Different localization in rat brain of the novel cytosolic ketone body-utilizing enzyme, acetoacetyl-CoA synthetase, as compared to succinyl-CoA:3-oxoacid CoA-transferase

In lipogenic tissue cytosol, ketone bodies are known to be activated by acetoacetyl-CoA synthetase (AACS) and incorporated into cholesterol and fatty acids. In order to investigate the physiological role of AACS in the brain, we examined the localization of AACS mRNA in rat brain by in situ hybridization using a labeled probe. High labeling was observed in the midbrain, pons/medulla, cerebral cortex, hippocampus and cerebellum, and the localization profile of AACS mRNA was different from that of succinyl-CoA:3-oxoacid CoA-transferase (SCOT), a mitochondrial ketone body-activating enzyme. In addition, the expression of AACS mRNA in the cerebellum was restricted primarily to glial cells, while in the cerebral cortex, it was restricted to neuronal cells. Streptozotocin treatment caused remarkable decreases in AACS mRNA levels in all regions where expression was observed, but changes in SCOT mRNA levels were not observed. These results suggest that the physiological role of AACS is different from that of SCOT and varies depending upon its localization in the brain.     

Genetic Obesity Affects Neural Ketone Body Utilization in the Rat Brain

Obesity causes various physiological disorders between the central nervous system and peripheral tissues. Ketone bodies have a neuro‐protective role and are strongly affected by obesity‐related metabolic disorders. To clarify the effects of obesity on ketone body utilization in brain, we examined the mRNA localization of acetoacetyl‐CoA synthetase (AACS), which activates ketone bodies for the synthesis of fatty acid and cholesterol, in various brain regions of Zucker fatty rats by in situ hybridization. The AACS mRNA level was increased in the paraventricular thalamic nucleus (PVT) but not affected in the cerebrum and hippocampus in Zucker fatty rats. In contrast, the AACS mRNA level was reduced in the arcuate hypothalamic nucleus (Arc) and ventromedial hypothalamic nucleus (VMH) in the hypothalamus. Succinyl‐CoA:3‐oxoacid CoA‐transferase (SCOT) mRNA level was decreased only in the PVT but not affected in the Arc and VMH. These data raise the possibility that AACS is regulated by the leptin signaling pathway in the hypothalamus but not in the PVT. As AACS was expressed in neural‐like cells, ketone bodies are assumed to be utilized for the synthesis of lipidic substances and to cause metabolic disorders in the nervous system.     

Transcriptional regulation of ketone body-utilizing enzyme,acetoacetyl-CoA synthetase,by C/EBPα during adipocyte differentiation

Acetoacetyl-CoA synthetase (AACS), an essential enzyme for the synthesis of fatty acid and cholesterol from ketone bodies, was found to be highly expressed in mouse adipose tissue, and GC box and C/EBPs motif were crucial for AACS promoter activity in 3T3-L1 adipocytes. Moreover, we found that AACS promoter activity was controlled mainly by C/EBPα during adipogenesis.     

Transcriptional regulation of ketone body-utilizing enzyme, acetoacetyl-CoA synthetase, by C/EBPalpha during adipocyte differentiation

Acetoacetyl-CoA synthetase (AACS), an essential enzyme for the synthesis of fatty acid and cholesterol from ketone bodies, was found to be highly expressed in mouse adipose tissue, and GC box and C/EBPs motif were crucial for AACS promoter activity in 3T3-L1 adipocytes. Moreover, we found that AACS promoter activity was controlled mainly by C/EBPalpha during adipogenesis.     

FGF-10 is a growth factor for preadipocytes in white adipose tissue

FGF-10 is a mesenchymal factor affecting epithelial cells during pattern formation. However, the expression and physiological role of FGF-10 in adults remains to be elucidated. We examined the expression of FGF-10 mRNA in a variety of adult rat tissues, and found to be most abundant in white adipose tissue. In white adipose tissue, FGF-10 mRNA was expressed in preadipocytes but not in mature adipocytes. The expression in white adipose tissue during postnatal development was also examined. The expression level was low at postnatal day 10 (P10). However, FGF-10 mRNA was abundantly detected later on (P28 and P48) when white adipose tissue growth was stimulated. We also examined the activity of recombinant FGF-10 for primary rat preadipocytes. FGF-10 showed significant mitogenic activity for primary preadipocytes, but did not affect the differentiation of preadipocytes. The expression profile of FGF-10 mRNA and the activity of FGF-10 reported here indicate that FGF-10, a unique secreted factor produced in white adipose tissue, acts as a growth factor for preadipocytes in white adipose tissues.     

