Liporegulation in diet-induced obesity. The antisteatotic role of hyperleptinemia

To test the hypothesis that the physiologic liporegulatory role of hyperleptinemia is to prevent steatosis during caloric excess, we induced obesity by feeding normal Harlan Sprague-Dawley rats a 60% fat diet. Hyperleptinemia began within 24 h and increased progressively to 26 ng/ml after 10 weeks, correlating with an approximately 150-fold increase in body fat (r = 0.91, p < 0.0001). During this time, the triacylglycerol (TG) content of nonadipose tissues rose only 1-2.7-fold implying antisteatotic activity. In rodents without leptin action (fa/fa rats and ob/ob and db/db mice) receiving a 6% fat diet, nonadipose tissue TG was 4-100 times normal. In normal rats on a 60% fat diet, peroxisome proliferator-activated receptor alpha protein and liver-carnitine palmitoyltransferase-1 (l-CPT-1) mRNA increased in liver. In their pancreatic islets, fatty-acid oxidation increased 30% without detectable increase in the expression of peroxisome proliferator-activated receptor-alpha or oxidative enzymes, whereas lipogenesis from [14C]glucose was slightly below that of the 4% fat-fed rats (p < 0.05). Tissue-specific overexpression of wild-type leptin receptors in the livers of fa/fa rats, in which marked steatosis is uniformly present, reduced TG accumulation in liver but nowhere else. We conclude that a physiologic role of the hyperleptinemia of caloric excess is to protect nonadipocytes from steatosis and lipotoxicity by preventing the up-regulation of lipogenesis and increasing fatty-acid oxidation.     

Soy protein suppresses gene expression of acetyl-coA carboxylase alpha from promoter PI in rat liver

Dietary soy protein isolate (SPI) reduces hepatic lipogenesis by suppressing gene expression of lipogenic enzymes, including acetyl-CoA carboxylase (ACC). In order to elucidate the mechanism of this regulation, the effect of dietary SPI on promoter (PI and PII) specific gene expression of ACC alpha was investigated. Rats were fed experimental diets containing SPI or casein as a nitrogen source. SPI feeding decreased the hepatic contents of total ACC mRNA as well as triglyceride (TG) content, but dietary SPI affected the amount of sterol-regulatory element binding protein (SREBP)-1 mRNA and protein very little. The amount of ACC mRNA transcribed from PII promoter containing SRE was not significantly affected by dietary protein, while a significant decrease in PI-generated ACC mRNA content was observed in rats fed the SPI diet. These data suggest that SPI feeding decreased the hepatic contents of ACC alpha mRNA mainly by regulating PI promoter via a nuclear factor(s) other than SREBP-1.     

Slow recovery of body fat lost during adenovirus-induced hyperleptinemia

In normal rats, adenovirus-induced hyperleptinemia causes disappearance of visible body fat, downregulation of lipogenic enzymes, and upregulation of oxidative enzymes and thermogenic proteins. In addition, preadipocyte markers replace mature adipocyte markers, suggesting dedifferentiation. In weight loss induced by caloric restriction, by contrast, the lipogenic machinery is essentially intact. To determine if the radical changes induced by leptin would slow the reappearance of body fat, we compared normal lean rats made hyperleptinemic by infusing an adenovirus-leptin construct with diet-matched littermates. Initially, in plasma leptin the hyperleptinemic rats averaged approximately 50x the controls and, although it declined progressively, it was still slightly elevated at 150 days (P < 0.05). In the hyperleptinemics, body fat mass, quantified by magnetic resonance spectroscopy, remained below the pretreatment value for 60 days, while in diet-matched controls it exceeded the pretreatment value. Epididymal fat pad weight in hyperleptinemics was still 28% below paired controls at 150 days posttreatment. Histologic examination revealed adipocytes of hyperleptinemic animals to be smaller 60 days after treatment. At 60 days, adipose tissue UCP-2 gene expression in hyperleptinemics was still above controls, but expression of other lipogenic and oxidative enzymes had returned to baseline expression levels. We conclude that in normal rats recovery of body fat following adenovirus-induced hyperleptinemia is much slower than after caloric restriction, possibly because of persistent upregulation of adipocyte UCP-2.     

