Rosiglitazone (PPARgamma-agonist) attenuates atherogenesis with no effect on hyperglycaemia in a combined diabetes-atherosclerosis mouse model

Background: The administration of peroxisome proliferator-activated receptor γ (PPARγ) agonists to low-density lipoprotein (LDL)-receptor-deficient mice resulted in a reduction in the atherosclerotic lesion area in male mice, but not in female mice. The male mice also exhibited reduction in insulin resistance while the female mice did not. To further examine the relationship between PPARγ agonists, insulin resistance and atherosclerosis, we used the model of accelerated atherosclerosis in male apolipoprotein E (apoE)-deficient mice rendered diabetic by low-dose streptozotocin (STZ).
Methods: Male, apoE-deficient mice ( n  = 48) were randomly divided into four groups. To induce diabetes, two groups received low-dose STZ and two groups served as controls. After diabetes induction, rosiglitazone (a PPARγ agonist) was administered by oral gavage to one of the diabetic and one of the non-diabetic groups.
Results: Rosiglitazone reduced significantly the atherosclerotic aortic plaque area in both diabetic and non-diabetic apoE-deficient mice: 340 ± 54 vs. 201 ± 27 μmol² (p = 0.001) in diabetic mice; 243 ± 22 vs. 158 ± 27 μmol² (p = 0.001) in non-diabetic mice. Also, rosiglitazone reduced the correlation coefficient between plasma glucose and the degree of atherosclerosis (p < 0.0025) without affecting plasma glucose levels. The rosiglitazone-treated mice, both diabetic and non-diabetic, had higher lipid levels.
Conclusions: Rosiglitazone-treated animals showed less atherosclerosis despite higher lipid levels and similar glucose levels. These data suggest a direct anti-atherogenic effect of rosiglitazone on the arterial wall.     

Effects of combined PPARgamma and PPARalpha agonist therapy on reverse cholesterol transport in the Zucker diabetic fatty rat

Aim:  We investigated the effects of the combined therapy of PPARγ and PPARα agonists on HDL metabolism, especially concerning reverse cholesterol transport (RCT), using Zucker diabetic fatty rats (ZDF/Crl-Lepr^fa rats) that are the rodent model for type 2 diabetes with obesity, hyperlipidaemia and insulin resistance.
Methods:  The ZDF rats were divided into four medicated groups as follows: pioglitazone as a PPARγ agonist (5 mg/kg/day; P group, n = 6), fenofibrate as a PPARα agonist (30 mg/kg/day; F group, n = 6), both these medications (P + F group, n = 6) and no treatment (UNT group, n = 6). Non-diabetic rats (ZDF/GmiCrl-lean, CON group, n = 6) served as controls. We evaluated HDL particle size and messenger RNA (mRNA) levels of the following factors: liver X receptor α (L × R α), ATP-binding cassette A1 (ABCA1) and ABCG1 which are regulated by PPARs and are related to early stage RCT.
Results:  The significant increase in HDL particle size was demonstrated in rats of the F and P + F groups, although changes in plasma HDL-cholesterol levels were not significant. In accordance with this finding, mRNA levels of ABCG1 in the liver increased significantly.
Conclusions:  These findings suggest the efficacy of combined therapy with PPARγ and PPARα in improving lipid metabolism, partly through the enhanced RCT, and insulin resistance in type 2 diabetes mellitus.     

Role of peroxisome proliferator-activated receptor-gamma (PPARgamma) during liver regeneration in rats

Background and Aim:  Peroxisome proliferator-activated receptor-gamma (PPARγ), a member of the nuclear receptor superfamily, is widely expressed in adipocytes and other tissues, including the liver. Several reports have shown that PPARγ activation induced cell-cycle arrest and apoptosis in tumor cells. We investigated the role of the PPARγ/ligand system and the effect of the PPARγ agonist during liver regeneration.
Methods:  Expression of PPARγ and serum levels of 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) by enzyme immunoassay were evaluated in rats following partial hepatectomy (PH group). Further, the effect of the PPARγ agonist, pioglitazone, on liver regeneration (PH + PGZ group) was evaluated by proliferating cell nuclear antigen labeling index, relative liver weight, and expression of cell-cycle regulators.
Results:  The number of PPARγ-stained hepatocytes decreased at 24 h (PH, 15.8 ± 2.2%; sham, 35.5 ± 2.4%; P  < 0.001) and increased in the late phase of liver regeneration compared to the sham-operated group ( P  < 0.001 at 48–120 h). The peaks of serum 15d-PGJ2 (627.0 ± 91.1 pg/ml) and PPARγ expression (90.6 ± 3.1%) coincided in the late phase of liver regeneration. Also, oral administration of pioglitazone inhibited hepatocyte proliferation, in terms of the proliferating cell nuclear antigen (PCNA) labeling index and p27 expression during the late phase of liver regeneration, and caused a transient reduction in liver mass when compared to the PH group.
Conclusions:  These results indicate that the PPARγ/ligand system may be one of the key negative regulators of hepatocyte proliferation and may be responsible for the inhibition of liver growth in the late phase of liver regeneration.     

