In vivo tissue specific modulation of rat insulin receptor gene expression in an experimental model of mineralocorticoid excess

Insulin receptor (IR) gene expression at the mRNA level was investigated in hindlimb skeletal muscle, epididymal adipose tissue and in the liver of rats exposed to prolonged in vivo administration of deoxycorticosterone acetate (DOCA). Following treatment, plasma insulin levels were reduced while glucose levels increased compared to values in control rats. DOCA-treated animals showed an increase in blood pressure and a reduction in body weight. This treatment also induced hypokalemia and decreased plasma protein levels. Sodium levels were unaffected. Moreover, no differences in DNA and protein content or in the indicator of cell size (protein/DNA) were observed in the skeletal muscle or adipose tissue of animals. In contrast, there was a clear increase in the protein and DNA contents of the liver with no change in the indicator of cell size. Northern blot assays revealed 2 major IR mRNA species of approximately 9.5 and 7.5 Kb in the 3 tissues from control animals. DOCA treatment induced no change in the levels of either RNA species in skeletal muscle. However, a decrease of approximately 22% was detected in the levels of both species in adipose tissue whereas the liver showed an increase of 64%. These results provide the first evidence for an in vivo tissue-specific modulation of IR mRNA levels under experimental conditions of mineralocorticoid excess.     

白藜芦醇对成年期追赶生长大鼠体成分的影响及机制

目的:观察白藜芦醇对成年期追赶生长大鼠体成分的影响及可能机制。方法:8周龄雄性 SD 大鼠分为6组(共2个时间点),即4周时间点3组:正常饮食4周 (NC4)组、热卡限制4周(R4)组,热卡限制同时白藜芦醇治疗(R4E)组;12周时间点3组:正常饮食12周(NC12)组,追赶生长(CUG)组,追赶生长白藜芦醇治疗(CUGE)组。每组含6只大鼠,白藜芦醇用生理盐水配制成一定浓度悬浊液,按100 mg/(kg·d)剂量予实验动物灌胃治疗。实验第4周、12周检测体重、躯干和全身的肌肉及脂肪含量、躯干与全身脂肪比例,实验第12周检测骨骼肌与附睾脂肪组织SIRT1的表达,附睾脂肪组织PPARγ的表达。结果:与NC12组相比,CUG组躯干及全身的脂肪含量、躯干与全身脂肪比例、附睾脂肪组织PPARγ的表达均明显升高(P＜0.05),肌肉含量、骨骼肌与附睾脂肪组织SIRT1的表达显著降低(P＜0.05或P＜0.01);与CUG组相比,经白藜芦醇干预后的CUGE组全身的脂肪含量、躯干与全身脂肪比例、附睾脂肪组织PPARγ的表达均明显降低(P＜0.05),肌肉含量、骨骼肌与附睾脂肪组织SIRT1的表达较CUG组显著提高(P＜0.05)。结论:白藜芦醇降低成年期追赶生长大鼠体脂含量,增加肌肉含量,改善腹部脂肪堆积,其机制可能与增加骨骼肌及内脏脂肪组织SIRT1表达,抑制内脏脂肪PPARγ的表达有关。     

A polyphenol rescues lipid induced insulin resistance in skeletal muscle cells and adipocytes

Skeletal muscle and adipose tissues are known to be two important insulin target sites. Therefore, lipid induced insulin resistance in these tissues greatly contributes in the development of type 2 diabetes (T2D). Ferulic acid (FRL) purified from the leaves of Hibiscus mutabilis, showed impressive effects in preventing saturated fatty acid (SFA) induced defects in skeletal muscle cells. Impairment of insulin signaling molecules by SFA was significantly waived by FRL. SFA markedly reduced insulin receptor β (IRβ) in skeletal muscle cells, this was affected due to the defects in high mobility group A1 (HMGA1) protein obtruded by phospho-PKCε and that adversely affects IRβ mRNA expression. FRL blocked PKCε activation and thereby permitted HMGA1 to activate IRβ promoter which improved IR expression deficiency. In high fat diet (HFD) fed diabetic rats, FRL reduced blood glucose level and enhanced lipid uptake activity of adipocytes isolated from adipose tissue. Importantly, FRL suppressed fetuin-A (FetA) gene expression, that reduced circulatory FetA level and since FetA is involved in adipose tissue inflammation, a significant attenuation of proinflammatory cytokines occurred. Collectively, FRL exhibited certain unique features for preventing lipid induced insulin resistance and therefore promises a better therapeutic choice for T2D.     

