Acid glycohydrolase in Chinese hamster with spontaneous diabetes. VII. The lack of short-term glucose-effect in cultured kidney cells.

An epithelial cell line, designated CHK-ACE, was established from the kidney of a spontaneously diabetic Chinese hamster from the highly inbred AC line. CHK-ACE was separated into two sublines, CHK-ACE-100 and CHK-ACE-400, by successive passages in 100 and 400 mg/dl glucose respectively. Extra- and intracellular activities of N-acetyl-beta-D-glucosaminidase and beta-D-galactosidase were measured in these cultures after exposure to varying concentrations of glucose (100, 200, 300 and 400 mg/dl) for one passage and 10% heated fetal calf serum for 6.5 h before enzyme measurements were taken; no apparent dependence on medium-glucose concentration was found. In serum-free medium, the time-dependent release of both N-acetyl-beta-D-glucosaminidase and beta-D-galactosidase was sustained for up to 24 h; no significant difference in their activities was found between CHK-ACE-100 cultures grown in 100 and 400 mg/dl glucose for one passage.

Insulin binding in cultured Chinese hamster kidney epithelial cells. The effects of glucose concentration in the medium and tunicamycin.

An epithelial cell line established from a Chinese hamster kidney, CHK-ACE, was separated into two sublines, CHK-ACE-100 and CHK-ACE-400, by 18 successive passages in medium containing 100 and 400 mg/dl glucose, respectively. Binding of CHK-ACE-100 and CHK-ACE-400 cell to 125I-labeled insulin showed similar pH and time dependency; 125I-labeled insulin, concentration differed in the two sublines, however. Degradation of 125I-labeled insulin, as determined by its ability to bind insulin antibody and cells, was more extensive when preincubated with CHK-ACE-400 cell than with CHK-ACE-100 cells. When CHK-ACE-100 cells were grown in 400 mg/dl glucose for six passages, these cells showed more insulin binding sites than cells grown parallel in 100 mg/dl glucose; whereas CHK-ACE-400 cells grown in 100 mg/dl glucose for six passages showed fewer insulin binding sites than those grown parallel in 400 mg/dl glucose. A slight increase in Kf/Ke ratio was observed in both sublines when grown in 400 mg/dl glucose as compared to 100 mg/dl glucose, indicating attenuated negative cooperativity of the binding sites in cells grown in 400 mg/dl glucose. Tunicamycin, at concentrations from 0.016 to 0.125 micrograms/ml, showed no direct effect on the assay of 125I-labeled insulin binding to CHK-ACE-100 cells; exposure of CHK-ACE-100 cells to tunicamycin, at concentrations from 0.01 to 0.2 micrograms/ml, for 24 h caused a dose-dependent decrease in insulin binding capacity and an increase in Kf/Ke ratio. These data indicate that the number of insulin binding sites in the cultured Chinese hamster kidney epithelial cells increased with high glucose concentrations in the culture medium, whereas tunicamycin, an inhibitor of protein glycosylation, lowered the number of insulin binding sites.

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.

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 adipocytes were used to estimate for 125I-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 number, 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.

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 beta-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 beta-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 ciglitazone-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 glycemic response to exogenous insulin. The hypoglycemic activity of ciglitazone was specific for obese-hyperglycemic and insulin-resistant animals.

Induction of uncoupling protein in brown adipose tissue. Synergy between norepinephrine and pioglitazone, an insulin-sensitizing agent.

