The effects of a high-fat or high-sucrose diet on serum lipid profiles,hepatic acyl-CoA synthetase,carnitine palmitoyltransferase-I,and the acetyl-CoA carboxylase mRNA levels in rats

The purpose of this study was to investigate the effects of altering relative intakes of fat and carbohydrates on serum lipid profiles, hepatic acyl-CoA synthetase (ACS), carnitine palmitoyltransferase-I (CPT-I), and the acetyl-CoA carboxlyase (ACC) mRNA level in Sprague-Dawley rats. For four weeks the rats were fed either an AIN-76 diet or one of its modified diets that were supplemented with 20% beef tallow (high-fat diet, HF) and 66.3% sucrose (high-sucrose diet, HS). The HS group had significantly higher serum triglyceride and total cholesterol concentrations when compared with the other groups. Serum LDL-cholesterol concentrations in the HS and HF groups were significantly higher when compared to the normal diet (ND) group. Serum HDL-cholesterol levels of the ND and HS groups were significantly higher than those of the HF group. The hepatic total lipid level of the HF group was significantly higher than those of other groups; triglyceride levels of the HS and HF groups were significantly higher than those of the ND group. Hepatic ACS mRNA levels of the HF group were significantly higher than those of the ND group. Hepatic CPT-I mRNA levels were higher in the HF group than other groups. Also, ACC mRNA levels in the liver increased in the HF group. In conclusion, changes in the composition of dietary fat and carbohydrates could affect the hepatic ACS, CPT-I, and ACC mRNA levels. These results facilitate our understanding of the coordinated regulation of the ACS, CPT-I, and ACC mRNA levels and will serve to enhance our understanding of the molecular mechanisms that underlie the regulation of fatty acid metabolism.     

Effects of fasting and re-feeding on the expression of Dec1, Per1, and other clock-related genes

To elucidate the food-entrainable oscillatory mechanism of peripheral clock systems, we examined the effect of fasting on circadian expression of clock genes including Dec1 and Dec2 in mice. Withholding of food for 2 days had these effects: the expression level of Dec1 mRNA decreased in all tissues examined, although Per1 mRNA level markedly increased; Per2 expression was reduced in the liver and heart only 42-46 h after the start of fasting; and expression profiles of Dec2 and Bmal1 were altered only in the heart and in the liver, respectively, whereas Rev-erbalpha mRNA levels did not change significantly. Re-feeding after 36-h starvation erased, at least in part, the effect of fasting on Dec1, Dec2, Per1, Per2, and Bmal1 within several hours, and restriction feeding shifted the phase of expression profiles of all examined clock genes including Dec1 and Dec2. These findings indicate that short-term fasting and re-feeding modulate the circadian rhythms of clock genes to different extents in peripheral tissues, and suggest that the expression of Dec1, Per1, and some other clock genes was closely linked with the metabolic activity of these tissues.     

Oxidation of hepatic carnitine palmitoyl transferase-I (CPT-I) impairs fatty acid beta-oxidation in rats fed a methionine-choline deficient diet

There is growing evidence that mitochondrial dysfunction, and more specifically fatty acid β-oxidation impairment, is involved in the pathophysiology of non-alcoholic steatohepatitis (NASH). The goal of the present study was to achieve more understanding on the modification/s of carnitinepalmitoyltransferase-I (CPT-I), the rate-limiting enzyme of the mitochondrial fatty acid β-oxidation, during steatohepatitis. A high fat/methionine-choline deficient (MCD) diet, administered for 4 weeks, was used to induce NASH in rats.We demonstrated that CPT-I activity decreased, to the same extent, both in isolated liver mitochondria and in digitonin-permeabilized hepatocytes from MCD-diet fed rats.At the same time, the rate of total fatty acid oxidation to CO(2) and ketone bodies, measured in isolated hepatocytes, was significantly lowered in treated animals when compared to controls. Finally, an increase in CPT-I mRNA abundance and protein content, together with a high level of CPT-I protein oxidation was observed in treated rats. A posttranslational modification of rat CPT-I during steatohepatitis has been here discussed.     

Regulation of phosphoenolpyruvate carboxykinase mRNA in mouse liver, kidney, and fat tissues by fasting, diabetes, and insulin.