Glucose transporters: structure, function, and regulation

Glucose is transported into the cell by facilitated diffusion via a family of structurally related proteins, whose expression is tissue-specific. One of these transporters, GLUT4, is expressed specifically in insulin-sensitive tissues. A possible change in the synthesis and/or in the amount of GLUT4 has therefore been studied in situations associated with an increase or a decrease in the effect of insulin on glucose transport. Chronic hyperinsulinemia in rats produces a hyper-response of white adipose tissue to insulin and resistance in skeletal muscle. The hyper-response of white adipose tissue is associated with an increase in GLUT4 mRNA and protein. In contrast, in skeletal muscle, a decrease in GLUT4 mRNA and a decrease (tibialis) or no change (diaphragm) in GLUT4 protein are measured, suggesting a divergent regulation by insulin of glucose transport and transporters in the 2 tissues. In rodents, brown adipose tissue is very sensitive to insulin. The response of this tissue to insulin is decreased in obese insulin-resistant fa/fa rats. Treatment with a beta-adrenergic agonist increases insulin-stimulated glucose transport, GLUT4 protein and mRNA. The data suggest that transporter synthesis can be modulated in vivo by insulin (muscle, white adipose tissue) or by catecholamines (brown adipose tissue).     

Protein synthesis in liver,skeletal muscle,and brown adipose tissue of rats fed a protein-deficient diet

Feeding protein-deficient diets to rats is known to stimulate diet-induced thermogenesis and activate brown adipose tissue (BAT). The fact that BAT protein content, unlike that of other tissues, is unnaffected by protein deficiency prompted us to measure tissue protein synthesis in vivo in animals maintained on normal- (18.8%) and low- (7.6%) protein (LP) diets. Protein synthesis was depressed in the liver of the LP rats due to a fall in RNA activity, with no change in RNA content, and synthesis was also reduced in skeletal muscle from the LP group, but this was due to decreased RNA content with no change in RNA activity. Conversely, protein synthesis, RNA, DNA, and protein content of interscapular BAT were all unaltered in protein-restricted animals. These data indicate that, unlike liver, skeletal muscle, and whole carcass, BAT protein synthesis is not reduced in protein-restricted rats, and this may be related to activation of thermogenesis in the tissue.     

Systemic administration of epidermal growth factor increases UCP3 mRNA levels in skeletal muscle and adipose tissue in rats

We have previously reported that systemic epidermal growth factor (EGF) treatment in rats reduces the amount of adipose tissue despite an unaltered food intake. The mitochondrial uncoupling proteins (UCP2 and UCP3) are thought to uncouple the respiratory chain and thus to increase energy expenditure. In order to find out whether the UCP system was involved in the EGF-induced weight loss, the effects of EGF on UCP2 and UCP3 in adipose tissue and skeletal muscle were investigated in the present study. Eight rats were treated with placebo or EGF (150 microg/kg/day) for seven days via mini-osmotic pumps. The EGF-treated rats gained significantly less body weight during the study period than the placebo-treated animals and had significantly less adipose tissue despite a similar food intake. The placebo group and the EGF group had similar UCP2 mRNA expression (in both adipose tissue and skeletal muscle), whereas the EGF-treated group compared to the placebo group had significantly higher UCP3 mRNA expression in both skeletal muscle (3.76 +/- 0.90 vs 8.41 +/- 0.87, P < 0.05) and in adipose tissue (6.38 +/- 0.71 vs 12.48 +/- 1.79, P < 0.05). In vitro studies with adipose tissue fragments indicated that the EGF effect probably is mediated indirectly as incubations with EGF (10 microM) were unable to affect adipose tissue UCP expression, whereas incubations with bromopalmitate stimulated both UCP2 and UCP3 mRNA expression twofold. Thus, EGF treatment in vivo was found to enhance UCP3 mRNA expression in both adipose tissue and skeletal muscle, which may indicate that the EGF effect on body composition might involve up-regulation of UCP3 in skeletal muscle and adipose tissue.     