Altered mRNA expression of hepatic lipogenic enzyme and PPARalpha in rats fed dietary levan from Zymomonas mobilis

Levan or high molecular beta-2,6-linked fructose polymer is produced extracellularly from sucrose-based substrates by bacterial levansucrase. In the present study, to investigate the effect of levan feeding on serum leptin, hepatic lipogenic enzyme and peroxisome proliferation-activated receptor (PPAR) alpha expression in high-fat diet-induced obese rats, 4-week-old Sprague-Dawley male rats were fed high-fat diet (beef tallow, 40% of calories as fat), and, 6 weeks later, the rats were fed 0%, 1%, 5% or 10% levan-supplemented diets for 4 weeks. Serum leptin and insulin level were dose dependently reduced in levan-supplemented diet-fed rats. The mRNA expressions of hepatic fatty acid synthase and acetyl CoA carboxylase, which are the key enzymes in fatty acid synthesis, were down-regulated by dietary levan. However, dietary levan did not affect the gene expression of hepatic malic enzyme, phosphatidate phosphohydrolase and HMG CoA reductase. Also, the lipogenic enzyme gene expression in the white adipose tissue (WAT) was not affected by the diet treatments. However, hepatic PPARalpha mRNA expression was dose dependently up-regulated by dietary levan, whereas PPARgamma in the WAT was not changed. The results suggest that the in vivo hypolipidemic effect of dietary levan, including anti-obesity and lipid-lowering, may result from the inhibition of lipogenesis and stimulation of lipolysis, accompanied with regulation of hepatic lipogenic enzyme and PPARalpha gene expression.     

Development of hepatic and adipose tissue lipogenic enzymes and insulinemia during suckling and weaning on to a high-fat diet in Zucker rats

This study was designed to monitor the developmental changes in insulinemia and lipogenic enzyme activities in both inguinal adipose tissue and liver during suckling (7, 9, 14, and 17 days of age) and weaning (22 and 30 days of age) on to either a low-fat or a high-fat diet in lean (Fa/fa) and obese (fa/fa) rats. Tissues were removed through surgery and genotypes were retrospectively determined. During suckling, there was no difference in liver enzyme activities between the two groups. In contrast, adipose tissue fatty acid synthetase was increased by 50% and citrate cleavage enzyme and malic enzyme by 30% by 9 days of age. By 17 days of age, there was a threefold elevation in these enzyme activities and 6-phosphogluconic dehydrogenase and a twofold increase in glucose-6-phosphate dehydrogenase per inguinal fat pad in fa/fa versus Fa/fa. Consistent with these results, fat pad weight was increased by 20%, 50%, and 100% at 9, 14, and 17 days of age, respectively, in obese as compared to lean pups. However only by 17 days of age could a slight but significant increase in insulin level be detected in obese pups. Enlargement of inguinal fat pad accelerated after weaning on to a low-fat diet and still more after weaning on to a high-fat diet. Weaning on to a low-fat diet elicited an induction of hepatic lipogenic enzymes two or three times greater in fa/fa than in lean pups, while weaning on to a high-fat diet blunted the differences between genotypes. The lipogenic enzyme activities displayed per total inguinal fat were three to ten times greater in obese than in lean pups, regardless of the diet. However, adipose tissue lipogenic enzyme activities were much lower after weaning on to a high-fat than on to a low-fat diet in obese pups. The high-fat diet was as effective as the low-fat diet in triggering hyperinsulinemia in obese pups. The increased adipose tissue capacity for lipogenesis, starting during the suckling period, could play an important etiologic role in the development and maintenance of obesity in the Zucker rat.-Bazin, R., and M. Lavau. Development of hepatic and adipose tissue lipogenic enzymes and insulinemia during suckling and weaning on to a high-fat diet in Zucker rats.     

Development of fatty acid-synthetic capacity in interscapular brown adipose tissue during suckling in genetically obese Zucker rats.