Previous investigations of the phosphoenolpyruvate carboxykinase (PEPCK) gene have been conducted using rats. In a recent comparative study, we investigated, for the first time, the effects of fasting, refeeding, alloxan-induced diabetes, and insulin treatment on the levels of PEPCK mRNA in mouse liver, kidney, and adipose tissues. As in rats, fasting and diabetes induced, while insulin repressed, hepatic PEPCK mRNA. In contrast, the response of renal PEPCK mRNA to fasting, refeeding, and diabetes in mice differed quantitatively with that in rats: fasting caused a twofold increase in mice and a fourfold increase in rats. Moreover, diabetes, which induces renal PEPCK mRNA indirectly by causing acidosis in rats, was without effect in mice. In adipose tissue, the results of previous studies in both rats and mice have shown that the amount of PEPCK protein and its rate of synthesis are increased by fasting and diabetes and decreased by refeeding and insulin treatment. Thus, it was surprising to find that fasting, refeeding, alloxan-induced diabetes, and insulin treatment had no effect on adipose tissue PEPCK mRNA in either rats or mice.     

Fatty acid oxidation and related gene expression in heart depleted of carnitine by mildronate treatment in the rat

The metabolic and genic effects induced by a 20-fold lowering of carnitine content in the heart were studied in mildronate-treated rats. In the perfused heart, the proportion of palmitate taken up then oxidized was 5-10% lower, while the triacylglycerol (TAG) formation was 100% greater than in controls. The treatment was shown to increase the maximal capacity of heart homogenates to oxidize palmitate, the mRNA level of carnitine palmitoyltransferase I (CPT-I) isoforms, the specific activity of CPT-I in subsarcolemmal mitochondria and the total carnitine content of isolated mitochondria. Concomitantly, the increased mRNA expression of lipoprotein lipase, fatty acid translocase and enzymes of TAG synthesis was associated with a 5- and 2-times increase in serum TAG and free fatty acid contents, respectively. The compartmentation of carnitine at its main functional location was expected to allow the increased CPT-I activity to ensure in vivo correct fatty acid oxidation rates. All the inductions related to fatty acid transport, oxidation and esterification most likely stem from the abundance of blood lipids providing cardiomyocytes with more fatty acids.     

Effect of Cheonggukjang supplementation upon hepatic acyl-CoA synthase, carnitine palmitoyltransferase I, acyl-CoA oxidase and uncoupling protein 2 mRNA levels in C57BL/6J mice fed with high fat diet

This study investigated the effect of Cheonggukjang on mRNA levels of hepatic acyl-CoA synthase (ACS), carnitine palmitoyltransferase I (CPT-I), acyl-CoA oxidase (ACO) and uncoupling protein 2 (UCP2), and on serum lipid profiles in C57BL/6J mice. Thirty male C57BL/6J mice were divided into three groups; normal diet (ND), high fat diet (HD) and high fat diet with 40% Cheonggukjang (HDC). Energy intake was significantly higher in the HDC group than in the ND and HD groups. The HDC group normalized in weight gain, epididymal and back fat (g/100 g) accumulation which are increased by high fat diet. Serum concentrations of triglyceride and total cholesterol in the HDC were significantly lower than those in the HD group. These results were confirmed by hepatic mRNA expression of enzymes and protein (ACS, CPT-1, ACO, UCP2) which is related with lipid metabolism by RT-PCR. Hepatic CPT-I, ACO and UCP2 mRNA expression was increased by Cheonggukjang supplementation. We demonstrated that Cheonggukjang supplement leads to increased mRNA expressions of enzymes and protein involved in fatty acid oxidation in liver, reduced accumulation of body fat and improvement of serum lipids in high fat diet fed mice.     