Increased rates of fatty acid uptake and plasmalemmal fatty acid transporters in obese Zucker rats

Giant vesicles were used to study the rates of uptake of long-chain fatty acids by heart, skeletal muscle, and adipose tissue of obese and lean Zucker rats. With obesity there was an increase in vesicular fatty acid uptake of 1.8-fold in heart, muscle and adipose tissue. In some tissues only fatty acid translocase (FAT) mRNA (heart, +37%; adipose, +80%) and fatty acid-binding protein (FABPpm) mRNA (heart, +148%; adipose, +196%) were increased. At the protein level FABPpm expression was not changed in any tissues except muscle (+14%), and FAT/CD36 protein content was altered slightly in adipose tissue (+26%). In marked contrast, the plasma membrane FAT/CD36 protein was increased in heart (+60%), muscle (+80%), and adipose tissue (+50%). The plasma membrane FABPpm was altered only in heart (+50%) and adipose tissues (+70%). Thus, in obesity, alterations in fatty acid transport in metabolically important tissues are not associated with changes in fatty acid transporter mRNAs or altered fatty acid transport protein expression but with their increased abundance at the plasma membrane. We speculate that in obesity fatty acid transporters are relocated from an intracellular pool to the plasma membrane in heart, muscle, and adipose tissues.     

Glutamine: fructose-6-phosphate amidotransferase activity and gene expression are regulated in a tissue-specific fashion in pregnant rats.

We examined whether regulation of glutamine: fructose-6-phosphate amidotransferase (GFA), the rate-limiting enzyme of the hexosamine pathway, is tissue specific and if so whether such regulation occurs at the level of gene expression. We compared GFA activity and expression and levels of UDP-hexosamines and UDP-hexoses between insulin-sensitive (liver and muscle) tissues and a glucose-sensitive (placenta) tissue from 19 day pregnant streptozotocin diabetic and non-diabetic rats. In pregnant non-diabetic rats GFA activities averaged (1521+/-75 pmol/mg protein x min) in the placenta, 895+/-74 in the liver and 81+/-11 in muscle (p<0.001 between each tissue). In the diabetic rats, GFA activities were approximately 50% decreased both in the liver (340+/-42 pmol/mg protein x min, p<0.05 vs control rats) and in skeletal muscle (46+/-3, p<0.05) compared to control rats. In the placenta, GFA activities were identical between diabetic (1519+/-112 pmol/mg protein x min) and non-diabetic (1521+/-75) animals. In the liver, the reduction in GFA activity could be attributed to a significant decrease in GFA mRNA concentrations, while GFA mRNA concentrations were similar in the placenta between diabetic and non-diabetic animals. UDP-N-acetylglucosamine (UDP-GlcNAc), the end product of the hexosamine pathway, was significantly reduced in the liver and in skeletal muscle but similar in the placenta between diabetic and non-diabetic rats. In summary, GFA activity and expression and the concentration of UDP-GlcNAc are decreased in the liver but unaltered in the placenta, although GFA activity is almost 2-fold higher in this tissue than in the liver. These data provide the first evidence for tissue specific regulation of GFA and for its regulation at the level of gene expression.     

Expression of glucose transporter 4 mRNA in adipose tissue and skeletal muscle of ovariectomized rats treated with sex steroid hormones

The effects of 17beta-estradiol (E) and/or progesterone (P) on glucose transporter 4 (GLUT4) expression in the adipose tissue and skeletal muscle of ovariectomized female rats were studied. The Sprague-Dawley rats received daily subcutaneous injections of various doses of E and/or P for 7 days (n=5-6 per dose). The expression of GLUT4 mRNA was assessed by performing ribonuclease protection assays. GLUT4 protein levels were assessed by Western blotting assays. The adipose tissue levels of GLUT4 mRNA were reduced by the administration of 50 microg E, which resulted in unphysiologically high serum E concentrations. Although the GLUT4 mRNA levels did not change after the administration of 10 microg E or 5 mg P, they were reduced significantly to approximately half the control group level by the administration of both hormones (p <0.01). The skeletal muscle GLUT4 mRNA levels were not changed significantly by hormone treatment. These findings suggest that E and P may be involved in the regulation of GLUT4 mRNA expression in adipose tissue.     

