SIRT1 and other sirtuins in metabolism

Objective FTO (Fat mass and obesity-associated) is associated with increased risk of obesity and type 2 diabetes incurrence. Studies have shown that the expression of FTO protein in skeletal muscle and adipose tissue is related to the oxidation rate of whole body substrate. With the increase of age, the body's carbohydrate oxidation rate decreases, the fat oxidation rate increases, and at the meanwhile the expression of FTO protein in skeletal muscle decreases and that in adipose increases. HIIT is very helpful for inhibiting obesity, insulin resistance and type 2 diabetes. So the purpose of this study is to investigate the effect of HIIT exercise on the expression of FTO protein in rats and its relationship to glucose and fat metabolism.
 Methods 20 Male, 3-week-old SD rats were randomly divided into two groups, each group has 10 rats. C group: sedentary; HIIT group: high-intensity intermittent training group (85% ~ 90% VO2max exercise for 6min, 50% VO2max exercise interval 4min, repeated 6 times. 5 times/week ,4 weeks). All subjects were maintained in a free facility with constant temperature of 25°C, light-dark cycle of 12/12 h and free access to water. 48 hours after the last exercise, all samples were taken with an overnight fast. The expression of FTO protein in skeletal muscle and adipose tissue was measured by Western Blot. Serum insulin was tested by ELISA; Estimation of blood glucose was tested by Glucose oxidase method.
 Results 1.The expression of FTO protein in skeletal muscle was significantly higher than that of group C (P <0.01); The expression of FTO protein in adipose tissue of HIIT group was significantly lower than that of group C (P <0.05); 2. Serum insulin levels of group HIIT was significantly lower than that of group C (p <0.01); And the blood glucose of group HIIT was significantly lower than that of group C (p <0.01). 3. Serum LDL-C of group HIIT was significantly lower than that of group C (p <0.01), and serum HDL-C of group HIIT was significantly higher than that of group C (p <0.01);4. Correlation analysis showed that serum insulin level was negatively correlated with skeletal muscle FTO protein expression (R = -0.454, p < 0.05). Correlation analysis showed that serum LDL-C levels was positively correlated with adipose tissue FTO protein expression (R=0.559, p < 0.05) and serum HDL-C levels was negatively correlated with adipose tissue FTO protein expression (R=-0.474, p < 0.05).
 Conclusions 1. HIIT can increase the protein expression of FTO in rat skeletal muscle and decrease the expression of FTO protein in adipose tissue; 2. HIIT can regulate glucose metabolism and lipid metabolism in rats; 3. The regulation of glucose metabolism by HIIT may be related to the increase of FTO protein expression in skeletal muscle. The regulation of lipid metabolism may be related to the reduction of FTO protein expression in adipose tissue.

Depletion of GLUT4, the primary glucose transporter protein in adipose tissue and skeletal muscle, is reported to contribute to insulin resistance in pregnancy or diabetes. To examine this phenomenon, the expression of GLUT4 protein was assessed by Western blotting in streptozotocin-induced diabetic pregnant rats. In adipose tissue, relative to control, it was decreased by 30% in the normal pregnant group (p<0.001), by 37% in the diabetic nonpregnant group (p<0.01) and by 65% in the diabetic pregnant group (p<0.001). On the other hand, no significant variation was evident among the groups in skeletal muscle. To assess the mechanisms responsible for depletion of GLUT4 protein in adipose tissue, we quantitated levels of GLUT4 mRNA with a RNase protection assay. It was decreased by 44% in the normal pregnant group (p<0.05) and by 55% in the diabetic pregnant group (p<0.05), but not altered in the diabetic nonpregnant group. These results suggest that the depletion of GLUT4 protein in adipose tissue is a factor contributing to insulin resistance in pregnancy or diabetes, especially when the two states exist in combination.

