Different sensitivity of PPARalpha gene expression to nutritional changes in liver of suckling and adult rats

The amount of peroxisome proliferator-activated receptor-alpha (PPARalpha) protein was markedly augmented in the liver of suckling rats compared to adult rats. This different PPARalpha abundance was used to study the sensitivity to nutritional changes in the expression and activity of this receptor. Thus, 10-day-old and adult rats were orally given either glucose, Intralipid or a combination of both diets, and liver mRNA levels of PPARalpha and the PPAR related genes, acyl-CoA oxidase (ACO) and phosphoenolpyruvate carboxykinase (PEPCK), and plasma metabolites were measured. In neonates, the expression of PPARalpha and ACO was seen to increase when the level of FFA in plasma was also high, unless an elevated level of insulin was also present. However, this fatty acid-induced effect was not detected in adult rats. On the contrary, the hepatic expression of PEPCK was modulated by the nutritional changes similarly in both neonates and adult rats. Thus, it may be concluded that the expression of the PPARalpha gene in adult rats seems to be less sensitive to nutritional changes than in neonates.     

Relationship between lipoprotein lipase and peroxisome proliferator-activated receptor-α expression in rat liver during development

The present study was addressed to determine whether the high expression of peroxisome proliferator-activated receptor-alpha (PPAR-alpha) in rat liver during the perinatal stage plays a role in the induction of liver lipoprotein lipase (LPL) expression and activity. Parallel increases in liver mRNA PPAR-alpha and LPL activity were found in newborn rats, and after a slight decline, values remained elevated until weaning. Anticipated weaning for 3 days caused a decline in those two variables as well as in the mRNA LPL level, and a similar change was also found in liver triacylglycerol concentration. Force-feeding with Intralipid in 10-day-old rats or animals kept fasted for 5 h showed high mRNA-PPARalpha and -LPL levels as well as LPL activity with low plasma insulin and high FFA levels, whereas glucose and a combination of glucose and Intralipid produced low mRNA-PPARalpha and -LPL levels as well as LPL activity. Under these latter conditions, plasma insulin and FFA levels were high in those rats receiving the combination of glucose and Intralipid, whereas plasma FFA levels were low in those force-fed with glucose. It is proposed that the hormonal and nutritional induction of liver PPAR-alpha expression around birth and its maintained elevated level throughout suckling is responsible for the induction of liver LPL-expression and activity during suckling.     

Insulin receptors in developing rat liver: Acquisition of down regulation in the immediate postnatal period

Alterations in the high and low affinity insulin receptor concentrations in developing rat liver were investigated. The number of high affinity receptors in partially purified plasma membranes from fetal rats increased from Days 19 through 22 of gestation, with no further increase in binding during the postnatal period. Fetuses of diabetic rats had approximately three times as many high affinity insulin receptors as age-matched fetuses of normal rats; however, by 1 day after birth the receptor number decreased to the normal level. Neither the number of low affinity receptors nor the affinity of insulin binding to high or low affinity receptors changed during development or between offspring of normal and diabetic rats. These changes in the number of high affinity hepatic insulin receptors from prenatal animals did not correlate with the concentration of plasma insulin. When suckling pups were rendered diabetic the changes in the number of high affinity insulin receptors correlated with alterations in plasma insulin concentrations. The number of high affinity sites/microgram DNA in hepatocytes from Day 18 fetal rats was not altered when cells were cultured for 48 h in medium containing 0, 250, or 5000 μU/ml of added insulin. When cultured hepatocytes derived from 1-day-old and adult rats were maintained in medium with added insulin concentrations of 250 or 5000 μU/ml the number of high affinity receptors/microgram DNA decreased as compared to the number of high affinity receptors in hepatocytes cultured in medium with no added insulin. This decrease in receptor number was accompanied by an increase in the affinity of insulin binding to its high affinity receptors. The data show that (i) only the high affinity insulin receptor number increases in rat liver during the prenatal period, (ii) fetuses of diabetic rats show a greater increase in high affinity receptors than do fetuses of normal animals, and (iii) the phenomenon of down regulation for high affinity insulin receptors is not observed in fetal rat liver, but is acquired in the immediate postnatal period.     