Expression of hepatic calcium-binding protein regucalcin mRNA is elevated by refeeding of fasted rats: Involvement of glucose,insulin and calcium as stimulating factors

The effect of refeeding on the expression of Ca²⁺-binding protein regucalcin mRNA in the liver of fasted rats was investigated. When rats were fasted overnight, the hepatic regucalcin mRNA level was reduced about 70% of that in feeding rats. Refeeding produced a remarkable elevation of hepatic regucalcin mRNA level (about 150–170% of fasted rats). Liver regucalcin concentration was appreciably increased by refeeding, although it was not altered by fasting. The oral administration of glucose (2 g/kg body weight) to fasted rats caused a significant increase in hepatic regucalcin mRNA level. Moreover, hepatic regucalcin mRNA level was clearly elevated by a single subcutaneous administration of insulin (10 and 100 U/kg) to fasted rats. The hormonal effect was not further enhanced by the simultaneous administration of calcium chloride (250 mg Ca/kg) to fasted rats, although calcium administration stimulated regucalcin mRNA expression in the liver. The present study suggests that the expression of hepatic regucalcin mRNA stimulated by refeeding is significantly involved in the action of insulin and/or calcium as stimulating factors.     

Fasting increases gene expressions of uncoupling proteins and peroxisome proliferator-activated receptor-gamma in brown adipose tissue of ventromedial hypothalamus-lesioned rats

Uncoupling proteins (UCPs) are supposed to be involved in diet-induced thermogenesis. Their activities are usually elevated by feeding and reduced by fasting in normal animals. To investigate whether fasting affects the expression of UCPs mRNA in brown adipose tissue (BAT) of bilateral ventromedial hypothalamus (VMH)-lesioned rats, we determined the gene expression of UCP1, UCP2 or UCP3 in BAT of VMH-lesioned rats and examined oxygen consumption in these rats under fed or 48-h fasted conditions. Northern blotting revealed no difference in the expression of UCPs mRNA in BAT between VMH-lesioned and sham-operated rats under the fed condition, however, expressions were increased markedly in BAT of VMH-lesioned rats under the fasted condition. Under the fed condition, no difference in oxygen consumption was observed between VMH-lesioned and sham-operated rats. Under the fasted condition, oxygen consumption decreased in both rats, however, it decreased in VMH-lesioned less than in sham operated rats. To explore the mechanism that fasting elevated BAT UCPs mRNA in VMH-lesioned rats, we measured peroxisome proliferator-activated receptor (PPAR)-gamma mRNA and protein in BAT, because PPAR-gamma agonist can elevate UCPs mRNA levels in BAT. Under the fed condition, no differences in the expression of PPAR-gamma mRNA and protein content were observed between in BAT of VMH-lesioned and sham-operated rats. Under the fasted condition, however, both increased in BAT of VMH-lesioned rats. These results suggest that VMH-lesions enhance the gene expression of UCPs in BAT under long-term fasting as a defensive reaction to inhibit the reduction of body temperature through an increase in PPAR-gamma activity.     

Adipose triglyceride lipase expression and fasting regulation are differently affected by cold exposure in adipose tissues of lean and obese Zucker rats

Adipose triglyceride lipase (ATGL) hydrolyzes triacylglycerols to diacylglycerols in the first step of lipolysis, providing substrates for hormone-sensitive lipase (HSL). Here we studied whether ATGL messenger RNA (mRNA) and protein levels were affected by 24-h cold exposure in different white adipose tissue depots and in interscapular brown adipose tissue of lean and obese Zucker rats submitted to feeding and 14-h fasting conditions. HSL mRNA expression was also studied in selected depots. In both lean and obese rats, as a general trend, cold exposure increased ATGL mRNA and protein levels in the different adipose depots, except in the brown adipose tissue of lean animals, where a decrease was observed. In lean rats, cold exposure strongly improved fasting up-regulation of ATGL expression in all the adipose depots. Moreover, in response to fasting, in cold-exposed lean rats, there was a stronger positive correlation between circulating nonesterified fatty acids (NEFA) and ATGL mRNA levels in the adipose depots and a higher percentage increase of circulating NEFA in comparison with control animals not exposed to cold. In obese rats, fasting-induced up-regulation of ATGL was impaired and was not improved by cold. The effects of obesity and cold exposure on HSL mRNA expression were similar to those observed for ATGL, suggesting common regulatory mechanisms for both proteins. Thus, cold exposure increases ATGL expression and improves its fasting-up-regulation in adipose tissue of lean rats. In obese rats, cold exposure also increases ATGL expression but fails to improve its regulation by fasting, which could contribute to the increased difficulty for mobilizing lipids in these animals.     

