Analysis of pancreatic islet cells and hormone content in the spontaneously diabetic KKAy mouse by morphometry, immunocytochemistry and radioimmunoassay

The splenic pancreas of 165 day old diabetic KKAy and age-matched nondiabetic C57BL/ 6 mice was examined by morphometry and immunocytochemistry at the light microscopic level and by radioimmunoassay to evaluate the morphology, surface area, endocrine cell composition and hormone content of the pancreatic islets. The insulin cells of the diabetic mice were severely degranulated and many of the glucagon, somatostatin and pancreatic polypeptide cells were displaced from the mantle to the core of the islet tissue where the non-insulin cells appeared to lose their continuity. The topography of some of the islets of KKAy mice was further deranged by acinar cells among the endocrine tissue. Morphometric analysis revealed that the surface area of the islets of KKAy mice was significantly expanded in comparison with that of C57BL/6 mice. The volume and numerical percents of the insulin cells were significantly increased whereas those of the glucagon and somatostatin cells were decreased in the KKAy mice. Since only the mean absolute number of insulin cells was elevated in the diabetic mice, the alteration in the relative proportions of the noninsulin cells and hypertrophy of the islets seemed to be a manifestation of insulin cell hyperplasia. Pancreatic insulin and somatostatin contents were markedly diminished in the islets of KKAy compared with those of C57BL/6 mice. These results demonstrate that the microscopic anatomy, endocrine cell populations and hormone content of the pancreatic islets are deranged in the KKAy mouse with severe hyperinsulinemia and hyperglycemia.

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.

Ciglitazone, a hypoglycemic agent: Early effects on the pancreatic islets of ob ob mice

Chronic administration of ciglitazone (5-4[1-methyl-cyclohexylmethoxy)-benzyl]-thiazolidine-2,4 dione) decreased both plasma glucose and insulin concentrations in ob/ob mice. When given as an admixture to the feed, blood glucose levels were reduced as early as 12 hours after initiation of treatment. Concomitant with the decrease in circulating insulin, there was an increased hormone content in the beta-cells as judged by RIA and aldehyde-fuchsin staining. Acute oral dosing with ciglitazone produced a 41% reduction in circulating insulin at a time when glucose concentrations were as yet unaffected. Ciglitazone also inhibited glucose-stimulated insulin secretion in vitro. The results suggest that the hypoglycemic agent, ciglitazone, may reduce plasma glucose and insulin concentrations at least partially as the result of independent mechanisms.