Changes in parameters of lipoprotein metabolism during rat hepatic development

Maintenance of whole body cholesterol homeostasis is determined in part by the liver. Thus, changes in expression of hepatic parameters important in the regulation of cholesterol metabolism may play key roles in determining how homeostasis is maintained. The expression of hepatic lipoprotein uptake systems was studied during development using as a ligand very-low density lipoproteins rich in apolipoprotein E that had been obtained from hypercholesterolemic adult rats. These lipoproteins can serve as ligands for cell surface receptors recognizing apolipoproteins B and/or E. Uptake was lowest in freshly isolated fetal rat hepatocytes, increased substantially in hepatocytes from neonates and was intermediate in those from adults. Binding of these lipoproteins to liver membranes prepared from fetal, neonatal, suckling, weaned and adult rats was lowest in fetal preparations, while those from suckling, weaned and adult livers behaved similarly. Numbers of binding sites in neonatal liver membranes were similar to those in adult, but showed a different affinity. On the basis of this data, the ability of hepatocytes to recognize and remove apolipoprotein B/E-containing lipoproteins from the plasma appears to be a function of the differential expression or regulation of lipoprotein-uptake systems during development.     

Apolipoprotein-E-gene expression in rat liver during development in relation to insulin and glucagon

An apolipoprotein-E (apo-E) cDNA probe, cloned by immunoscreening of a lambda GT11 rat liver cDNA library, was used to further characterize the expression of the apo-E gene in rat liver during development, in relation to plasma insulin and glucagon levels. The apo-E mRNA level was low in fetus liver, then abruptly increased at birth and rose further during the suckling period. It returned to the level at birth in 10-week-old adults. These variations were paralleled with dramatic changes in plasma glucagon, which rose at birth and remained high during suckling. At the same time, the insulin/glucagon molar ratio fell. Administration of N6,O2-dibutyryl cAMP to 5-day-old rats resulted in a significant induction of liver apo-E mRNA. Moreover, liver apo-E mRNA rose in 10-h-fasted suckling rats as compared to controls, while plasma glucagon increased and the insulin/glucagon ratio decreased. Conversely, glucose feeding of suckling rats did not induce any increase in liver apo-E mRNA, the insulin/glucagon ratio was 10-fold higher than in fasted animals. Our results are consistent with liver apo-E gene expression being under the control of plasma glucagon and of the glucagon/insulin balance.     

Traditional Chinese Medicine improves dysfunction of peroxisome proliferator-activated receptor alpha and microsomal triglyceride transfer protein on abnormalities in lipid metabolism in ethanol-fed rats

We report the effects of Traditional Chinese Medicine (TCM) on alcohol-induced fatty liver in rats. TCM consists of Astragalus membranaceus, Morus alba, Crataegus pinnatifida, Alisma oriental, Salvia miltiorrhiza and Pueraria lobata. The rats were separated randomly into five groups; the CD group (n=10), which was fed a control diet for 10 weeks, the ED group (n=10), which was fed an isocaloric liquid diet containing ethanol for 10 weeks and given daily oral doses of TCM (0.222 g/kg/day; TCM222, 0.667 g/kg/day; TCM667, and 2.000 g/kg/day; TCM2000, n=10, respectively) over the last four weeks of the study. The ED group developed fatty livers, as determined by their lipid profiles and liver histological findings. Compared with the control group, liver/body weight, plasma triglyceride (TG) and total cholesterol (TC), liver TG and TC, plasma alanine aminotransferase (ALT) and aspartic aminotransferase (AST) significantly increased in the ED group. Also, free fatty acids (FFA) levels increased in both plasma and liver during the administration of ethanol. On the other hand, when rats were administrated with TCM, their liver/body weight, plasma TG, TC and FFA, liver TG, TC and FFA, plasma ALT and AST decreased significantly and the degree of hepatic lipid droplets was markedly improved compared with those in the ED group. Proper function of the peroxisome proliferator-activated receptor alpha (PPARalpha) is essential for the regulation of hepatic fatty acid metabolism. Microsomal triglyceride transfer protein (MTP) is essential for the secretion of triglycerides from the liver. mRNAs for PPARalpha and MTP were reduced in the livers of ethanol-fed rats. TCM restored the mRNA levels of PPARalpha and MTP, and prevented development of fatty livers in ethanol-fed rats. Impairment of PPARalpha and MTP function during ethanol consumption contributes to the development of alcohol-induced fatty liver, which can be overcome by TCM.     