The development of the lipogenic capacity in brown adipose tissue was studied in suckling lean (Fa/fa) and obese (fa/fa) Zucker pups aged from 7 to 22 days. In both lean and obese pups, activities of the two key lipogenic enzymes, fatty acid synthetase and acetyl-CoA carboxylase, and of citrate cleavage enzyme rose from the early to the late suckling period. Compared with lean pups, 7-day-old fa/fa pups showed a 35% increase in fat accumulation in interscapular brown adipose tissue and a 25% increase in fatty acid synthetase activity. By 10 days of age, fat deposition, lipogenesis in vivo (assessed by the incorporation of 3H from 3H2O into fatty acids) and fatty acid synthetase activity were 1.5-2-fold higher in pre-obese than in lean pups. Compared with lean pups, the increased lipogenesis in vivo observed in brown adipose tissue of 10-day-old pre-obese pups could not entirely account for the difference in fat deposition observed in this tissue, suggesting that additional mechanisms are at play to explain the increased fat content of this tissue.     

Interaction between growth hormone and insulin in the regulation of lipoprotein metabolism in the rat

The importance of insulin for the in vivo effects of growth hormone (GH) on lipid and lipoprotein metabolism was investigated by examining the effects of GH treatment of hypophysectomized (Hx) female rats with and without concomitant insulin treatment. Hypophysectomy-induced changes of HDL, apolipoprotein (apo)E, LDL, and apoB levels were normalized by GH treatment but not affected by insulin treatment. The hepatic triglyceride secretion rate was lower in Hx rats than in normal rats and increased by GH treatment. This effect of GH was blunted by insulin treatment. The triglyceride content in the liver changed in parallel with the changes in triglyceride secretion rate, indicating that the effect of the hormones on triglyceride secretion was dependent on changed availability of triglycerides for VLDL assembly. GH and insulin independently increased editing of apoB mRNA, but the effects were not additive. The expression of fatty-acid synthase (FAS), stearoyl-CoA desaturase-1 (SCD-1), and sterol regulatory element-binding protein-1c (SREBP-1c) was increased by GH treatment. Insulin and GH had no additive effects on these genes; instead, insulin blunted the effect of GH on SREBP-1c mRNA. In contrast to the liver, adipose tissue expression of SREBP-1c, FAS, or SCD-1 mRNA was not influenced by GH. In conclusion, the increased hepatic expression of lipogenic enzymes after GH treatment may be explained by increased expression of SREBP-1c. Insulin does not mediate the effects of GH but inhibits the stimulatory effect of GH on hepatic SREBP-1c expression and triglyceride secretion rate.     

Chronic food restriction up-regulates 11-β-hydroxysteroid dehydrogenase

Corticosterone — product of 11-β-hydroxysteroid dehydrogenase type I (11βHSD1) stimulates adipocytes differentiation and activates lipogenic enzymes gene expression in white adipose tissue (WAT) of rats. The aim of the study was to examine the effect of chronic food restriction, often practised by obese individuals trying to lose body mass, on: a) 11βHSD1 gene expression, b) expression of genes associated with adipocyte differentiation (PPARg, SREBP-1, adiponectin), and c) expression of genes associated with lipogenesis in WAT of rats. Two-month old rats were divided into a control and a food restricted group obtaining 50% of food consumed by controls for 30 days. mRNA levels of studied genes in perirenal WAT were analysed by real-time PCR. 11βHSD1 and lipogenic enzymes activities were measured by radiometric conversion assay and by spectrophotometric assay respectively. Food restriction caused significant increase of 11βHSD1, PPARg, SREBP1, adiponectin and lipogenic enzymes mRNA levels in perirenal WAT. 11βHSD1 and some lipogenic enzymes activities were also increased by food restriction. The coordinated up-regulation of 11βHSD1, and genes associated with adipocyte differentiation and lipogenesis by food restriction suggests that such nutritional condition shifts WAT metabolism, that would permit this tissue to synthesize and accumulate triacylglycerols immediately after refeeding.     

