Up-regulation of stearoyl-CoA desaturase 1 and elongase 6 genes expression in rat lipogenic tissues by chronic food restriction and chronic food restriction/refeeding

Successful treatment of obesity and related diseases by chronic food restriction requires the understanding of the effect of such nutritional therapy on the expression of genes which have been implicated to be involved in some diseases associated with obesity. The purpose of this study was to examine the effect of chronic food restriction and chronic food restriction/refeeding on lipogenic enzymes, especially the expression of genes encoding the stearoyl-CoA desaturase 1 (Scd1) and elongase6 (Elovl6) in rat liver and adipose tissue. We found that both chronic food restriction and chronic food restriction/refeeding caused increased expression of the Scd1 and Elovl6 genes in both the liver and adipose tissue. The increase was more pronounced in case of chronic food restriction/refeeding (several-fold increase) than that in chronic food restriction alone (two to threefold increase). Essentially, similar results were obtained when the expression of fatty acid synthase, acetyl-CoA carboxylase, ATP-citrate lyase, and malic enzyme genes was studied. Moreover, we found that chronic food restriction and short-term fasting exert opposite effects on the expression of lipogenic enzymes genes. The increased expression of the genes encoding Scd1, Elovl6, and other key lipogenic enzymes may favor fat storage after chronic food restriction/refeeding and may be part of the molecular mechanism by which food restriction/refeeding increases body weight and enhances susceptibility to insulin resistance.     

Molecular characterization of lipoprotein lipase, hepatic lipase and pancreatic lipase genes: effects of fasting and refeeding on their gene expression in red sea bream Pagrus major

To investigate the nutritional regulation of lipid metabolism in fish, molecular characterization of lipases was conducted in red sea bream Pagrus major, and the effects of fasting and refeeding on their gene expression was examined. Together with data from a previous study, a total of four lipase genes were identified and characterized as lipoprotein lipase (LPL), hepatic lipase (HL) and pancreatic lipase (PL). These four lipase genes, termed LPL1, LPL2, HL and PL, share a high degree of similarity. LPL1 and LPL2 genes were expressed in various tissues including adipose tissue, gill, heart and hepatopancreas. HL gene was exclusively expressed in hepatopancreas. PL gene expression was detected in hepatopancreas and adipose tissue. Red sea bream LPL1 and LPL2 gene expression levels in hepatopancreas were increased during 48 h of fasting and decreased after refeeding, whereas no significant change in the expression levels of LPL1 and LPL2 was observed in adipose tissue, indicating that LPL1 and LPL2 gene expression is regulated in a tissue-specific manner in response to the nutritional state of fish. HL and PL gene expression was not affected by fasting and refeeding. The results of this study suggested that LPL, HL and PL gene expression is under different regulatory mechanisms in red sea bream with respect to the tissue-specificities and their nutritional regulation.     

Filling the gap - COI barcode resolution in eastern Palearctic birds

Background

Many fish species experience long periods of fasting in nature often associated with seasonal reductions in water temperature and prey availability or spawning migrations. During periods of nutrient restriction, changes in metabolism occur to provide cellular energy via catabolic processes. Muscle is particularly affected by prolonged fasting as myofibrillar proteins act as a major energy source. To investigate the mechanisms of metabolic reorganisation with fasting and refeeding in a saltwater stage of Atlantic salmon (Salmo salar L.) we analysed the expression of genes involved in myogenesis, growth signalling, lipid biosynthesis and myofibrillar protein degradation and synthesis pathways using qPCR.

Results

Hierarchical clustering of gene expression data revealed three clusters. The first cluster comprised genes involved in lipid metabolism and triacylglycerol synthesis (ALDOB, DGAT1 and LPL) which had peak expression 3-14d after refeeding. The second cluster comprised ADIPOQ, MLC2, IGF-I and TALDO1, with peak expression 14-32d after refeeding. Cluster III contained genes strongly down regulated as an initial response to feeding and included the ubiquitin ligases MuRF1 and MAFbx, myogenic regulatory factors and some metabolic genes.