Expression of neuropeptide W in rat stomach mucosa: regulation by nutritional status, glucocorticoids and thyroid hormones

Neuropeptide W (NPW) is a recently identified neuropeptide that binds to G-protein-coupled receptor 7 (GPR7) and 8 (GPR8). In rodent brain, NPW mRNA is confined to specific nuclei in hypothalamus, midbrain and brainstem. Expression of NPW mRNA has also been confirmed in peripheral organs such as stomach. Several reports suggested that brain NPW is implicated in the regulation of energy and hormonal homeostasis, namely the adrenal and thyroid axes; however the precise physiological role and regulation of peripheral NPW remains unclear. In this study, we examined the effects of nutritional status on the regulation of NPW in stomach mucosa. Our results show that in this tissue, NPW mRNA and protein expression is negatively regulated by fasting and food restriction, in all the models we studied: males, females and pregnant females. Next, we examined the effect of glucocorticoids and thyroid hormones on NPW mRNA expression in the stomach mucosa. Our data showed that NPW expression is decreased in this tissue after glucocorticoid treatment or hyperthyroidism. Conversely, hypothyroidism induces a marked increase in the expression of NPW in rat stomach. Overall, these data indicate that stomach NPW is regulated by nutritional and hormonal status.     

Peroxisomal proliferator-activated receptor-gamma coactivator-1 alpha (PGC-1 alpha) enhances the thyroid hormone induction of carnitine palmitoyltransferase I (CPT-I alpha)

Carnitine palmitoyltransferase I (CPT-I) catalyzes the rate-controlling step in the pathway of mitochondrial fatty acid oxidation. Thyroid hormone will stimulate the expression of the liver isoform of CPT-I (CPT-I alpha). This induction of CPT-I alpha gene expression requires the thyroid hormone response element in the promoter and sequences within the first intron. The peroxisomal proliferator-activated receptor-gamma coactivator-1 alpha (PGC-1 alpha) is a coactivator that promotes mitochondrial biogenesis, mitochondrial fatty acid oxidation, and hepatic gluconeogenesis. In addition, PGC-1 alpha will stimulate the expression of CPT-I alpha in primary rat hepatocytes. Here we report that thyroid hormone will increase PGC-1 alpha mRNA and protein levels in rat hepatocytes. In addition, overexpression of PGC-1 alpha will enhance the thyroid hormone induction of CPT-I alpha indicating that PGC-1 alpha is a coactivator for thyroid hormone. By using chromatin immunoprecipitation assays, we show that PGC-1 alpha is associated with both the thyroid hormone response element in the CPT-I alpha gene promoter and the first intron of the CPT-I alpha gene. Our data demonstrate that PGC-1 alpha participates in the stimulation of CPT-I alpha gene expression by thyroid hormone and suggest that PGC-1 alpha is a coactivator for thyroid hormone.     

禁食及胰岛素水平对大鼠解偶联蛋白-1、2、3基因表达的影响

 为探讨禁食和胰岛素对解偶联蛋白 - 1、2、3基因 (UCP1 ,2 ,3)表达的影响 ,应用 RT- PCR方法观察了在不同禁食时间和应用胰岛素条件下大鼠白色脂肪组织、棕色脂肪组织和骨骼肌中 UCP1 ,2 ,3m RNA水平的变化 .UCP1基因只在大鼠棕色脂肪组织中表达 .UCP2 ,3基因在三种组织中均有表达 ,在白色脂肪组织中以 UCP2表达为主 ;在骨骼肌中以 UCP3表达为主 .过夜禁食使棕色脂肪组织 UCP1 ,3m RNA水平明显下降 (P<0 .0 1 ) ;UCP2 m RNA水平在三种组织中均呈上升反应 ,以白色脂肪组织中表现最为明显 (P<0 .0 5) ;而对白色脂肪组织和骨骼肌中 UCP3基因表达无明显影响 .禁食时间延长至 48h,除棕色脂肪组织中 UCP2 ,3基因有明显下降外 ,各组织中UCPs基因表达基本调节至正常或高于对照组水平 .胰岛素对 UCPs基因表达水平有一定的上调作用 ,这一作用对棕色脂肪组织 UCPs各基因及骨骼肌中 UCP3基因表现得尤为明显 (P<0 .0 5) .大鼠 UCPs基因表达有一定的组织特异性 ;禁食时间对三种组织中 UCPs各成员基因表达的影响有时相上的区别 ;胰岛素可以调 UCPs各成员基因的表达 .结果反映了 UCPs各成员在能量代谢调节上的不同作用 ,这为理解膳食 -产热与体重调节的关系 ,及其能量代谢平衡与疾病关系提供了实验依据     