Central leptin regulates the UCP1 and ob genes in brown and white adipose tissue via different beta-adrenoceptor subtypes

The three known subtypes of beta-adrenoreceptors (beta(1)-AR, beta(2)-AR, and beta(3)-AR) are differentially expressed in brown and white adipose tissue and mediate peripheral responses to central modulation of sympathetic outflow by leptin. To assess the relative roles of the beta-AR subtypes in mediating leptin's effects on adipocyte gene expression, mice with a targeted disruption of the beta(3)-adrenoreceptor gene (beta(3)-AR KO) were treated with vehicle or the beta(1)/beta(2)-AR selective antagonist, propranolol (20 microgram/g body weight/day) prior to intracerebroventricular (ICV) injections of leptin (0.1 microgram/g body weight/day). Leptin produced a 3-fold increase in UCP1 mRNA in brown adipose tissue of wild type (FVB/NJ) and beta(3)-AR KO mice. The response was unaltered by propranolol in wild type mice, but was completely blocked by this antagonist in beta(3)-AR KO mice. In contrast, ICV leptin had no effect on leptin mRNA in either epididymal or retroperitoneal white adipose tissue (WAT) from beta(3)-AR KOs. Moreover, propranolol did not block the ability of exogenous leptin to reduce leptin mRNA in either WAT depot site of wild type mice. These results demonstrate that the beta(3)-AR is required for leptin-mediated regulation of ob mRNA expression in WAT, but is interchangeable with the beta(1)/beta(2)-ARs in mediating leptin's effect on UCP1 mRNA expression in brown adipose tissue.     

The gender- and fat depot-specific regulation of leptin, resistin and adiponectin genes expression by progesterone in rat

Progesterone affects lipid metabolism in adipose tissue and influences fat distribution in human. The aim of the study was to analyze the effect of progesterone on rat body and fat mass and on expression of genes encoding adipokines involved in the regulation of energy homeostasis. The results presented here indicate that progesterone administration to females caused increase in body and inguinal white adipose tissue mass. The increase of inguinal white adipose tissue mass is associated with the hypertrophy of adipocyte. The same dose of progesterone caused increase of its circulating concentration in males, however it barely reached the value observed in non-treated control females and did not have any effect on body and fat mass. The elevated circulating progesterone concentration was associated with an approximately 6- and 2-fold increase of leptin and resistin mRNA level respectively, and 2-fold decrease of adiponectin mRNA level only in inguinal white adipose tissue of females. RU 486, specific antagonist of progesterone receptor, abolished the effect of progesterone on the adipokine mRNA level in inguinal adipose tissue. In males, the elevated circulating progesterone concentration showed no effects on leptin, resistin or adiponectin mRNA level in inguinal, retroperitoneal or epididymal adipose tissue. Moreover, the results presented in this paper demonstrate a relatively high level of progesterone receptor mRNA in inguinal white adipose tissue of females, which was down-regulated in response to progesterone administration. In retroperitoneal adipose tissue of control females progesterone receptor mRNA level was approximately 3-fold lower as compared to inguinal adipose tissue. In inguinal, epididymal and retroperitoneal white adipose tissue of males progesterone receptor mRNA was hardly detected. Our results suggest that depot- and sex-dependent responsiveness of adipose tissue to the pharmacological dose of progesterone is controlled by both circulating concentration of progesterone and the white adipose tissue progesterone receptor level.     