Peroxisome proliferator-activated receptor-alpha expression in rat liver during postnatal development

The expression of the peroxisome proliferator-activated receptor-alpha (PPARalpha) as well as of some related genes was studied in rat liver at different stages of development (from 19-day-old fetuses to 1 month-old rats). The level of PPARalpha mRNA appeared higher in neonates than in fetuses or 1 month-old rats. Whereas the pattern for phosphoenolpyruvate carboxykinase (PEPCK) mRNA level was similar to that of PPARalpha, the mRNA level of both acyl-CoA oxidase (ACO) and apolipoprotein CIII (apo CIII) showed diverse profiles. Western blotting analysis also revealed an increased level of PPARalpha protein in liver of suckling rats. Similarities of mRNA PEPCK and PPARalpha expression indicate a common control mechanism, where both nutritional and hormonal factors may be involved.     

PPARalpha /gamma ragaglitazar eliminates fatty liver and enhances insulin action in fat-fed rats in the absence of hepatomegaly

Peroxisome proliferator-activated receptor (PPAR)alpha and PPARgamma agonists lower lipid accumulation in muscle and liver by different mechanisms. We investigated whether benefits could be achieved on insulin sensitivity and lipid metabolism by the dual PPARalpha/gamma agonist ragaglitazar in high fat-fed rats. Ragaglitazar completely eliminated high-fat feeding-induced liver triglyceride accumulation and visceral adiposity, like the PPARalpha agonist Wy-14643 but without causing hepatomegaly. In contrast, the PPARgamma agonist rosiglitazone only slightly lessened liver triglyceride without affecting visceral adiposity. Compared with rosiglitazone or Wy-14643, ragaglitazar showed a much greater effect (79%, P < 0.05) to enhance insulin's suppression of hepatic glucose output. Whereas all three PPAR agonists lowered plasma triglyceride levels and lessened muscle long-chain acyl-CoAs, ragaglitazar and rosiglitazone had greater insulin-sensitizing action in muscle than Wy-14643, associated with a threefold increase in plasma adiponectin levels. There was a significant correlation of lipid content and insulin action in liver and particularly muscle with adiponectin levels (P < 0.01). We conclude that the PPARalpha/gamma agonist ragaglitazar has a therapeutic potential for insulin-resistant states as a PPARgamma ligand, with possible involvement of adiponectin. Additionally, it can counteract fatty liver, hepatic insulin resistance, and visceral adiposity generally associated with PPARalpha activation, but without hepatomegaly.     

Roles of plasma interleukin-6 and tumor necrosis factor-alpha and FFA and TG in the development of insulin resistance induced by high-fat diet

The role of adipokines in development of insulin resistance still remains controversial. The purpose of the present study was to examine the dynamic changes of fasting plasma levels of interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha), free fatty acids (FFA) and insulin in a Sprague-Dawley rat insulin resistant model induced by high-fat diet. Heterotopic deposition of triglycerides (TG) in liver, skeletal muscles and pancreatic islet was also investigated. The fasting plasma level of insulin in rats in the high-fat diet group was significantly higher than that in the normal diet group on day 21 (P<0.01), suggesting that an increased insulin resistance developed in the high-fat diet group. However, no significant difference in the plasma IL-6 level was observed between the two groups (P>0.05), although in both groups, the plasma IL-6 level was significantly higher on day 21 than that of the day 0 (P<0.05). The plasma FFA level in the high-fat diet group began to increase significantly on day 21 (P<0.05), and elevated markedly on day 28, was positively correlated to the fasting plasma insulin level. Histological study revealed a more abundant TG deposition in liver and skeletal muscles (from quadriceps femoris) in the high-fat diet group than in the normal diet group on day 21, and the liver deposition was even higher on day 28. However, no deposition was observed in pancreatic islets. The plasma TNF-alpha level remained unchanged throughout the duration of the experiment. These results indicate that the progression of insulin resistance in high-fat diet rats is closely related to the plasma FFA elevation and the heterotopic deposition of TG in liver and skeletal muscles, but is unrelated to the plasma TNF-alpha and IL-6 levels.     