Effects of rosiglitazone and oleic acid on UCP-3 expression in L6 myotubes

Aims: The regulation of uncoupling protein-3 (UCP-3) expression in muscle remains unclear, specifically in relation to dietary and drug treatments. The present study evaluated the effects of oleic acid and rosiglitazone on UCP-3 mRNA expression in differentiated L6 myotubes.
Methods: L6 myocytes were cultured and differentiated prior to exposure to rosiglitazone 10 µmol/l, oleic acid 100 µmol/l, or the combination, for 24 h, prior to semiquantitative evaluation of UCP-3 mRNA relative to GAPDH mRNA by RT-PCR.
Results: Exposure to oleic acid produced a significant increase in UCP-3 mRNA (0.012 ± 0.007 vs. 0.0011 ± 0.0006 for untreated cells, relative to GAPDH mRNA, p < 0.001). Rosiglitazone alone had no effect on UCP-3 expression and nor did the glitazone affect oleic-acid-induced upregulation of UCP-3.
Conclusions: In L6 myotubes, 24-h exposure to oleic acid produced a 10-fold increase in UCP-3 mRNA expression, but rosiglitazone had no effect. Oleic-acid-induced upregulation of UCP-3 was not affected (positively or negatively) by glitazone exposure.     

Administration of myostatin does not alter fat mass in adult mice

Aim:   Myostatin, a member of the TGF-beta superfamily, is produced by skeletal muscle and acts as a negative regulator of muscle mass. It has also been suggested that low-dose administration of myostatin (2 μg/day) in rodents can reduce fat mass without altering muscle mass. In the current study, we attempted to further explore the effects of myostatin on adipocytes and its potential to reduce fat mass, since myostatin administration could potentially be a useful strategy to treat obesity and its complications in humans.
Methods:   Purified myostatin protein was examined for its effects on adipogenesis and lipolysis in differentiated 3T3-L1 adipocytes as well as for effects on fat mass in wild-type, myostatin null and obese mice.
Results:   While myostatin was capable of inhibiting adipogenesis in 3T3-L1 cells, it did not alter lipolysis in fully differentiated adipocytes. Importantly, pharmacological administration of myostatin over a range of doses (2–120 μg/day) did not affect fat mass in wild-type or genetically obese (ob/ob, db/db) mice, although muscle mass was significantly reduced at the highest myostatin dose.
Conclusions:   Our results suggest that myostatin does not reduce adipose stores in adult animals. Contrary to prior indications, pharmacological administration of myostatin does not appear to be an effective strategy to treat obesity in vivo .     

Multiple molecular targets underlie the antidiabetic effect of Nigella sativa seed extract in skeletal muscle, adipocyte and liver cells

Aim: Nigella sativa ( N. sativa ) is a plant widely used in traditional medicine of North African countries. During the last decade, several studies have shown that extracts from the seeds of N. sativa have antidiabetic effects.
Methods: Our group has recently demonstrated that N. sativa seed ethanol extract (NSE) induces an important insulin-like stimulation of glucose uptake in C2C12 skeletal muscle cells and 3T3-L1 adipocytes following an 18 h treatment. The purpose of the present study was to elucidate the pathways mediating this insulin-like effect and the mechanisms through which these pathways are activated.
Results: Results from western immunoblot experiments indicate that in C2C12 cells as well as in H4IIE hepatocytes, but not in 3T3-L1 cells, NSE increases activity of Akt, a key mediator of the effects of insulin, and activity of AMP-activated protein kinase (AMPK), a master metabolic regulating enzyme. To test whether the activation of AMPK resulted from a disruption of mitochondrial function, the effects of NSE on oxygen consumption were assessed in isolated liver mitochondria. NSE was found to exhibit potent uncoupling activity.
Conclusion: Finally, to provide an explanation for the effects of NSE in adipocytes, PPARγ stimulating activity was tested using a reporter gene assay. Results indicate that NSE behaves as an agonist of PPARγ. The data supports the ethnobotanical use of N. sativa seed oil as a treatment for diabetes, and suggests potential uses of this product, or compounds derived thereof, against obesity and the metabolic syndrome.     

PPARγ Pro12Ala Pro/Pro and resistin SNP-420 G/G genotypes are synergistically associated with plasma resistin in the Japanese general population