Protein-mediated trans-membrane and intracellular fatty acid trafficking are becoming increasingly recognised as biochemically and physiologically important concepts. Obesity and insulin resistance are polygenic disorders, heavily influenced by environmental and life-style factors, and are virtually always associated with disturbed fatty acid metabolism in adipose and other tissues. The aim of this study was to investigate mRNA expression levels of fatty-acid-handling proteins in adipose tissue in relation to markers of genetic and acquired obesity and insulin resistance. We quantified mRNA expression of subcutaneous adipose tissue fatty-acid-handling proteins (ALBP, KLBP, FATP1, FATP4, CD36, ACS1) in 17 monozygotic twin-pairs with a range of intra-pair differences (Delta) in BMI and detailed measures of obesity and insulin resistance, allowing influences of genetic and non-genetic factors to be distinguished. In acquired obesity FATP4 expression was up-regulated independently of genetic background (DeltaFATP4 versus DeltaBMI; r=0.50, p=0.04; DeltaFATP4 versus Deltabody fat; r=0.59, p=0.01). Similarly, CD36 and FATP1 expression correlated with acquired differences in HDL cholesterol and non-esterified fatty acid concentrations respectively. Moreover, FATP4 and CD36 expression levels correlated with measures of obesity and insulin resistance that are influenced by both genetic and non-genetic factors (FATP4 versus BMI: r=0.53, p=0.0001; FATP4 versus body fat: r=0.51, p=0.002; FATP4 versus homeostasis model assessment [HOMA]: r=0.49, p=0.001; CD36 versus BMI: r=0.50, p=0.02; CD36 versus body fat: r=0.63, p=0.001; CD36 versus HOMA: r=0.34, p=0.06). These findings indicate that expression of specific adipose tissue fatty-acid-handling proteins is related to obesity and insulin resistance, and that, in particular, FATP4 plays a role in acquired obesity. Our results suggest that facilitated fatty acid trafficking is a physiologically and pathologically relevant phenomenon in man.

Mitochondrial enzyme expression is reduced in adipose tissue from old mice, yet little is known regarding mechanisms that could be mediating, or interventions that could be used, to reverse these changes. The purpose of this study was to examine the relationship between lipolytic and fatty acid reesterification enzymes, 5' adenosine monophosphate-activated protein kinase and mitochondrial proteins in adipose tissue from young versus old mice. A second aim was to determine whether metformin treatment could rescue the age-associated decline in adipose tissue mitochondrial proteins. Approximately 22-month-old male C57BL/6 mice were fed a diet with or without 0.5% metformin for 8 weeks. Compared with young mice (~11 wk of age), the protein content/phosphorylation of hormone-sensitive lipase, adipose tissue triglyceride lipase, and phosphoenolpyruvate carboxykinase were reduced in old mice. This was paralleled by increases in the plasma nonesterified fatty acid:glycerol ratio and reductions in adipose tissue 5' adenosine monophosphate-activated protein kinase activity and select mitochondrial proteins in old mice. There were no differences in these variables when comparing adipose tissue from young and 6-month-old mice. While metformin improved glucose homeostasis, it did not increase 5' adenosine monophosphate-activated protein kinase phosphorylation or mitochondrial enzymes. Our findings demonstrate a co-ordinated down regulation of lipolytic, reesterification, and mitochondrial enzymes in adipose tissue with aging that is unresponsive to metformin treatment.