High fat feeding and dietary <Emphasis Type="SmallCaps">l</Emphasis>-arginine supplementation differentially regulate gene expression in rat white adipose tissue

Dietary l-arginine (Arg) supplementation reduces white-fat gain in diet-induced obese rats but the underlying mechanisms are unknown. This study tested the hypothesis that Arg treatment affects expression of genes related to lipid metabolism in adipose tissue. Four-week-old male Sprague–Dawley rats were fed a low-fat (LF) or high-fat (HF) diet for 15 weeks. Thereafter, lean or obese rats continued to be fed their same respective diets and received drinking water containing 1.51% Arg–HCl or 2.55% l-alanine (isonitrogenous control). After 12 weeks of Arg supplementation, rats were euthanized to obtain retroperitoneal adipose tissue for analyzing global changes in gene expression by microarray. The results were confirmed by RT-PCR analysis. HF feeding decreased mRNA levels for lipogenic enzymes, AMP-activated protein kinase, glucose transporters, heme oxygenase 3, glutathione synthetase, superoxide dismutase 3, peroxiredoxin 5, glutathione peroxidase 3, and stress-induced protein, while increasing expression of carboxypeptidase-A, peroxisome proliferator activated receptor (PPAR)-α, caspase 2, caveolin 3, and diacylglycerol kinase. In contrast, Arg supplementation reduced mRNA levels for fatty acid binding protein 1, glycogenin, protein phosphates 1B, caspases 1 and 2, and hepatic lipase, but increased expression of PPARγ, heme oxygenase 3, glutathione synthetase, insulin-like growth factor II, sphingosine-1-phosphate receptor, and stress-induced protein. Biochemical analysis revealed oxidative stress in white adipose tissue of HF-fed rats, which was prevented by Arg supplementation. Collectively, these results indicate that HF diet and Arg supplementation differentially regulate gene expression to affect energy-substrate oxidation, redox state, fat accretion, and adipocyte differentiation in adipose tissue. Our findings provide a molecular mechanism to explain a beneficial effect of Arg on ameliorating diet-induced obesity in mammals.     

Marked enhancement of acyl-CoA synthetase activity and mRNA, paralleled to lipoprotein lipase mRNA, in adipose tissues of Zucker obese rats (fa/fa).

To clarify the role of acyl-CoA synthetase in development of obesity, the mRNA levels and activities were studied in Zucker fatty rats (fa/fa). In Zucker fatty rats compared with their lean littermates, marked enhancement of ACS were observed in adipose tissues. Obese/lean rats ratio of ACS activity and mRNA in abdominal subcutaneous fat (3.3- and 3.9-fold, respectively) were greater than in mesenteric fat (2.0- and 2.2-fold). The enhancement of ACS activity and mRNA in the liver of fatty rats (1.2- and 1.8-fold) were less than those in the adipose tissues. There were no enhancement of ACS activities and mRNA levels in heart tissue of the obese rats. LPL mRNA levels were also enhanced in adipose tissue of fatty rats and obese/lean ratio of LPL mRNA was also higher in abdominal subcutaneous fat than mesenteric fat (6.2- vs 3.1-fold). The larger obese/lean rats ratio of LPL and ACS parameters in abdominal subcutaneous fat than mesenteric fat may be related to the observation that the increase of subcutaneous fat weight was larger than that of mesenteric fat weight in fatty rats (21.1- vs 4.9-fold). Integrated enhancement of LPL and ACS gene expression in adipose tissue may play an important role in the development of obesity.     

Effect of diets rich in medium-chain and long-chain triglycerides on lipogenic-enzyme gene expression in liver and adipose tissue of the weaned rat.

The activity and mRNA concentrations of two lipogenic enzymes, fatty-acid synthase and acetyl-CoA carboxylase were measured in the liver and white adipose tissue of rats weaned to a carbohydrate-rich diet containing either long-chain or medium-chain fatty acids, and compared to those of rats weaned on a diet containing less than 1% (total energy) fat (high-carbohydrate diet). In the liver, the diet containing long-chain fatty acids inhibited the increase of both lipogenic-enzyme mRNA concentrations and activities seen at weaning on the high-carbohydrate diet but did not prevent the decrease in phosphoenolpyruvate carboxykinase mRNA and activity. In contrast, the diet containing medium-chain fatty acids induced a slower but finally similar increase in lipogenic-enzyme mRNA concentrations and activities. In adipose tissue, a similar trend was observed, although the inhibitory effect of the diet containing long-chain fatty acids was considerably less marked than in liver. It is concluded that medium-chain and long-chain fatty acids have not the same inhibitory potency of the gene expression of lipogenic enzymes, and that long-chain fatty acids have a more marked effect in the liver.     