Insulin resistance and obesity in rodent models of non-insulin-dependent diabetes mellitus have been correlated with ablated or defective brown adipose tissue (BAT) function. The mitochondrial uncoupling protein (UCP) allows BAT to perform its unique role in facultative energy expenditure. In this study, we observed an increase in both BAT mass and the expression of UCP mRNA in BAT from obese diabetic mice and their lean littermates following treatment with the thiazolidinedione pioglitazone, a novel insulin-sensitizing agent. Thus, we wanted to ascertain if pioglitazone directly induces BAT differentiation. We found that treatment for 48 hr with pioglitazone caused a 32-fold increase in UCP mRNA, whereas a 7-hr treatment with norepinephrine caused a 24-fold increase in expression. Cells treated with pioglitazone for 48 hr, with norepinephrine added during the last 7 hr, demonstrated a 59-fold increase in UCP mRNA. However, simultaneous treatment with pioglitazone and repeated treatment norepinephrine for 48 hr yielded a greater than 200-fold increase in UCP mRNA. Examination of UCP protein levels demonstrated a similar time-dependent increase with pioglitazone and/or norepinephrine treatment, as well as a synergistic increase with concurrent pioglitazone and norepinephrine treatment. This study shows that pioglitazone exerts a direct effect on BAT cells in vitro by increasing UCP mRNA and protein levels, and that it also synergizes with norepinephrine perhaps by inducing and stabilizing UCP mRNA and/or preventing proteolysis of UCP protein.

Morphometric evaluation of capillary basement membrane thickness in the quadriceps muscle of diabetic and nondiabetic Chinese hamsters.

Quadriceps muscle capillaries from 19-23 month old genetically diabetic (XA and AC) and nondiabetic (M) subline Chinese hamsters were morphometrically evaluated to determine if capillary basement membrane thickening (CBMT) is a quantifiable complication of diabetes. Significant CBMT was present in the diabetic XA Chinese hamsters (49.37 nm +/- 17.81, p less than 0.007) in comparison with the nondiabetic M hamsters (34.08 nm +/- 9.98). Although there was a trend towards expansion of the muscle capillary basement membranes in the diabetic AC Chinese hamsters, the value was not statistically significant. A nested analysis of variance showed that the greatest source of variation in basement membrane thickness occurred among capillaries within each animal. In addition, a positive correlation (r = 0.62; p less than 0.002) existed between blood glucose levels and CBMT in the XA subline. These data should serve as guidelines for evaluation of antimicrovascular disease compounds which will be tested to determine if they prevent or retard microangiopathy in the diabetic Chinese hamster.

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.

Insulin binding in cultured Chinese hamster kidney epithelial cells: the effect of serum in the medium.

[125I] Insulin (porcine) binding to an epithelial cell line established from a Chinese hamster kidney, CHK-AC E-100, showed an optimum at pH 8.0 and reached a maximum after 2.5 h incubation at 25 degrees C. Dissociation of bound [125I] insulin was facilitated by the addition of unlabeled insulin in the dilution buffer. Porcine insulin effectively competed for [125I] insulin binding to the cultured cells and was 30 and 90 times as potent as guinea pig insulin and porcine proinsulin in causing 50% inhibition of [125I] insulin binding; glucagon was completely ineffective. Scatchard analysis of the binding data yielded a curvilinear plot and a capacity of 0.6 ng/10(6) cells; the average affinity of the empty receptor, Ke, was calculated to be 1.78 X 10(6) M-1 and that of the filled receptor, Kf, 0.57 X 10(8) m-1, Substitution of fetal bovine serum (FBS) in the culture medium with bovine calf, bovine newborn, of bovine calf serum altered insulin binding characteristics in the cells and reduced cell growth. Insulin binding characteristics of cells grown in hormone-supplemented medium containing 0 to 0.1% FBS were similar to those of cells grown in minimum essential medium (MEM) containing 2 to 5% FBS. The data indicated that the established Chinese hamster kidney epithelial cell line CHK-AC E-100 possessed specific insulin receptors and the characteristics of the receptors could be manipulated by changing the serum in culture medium.

Ciglitazone, a new hypoglycaemic agent. 3. Effect on glucose disposal and gluconeogenesis in vivo in C57BL/6J-ob/ob and -+/? mice.

Ciglitazone 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.