What is the central question of this study? What is the relationship between proteins in skeletal muscle and adipose tissue determined at rest and at peak rates of fat oxidation in men and women? What is the main finding and its importance? The resting contents of proteins in skeletal muscle involved in triglyceride hydrolysis and mitochondrial lipid transport were more strongly associated with peak fat oxidation rates than proteins related to lipid transport or hydrolysis in adipose tissue. Although females displayed higher relative rates of fat oxidation than males, this was not explained by the proteins measured in this study, suggesting that other factors determine sex differences in fat metabolism. We explored key proteins involved in fat metabolism that might be associated with peak fat oxidation (PFO) and account for sexual dimorphism in fuel metabolism during exercise. Thirty-six healthy adults [15 women; 40±11years of age; peak oxygen consumption 42.5±9.5ml(kg body mass)-1 min-1 ; mean±SD] completed two exercise tests to determine PFO via indirect calorimetry. Resting adipose tissue and/or skeletal muscle biopsies were obtained to determine the adipose tissue protein content of PLIN1, ABHD5 (CGI-58), LIPE (HSL), PNPLA2 (ATGL), ACSL1, CPT1B and oestrogen receptorα (ERα) and the skeletal muscle protein content of FABP 3 (FABPpm), PNPLA2 (ATGL), ACSL1, CTP1B and ESR1 (ERα). Moderate strength correlations were found between PFO [in milligrams per kilogram of fat-free mass (FFM) per minute] and the protein content of PNPLA2 (ATGL) [rs =0.41 (0.03-0.68), P<0.05] and CPT1B [rs =0.45 (0.09-0.71), P<0.05] in skeletal muscle. No other statistically significant bivariate correlations were found consistently. Females had a greater relative PFO than males [7.1±1.9 vs. 4.5±1.3 and 7.3±1.7 vs. 4.8±1.2mg(kg FFM)-1 min-1 in the adipose tissue (n=14) and skeletal muscle (n=12) subgroups, respectively (P<0.05)]. No statistically significant sex differences were found in the content of these proteins. The regulation of PFO might involve processes relating to intramyocellular triglyceride hydrolysis and mitochondrial fatty acid transport, and adipose tissue is likely to play a more minor role than muscle. Sex differences in fat metabolism are likely to be attributable to factors other than the resting content of proteins in skeletal muscle and adipose tissue relating to triglyceride hydrolysis and fatty acid transport.

Although obesity is a risk factor for development of type 2 diabetes and chemical modification of proteins by advanced glycoxidation and lipoxidation end products is implicated in the development of diabetic complications, little is known about the chemical modification of proteins in adipocytes or adipose tissue. In this study we show that S-(2-succinyl)cysteine (2SC), the product of chemical modification of proteins by the Krebs cycle intermediate, fumarate, is significantly increased during maturation of 3T3-L1 fibroblasts to adipocytes. Fumarate concentration increased > or =5-fold during adipogenesis in medium containing 30 mm glucose, producing a > or =10-fold increase in 2SC-proteins in adipocytes compared with undifferentiated fibroblasts grown in the same high glucose medium. The elevated glucose concentration in the medium during adipocyte maturation correlated with the increase in 2SC, whereas the concentration of the advanced glycoxidation and lipoxidation end products, N(epsilon)-(carboxymethyl)lysine and N(epsilon)-(carboxyethyl)lysine, was unchanged under these conditions. Adipocyte proteins were separated by one- and two-dimensional electrophoresis and approximately 60 2SC-proteins were detected using an anti-2SC polyclonal antibody. Several of the prominent and well resolved proteins were identified by matrix-assisted laser desorption ionization time-of-flight/time-of-flight mass spectrometry. These include cytoskeletal proteins, enzymes, heat shock and chaperone proteins, regulatory proteins, and a fatty acid-binding protein. We propose that the increase in fumarate and 2SC is the result of mitochondrial stress in the adipocyte during adipogenesis and that 2SC may be a useful biomarker of mitochondrial stress in obesity, insulin resistance, and diabetes.