Seasonal changes in critical enzymes of lipogenesis and triacylglycerol synthesis in the marmot (Marmota flaviventris)

Fatty acid metabolism and triacylglycerol synthesis are critical processes for the survival of hibernating mammals that undergo a prolonged fasting period. Fatty acid synthase, fatty-acid-CoA ligase, diacylglycerol acyltransferase, and monoacylglycerol acyltransferase activities were measured in liver and in white and brown adipose tissue, in order to determine whether enzymes of lipogenesis and triacylglycerol synthesis vary seasonally during hibernation in the yellow-bellied marmot (Marmota flaviventris). Compared with mid-winter hibernation, fatty acid synthase activity was higher in all three tissues during early spring when marmots emerged from hibernation and in mid-summer when they were feeding, consistent with the synthesis of fatty acids from the carbohydrate-rich summer diet. Fatty-acid-CoA ligase and diacylglycerol acyltransferase activities were highest in summer in white adipose tissue when triacylglycerol synthesis would be expected to be high; diacylglycerol acyltransferase activity was also high in brown adipose tissue during spring and summer. In liver, however, diacylglycerol acyltransferase specific activity was highest during hibernation, suggesting that triacylglycerol synthesis may be prominent in liver in winter. Monoacylglycerol acyltransferase activity, which may aid in the retention of essential fatty-acids, was 80-fold higher in liver than in white or brown adipose tissue, but did not vary seasonally. Its dependence on palmitoyl-CoA suggests that a divalent cation might play a role in enzyme activation. The high hepatic diacylglycerol acyltransferase activity during hibernation suggests that the metabolism of very low density lipoprotein may be important in the movement of adipose fatty acids to brown adipose tissue and muscle during the rewarming that occurs periodically during hibernation. These studies suggest that enzymes of lipid metabolism vary seasonally in the marmot, consistent with requirements of this hibernator for triacylglycerol synthesis and metabolism.Abbreviations BAT brown adipose tissue - DGAT diacylglycerol acyltransferase - FAS fatty acid synthase - K _m Michaelis constant - MGAT monoacylglycerol acyltransferase - RQ respiratory quotiant - VLDL very low density lipoprotein - WAT white adipose tissue     

IL-6 Indirectly Modulates the Induction of Glyceroneogenic Enzymes in Adipose Tissue during Exercise

Background

Glyceroneogenesis is an important step in the control of fatty acid re-esterification with PEPCK and PDK4 being identified as key enzymes in this process. We have previously shown that glyceroneogenic enzymes such as PDK4 are rapidly induced in white adipose tissue during exercise. Recent studies have suggested that IL-6 regulates adipose tissue metabolism and gene expression during exercise. Interestingly, IL-6 has been reported to directly decrease PEPCK expression. The purpose of this investigation was to determine the role of IL-6 in modulating the effects of exercise on the expression of glyceroneogenic enzymes in mouse adipose tissue. We hypothesized that the exercise-mediated induction of PDK4 and PEPCK would be greater in adipose tissue from IL-6 deficient mice compared to wild type controls.

Methodology and Principle Findings

Treatment of cultured epididymal adipose tissue (eWAT) with IL-6 (150 ng/ml) increased the phosphorylation of AMPK, ACC and STAT3 and induced SOCS3 mRNA levels while decreasing PEPCK and PDK4 mRNA. AICAR decreased the expression of PDK4 and PEPCK. The activation of AMPK by IL-6 was independent of increases in lipolysis. An acute bout of treadmill running (15 meters/minute, 5% incline, 90 minutes) did not induce SOCS3 or increase phosphorylation of STAT3 in eWAT, indicating that IL-6 signalling was not activated. Exercise-induced increases in PEPCK and PDK4 mRNA expression were attenuated in eWAT from IL-6^−/− mice in parallel with a greater relative increase in AMPK phosphorylation compared to exercised WT mice. These changes occurred independent of alterations in beta-adrenergic signalling in adipose tissue from IL-6^−/− mice.

Conclusions and Significance

Our findings question the role of IL-6 signalling in adipose tissue during exercise and suggest an indirect effect of this cytokine in the regulation of adipose tissue gene expression during exercise.     