Beyond lipids, pharmacological PPARalpha activation has important effects on amino acid metabolism as studied in the rat

PPARalpha agonists have been characterized largely in terms of their effects on lipids and glucose metabolism, whereas little has been reported about effects on amino acid metabolism. We studied responses to the PPARalpha agonist WY 14,643 (30 micromol x kg(-1) x day(-1) for 4 wk) in rats fed a saturated fat diet. Plasma and urine were analyzed with proton NMR. Plasma amino acids were measured using HPLC, and hepatic gene expression was assessed with DNA arrays. The high-fat diet elevated plasma levels of insulin and triglycerides (TG), and WY 14,643 treatment ameliorated this insulin resistance and dyslipidemia, lowering plasma insulin and TG levels. In addition, treatment decreased body weight gain, without altering cumulative food intake, and increased liver mass. WY 14,643 increased plasma levels of 12 of 22 amino acids, including glucogenic and some ketogenic amino acids, whereas arginine was significantly decreased. There was no alteration in branched-chain amino acid levels. Compared with the fat-fed control animals, WY 14,643-treated animals had raised plasma urea and ammonia levels as well as raised urine levels of N-methylnicotinamide and dimethylglycine. WY 14,643 induced changes in a number of key genes involved in amino acid metabolism in addition to expected effects on hepatic genes involved in lipid catabolism and ketone body formation. In conclusion, the present results suggest that, in rodents, effects of pharmacological PPARalpha activation extend beyond control of lipid metabolism to include important effects on whole body amino acid mobilization and hepatic amino acid metabolism.     

Effect of neonatal hypoxia on the development of hepatic lipase in the rat

Increases in plasma lipids occur during hypoxia in suckling but not in weaned rats and may result from altered hepatic enzyme activity. We exposed rats to 7 days of hypoxia from birth to 7 days of age (suckling) or from 28 to 35 days of age (weaned at day 21). Hypoxia led to an increase in hepatic lipid content in the suckling rat only. Hepatic lipase was decreased to approximately 45% of control in 7-day-old rats exposed to hypoxia but not in hypoxic 35-day-old rats. Hypoxic suckling rats also had a 50% reduction in lactate dehydrogenase activity, whereas transaminase activity and CYP1A and CYP3A protein content were not different between hypoxic and normoxic groups. Additional rats were studied 7 and 14 days after recovery from hypoxic exposure from birth to 7 days of age; hepatic lipase activity had recovered to 85% by 7 days and to 100% by 14 days in the rats previously exposed to hypoxia. Administration of dexamethasone to neonatal rats to simulate the hyperglucocorticoid state found in hypoxic 7-day-old rats led to a moderate decrease ( approximately 75% of control) in hepatic lipases. Developmentally, in the normoxic state, hepatic lipases increased rapidly after birth and reached levels more than twofold that of the newborn by 7 days of age. Hypoxia delays the maturation of hepatic lipases. We suggest that the decrease in hepatic lipase activity contributes to hyperlipemia in the hypoxic newborn rats.     