Lipoprotein lipase and lipogenic enzyme activities in adipose tissue from rats fed different lipid sources

This work was designed to study the effect of different lipid sources on the activities of lipoprotein lipase and lipogenic enzymes in adipose tissue from rats fedad libitum or energy-controlled diets. Male Wistar rats were fed diets containing 40% of energy as fat (olive oil, sunflower oil, palm oil or beef tallow), for 4 wk. Underad libitum feeding no differences were found among dietary fat groups in final body weight, adipose tissue weights and total body fat. Under energy-controlled feeding, despite isoenergetic intake, rats fed the beef tallow diet gained significantly less weight than rats fed the other three diets. Beef tallow fed rats showed the lowest values for adipose tissue weights and total body fat. When rats had free access to food no effect of dietary lipid source on lipogenic enzyme activities was found. In contrast, under energy-controlled feeding rats fed the beef tallow diet showed significantly higher activities of glucose-6-phosphate dehydrogenase and fatty acid synthase than rats fed the other three diets. Heparin-releasable lipoprotein lipase activity in perirenal and subcutaneous adipose tissues was not different among rats fed olive oil, safflower oil, palm oil or beef tallow. When comparing both adipose tissue anatomical locations, significantly higher activities were found in subcutaneous than in perirenal fat pad independently of dietary fat. In conclusion, under our experimental protocol, lipogenesis in rat adipose tissue does not seem to be affected by dietary fat type.     

Adipose-tissue-specific increase in glyceraldehyde-3-phosphate dehydrogenase activity and mRNA amounts in suckling pre-obese Zucker rats. Effect of weaning.

The regulation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene expression was studied during the onset of obesity in the genetically obese (fa/fa) rat by determination of GAPDH activity and hybridizable mRNA amounts in adipose tissue and liver from suckling and weanling rats. GADPH activity remained low throughout the suckling period, and a burst of activity occurred after weaning in both lean and obese pups. As early as 7 days of age, adipose tissue from pre-obese rats displayed a significant increase in enzyme activity, whereas no difference could be detected in the liver. In both suckling (16 days of age) and weanling (30 days of age) obese rats a proportionate increase in GAPDH activity and mRNA amounts was observed in adipose tissue, but not in liver. It is concluded that the obese genotype influences GAPDH gene expression at a pretranslational level and in a tissue-specific manner. This phenomenon could partly contribute to the hyperactive fat accretion in the obese rat, since glycolysis is the major metabolic pathway for lipogenic substrates in adipose tissue.     

Fat metabolism is regulated by altered gene expression of lipogenic enzymes and regulatory factors in liver and adipose tissue but not in semimembranosus muscle of pigs during the fattening period

It has been shown previously that lipid metabolism is regulated by fatty acids (FA) and that thyroid hormones are important regulators of energy metabolism. The effects of weight, dietary fat level and dietary FA profile on thyroid hormone levels and expression of lipogenic genes and tissue FA composition were studied. Sixty-one crossbred gilts weighing 62 ± 5.2 kg BW average were either slaughtered at the beginning of the trial (n = 5) or fed one of seven diets (n = 8 pigs per diet): a semi-synthetic diet formulated to contain a very low level of fat (NF) and six diets based on barley-soybean meal supplemented with approximately 10% fat of different origin and slaughtered at 100 kg BW. The supplemental fats were tallow, high-oleic sunflower oil, sunflower oil (SFO), linseed oil, fat blend (55% tallow, 35% sunflower oil, 10% linseed oil) and fish oil blend (40% fish oil, 60% linseed oil). In general, the dietary FA profiles altered the FA composition of liver, semimembranosus muscle and adipose tissues. Pigs fed the NF diet had the highest free and total triiodothyronine (T3) values followed by pigs fed SFO. Total T3 levels were higher in pigs at 60 kg than in pigs at 100 kg. Correlations between thyroid hormones and genes encoding enzymes of fat synthesis in adipose tissue (acetyl CoA carboxylase (ACACA), fatty acid synthase and stearoyl CoA desaturase (SCD)) and the large differences in expression of lipogenic genes at different weights (60 and 100 kg BW), suggest a role for thyroid hormones and for T3, in particular, in regulating whole animal fat metabolism, with effects brought about by altered expression of lipogenic genes. Liver sterol receptor element binding protein-1 (SREBP1) mRNA content was affected by dietary treatment (P < 0.001) and was correlated with ACACA and SCD, whereas adipose tissue SREBP1 was not correlated with the mRNA abundance of any lipogenic enzyme. Weight and tissue factors showed greater influence on mRNA abundance of genes related with lipid metabolism than diet and tissue FA composition. In the pig, FA synthesis appear to be of greater magnitude in adipose tissue than in the liver as suggested by the higher expression of lipogenic genes in adipose tissue.     

