Pyruvate decarboxylase III. Specificity restrictions for thiamine pyrophosphate in the protein association step, sub-unit structure.

Pyruvate decarboxylase dissociates into sub-units of one half the molecular weight at alkaline pH. At the same conditions the cofactors thiamine pyrophosphate and Mg2+ are released and can be separated from the protein. Thiamine pyrophosphate is an obligatory cofactor for reconstitution to the oligomer [1]. In this study the effect of thiamine pyrophosphate derivatives (thiamine monophosphate, thiamine, and thiazole pyrophosphate) upon the reconstitution procedure was evaluated. The complete association of sub-units to form active oligomer was attained only when thiamine pyrophosphate was present. It is concluded that both the pyrimidine ring and the pyrophosphate group are required for productive co-enzyme binding and it is proposed that this interaction effects a conformational change which promotes protomer aggregation to form the enzymatically active holoenzyme. In addition data are presented which indicate that the monomer unit is 60 000 +/- 3000 daltons and that the N-terminal amino acid is histidine. Since the molecular weight of the active oligomer is 230 000 it is proposed that pyruvate decarboxylase is a tetramer comprised of four identical or nearly identical monomer units.

Gluconeogenesis in developing rat kidney cortex.

1. Gluconeogenesis in developing rat kidney cortex was studied by assaying the activities of two enzymes, glucose 6-phosphatase and phosphoenolpyruvate carboxykinase, and by measuring glucose formation in tissue slices. 2. Glucose 6-phosphatase and phosphoenolpyruvate carboxykinase are present in late foetal (21-22-day-old) tissue and increase rapidly postnatally. Maximum activity of phosphoenolpyruvate carboxykinase occurs at 7 days of age, followed by a decline to the adult level. Glucose 6-phosphatase activity rises during the first 2 postnatal weeks and then declines. 3. Late foetuses synthesize glucose from both pyruvate and l-glutamate. The rate increases during the first 2 weeks to above adult levels. Synthesis is always higher from pyruvate than from glutamate. 4. The effect of 24hr. starvation was studied in perinatal animals. The results indicate that the ability to increase the rate of glucose synthesis as a result of starvation is not present at birth, but develops some time after the second postnatal day.

Effect of high fat weanling diets containing either medium-chain triglycerides or long-chain triglycerides on the development of obesity in the Zucker rat.

Zucker rats were early weaned onto either medium-chain (MCT) or long-chain triglycerides (LCT) to examine the effect on the development of obesity. Preobese and lean pups were weaned at 16 days to isocaloric, isonitrogenous liquid diets containing either 65% MCT or LCT (by calories) or to a "stocklike" (5.5% fat, 72.6% carbohydrate) control diet or were pair-fed stocklike diet to MCT-fed rats until day 45. MCT-feeding lowered body weight gain and fat pad weight in obese and lean rats compared to stocklike-fed controls. Additionally, fat cell size and lipoprotein lipase (LPL) activity and hepatic acetyl CoA carboxylase activity were reduced in obese MCT-fed rats compared to obese controls fed stocklike diet. Except for altered LPL activity the effects produced by MCT-feeding were attributable to its anorectic effect. However, all obese rats, including the MCT group, developed an obese body composition and were hyperinsulinemic. The development sequence leading to obesity may be derived from a fundamental cellular defect that results in metabolic alterations in different tissues at critical periods of development. Thus, effective treatment of this genetic obesity requires a better understanding of fa gene action.

Development of pancreatic and plasma insulin in prenatal and suckling Zucker rats.

Pancreatic and plasma insulin concentrations in preobese and lean Zucker rats were determined during three developmental periods: 1) gestation, 2) suckling, and 3) postweaning. Individual fetal samples were derived from two types of matings: 1) homozygous obese males (fafa) with heterozygous females (Fafa), and 2) homozygous lean males and females (FaFa). Suckling and postweaned pups from similar matings were partially pancreatectomized, tail bled, and identified retrospectively. In 21-day-old fetuses bearing the fa gene, plasma insulin concentration was elevated (P less than 0.001) and pancreatic concentration was slightly lower (P less than 0.05) compared to homozygous lean fetuses. Neither pancreatic nor plasma insulin concentration differed between preobese and lean pups during suckling, except that pancreatic concentration became elevated in preobese pups on postnatal day 20 (P less than 0.05). Plasma insulin concentration was elevated 24 h postweaning (P less than 0.01). These data support the hypothesis that the fa gene initiates metabolic changes during gestation that are modulated during suckling, but reappear at weaning. The data also establish that increased adipocyte size and lipoprotein lipase activity in 7- to 12-day-old preobese pups are not dependent on concomitant hyperinsulinemia.

Regional and genotypic differences in stromal-vascular cells from obese and lean Zucker rats.

LPL activity, total lipogenesis and rates of growth were determined for stromal-vascular cells derived from epididymal and inguinal depots of 13 1/2-week-old obese and lean Zucker rats. LPL activity, in cells of both depots, was found to increase between days 4 and 6 and decrease by day 8 in the presence of insulin. Inguinal derived fatty cell LPL activity increased between days 4 and 8 in contrast to lean cells which peaked at day 6 under basal growth conditions. LPL activity was elevated in fatty versus lean cells at days 6 and 8 in inguinal derived stromal vascular cells while in epididymal derived stromal vascular cells, LPL activity was elevated in lean versus fatty derived cells at day 4 and 6 but by day 8 the genotypic effect was reversed. Lipogenesis was elevated in lean versus fatty derived epididymal and inguinal cells at all concentrations of insulin and lean cells showed a dose-dependent response to insulin in contrast to fatty cells. There were no effects of genotype on the proliferative capacity of cells from either depot but some regional differences in growth were observed. These data illustrate that fa gene effects can be studied in primary cell culture.