High glucose-induced enzyme activity changes in cultured cell lines established from the kidneys of Chinese hamsters with aglycosuria or spontaneous glycosuria.

Chinese hamster kidney epithelial-like cells derived from highly inbred nondiabetic (AV) and diabetic (XA) genetic sublines were passaged in medium containing 100 or 400 mg/dl glucose. The effect of high medium glucose on the activities of 5 enzymes involved in glucose metabolism was followed and significant glucose-dependent difference was observed. The effects, however, were opposite in cells derived from AV and XA sublines.

Alterations in glycohydrolase activities in streptozotocin-diabetic Chinese hamsters (Cricetulus griseus).

1. Six weeks after the injection of streptozotocin at 125 mg/kg i.p. in the AV line nondiabetic Chinese hamsters, the animals showed hyperglycemia, increased kidney, pancreas and stomach weights and stomach glucagon contents and depletion of insulin and glucagon in the pancreas. 2. Plasma beta-D-galactosidase and N-acetyl-beta-D-glucosaminidase were elevated; whereas alpha-D-glucosidase was decreased and alpha-D-galactosidase remained unchanged in the plasma. 3. In the kidney, streptozotocin-diabetes led to depression of alpha-D-mannosidase, beta-D-fucosidase and N-acetyl-beta-D-glucosaminidase activities in both 12,000 g supernatant and precipitate fractions, decreases in alpha-D-glucosidase in the supernatant only and no change in alpha-L-fucosidase, alpha-D-galactosidase, beta-D-galactosidase and beta-D-glucuronidase. 4. In the liver, significant increases in N-acetyl-beta-D-glucosaminidase, alpha-D-galactosidase, beta-D-galactosidase, beta-D-fucosidase, beta-D-glucosidase and alpha-D-mannosidase were found in either the supernatant or the precipitate fraction of the diabetic animals. The data indicate diabetes-dependent tissue-specific changes in glycohydrolases in the Chinese hamster.

Streptozotocin-induced diabetes in the Chinese hamster. Biochemical and endocrine disorders.

Streptozotocin treatment (125 mg/kg) in the Chinese hamster induced hyperglycaemia, hypoinsulinaemia, hyperglucagonaemia and changes in body, liver, pancreas, stomach, kidney and adipose tissue weights. The pancreatic reserves of insulin and glucagon in the diabetic animals were low, but stomach glucagon high. These animals showed high levels of phosphoenolpyruvate carboxykinase and low levels of glucokinase, hexokinase, isocitrate dehydrogenase and malic enzyme, but normal levels of pyruvate kinase in the liver. Increases in lactate dehydrogenase subunit B and isozymes 2, 3 and 4 were also observed in the liver, but not in the epididymal fat pad, of the diabetic animals. N-Acetyl-beta-D-glucosaminidase was elevated in plasma, liver and heart, but not in the kidney of the treated animals. Renal alpha-galactosidase and beta-glucosidase were depressed, whereas beta-galactosidase and alpha-glucosidase remained essentially normal. These features indicated that there were considerable differences between the biochemical disorders associated with streptozotocin-diabetes in the Chinese hamster and the published observations in the rat.

Adipocyte fatty acid-binding protein: regulation of gene expression in vivo and in vitro by an insulin-sensitizing agent.