Impact of α-lipoic acid on liver peroxisome proliferator-activated receptor-α, vascular remodeling, and oxidative stress in insulin-resistant rats

This study sought to determine the impact of α-lipoic acid (LA) on superoxide anion (O(2)(?-)) production and peroxisome proliferator-activated receptor-α (PPARα) expression in liver tissue, plasma free fatty acids (FFA), and aortic remodeling in a rat model of insulin resistance. Sprague-Dawley rats (50-75 g) were given either tap water or a drinking solution containing 10% D-glucose for 14 weeks, combined with a diet with or without LA supplement. O(2)(?-) production was measured by lucigenin chemiluminescence, and PPAR-α expression by Western blotting. Cross-sectional area (CSA) of the aortic media and lumen and number of smooth muscle cells (SMC) were determined histologically. Glucose increased systolic blood pressure (SBP), plasma levels of glucose and insulin, and insulin resistance (HOMA index). All of these effects were attenuated by LA. Whereas glucose had no effect on liver PPAR-α protein level, it decreased plasma FFA. LA decreased the aortic and liver O(2)(?-) production, body weight, and plasma FFA levels in control and glucose-treated rats. Liver PPAR-α protein levels were increased by LA, and negatively correlated with plasma FFA. Medial CSA was reduced in all glucose-treated rats, and positively correlated with plasma FFA but not with SBP or aortic O(2)(?-) production. Glucose also reduced aortic lumen area, so that the media-to-lumen ratio remained unchanged. The ability of LA to lower plasma FFA appears to be mediated, in part, by increased hepatic PPAR-α expression, which may positively affect insulin resistance. Glucose-fed rats may serve as a unique model of aortic atrophic remodeling in hypertension and early metabolic syndrome.     

Variable effect of ghrelin administration on pancreatic development in young rats. Role of insulin-like growth factor-1.

Ghrelin, an endogenous ligand of the growth hormone secretagogue receptor, has been primarily isolated from the human and rat stomach. Ghrelin has been shown to stimulate appetite and fat deposition in adult rats and humans. The aim of this study was to investigate the effect of ghrelin administration on pancreatic growth in suckling, weaned and peripubertal seven week old rats. Rats were treated with saline or ghrelin (4, 8 or 16 nmol/kg/dose) intraperitoneally twice a day: suckling rats were treated for 7 or 14 days starting from the first postnatal day, three week old weaned rats and seven weeks old rats were treated for 5 days. Treatment with ghrelin did not affect animal weight in suckling or weaned rats, whereas in young seven week old rats, ghrelin caused a significant increase in body weight. Ghrelin decreased food intake in weaned rats; whereas in seven week old rats, food intake was enhanced. In suckling rats, ghrelin decreased the pancreatic weight, pancreatic amylase content, DNA synthesis and DNA content. In contrast, ghrelin increased pancreatic weight, DNA synthesis, DNA content and amylase content in weaned or young seven week old rats. Pancreatic blood flow was not affected by ghrelin in any group of rats tested. Ghrelin increased serum level of growth hormone in all rats. This effect was weak in suckling rats, higher in weaned and the highest in seven week old animals. Ghrelin did not affect serum level of insulin-like growth factor-1 (IGF-1) in suckling rats. In weaned and in seven week old rats, treatment with ghrelin caused increase in serum level of IGF-1. We conclude that ghrelin reduces pancreatic growth in suckling rats; whereas in weaned and young seven week old animals, treatment with ghrelin increases pancreatic growth. This biphasic effect of ghrelin in young animals on pancreatic growth seems to be related to age-dependent changes of the release of anabolic IGF-1.     

高脂饮食和地塞米松联合诱导胰岛素抵抗大鼠模型

目的用高脂饲料＋地塞米松（dexamethasone,DEX）隔日腹腔注射建立实验性胰岛素抵抗大鼠模型,研究该模型糖代谢、脂代谢和激素水平等方面的变化。方法采用Wistar雄性大鼠,分为正常对照组、高脂组、DEX组（1mg/kg,i.p.）和高脂＋DEX组（1mg/kg,i.p.）,连续观察8周,每周测定大鼠空腹血糖,分别于造模第2周和第8周测糖耐量,8周后处死大鼠,测定胸腺、脾脏、肝脏等脏器重量。结果高脂饲料能加重腹腔注射DEX造成的空腹血糖升高,造模第8周空腹血糖（7.7±0.7）较空白组（6.5±0.6）显著升高。使模型动物糖耐量明显异常,肝糖原、肌糖原含量显著增加,血浆胰岛素及游离脂肪酸水平显著升高,各脏器指数明显增加。结论高脂＋DEX隔日腹腔注射能成功诱导胰岛素抵抗大鼠模型,这种造模方法较单纯注射DEX或单纯高脂饲养成模率高,造模周期短。     