Tissue-specific effect of refeeding after short- and long-term caloric restriction on malic enzyme gene expression in rat tissues

Restricting food intake to a level below that consumed voluntarily (85%, 70% and 50% of the ad libitum energy intake for 3 or 30 days) and re-feeding ad libitum for 48 h results in an increase of malic enzyme (ME) gene expression in rat white adipose tissue. The increase of ME gene expression was much more pronounced in rats maintained on restricted diet for 30 days than for 3 days. The changes in ME gene expression resembled the changes in the content of SREBP-1 in white adipose tissue. A similar increase of serum insulin concentration was observed in all groups at different degrees of caloric restriction and refed ad libitum for 48 h. Caloric restriction and refeeding caused on increase of ME activity also in brown adipose tissue (BAT) and liver. However, in liver a significant increase of ME activity was found only in rats maintained on the restricted diet for 30 days. No significant changes after caloric restriction and refeeding were found in heart, skeletal muscle, kidney cortex, and brain. These data indicate that the increase of ME gene expression after caloric restriction/refeeding occurs only in lipogenic tissues. Thus, one can conclude that caloric restriction/refeeding increases the enzymatic capacity for fatty acid biosynthesis.     

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     

Visfatin mRNA expression in human subcutaneous adipose tissue is regulated by exercise

Visfatin [pre-beta-cell colony-enhancing factor (PBEF)] is a novel adipokine that is produced by adipose tissue, skeletal muscle, and liver and has insulin-mimetic actions. Regular exercise enhances insulin sensitivity. In the present study, we therefore examined visfatin mRNA expression in abdominal subcutaneous adipose tissue and skeletal muscle biopsies obtained from healthy young men at time points 0, 3, 4.5, 6, 9, and 24 h in relation to either 3 h of ergometer cycle exercise at 60% of Vo(2 max) or rest. Adipose tissue visfatin mRNA expression increased threefold at the time points 3, 4.5, and 6 h in response to exercise (n = 8) compared with preexercise samples and compared with the resting control group (n = 7, P = 0.001). Visfatin mRNA expression in skeletal muscle was not influenced by exercise. The exercise-induced increase in adipose tissue visfatin was, however, not accompanied by elevated levels of plasma visfatin. Recombinant human IL-6 infusion to mimic the exercise-induced IL-6 response (n = 6) had no effect on visfatin mRNA expression in adipose tissue compared with the effect of placebo infusion (n = 6). The finding that exercise enhances subcutaneous adipose tissue visfatin mRNA expression suggests that visfatin has a local metabolic role in the recovery period following exercise.     

Reduced expression of PGC-1 and insulin-signaling molecules in adipose tissue is associated with insulin resistance

Peroxisome proliferator-activated receptor gamma (PPAR gamma) co-activator 1 (PGC-1) regulates glucose metabolism and energy expenditure and, thus, potentially insulin sensitivity. We examined the expression of PGC-1, PPAR gamma, insulin receptor substrate-1 (IRS-1), glucose transporter isoform-4 (GLUT-4), and mitochondrial uncoupling protein-1 (UCP-1) in adipose tissue and skeletal muscle from non-obese, non-diabetic insulin-resistant, and insulin-sensitive individuals. PGC-1, both mRNA and protein, was expressed in human adipose tissue and the expression was significantly reduced in insulin-resistant subjects. The expression of PGC-1 correlated with the mRNA levels of IRS-1, GLUT-4, and UCP-1 in adipose tissue. Furthermore, the adipose tissue expression of PGC-1 and IRS-1 correlated with insulin action in vivo. In contrast, no differential expression of PGC-1, GLUT-4, or IRS-1 was found in the skeletal muscle of insulin-resistant vs insulin-sensitive subjects. The findings suggest that PGC-1 may be involved in the differential gene expression and regulation between adipose tissue and skeletal muscle. The combined reduction of PGC-1 and insulin signaling molecules in adipose tissue implicates adipose tissue dysfunction which, in turn, can impair the systemic insulin response in the insulin-resistant subjects.     