Increasing protein at the expense of carbohydrate in the diet down-regulates glucose utilization as glucose sparing effect in rats

High protein (HP) diet could serve as a good strategy against obesity, provoking the changes in energy metabolic pathways. However, those modifications differ during a dietary adaptation. To better understand the mechanisms involved in effect of high protein diet (HP) on limiting adiposity in rats we studied in parallel the gene expression of enzymes involved in protein and energy metabolism and the profiles of nutrients oxidation. Eighty male Wistar rats were fed a normal protein diet (NP, 14% of protein) for one week, then either maintained on NP diet or assigned to a HP diet (50% of protein) for 1, 3, 6 and 14 days. mRNA levels of genes involved in carbohydrate and lipid metabolism were measured in liver, adipose tissues, kidney and muscles by real time PCR. Energy expenditure (EE) and substrate oxidation were measured by indirect calorimetry. Liver glycogen and plasma glucose and hormones were assayed. In liver, HP feeding 1) decreased mRNA encoding glycolysis enzymes (GK, L-PK) and lipogenesis enzymes(ACC, FAS), 2) increased mRNA encoding gluconeogenesis enzymes (PEPCK), 3) first lowered, then restored mRNA encoding glycogen synthesis enzyme (GS), 4) did not change mRNA encoding β-oxidation enzymes (CPT1, ACOX1, βHAD). Few changes were seen in other organs. In parallel, indirect calorimetry confirmed that following HP feeding, glucose oxidation was reduced and fat oxidation was stable, except during the 1(st) day of adaptation where lipid oxidation was increased. Finally, this study showed that plasma insulin was lowered and hepatic glucose uptake was decreased. Taken together, these results demonstrate that following HP feeding, CHO utilization was increased above the increase in carbohydrate intake while lipogenesis was decreased thus giving a potential explanation for the fat lowering effect of HP diets.     

A low fish oil inhibits SREBP-1 proteolytic cascade,while a high-fish-oil feeding decreases SREBP-1 mRNA in mice liver: relationship to anti-obesity

Rodents fed fish oil showed less obesity with a reduction of triglyceride synthesis in liver, relative to other dietary oils, along with a decrease of mature form of sterol regulatory element binding protein-1 (SREBP-1) and activation of peroxisome proliferator-activated receptor alpha (PPARalpha). Decrease of mature SREBP-1 protein by fish oil feeding was due to either inhibition of SREBP-1 proteolytic cascade or to decrease of its mRNA. To clarify its mechanism and relation to antiobesity effect, mice were fed fish oil in a range from 10 to 60 energy percent (en%). Fish oil feeding decreased body weight and fat mass in a dose-dependent manner, in parallel with PPARalpha activation and a decrease of SREBP-1 mRNA. However, compared with 0 en% fish oil feeding, 10 en% fish oil feeding decreased mature SREBP-1 protein by 50% with concomitant decreases of lipogenic genes, while precursor SREBP-1 protein rather increased by 1.3-fold. These data suggest that physiological doses of fish oil feeding effectively decrease expression of liver lipogenic enzymes by inhibiting SREBP-1 proteolytic cascade, while substantial decrease of SREBP-1 expression is observed in its pharmacological doses, and that activation of PPARalpha rather than SREBP-1 decrease might be related to the antiobesity effect of fish oil feeding.