Conclusion

Early responses to refeeding in fasted salmon included the synthesis of triacylglycerols and activation of the adipogenic differentiation program. Inhibition of MuRF1 and MAFbx respectively may result in decreased degradation and concomitant increased production of myofibrillar proteins. Both of these processes preceded any increase in expression of myogenic regulatory factors and IGF-I. These responses could be a necessary strategy for an animal adapted to long periods of food deprivation whereby energy reserves are replenished prior to the resumption of myogenesis.     

Major Facilitator Superfamily Domain-Containing Protein 2a (MFSD2A) Has Roles in Body Growth,Motor Function,and Lipid Metabolism

The metabolic adaptations to fasting in the liver are largely controlled by the nuclear hormone receptor peroxisome proliferator-activated receptor alpha (PPARα), where PPARα upregulates genes encoding the biochemical pathway for β-oxidation of fatty acids and ketogenesis. As part of an effort to identify and characterize nutritionally regulated genes that play physiological roles in the adaptation to fasting, we identified Major facilitator superfamily domain-containing protein 2a (Mfsd2a) as a fasting-induced gene regulated by both PPARα and glucagon signaling in the liver. MFSD2A is a cell-surface protein homologous to bacterial sodium-melibiose transporters. Hepatic expression and turnover of MFSD2A is acutely regulated by fasting/refeeding, but expression in the brain is constitutive. Relative to wildtype mice, gene-targeted Mfsd2a knockout mice are smaller, leaner, and have decreased serum, liver and brown adipose triglycerides. Mfsd2a knockout mice have normal liver lipid metabolism but increased whole body energy expenditure, likely due to increased β-oxidation in brown adipose tissue and significantly increased voluntary movement, but surprisingly exhibited a form of ataxia. Together, these results indicate that MFSD2A is a nutritionally regulated gene that plays myriad roles in body growth and development, motor function, and lipid metabolism. Moreover, these data suggest that the ligand(s) that are transported by MFSD2A play important roles in these physiological processes and await future identification.     

Gene Expression Profile Analysis of Type 2 Diabetic Mouse Liver

Liver plays a key role in glucose metabolism and homeostasis, and impaired hepatic glucose metabolism contributes to the development of type 2 diabetes. However, the precise gene expression profile of diabetic liver and its association with diabetes and related diseases are yet to be further elucidated. In this study, we detected the gene expression profile by high-throughput sequencing in 9-week-old normal and type 2 diabetic db/db mouse liver. Totally 12132 genes were detected, and 2627 genes were significantly changed in diabetic mouse liver. Biological process analysis showed that the upregulated genes in diabetic mouse liver were mainly enriched in metabolic processes. Surprisingly, the downregulated genes in diabetic mouse liver were mainly enriched in immune-related processes, although all the altered genes were still mainly enriched in metabolic processes. Similarly, KEGG pathway analysis showed that metabolic pathways were the major pathways altered in diabetic mouse liver, and downregulated genes were enriched in immune and cancer pathways. Analysis of the key enzyme genes in fatty acid and glucose metabolism showed that some key enzyme genes were significantly increased and none of the detected key enzyme genes were decreased. In addition, FunDo analysis showed that liver cancer and hepatitis were most likely to be associated with diabetes. Taken together, this study provides the digital gene expression profile of diabetic mouse liver, and demonstrates the main diabetes-associated hepatic biological processes, pathways, key enzyme genes in fatty acid and glucose metabolism and potential hepatic diseases.     