Pioglitazone, a thiazolidinedione, is a novel antidiabetic compound that can lower blood glucose in diabetic rodents by increasing insulin sensitivity in target tissues. We have previously demonstrated that pioglitazone can enhance the insulin- or insulin-like growth factor-1-regulated differentiation of 3T3-L1 cells, a cell line that undergoes morphological and biochemical differentiation to mature adipocytes [Mol. Pharmacol. 41:393-398 (1992)]. In this study, we have examined the effect of pioglitazone on the expression of the adipocyte fatty acid-binding protein (aFABP) in ob/ob mice and 3T3-L1 cells. Administration of the drug to mice was observed to cause a dose-dependent increase in aFABP mRNA expression in epididymal fat, which was correlated with a decrease in blood glucose and insulin levels. Treatment of 3T3-L1 cells with pioglitazone enhanced aFABP expression in a time-dependent fashion. To explore a possible direct effect of pioglitazone on aFABP expression, a chimeric gene was constructed containing the aFABP promoter fused upstream of the bacterial reporter gene for chloramphenicol acetyltransferase. After transfection into 3T3-L1 cells and selection of stable transformants, regulation of the chimeric gene was studied. Pioglitazone, in combination with insulin or insulin-like growth factor-1, was observed to elicit a dose-dependent increase in expression, indicating a role for pioglitazone in regulating transcription of the aFABP gene. Several thiazolidinedione analogs were tested for their ability to induce the expression of the chimeric gene, and it was found that activity in this assay paralleled the structure-activity relationships observed for enhancement of 3T3-L1 cell differentiation. These observations on control of aFABP gene expression by pioglitazone suggest possible mechanisms by which cellular sensitivity to insulin may be regulated.

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 non-insulin 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 hypoglycemic agent. 5. Effect on renal lesions in C57BL/KsJ-db/db mice.

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 and of PAS-positive material in the glomerular mesangium and renal tubules. Long-term administration (12 and 20 weeks) of ciglitazone significantly improved the blood glucose of most of the treated db/db mice. There appeared to be a reduction of glomerular IgM and IgG in the treated compared with the control group, although IgM did not achieve statistical significance due to heavy stain deposition in two of the treated mice with continuous and severe hyperglycemia. Treated and control mice displayed a similar diffuse expansion and mild thickening of the glomerular mesangium characterized by moderate deposition of PAS-positive material. Expansion of the mesangium was probably not retarded or prevented by ciglitazone therapy since this pathologic process may be controlled by an interaction of metabolic factors other than hyperglycemia per se in the db/db mouse. Glycogen vacuolization (Armeni-Epstein lesion) of the renal tubules was completely ameliorated in the treated mice which showed a reduction of hyperglycemia. The results of this study suggest that prolonged treatment with ciglitazone elicits an improvement of hyperglycemia which seems to retard or reverse glomerular immunopathology and completely reverse tubular derangement but does not prevent expansion of the glomerular matrix in severely diabetic C57BL/KsJ-db/db 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.

Systematic evaluation of microangiopathy in diabetic Chinese hamsters. I. Morphometric analysis of minimal glomerular basement membrane thickness in 11- to 15- and 19- to 23-month-old Chinese hamsters.

Renal glomeruli of 11- to 15- and 19- to 23-month-old nondiabetic (M) and diabetic (XA and AC) genetic sublines of Chinese hamsters were morphometrically analyzed to determine if minimal capillary basement membrane thickening (CBMT) is a microvascular complication in this animal model. Minimal glomerular capillary basement membrane thickness was significantly elevated in the AC diabetic subline (117.2 nm +/- 5.0, P less than 0.004) compared with the M nondiabetic subline (99.0 nm +/- 14.0) in the 11- to 15-month age span. However, in the 19- to 23-month age range, both the XA (140.2 nm +/- 20.0, P less than 0.02) and AC (140.1 nm +/- 12.4, P less than 0.04) diabetic sublines displayed significantly greater glomerular capillary basement membrane thickness in comparison with the M nondiabetic subline (119.0 nm +/- 13.4). The greatest influence on CBMT in the diabetics was shown to be a combination of the aging process and severity of hyperglycemia. This initial systematic morphometric study has demonstrated that glomerular CBMT is a characteristic microvascular lesion in 11- to 15-month-old diabetic AC and 19- to 23-month-old diabetic XA and AC Chinese hamsters in comparison with age-matched nondiabetics. Furthermore, this investigation suggests that the Chinese hamster appears to be an acceptable model for the study of chronic complications associated with diabetic nephropathy.