Reduced adipsin mRNA and circulating adipsin protein are modulated by adrenal steroids in obese Zucker rats.

We investigated expression of the adipose-specific serine protease adipsin in genetically obese Zucker rats and whether adrenalectomy modifies expression. Adipsin mRNA levels were determined by slot blot and Northern blot analysis of total RNA samples extracted from epididymal adipose tissue and isolated retroperitoneal adipocytes of obese (fa/fa) and homozygous lean (Fa/Fa) Zucker rats. Both 30-day-old and 4-mo-old animals were analyzed in experiment 1. In experiment 2, 10-wk-old obese and lean rats were either bilaterally adrenalectomized or sham operated, and adipsin mRNA levels were determined on tissue and cell samples 2 wk postsurgery. In both experiments, serum adipsin protein was determined by Western blot analysis and plasma insulin by radioimmunoassay. The data show that both adipsin mRNA and adipsin protein are reduced in obese compared with lean rats and that adrenalectomy restores these values toward normal in obese rats. The data thus suggest that adrenal steroids are involved in modulating adipsin expression in obese Zucker rats and that insulin may be an intermediary factor in such modulation.

Zucker and Wistar diabetic fatty rats show different response to adrenalectomy.

The short-term effects of adrenalectomy on certain aspects of glucose homeostasis and adiposity were examined in Zucker and Wistar diabetic fatty (WDF) rats. Ten-week-old male obese and lean WDF and Zucker rats were adrenalectomized or underwent sham operation. Obese rats of each strain were pair fed the intake of obese adrenalectomized rats. Intragastric glucose tolerance tests showed that sham-operated obese rats of both strains were severely hyperinsulinemic compared with leans; adrenalectomy and pair feeding reduced palsma insulin to lean levels in Zucker but not WDF rats. At the time they were killed, sham-operated obese WDF rats were significantly hyperglycemic and hyperinsulinemic compared with other groups, but adrenalectomy reduced plasma glucose and insulin to lean levels in both strains. Adrenalectomy reduced inguinal and retroperitoneal fat pad weights more in Zucker than WDF obese rats. Although adrenalectomy decreased epididymal and inguinal fat cell size in both obese rat strains, the effect was greater in Zucker compared with WDF rats. These data suggest that the basis for the differential response to adrenalectomy in obese WDF and Zucker rats may reside in their different genetic backgrounds.

Adipoblasts from the Zucker fafa rat.

In vitro experiments using both primary fetal hepatocyle cultures and adipoblast cultures have demonstrated that the presence of the fa gene is associated with decreased synthetic capacity, when compared to wild-type cultures. These results are in contrast to the elevated lipogensis and lipoprotein-lipase activities found in vivo in young adult obses (fafa) Zucker rats compared to their lean littermates. These studies used adipoblast cultures to address three possible explanations for these in vitro-in vivo differences: 1) FaFa and fafa adipoblast cultures represent different cell populations with intrinsically different abilities to differentiate, ie, to lipid-fill. 2) The decreased synthetic capacities in fafa vs FaFa adipoblast cultures are specific to cultures derived from the epididymal pad. 3) Cultured adipoblasts produce factor(s) that affect adipoblast differentiation in vitro. Results indicate that 1) the rate of differentiation is slower in fafa than in FaFa adipoblasts 2) there are depot-related differences in lipid metabolism, but these differences do not negate the in vitro association between the fa gene and decreased synthetic capacity and 3) FaFa epididymal-derived adipoblasts produce a factor(s) that affects inguinal-derived adipoblast differentiation and/or growth in vitro. Thus it is important to take both the site of cell origin and culture conditions into consideration when using in vitro systems as an approach to understanding complex in vivo disorders, such as obesity in the Zucker fafa rat.

Mechanisms of increased lipoprotein lipase in fat cells of obese Zucker rats.

The mechanisms underlying the increased activity of lipoprotein lipase (LPL) in adipocytes of genetically obese Zucker rats was studied. Relative rates of LPL synthesis (percent of total protein synthesis) determined by biosynthetic labeling and specific immunoprecipitation were similar in isolated fat cells from lean and obese rats, in the absence or presence of insulin. Insulin stimulated LPL synthesis as a result of a general increase in protein synthesis, and this effect was more marked in the obese fat cells. Levels of LPL mRNA, as a percent of total RNA, were also similar in fat cells from lean and obese rats. In contrast, when the data are calculated on a per fat cell basis, rates of LPL synthesis per fat cell are ninefold higher in obese compared with lean cells, accounting for the increase in LPL activity per fat cell. Fat cells from lean and obese rats showed similar rates of binding and degradation of purified bovine milk 125I-labeled LPL per unit fat cell surface area. Thus, on a per cell basis, rates of LPL turnover are increased in enlarged Zucker rat adipocytes, but there is no specific abnormality in the cellular regulation of LPL. Increases in LPL activity in obese rat adipocytes are related to an overall hyperresponsiveness to insulin effects on protein synthesis.