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Previous studies have shown that the adipose tissue of young genetically obese Zucker rats was characterized by a coordinate overtranscription of lipogenic genes, suggesting that the fa mutation triggers transcription factor(s) acting in common on the promoters of these genes. To test this hypothesis, we developed a system of transient transfection of rat adipocytes with constructs containing glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and fatty acid synthetase (FAS) promoters fused to gene reporter CAT. Those transfected cells expressed high levels of promoter-driven chloramphenicol acetyltransferase (CAT) activity through correctly initiated transcription as shown by primer extension analysis. Using this system we found a direct effect of insulin on GAPDH and FAS gene expression in rat adipocytes. In transfected adipocytes from obese compared to lean rats, activity of GAPDH and FAS promoters fused to CAT, was 2.6- and 8-fold increased, respectively. In contrast when reporter gene activity was driven by either phosphoenolpyruvate carboxykinase or beta-actin promoter, no difference could be observed between lean and obese, pointing out the promoter specificity of genotype effect. 5' deletion analysis of GAPDH promoter allowed us to narrow down the fa responsive region to nucleotide -488-329. As assessed by gel retardation and DNase I footprinting analysis, adipocyte nuclear protein interactions to this 159-bp fragment were found to be identical and to footprint the same 20-bp sequence. This study pointed out that overexpression of GAPDH and FAS genes in adipose tissue of genetically obese rats relies on promoter activation, through a 159-bp cis-acting region within the GAPDH promoter. The effects of the fa mutation on trans-acting factors binding to this region remain to be identified.

The influence of insulin on [6-l4 C] glucose metabolism was assessed in vitro and in vivo in epididymal adipose tissue and diaphragm of rats fed either a low-fat (9 per cent fat cal.) or a high-fat diet (72 per cent fat cal.). In vitro, diaphragm of fat-fed rats showed a lower glucose uptake than that of rats fed the low-fat diet, but had identical glycogen labeling and lactic acid production and a strongly reduced I4 CO2 production. Responsiveness of these pathways to insulin was unaltered by the fat content of the diet. The adipose tissue of fat-fed rats versus that of rats fed the low-fat diet showed: a higher lactic acid production and more efficient glycerogenesis and glycogenesis, all of these pathways being responsive to insulin; a lower glucose uptake and a strongly depressed fatty acid labeling, these two pathways being unresponsive to insulin. In-vivo labeling of glycogen in diaphragm in both basal and insulin-stimulated conditions was identical in the two groups of rats. In adipose tissue the amount of t4 C sequestered in the gh/ceride-glycerol moiety was the same in the two groups in basal and insulin-stimulated conditions, whereas the labeling of the fatty acid moiety and its increment with insulin were reduced by more than 99 per cent by the high-fat diet.These results show that alterations in fat content of the diet lead to differences in response to insulin that are pathway-and organspecific. DIABETES 27:114-20, February, 1978.A high-fat diet in rats causes dramatic changes in peripheral tissue glucose metabolism both in vitro and in vivo. 1 " 5 In addition, an impaired tolerance to intravenously 6 or orally 7 administered glucose and a reduced effect of exogenous 6 or endogenous 7 insulin

The effects of a fish oil concentrate on blood lipids and lipoproteins were examined in relation to their effects on liver fatty acid synthase (FAS), 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, adipose tissue lipoprotein lipase (LPL), and hepatic triglyceride lipase (H-TGL). For 15 days, 2-mo-old rats were fed a control diet (10% of calories from fat, 4% fat by weight) or diets with 50% of calories (25% wt/wt) provided by lard, lard and fish oil calories (35%/15%), or lard and corn oil (35%/15%). The high-lard diet increased plasma chylomicron and liver triglycerides. The high-lard diet greatly decreased FAS, HMG-CoA reductase, and LPL activities; it also reduced H-TGL activity. Compared with the lard diet, the lard-fish oil diet decreased plasma TG by drastically lowering chylomicron (4-fold, P < 0.001) and very-low-density lipoprotein levels (P < 0.001). It also reduced high-density lipoprotein levels. The lard-fish oil diet prevented hepatic triglyceride accumulation and decreased FAS activity and mass by 3.5-fold (P < 0.001) but did not further decrease HMG-CoA reductase activity. Adipose tissue LPL activity was 2.5-fold (P < 0.001) higher with the lard-fish oil diet than with the lard diet, and H-TGL activity decreased significantly (-32%, P < 0.01), despite unaltered levels of H-TGL mRNA. These effects were significant with only 10% fish oil concentrate in the lard diet. They were not observed with the lard-corn oil diet.(ABSTRACT TRUNCATED AT 250 WORDS)

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This study was undertaken to examine whether there were sex-associated differences in the action of insulin on glucose metabolism in adipocytes. Insulin binding and the dose-response curves for glucose transport (assessed by measuring the cellassociated radioactivity after 15-s incubation with 50 ;&M 16-'4Clglucose) and [U-'4Cjglucose (5 mM) metabolism into CO2 and lipids were compared in retroperitoneal adipocytes from age-matched (84 d) male and female rats. In addition, the activity of fatty acid synthetase, one of the key lipogenic enzymes, was determined. Fat cell size was not significantly larger in females than in males (0.238 vs. 0.209 jig lipid per cell). At insulin concentrations < 1.6 nM, adipocytes from females bound significantly more insulin than did adipocytes from males, due to an increased apparent affinity of the receptors for insulin. Accordingly, the sensitivity of glucose transport to insulin was greater in females than in males: insulin concentration eliciting half-maximal stimulation (ED50) = 0.19 nM vs. 0.41 nM. At maximal insulin stimulation the rates of glucose transport (12 times the basal values) were similar in the two sexes. In contrast, the maximal effect of insulin on glucose conversion to CO2 plus lipids was much greater in the adipocytes from females than males (increment over basal: 472 vs. 249 nmol/106 cells per 2 h). Fatty acid synthesis contributed -40% of the incremental difference between the two types of adipocytes, while glyceride-glycerol synthesis contributed <10%. The insulin dose-response curves for adipocytes from females were shifted to the left for all the metabolic pathways investigated. The mean ED50 for total glucose metabolism in females was 50% of that in males (0.07 nM vs. 0.15 nM). Marked sex-associated differences in the action of insulin on glucose metabolism were also observed in subcutaneous inguinal adipocytes (increment over basal: 137 and 56 nmol/106 cells per 2 h, ED50 = 0.13 nM and 0.30 nM in females and males, respectively). The intracellular capacity to metabolize glucose through the fatty acid synthesis pathway, as assessed by FAS activity, was higher in adipocytes from females than in those from males and was greater in retroperitoneal than in i guinal adipocytes. Furthermore, by plotting the individual data, a highly significant correlation (r = 0.92, P < 0.001) was found between the absolute effect of insulin on glucose metabolism at maximal stimulation and the fatty acid synthetase activity of the cells.These results indicate that the response of glucose metabolism to insulin in adipocytes from female as compared with male rats is characterized by two main features: (a) an increased sensitivity primarily due to an increase in insulin binding, and (b) an increased responsiveness closely associated with a postreceptor increase in the lipogenic capacity of the cell. These findings might be relevant to the differential disposition of male and female rats to develop fatness.