To investigate the expression of eNOS and iNOS mRNA and protein in adipose tissue from subcutaneous (s.c.) and omental adipose tissue of obese subjects. Subcutaneous and omental adipose tissue was obtained from subjects undergoing weight reduction surgery. Messenger RNA and protein levels were measured in tissue extracts and related to basal lipolysis, which was measured in isolated adipocytes from the same subjects. Eight overweight but otherwise healthy male subjects (age 43.4+/-10.3 y, BMI 39+/-3.5 kg/m(2), mean+/-s.e.m.). For mRNA detection a competitive reverse transcription polymerase chain reaction method was used while protein was detected by Western blot. Glycerol release was determined in isolated adipocytes using a standard luminometric assay. Tissue mRNA levels for eNOS in s.c. tissue were 6098+/-1969 amol/mg RNA and in omental tissue 6987+/-2914 amol/mg RNA (mean+/-s.e.m., P=0.75). iNOS mRNA levels were substantially lower; in s.c. tissue 227+/-127 amol/mg RNA and in omental tissue 245+/-162 amol/mg RNA (P=0.8). In Western blot, eNOS protein levels in s.c. and omental tissue were 1.88+/-2.0 and 7.47+/-4.11 (OD/mm(2) 100 microg total protein, P=0.0063), respectively. iNOS protein was expressed at significantly lower levels and barely detectable in both s.c. and omental tissue. Basal rate of lipolysis was two times higher in s.c. compared to omental fat cells (P=0.028). eNOS protein is markedly increased in omental compared to s.c. adipose tissue in human obese subjects, probably due to post-transcriptional mechanisms. Since basal lipolysis is much lower in omental vs s.c. adipose tissue it is possible that regionally increased NO production, primarily by eNOS, may be involved in the site difference of basal lipolysis in obese subjects.

PurposeThe aim of this study was to investigate the relationship between gestational diabetes mellitus (GDM) and Chemerin by analyzing chemerin levels in peripheral blood and cord blood, and chemerin mRNA and its protein expression in placenta and adipose tissue.MethodsA case-control study was conducted in 110 women with GDM and 110 controls without GDM who received regular prenatal services and delivered at Shanghai Pudong New Area Healthcare Hospital for Women and Children from June 2019 to December 2020.ResultsThe age, pre-pregnancy weight, pre-pregnancy BMI, antepartum BMI, TG/HDL ratio and TG levels in pregnant women with GDM were significantly higher than those in women without GDM, and HDL levels were significantly lower than those in the normal group. Chemerin in the umbilical cord blood of the GDM group was significantly higher than in that of the normal group, but there was no difference in chemerin levels in peripheral blood. In the two groups, the chemerin concentration in peripheral blood was significantly higher than that in umbilical cord blood (P<0.001). The Chemerin mRNA and protein expression levels in the placenta and adipose tissue of pregnant women in the GDM group were significantly higher than those in the normal group (P <0.001). In the GDM group, the expression of chemerin protein in adipose tissue was significantly higher than that in placental tissue. Regression analysis showed that the expression level of chemerin protein in placental tissue and adipose tissue was positively correlated with the risk of GDM.ConclusionElevated chemerin is closely related to the risk of GDM, and the placenta may be an important secretion of chemotactic factor sources in addition to adipose tissue and participate in the development of GDM.

Background: PPAR-γ and PRDM16 proteins have key role in the metabolism of adipose tissue and the conversion of white tissue to brown adipose tissue. But, the role of exercise on these two important proteins has not been studied in subcutaneous adipose tissue. Objective: The aim of this study was to investigate the effect of continuous training on the level of PPAR-γ and PRDM16 proteins in the adipose tissue in overweight male Sprague-Dawley rats with diabetes. Methods: In this study, 16 two-month old Sprague-Dawley rats with an average weight of 270±20 g were selected and randomly divided into two groups: control (n=8) and continuous training (n=8). The training group exercised according to the training program 4 days a week for 8 weeks while the control group did not have a training program. Independent t-test was used to analyze the data. Findings: There was a significant increase in the expression of PPAR-γ (P=0.004) and PRDM16 (P=0.0001) proteins in the training group compared to control group. Conclusion: Considering the increase of PPAR-γ and PRDM16 proteins in adipose tissue after continuous exercise and the important role of these two proteins in the fat metabolism, aerobic exercise can be an important mechanism for reducing this tissue in obese individuals and converting white tissue to brown. Keywords: Adipose tissue, Rats, Exercise, PPAR gamma, PRDM16

Expression of GLUT4 Glucose Transporter mRNA and Protein in Skeletal Muscle and Adipose Tissue from Rats in Late Pregnancy

Adipocyte Maturation Arrest: A Determinant of Systemic Insulin Resistance to Glucose Disposal