Selective detection of UCP 3 expression in skeletal muscle: effect of thyroid status and temperature acclimation

A novel peptide antibody to UCP 3 is characterized which is sensitive and discriminatory for UCP 3 over UCP 2, UCP 1 and other mitochondrial transporters. The peptide antibody detects UCP 3 expression in E. coli, COS cells and yeast expression systems. The peptide antibody detects a single approximately 33 kDa protein band in mitochondria from isolated rat skeletal muscle, mouse and rat brown adipose tissue, and in whole muscle groups (soleus and extensor digitorum longus) from mice. No 33 kDa band is detectable in isolated mitochondria from liver, heart, brain, kidney and lungs of rats, or gastrocnemius mitochondria from UCP 3 knock-out mice. From our data, we conclude that the peptide antibody is detecting UCP 3 in skeletal muscle, skeletal muscle mitochondria and brown adipose tissue mitochondria. It is also noteworthy that the peptide antibody can detect human, mouse and rat forms of UCP 3. Using the UCP 3 peptide antibody, we confirm and quantify the increased (2.8-fold) UCP 3 expression observed in skeletal muscle mitochondria isolated from 48-h-starved rats. We show that UCP 3 expression is increased (1.6-fold) in skeletal muscle of rats acclimated over 8 weeks to 8 degrees C and that UCP 3 expression is decreased (1.4-fold) in rats acclimated to 30 degrees C. Furthermore, UCP 3 expression is increased (2.3-fold) in skeletal muscle from hyperthyroid rats compared to euthyroid controls. In addition, we show that UCP 3 expression is only coincident with the mitochondrial fraction of skeletal muscle homogenates and not peroxisomal, nuclear or cytosolic and microsomal fractions.     

Tissue specific,sex and age--related differences in the 6-phosphogluconate dehydrogenase gene expression

Data presented in thid paper indicate that: (1) the age-related changes in 6-phosphogluconate dehydrogenase (6PGDH) activity depend on sex and tissue. No differences in the liver 6PGDH activity between young (1-month-old) males and females were found. In adult males, the activity was the same as in young animals but, in adult females, it reached the value twice as high as in the young. In adipose tissue (both white and brown) and kidney cortex, the enzyme activity decreased with age both in males and females. There were no differences between males and females 6PGDH activity in brain, heart and skeletal muscle. (2) The sex and age-related changes in the liver 6PGDH activity occur predominantly at the level of mRNA cellular concentration. (3) In the liver of ovariectomized rats decrease of 6PGDH activity and mRNA level was observed. Oestradiol administration to ovariectomized rats restored liver 6PGDH activity and liver 6PGDH mRNA levels to that observed in controls. No changes in 6PGDH activity and mRNA levels were found in white adipose tissue (WAT) of ovariectomized adult rats and in ovariectomized rats treated with oestradiol. (4) Oestradiol administration to males caused an increase of liver 6PGDH activity and mRNA levels to values observed in females, but was without an effect on WAT 6PGDH activity and mRNA level. (5) These results suggest that 6PGDH activity in different tissues is not regulated in coordinate fashion and that oestradiol plays an important role in the liver enzyme activity regulation.     

Food restriction improves glucose and lipid metabolism through Sirt1 expression: a study using a new rat model with obesity and severe hypertension

AimsTo determine the effects of food restriction (FR) on the expression of Sirt1 and its down-stream factors related to lipid and glucose metabolism in obese and hypertensive rats (SHRSP/IDmcr-fa), as a model of human metabolic syndrome.Main methodsMale, 10-week-old SHRSP/IDmcr-fa rats were treated with 85% FR for 2 weeks. Metabolic parameters, serum adipocytokines and distribution of serum adiponectin multimers were investigated. Sirt1 expression was determined in epididymal adipose tissue, liver and skeletal muscle. We also determined the expression of PPARα, γ and other adipocyte-related genes in epididymal adipose tissue, and glucose transporters (GLUT2 and GLUT4) in the liver and skeletal muscle.Key findingsFR improved the general conditions as well as blood chemistry of SHRSP/IDmcr-fa rats. In the epididymal adipose tissue of the FR rats, Sirt1 expression was enhanced, as was adiponectin, whereas leptin was downregulation, findings that were paralleled by the serum protein levels. Furthermore, the serum ratio of high to total adiponectin was increased in the FR group. The mRNA expression of Sirt1 was upregulated in the adipose tissue in the FR group. Sirt1 mRNA expression was downregulated, while PPARα and GLUT2 expression was enhanced in the liver. No differences were found in terms of Sirt1, PPAR or GLUT4 expression in skeletal muscle.SignificanceThese results indicate that FR corrects adipokine dysfunction by activating PPARγ via Sirt1 in adipose tissue. Furthermore, glucose and lipid metabolism are activated by upregulation of GLUT2 via the activation of PPARα in the liver.