Evidence that triiodothyronine decreases rat serum leptin concentration by down-regulation of leptin gene expression in white adipose tissue

Conflicting results have been reported regarding the effect of triiodothyronine (T(3)) on serum leptin and adipose tissue leptin gene expression in human and animals. The aim of the present study was to evaluate the effect of administration of increasing doses of T(3) on serum leptin concentration and on leptin mRNA abundance in white adipose tissue of rats. The results presented in this paper indicate that administration of single different doses of T(3) to euthyroid rats resulted dose dependent increases of serum total T(3) concentrations which are associated with a decrease in white adipose tissue leptin mRNA level. The leptin mRNA level in white adipose tissue was negatively correlated with serum total T(3) concentration (r=-0.8, p<0.001). Like white adipose tissue leptin mRNA level, serum leptin concentration decreased after T(3) administration, and was also negatively correlated with the serum T(3) concentration (r=-0.8, p<0.001). In contrast, administration of T(3) to the same rats led to a significant increase in white adipose tissue expression of the malic enzyme gene (malic enzyme activity and malic enzyme mRNA level), a known target gene for T(3). The results indicate that T(3) exerts a selective inhibitory effect on white adipose tissue leptin gene expression in vivo. A conclusion is that T(3) decreases rat serum leptin concentration by down-regulation of leptin gene expression in white adipose tissue.     

The role of the sympathetic nervous system in the regulation of leptin synthesis in C57BL/6 mice

The objectives of this study were to determine whether leptin synthesis is regulated by the sympathetic nervous system and if so whether beta-adrenergic receptors mediate this effect. We show that sympathetic blockade by reserpine increases leptin mRNA levels in brown but not white adipose tissue, while acute cold-exposure decreases leptin expression 10-fold in brown adipose tissue and 2-fold in white adipose tissue. The cold-induced reduction in leptin mRNA can be prevented by a combination of propranolol and SR 59230A but not by either antagonist alone, indicating that beta3-adrenergic receptors and classical beta1/beta2-adrenergic receptors both mediate responses to sympathetic stimulation. Circulating leptin levels reflect synthesis in white adipose tissue but not in brown adipose tissue.     

Dietary conjugated linoleic acid reduces body fat mass and affects gene expression of proteins regulating energy metabolism in mice

ICR and C57BL/6J mice were fed experimental diets containing either a 2% fatty acid preparation rich in conjugated linoleic acid (CLA) or a preparation rich in linoleic acid and free of CLA for 21 days. CLA greatly decreased weights of white adipose tissue and interscapular brown adipose tissue in the two strains. CLA reduced mRNA levels of glucose transporter 4 (Glut 4) in white and brown adipose tissue of both strains. A CLA-dependent decrease in mRNA levels of peroxisome proliferator activated receptor (PPAR) gamma was seen in interscapular brown adipose tissue of both strains and in white adipose tissue of C57BL/6J but not ICR mice. Dietary CLA was found to cause a decrease in the mRNA levels of uncoupling protein (UCP) 1 in brown adipose tissue when the value was corrected for the expression of a house-keeping gene (beta-actin) in the two strains. Uncorrected values were, however, indistinguishable between the animals fed the CLA diet and CLA-free diet. UCP 3 expression in brown adipose tissue was much lower in mice fed the CLA diet than in those fed the control diet in both strains. In contrast, CLA greatly up-regulated the gene expression of UCP 2 in brown adipose tissue. Dietary CLA also increased UCP 2 mRNA level in skeletal muscle. It is apparent that dietary CLA decreases white and brown adipose tissue mass, accompanying changes in the gene expression of proteins regulating energy metabolism in white and brown adipose tissues, and skeletal muscle of mice.     

Triiodothyronine induces UCP-1 expression and mitochondrial biogenesis in human adipocytes

Uncoupling protein (UCP)-1 expressed in brown adipose tissue plays an important role in thermogenesis. Recent data suggest that brown-like adipocytes in white adipose tissue (WAT) and skeletal muscle play a crucial role in the regulation of body weight. Understanding of the mechanism underlying the increase in UCP-1 expression level in these organs should, therefore, provide an approach to managing obesity. The thyroid hormone (TH) has profound effects on mitochondrial biogenesis and promotes the mRNA expression of UCP in skeletal muscle and brown adipose tissue. However, the action of TH on the induction of brown-like adipocytes in WAT has not been elucidated. Thus we investigate whether TH could regulate UCP-1 expression in WAT using multipotent cells isolated from human adipose tissue. In this study, triiodothyronine (T(3)) treatment induced UCP-1 expression and mitochondrial biogenesis, accompanied by the induction of the CCAAT/enhancer binding protein, peroxisome proliferator-activated receptor-γ coactivator-1α, and nuclear respiratory factor-1 in differentiated human multipotent adipose-derived stem cells. The effects of T(3) on UCP-1 induction were dependent on TH receptor-β. Moreover, T(3) treatment increased oxygen consumption rate. These findings indicate that T(3) is an active modulator, which induces energy utilization in white adipocytes through the regulation of UCP-1 expression and mitochondrial biogenesis. Our findings provide evidence that T(3) serves as a bipotential mediator of mitochondrial biogenesis.     