Weaning and metabolic regulation in the rat

Premature weaning of rats to high carbohydrate diets causes a variety of short- and long-term changes in lipid metabolism, but the mechanisms involved are unclear. It is likely that interaction of diet with certain emerging hormonal control patterns during weaning might condition metabolic control and (or) subsequent adaptations in the adult organism. This implies that the adaptive responses of infant animals to diet may differ from those of the mature organism. For example, premature weaning leads to early appearance of rat liver malic enzyme (ME), even when fat supplies as much as 65% of the dietary energy; the same diet suppresses ME activity in 45-day-old rats. The levels of plasma glucagon and thyroid hormones are elevated during the weaning period. Several studies have shown that triiodothyronine evokes hepatic ME in suckling rats. Conversely, glucagon infusion into prematurely weaned rats suppresses the early appearance of the enzyme. Premature weaning, regardless of fat intake, leads to a rapid decline in plasma glucagon levels. Since glucagon is known to antagonize the actions of triiodothyronine on liver ME, the interaction of diet with glucagon and thyroid hormones is conceivably part of the mechanism responsible for the early appearance of hepatic malic enzyme, whereby the decline of plasma glucagon permits triiodothyronine to act on liver ME. Insulin probably exerts a permissive action subsequently. The manner in which these events relate to the long-term consequences of premature weaning is unknown.     

Development of insulin sensitivity in white adipose tissue during the suckling-weaning transition in the rat. Involvement of glucose transport and lipogenesis.

The changes of insulin responsiveness of white adipose tissue during the suckling-weaning transition in the rat were investigated in vitro on isolated adipocytes. Insulin binding, glucose transport and glucose metabolism in adipocytes from suckling rats and from rats weaned on to a high-carbohydrate (HC) or a high-fat (HF) diet were compared. Despite similar insulin binding, insulin-stimulated glucose transport rate is lower in adipocytes from suckling rats and HF-weaned rats than in adipocytes from HC-weaned rats. Moreover, whereas insulin markedly stimulates glucose metabolism in adipocytes from HC-weaned rats, glucose metabolism is totally unresponsive to insulin in adipocytes from suckling and HF-weaned rats. This insulin resistance is associated with a very low rate of lipogenesis and low activities of acetyl-CoA carboxylase, fatty acid synthase and pyruvate dehydrogenase.     

Maternal chitosan oligosaccharide supplementation affecting expression of circadian clock genes,and possible association with hepatic cholesterol accumulation in suckling piglets

Chitosan oligosaccharide (COS) has been shown to reduce lipid accumulation in liver in mice and rats. The purpose of this study was to investigate whether maternal COS feeding affects hepatic lipid metabolism via influencing the expression of circadian clock genes in piglets. From day (d) 85 of gestation to d 14 of lactation, sixteen pregnant sows were divided into a control group (basal diet without COS supplementation) and a COS group (30 mg COS/kg basal diet). After farrowing, one piglet per litter in each group was selected for the collection of plasma and liver samples on d 0 and d 14 of age, respectively. Interestingly and significantly, we found that maternal COS supplementation promoted plasma and hepatic cholesterol accumulation and up-regulated the mRNA level of negative-regulated element period 1 (Per1), and reduced the abundance of the positive elements, circadian locomotor output cycles kaput (CLOCK), and brain muscle Arnt-like 1 (BMAL1) in the suckling piglets on d 14. These alterations may promote the hepatic cholesterol accumulation, which, in turn, activates hepatic bile acid metabolism and attenuates the relative expression levels of lipid metabolism-associated genes in the liver. However, the expression of CLOCK and BMAL1 and the lipid profile in the plasma and liver were not affected by COS supplementation on d 0. Collectively, our results indicate that maternal supplementation with COS postpartum up-regulates cholesterol accumulation in suckling piglets at age d 14, in part, by the regulation of circadian clock genes.