Feeding conditions control the expression of genes involved in sterol metabolism in peripheral blood mononuclear cells of normoweight and diet-induced (cafeteria) obese rats

Peripheral blood mononuclear cells (PBMC) are easily obtainable cells from blood whose gene expression profiles have been proven to be highly robust in distinguishing a disease state from healthy state. Sterol metabolism is of physiological importance, and although its nutritional response in liver has been described, it is poorly studied in PBMC. To examine if PBMC sterol metabolism reflects diet-induced physiological responses, we analysed the whole genome gene expression response of PBMC and focused on sterol metabolism-related genes affected by different feeding conditions (ad libitum feeding, fasting, and refeeding) in normoweight (control) rats and in diet-induced (cafeteria) obese rats.Our results of microarray analysis show that, in control rats, 21 genes involved in sterol metabolism were regulated by the different feeding conditions, whereas in cafeteria-obese rats, only seven genes showed a changed expression. Most of the genes identified were classified into three pathways: sterol biosynthesis, cholesterol transport and uptake and sterol signaling. The expression profile of these genes was similar to that previously described for liver, decreasing in response to fasting conditions and recovering the levels found in fed animals after 6-h-refeeding. In addition, our data and the comparable expression pattern of sterol metabolism-related genes between PBMC and liver suggest similar sterol regulatory element-binding protein-mediated regulatory mechanisms in response to feeding conditions in both tissues.In conclusion, the expression of genes involved in sterol metabolism is highly controlled by feeding conditions in PBMC of control rats, but this control is impaired in cafeteria-obese animals. The pathophysiological significance of this impairment requires further investigation.     

The Hepatic Genes for Immunoproteasome Are Upregulated by Refeeding after Fasting in the Rat

Considering that animals maintain energy homeostasis in response to nutrient levels, experiments were done to elucidate the temporal effects of refeeding after fasting on gene expression profiles in the rat liver. Using DNA microarray technology, we first compared gene expression profiles in the livers of rats allowed to feed for 6 h after fasting for 18 h and those in 24-h fasting rats, and found that the expression levels of energy metabolism-related genes in the two groups were different. In addition, refeeding induced upregulation of the genes encoding immunoproteasome components. Finally, immunoblot analysis confirmed changes in protein levels, suggesting that refeeding after fasting enhanced immune function.     

Metallothioneins 1 and 2, but not 3, are regulated by nutritional status in rat white adipose tissue

Background

Cumulating evidence underlines the role of adipose tissue metallothionein (MT) in the development of obesity and type 2 diabetes. Fasting/refeeding was shown to affect MT gene expression in the rodent liver. The influence of nutritional status on MT gene expression in white adipose tissue (WAT) is inconclusive. The aim of this study was to verify if fasting and fasting/refeeding may influence expression of MT genes in WAT of rats.

Results

Fasting resulted in a significant increase in MT1 and MT2 gene expressions in retroperitoneal, epididymal, and inguinal WAT of rats, and this effect was reversed by refeeding. Altered expressions of MT1 and MT2 genes in all main fat depots were reflected by changes in serum MT1 and MT2 levels. MT1 and MT2 messenger RNA (mRNA) levels in WAT correlated inversely with serum insulin concentration. Changes in MT1 and MT2 mRNA levels were apparently not related to total zinc concentrations and MTF1 and Zn transporter mRNA levels in WAT. Fasting or fasting/refeeding exerted no effect on the expression of MT3 gene in WAT. Addition of insulin to isolated adipocytes resulted in a significant decrease in MT1 and MT2 gene expressions. In contrast, forskolin or dibutyryl-cAMP (dB-cAMP) enhanced the expressions of MT1 and MT2 genes in isolated adipocytes. Insulin partially reversed the effect of dB-cAMP on MT1 and MT2 gene expressions.

Conclusions

This study showed that the expressions of MT1 and MT2 genes in WAT are regulated by nutritional status, and the regulation may be independent of total zinc concentration.

     

Liver cancer is induced by a subnecrogenic dose of DENA when associated with fasting/refeeding: role of glutathione-transferase and lipid peroxidation.