The lipogenic enzyme acetoacetyl-CoA synthetase and ketone body utilization for denovo lipid synthesis,a review

Acetoacetyl-CoA synthetase (AACS) is the key enzyme in the anabolic utilization of ketone bodies (KBs) for denovo lipid synthesis, a process that bypasses citrate and ATP citrate lyase. This review shows that AACS is a highly regulated, cytosolic, and lipogenic enzyme and that many tissues can readily use KBs for denovo lipid synthesis. AACS has a low micromolar K_m for acetoacetate, and supply of acetoacetate should not limit its activity in the fed state. In many tissues, AACS appears to be regulated in conjunction with the need for cholesterol, but in adipose tissue, it seems tied to fatty acid synthesis. KBs are readily utilized as substrates for lipid synthesis in lipogenic tissues, including liver, adipose tissue, lactating mammary gland, skin, intestinal mucosa, adrenals, and developing brain. In numerous studied cases, KBs served several-fold better than glucose as substrates for lipid synthesis, and when present, KBs suppressed the utilization of glucose for lipid synthesis. Here, it is hypothesized that a physiological role for the utilization of KBs for lipid synthesis is a metabolic process of lipid interconversion. Fatty acids are converted to KBs in liver, and then, the KBs are utilized to synthesize cholesterol and other long-chain fatty acids in liver and nonhepatic tissues. The conversion of fatty acids to cholesterol via the KBs may be a particularly important example of lipid interconversion. Utilizing KBs for lipid synthesis is glucose sparing and probably is important with low carbohydrate diets. Metabolic situations and tissues where this pathway may be important are discussed.     

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.     

Increased norepinephrine by medium-chain triglyceride attributable to lipolysis in white and brown adipose tissue of C57BL/6J mice

A further investigation of the lipolysis induced by medium-chain triglyceride (MCT) was conducted on C57BL/6J mice fed with a diet containing 2% MCT or 2% long-chain triglyceride (LCT). Blood norepinephrine, body fat and blood lipid variables, and the protein or mRNA expression of the genes relevant to lipolysis were measured and analyzed in the white and brown adipose tissue (WAT, BAT). Decreased body fat and improved blood lipid profiles attributable to MCT were confirmed. A higher level of blood norepinephrine was observed with the MCT diet. The adipose triglyceride lipase (ATGL) activity and its mRNA expression, the expression of protein and mRNA of the beta 3 adrenergic receptor (β3-AR) in both WAT and BAT, and the hormone-sensitive lipase (HSL) activity and its mRNA expression in BAT were significantly increased in the mice with MCT feeding. The lipolysis induced by MCT might be partially mediated by increasing norepinephrine, thereafter signaling the up-regulation of β3-AR, ATGL, and HSL in WAT and BAT.     

Feeding scallop shell powder induces the expression of uncoupling protein 1 (UCP1) in white adipose tissue of rats

Previously we found that the organic components in scallop shell promote lipolysis in differentiated 3T3-L1 and C3H10T1/2 adipocyte cells, and that incorporating scallop shell powder into the diet of rats reduced the amount of white adipose tissue. In this study, we used RT-PCR to investigate the effect of ingesting scallop shell powder on the gene expression profile of uncoupling proteins (UCPs) regulating energy metabolism in rats.Feeding of scallop shell powder increased mRNA levels of UCP1 and UCP2 in white adipose tissue. By contrast, scallop shell powder had no effect on the expression of UCP1 in brown adipose tissue, although the expression level of UCP2 mRNA decreased significantly. These results suggest that feeding scallop shell powder increases gene expression of UCP1 that may regulate energy metabolism in white adipose tissue, resulting in the observed reduction in weight of white adipose tissue.     

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