Radiation attenuates physiological angiogenesis by differential expression of VEGF, Ang-1, tie-2 and Ang-2 in rat brain

The etiology of radiation-induced cerebrovascular rarefaction remains unknown. In the present study, we examined the effect of whole-brain irradiation on endothelial cell (EC) proliferation/apoptosis and expression of various angiogenic factors in rat brain. F344 × BN rats received either whole-brain irradiation (a single dose of 10 Gy γ rays) or sham irradiation and were maintained for 4, 8 and 24 h after irradiation. Double immunofluorescence staining was employed to visualize EC proliferation/apoptosis in brain. The mRNA and protein expression levels of vascular endothelial growth factor (VEGF), angiopoietin-1 (Ang-1), endothelial-specific receptor tyrosine kinase (Tie-2), and Ang-2 in brain were determined by real-time RT-PCR and immunofluorescence staining. A significant reduction in CD31-immunoreactive cells was detected in irradiated rat brains compared with sham-irradiated controls. Whole-brain irradiation significantly suppressed EC proliferation and increased EC apoptosis. In addition, a significant decrease in mRNA and protein expression of VEGF, Ang-1 and Tie-2 was observed in irradiated rat brains. In contrast, whole-brain irradiation significantly upregulated Ang-2 expression in rat brains. The present study provides novel evidence that whole-brain irradiation differentially affects mRNA and protein expression of VEGF, Ang-1, Tie-2 and Ang-2. These changes are closely associated with decreased EC proliferation and increased EC apoptosis in brain.     

Interactions between the consumption of a high-fat diet and fasting in the regulation of fatty acid oxidation enzyme gene expression: an evaluation of potential mechanisms

The consumption of high-fat diets (HFDs) and fasting are known to increase the expression of enzymes involved in fatty acid oxidation (FAO). However, it has been reported that the ability of physiological stressors to induce enzymes of FAO in skeletal muscle is blunted with obesity. In this regard, we sought to explore the effects and potential mechanisms of an HFD on the expression of FAO enzymes in the fed and fasted state. The consumption of an HFD increased the mRNA expression or protein content of medium-chain acyl-CoA dehydrogenase (MCAD), uncoupling protein-3 (UCP3), and pyruvate dehydrogenase kinase 4 (PDK4) in the fed state. Fasting increased the mRNA expression of PDK4, MCAD, and UCP-3, and the protein content of UCP-3 in chow but not HFD rats. HFDs did not increase carnitine palmitoyl transfer-1 (CPT-1) mRNA levels in the fed state and the effects of fasting were markedly reduced compared with chow-fed rats. The expression of peroxisome-proliferator-activated receptor-γ coactivator-1β (PGC-1β) was increased in muscle from HFD rats in the fed state, while PGC-1-related coactivator (PRC) was increased with fasting in chow-fed but not HFD rats. Plasma fatty acid levels were elevated in the fed state from HFD rats but not increased further with fasting, whereas fasting increased plasma fatty acids in chow-fed animals. Fasting-mediated increases in plasma epinephrine, and the activation of PKA and AMPK in skeletal muscle were similar between chow and HFD rats. p38 MAPK phosphorylation was increased with fasting in chow-fed but not HFD rats. Our findings suggest that a blunted effect of fasting on the induction of PDK4, MCAD, and UCP3 in skeletal muscle from HFD rats is likely a result of already elevated levels of these enzymes, the induction of which is associated with increases in plasma fatty acid and PGC-1β. On the other hand, a blunted induction of PRC and CPT-1 mRNA may be explained by decreases in p38 MAPK signaling.     