In previous studies, we reported that fasting/refeeding has a role in sustaining the initiation of liver cancer by a subnecrogenic (noninitiating) dose of diethylnitrosamine (DENA). This research investigated whether the metabolic alterations imposed by fasting/refeeding provide an imbalance between the generation of carcinogenic molecules and the scavenger defense mechanisms in rat liver. Metabolism of DENA, levels of reduced glutathione (GSH) and GSH transferase (GST) activity, as well as basal and stimulated malondialdehyde (MDA) production, were examined. Rats fasted for 4 days showed a decrease in the liver levels of GSH, GST activity, monounsaturated fatty acids and % of labeled nuclei. After 1 day of refeeding, at which point DENA was administered, the levels of GSH recovered, GST activity remained below control values, basal and stimulated MDA production and content of total polyunsaturated fatty acids in liver phospholipids decreased. One day after DENA treatment, MDA production further decreased, although the % of labeled nuclei increased. No significant changes in the content of arachidonic acid, the main target of peroxidation, were observed at any time. The results indicated that the induction of the hepatocellular carcinoma was associated with a depression of GST activity and lipid peroxidation when rats were given 20 mg/kg of DENA after 1 day of refeeding after 4-day fasting.     

Involvement of the peroxisome proliferator-activated receptor alpha in regulating long-chain acyl-CoA thioesterases

Long-chain acyl-CoA thioesterases catalyze the hydrolysis of acyl-CoAs to the corresponding free fatty acid and CoA. We recently cloned four members of a novel multi-gene family of peroxisome proliferator-induced genes encoding cytosolic (CTE-I), mitochondrial (MTE-I), and peroxisomal (PTE-Ia and PTE-Ib) acyl-CoA thioesterases (Hunt et al. 1999. J. Biol. Chem. 274: 34317-34326). As the peroxisome proliferator-activated receptor alpha (PPARalpha) plays a central role in regulating genes involved in lipid metabolism, we examined the involvement of this receptor in regulation of the thioesterases, particularly CTE-I and MTE-I. Northern blot analysis shows that the induction of these thioesterases by clofibrate is mediated through a strictly PPARalpha-dependent mechanism. All four acyl-CoA thioesterases are induced at mRNA level by fasting and using PPARalpha-null mice, it is evident that the increase in CTE-I due to fasting is mainly independent of the PPARalpha in liver and heart. The CTE-I gene responds rapidly to fasting, with induction of mRNA and protein evident after 6 h. This fasting effect is rapidly reversible, with CTE-I mRNA returning almost to control levels after 3 h refeeding, and being further repressed to 20% of control after 9 h refeeding. Although CTE-I mRNA shows a low basal expression in liver, it can be suppressed 90% by feeding a fat-free diet.These data demonstrate that the nutritional regulation of the thioesterases involves the PPARalpha and other signaling pathways responsible for activation and repression. Putative physiological functions for the acyl-CoA thioesterases are discussed.     

Physiological response in the even-toothed shrew <Emphasis Type="Italic">Sorex isodon</Emphasis> to fasting and refeeding

The red-toothed shrews (genus Sorex) are one of the smallest mammals. The amount of food they consume a day exceeds their own weight, and without food they can survive for only few hours. Representatives of this genus have extremely high metabolic rates. This study addressed the effect of 8-h fasting and 13-h refeeding on the body weight, blood glucose level, liver glycogen and lipid levels, and relative weight of inguinal and interscapular adipose tissues in the even-toothed shrews (S. isodon). Fasting led to a decrease in the body weight, blood glucose and liver glycogen levels. The relative weight of adipose tissue also decreased, while the liver lipid level increased significantly. After refeeding, blood glucose and liver glycogen levels were considerably higher than in control, while other parameters remained almost the same as in control. Physiological response to fasting develops in S. isodon quite rapidly, promoted by the high metabolic rate.     