The purpose of this project was to provide a profile of DNA, RNA, and protein content in adipose tissue, which is relatively understudied in humans, to gain more insight into the amount of tissue that may be required for various analyses. Skeletal muscle tissue was also investigated to provide a direct comparison into potential differences between these two highly metabolically active tissues. Basal adipose and skeletal muscle tissue samples were obtained from 10 (7 M, 3 W) recreationally active participants [25 ± 1 yr; 84 ± 3 kg, maximal oxygen consumption (V̇o2max): 3.5 ± 0.2 L/min, body fat: 29 ± 2%]. DNA, RNA, and protein were extracted and subsequently analyzed for quantity and quality. DNA content of adipose and skeletal muscle tissue was 52 ± 14 and 189 ± 44 ng DNA·mg tissue-1, respectively (P < 0.05). RNA content of adipose and skeletal muscle tissue was 46 ± 14 and 537 ± 72 ng RNA·mg tissue-1, respectively (P < 0.05). Protein content of adipose and skeletal muscle tissue was 4 ± 1 and 177 ± 10 µg protein·mg tissue-1, respectively (P < 0.05). In summary, human adipose had 28% of the DNA, 9% of the RNA, and 2% of the protein found in skeletal muscle per mg of tissue. This information should be useful across a wide range of human clinical investigation designs and various laboratory analyses.NEW & NOTEWORTHY This investigation studied DNA, RNA, and protein contents of adipose and skeletal muscle tissues from young active individuals. A series of optimization steps were investigated to aid in determining the optimal approach to extract high-yield and high-quality biomolecules. These findings contribute to the knowledge gap in adipose tissue requirements for molecular biology assays, which is of increasing importance due to the growing interest in adipose tissue research involving human exercise physiology research.

The effect of catecholamines on the levels of S-100 protein and nervous system-specific enolase (NSE) in epididymal adipose tissue of Wistar rats in vivo was examined by sensitive enzyme immunoassay methods. Soluble S-100 protein levels in the adipose tissue of 9-12-week-old rats (1.46 +/- 0.19 microgram/mg protein) were decreased to less than 50% of those of controls by serial injection (for 4-7 days) of epinephrine (0.1 mg/day) or norepinephrine (0.15 mg) with, however, little effect on the levels of membrane-bound (pentanol-extractable) S-100 protein. A significant decrease in the soluble S-100 protein levels was observed at 2 h after a single injection of epinephrine (1.04 +/- 0.13 microgram/mg protein). On the other hand, levels of NSE subunit (gamma subunit or 14-3-2 protein) in adipose tissue (0.51 +/- 0.03 gamma gamma-equivalent pmol/mg protein) were increased to 170% of control by serial injection (for 7 days) of epinephrine or norepinephrine with little change of the level of enolase alpha subunit on a mg protein basis. Isoproterenol had no apparent effect on the levels of soluble S-100 protein and NSE subunit. These results suggest that the levels of S-100 protein and NSE in adipose tissue are regulated by catecholamines.

Measuring body composition and regional fat mass accurately

Myricetin is a biologically active natural polyphenol with beneficial effects on metabolic health. This study aimed to examine the effects of myricetin on the expression levels of genes involved in lipolysis and mitochondrial respiration in adipocytes and the anti-obesity potential of myricetin. The results indicated that myricetin reduced triglyceride (TG) content and increased mitochondrial content and oxygen consumption rate (OCR) in adipocytes in vitro. To determine anti-obesity effect of myricetin, C57BL6/J mice were fed a high-fat diet (HFD) for eight weeks and then treated with myricetin (10 mg/kg) for 2 weeks. The in vivo treatment of myricetin reduced body weight by 11%. Furthermore, it improved the glucose tolerance, and increased fatty acid consumption of HFD-fed mice. Myricetin treatment increased Sirt3 expression and reduced the acetylation of mitochondrial proteins in adipose tissue. Finally, the knockdown of Sirt3 in adipocytes reduced the myricetin-induced increase in mitochondrial oxygen consumption rate by about 27% compared to controls. Our results indicated that myricetin exerted anti-obesity effects through the upregulation of Sirt3 expression and mitochondrial metabolism in adipose tissue.