Impact of high altitude on the hepatic fatty acid oxidation and synthesis in rats

High altitude (HA) affects energy metabolism. The impact of acute and chronic HA acclimatization on the major metabolic pathways is still controversial. In this study, we aimed to unveil the impact of HA on the key enzymes involved in the fatty acid (FA) metabolism in liver. Rats were exposed to an altitude of 4300 m for 30 days and the expressions of two key proteins involved in FA β-oxidation (carnitine palmitoyl transferase I, CPT-I; and peroxisome proliferator-activated receptor alpha, PPARα), two proteins involved in FA synthesis (acetyl CoA carboxylase-1, ACC-1; and AMP-activated protein kinase, AMPK), as well as the total ketone body in the liver and the plasma FFAs were examined. Rats without HA exposure were used as controls. We observed that the acute exposure of rats to HA (3 days) led to a significant increase in the expressions of CPT-I and PPARα and in the total hepatic ketone body. Longer exposure (15 days) caused a marked decrease in the expression of CPT-I and PPARα. By 30 days after HA exposure, the expression levels of CPT-I and PPARα returned to the control level. The hepatic ACC-1 level showed a significant increase in rats exposed to HA for 1 and 3 days. In contrast, the hepatic level of AMPK showed a significant reduction throughout the experimental period. Plasma FFA concentrations did not show any significant changes following HA exposure. Thus, increased hepatic FA oxidation and synthesis in the early phase of HA exposure may be among the important mechanisms for the rats to respond to the hypoxic stress in order to acclimatize themselves to the stressful environments.     

Macronutrient composition of the diet differentially affects leptin and adiponutrin mRNA expression in response to meal feeding

A number of adipose-specific genes, including adiponutrin and the adipocytokines, appear to be involved in regulating overall energy balance, as their expression is dysregulated in various obese states and is responsive to feeding. This study determined the effect of meal-feeding diets of markedly different macronutrient composition (70% by weight protein or fat) on the expression of adiponutrin and several adipocytokines in white adipose tissue of rats. Adiponutrin mRNA rapidly increased by at least 8-fold within 3 hours after the high-protein meal. This response was similar to that seen after a high-sucrose meal (70% by weight of sucrose). In contrast, leptin mRNA was unchanged after the high-protein meal, whereas it increased more than 5-fold after a high-sucrose meal. On the high-protein diet the leptin mRNA did not decline upon fasting after the meal, whereas on the high-sucrose diet fasting brought about a rapid decline in leptin mRNA, suggesting that the composition of the diet had altered mRNA turnover. In rats on diets high in either saturated or polyunsaturated fats, adiponutrin mRNA remained at fasting levels even after the meals. Leptin mRNA was unchanged and was maintained at post-meal levels. Resistin and acrp30/adiponectin mRNAs remained unchanged regardless of the macronutrient composition of the diet. The mechanism by which macronutrient composition of the diet is able to differentially influence the expression of adiponutrin and the adipocytokines, leptin, resistin, and acrp30/adiponectin remains to be determined.     

Blood-Brain Barrier Permeability Is Exacerbated in Experimental Model of Hepatic Encephalopathy via MMP-9 Activation and Downregulation of Tight Junction Proteins

The present study was designed to investigate the mechanisms involved in blood-brain barrier (BBB) permeability in bile duct ligation (BDL) model of chronic hepatic encephalopathy (HE). Four weeks after BDL surgery, a significant increase was observed in serum bilirubin levels. Masson trichrome staining revealed severe hepatic fibrosis in the BDL rats. ^99mTc-mebrofenin retention was increased in the liver of BDL rats suggesting impaired hepatobiliary transport. An increase in permeability to sodium fluorescein, Evans blue, and fluorescein isothiocyanate (FITC)-dextran along with increase in water and electrolyte content was observed in brain regions of BDL rats suggesting disrupted BBB. Increased brain water content can be attributed to increase in aquaporin-4 mRNA and protein expression in BDL rats. Matrix metalloproteinase-9 (MMP-9) mRNA and protein expression was increased in brain regions of BDL rats. Additionally, mRNA and protein expression of tissue inhibitor of matrix metalloproteinases (TIMPs) was also increased in different regions of brain. A significant decrease in mRNA expression and protein levels of tight junction proteins, viz., occludin, claudin-5, and zona occluden-1 (ZO-1) was observed in different brain regions of BDL rats. VCAM-1 mRNA and protein expression was also found to be significantly upregulated in different brain regions of BDL animals. The findings from the study suggest that increased BBB permeability in HE involves activation of MMP-9 and loss of tight junction proteins.