Unusual increase of Scd1 and Elovl6 expression in rat inguinal adipose tissue

It is generally accepted that the location of body fat deposits may play an important role in the risk of developing some endocrine and metabolic diseases. We have studied the effect of food restriction and food restriction/refeeding, often practiced by individuals trying to lose body weight, on the expression of genes which are associated with obesity and certain metabolic disorders in inguinal, epididymal, and perirenal rat white adipose tissues. Gene expression was analyzed by real time semi-quantitative polymerase chain reaction and by Western blot. We found that prolonged food restriction caused a significant decrease of body and adipose tissue mass as well as the increase of Scd1 and Elovl6 gene expressions in all main rat adipose tissue deposits. Food restriction/refeeding caused increases of: a) Scd1 and Elovl6 mRNA levels in adipose tissue, b) Scd1 protein level and c) desaturation index in adipose tissue. The increased expression of both genes was unusually high in inguinal adipose tissue. The results suggest that the increase of Scd1 and Elovl6 gene expressions in white adipose tissue by prolonged food restriction and prolonged food restriction/refeeding may contribute to accelerated fat recovery that often occurs in individuals after food restriction/refeeding.     

Activation of peroxisome proliferator-activated receptor-α (PPARα) suppresses postprandial lipidemia through fatty acid oxidation in enterocytes

Activation of peroxisome proliferator-activated receptor (PPAR)-α which regulates lipid metabolism in peripheral tissues such as the liver and skeletal muscle, decreases circulating lipid levels, thus improving hyperlipidemia under fasting conditions. Recently, postprandial serum lipid levels have been found to correlate more closely to cardiovascular diseases than fasting levels, although fasting hyperlipidemia is considered an important risk of cardiovascular diseases. However, the effect of PPARα activation on postprandial lipidemia has not been clarified. In this study, we examined the effects of PPARα activation in enterocytes on lipid secretion and postprandial lipidemia. In Caco-2 enterocytes, bezafibrate, a potent PPARα agonist, increased mRNA expression levels of fatty acid oxidation-related genes, such as acyl-CoA oxidase, carnitine palmitoyl transferase, and acyl-CoA synthase, and oxygen consumption rate (OCR) and suppressed secretion levels of both triglycerides and apolipoprotein B into the basolateral side. In vivo experiments revealed that feeding high-fat-diet containing bezafibrate increased mRNA expression levels of fatty acid oxidation-related genes and production of CO₂ and acid soluble metabolites in enterocytes. Moreover, bezafibrate treatment suppressed postprandial lipidemia after oral administration of olive oil to the mice. These findings indicate that PPARα activation suppresses postprandial lipidemia through enhancement of fatty acid oxidation in enterocytes, suggesting that intestinal lipid metabolism regulated by PPARα activity is a novel target of PPARα agonist for decreasing circulating levels of lipids under postprandial conditions.     

Food restriction and refeeding have no effect on cellular and humoral immunity in Mongolian gerbils (Meriones unguiculatus)

Small mammals in the temperate area often face fluctuations in food availability. Changes in food availability may have a great influence on an animals' immunity, which is important to their survival. We tested the hypothesis that cellular and humoral immunity would be suppressed by food restriction and restored to control levels by refeeding in Mongolian gerbils Meriones unguiculatus. Forty adult male gerbils were randomly divided into food-restricted (80% of baseline food intake) and food ad lib. groups. Similarly, another 40 adult male gerbils were also randomly assigned to two groups: a group for which food was restricted for 36 d and then provided ad lib. and a group that was continuously fed ad lib. Half of the gerbils in each group were injected with phytohemagglutinin (PHA) and keyhole limpet hemocyanin solution to assess cellular and humoral immunity, respectively; the others were injected with sterile saline as control groups. Food-restricted gerbils had significantly lower body mass, body fat mass, dry thymus mass, wet and dry spleen mass, and serum leptin levels than those of the controls, whereas refeeding restored these parameters to the controls. Both food restriction and refeeding had no significant effect on PHA response indicative of cellular immunity, immunoglobulin G and immunoglobulin M concentrations, and white blood cells. We also found that food restriction decreased corticosterone levels in food-restricted gerbils, while refeeding increased corticosterone levels in refed gerbils compared with the controls. Our results suggest that cellular and humoral immunity were not affected by food restriction and refeeding in gerbils.