Objective  The Ala allele of the Pro12Ala polymorphism (rs1801282) of peroxisome proliferator-activated receptor γ (PPARγ) is protective against type 2 diabetes (T2DM). Resistin, secreted from adipocytes, causes insulin resistance in rodents. Resistin gene expression is reduced by the PPARγ ligand. We previously reported that subjects with the G/G genotype of a resistin gene single nucleotide polymorphism (SNP) at –420 (rs1862513) had the highest circulating resistin levels, followed by C/G and C/C. The aim of this study was to determine the relationship among PPARγ Pro12Ala polymorphism, resistin SNP-420, and plasma resistin.
Design, patients and measurements  We cross-sectionally analysed 2077 community-dwelling subjects attending an annual medical check-up. Genotypes were determined by TaqMan analysis. Fasting plasma resistin was measured using ELISA.
Results  Plasma resistin appeared to be higher in subjects with the Pro/Pro genotype of PPARγ than those with Pro/Ala and Ala/Ala genotypes (mean ± SE, 11·6 ± 0·2 vs. 10·4 ± 0·5 μg/l). Multiple regression analysis, adjusted for age, gender, BMI, and resistin SNP-420, revealed that the Pro/Pro genotype was a positive predictor of plasma resistin (PPARγ ,  Pro/Pro vs. Pro/Ala + Ala/Ala, unstandardized regression coefficient (β) = 1·03, P  = 0·0384). The effects of the Pro/Pro genotype of PPARγ (Pro/Pro vs. Pro/Ala + Ala/Ala) and the G/G genotype of resistin SNP-420 (G/G vs. C/C) on plasma resistin were synergistic (β = 4·76, P  = 0·011).
Conclusions  The PPARγ Pro12Ala Pro/Pro and resistin SNP-420 G/G genotypes were synergistically associated with plasma resistin, when adjusted for age, gender, and BMI, in the Japanese general population.     

Insulin-sensitizing effect of rosiglitazone (BRL-49653) by regulation of glucose transporters in muscle and fat of Zucker rats.

Thiazolidinediones (TZDs), a class of antidiabetic agents, are specific agonists of peroxisome proliferator activator receptor (PPARgamma). However, their mechanisms of action, and the in vivo target tissues that mediate insulin sensitization are not well understood. The aim of this study was to investigate the role of glucose transporters (GLUT-1 and GLUT-4) in the TZD insulin-sensitizer action. The effects of rosiglitazone treatment were studied using Zucker (fa/fa) rats after 7 days of oral dosing (3.6 mg/kg/d). Rosiglitazone lowered (approximate 80%) basal plasma insulin levels in obese rats and substantially corrected (approximately 50%) insulin resistance based upon results from hyperinsulinemic euglycemic clamp studies. GLUT-4 protein levels were reduced (approximately 75%) in adipose tissue of obese rats and treatment with rosiglitazone normalized them. Interestingly, GLUT-1 protein content was increased in adipose tissue ( thick approximate 150%) and skeletal muscle (approximately 50%) of obese rats and treatment with rosiglitazone increased it even more by 5.5-fold in fat and by 2.5-fold in muscle. Consistent with these results, basal (GLUT-1-mediated) transport rate of 3-O-methyl-D-glucose into isolated epitrochlearis muscle was elevated in response to rosiglitazone. Incubation of fully differentiated 3T3-L1 adipocytes with the drug for 7 days increased the levels of GLUT-1 protein, but did not affect GLUT-4 levels. In conclusion, rosiglitazone may improve insulin resistance in vivo by normalizing GLUT-4 protein content in adipose tissue and increasing GLUT-1 in skeletal muscle and fat. While the drug has a direct effect on GLUT-1 protein expression in vitro without a direct effect on GLUT-4 suggests that direct and indirect effects of rosiglitazone on glucose transporters may have an important role in improving insulin resistance in vivo.     

Peroxisome proliferator-activated receptor γ (PPAR) agonism reduces the insulin-stimulated increase in circulating interleukin-6 in GH replaced GH-deficient adults

Context  Peroxisome proliferator-activated receptor γ (PPARγ) agonists modify cardiovascular risk factors and inflammatory markers in patients with type 2 diabetes. GH treatment in GH-deficient (GHD) patients may cause insulin resistance and exerts ambiguous effects on inflammatory markers.
Objective  To investigate circulating markers of inflammation and endothelial function in GH replaced GHD patients before and after 12 weeks administration of either pioglitazone 30 mg/day ( N  = 10) or placebo ( N  = 10) in a randomized double-blind parallel design.
Methods  Circulating levels of interleukins (ILs)-1β, IL-2, IL-4, IL-6, IL-8, IL-10, tumour necrosis factor (TNF)-α, high sensitivity C-reactive protein, vascular cell adhesion molecule-I, and osteoprotegerin (OPG) were measured in the basal state and after a 2·5 h hyperinsulinaemic euglycaemic clamp.
Results  Insulin sensitivity improved in the group receiving PPARγ agonist ( P =  0·03). Serum IL-6 levels increased by 114 ± 31% (mean ± SE) in the entire group ( N  = 20) following the hyperinsulinaemic euglycaemic clamp ( P =  0·01) performed at study start. Twelve weeks of PPARγ agonist treatment significantly abrogated this insulin-stimulated increment in IL-6 levels compared to placebo ( P =  0·01). Furthermore PPARγ agonist treatment significantly lowered basal IL-4 levels ( P <  0·05).
Conclusions  (i) IL-6 levels increase during a hyperinsulinaemic clamp in GH replaced patients (ii) This increase in IL-6 is abrogated by PPARγ agonist treatment (iii) we hypothesize that PPARγ agonist-induced improvement of insulin sensitivity may obviate a compensatory rise in IL-6.     

Acetate and propionate short chain fatty acids stimulate adipogenesis via GPCR43

It has recently been discovered that G protein-coupled receptors (GPCR) 41 and 43 are characterized by having the short chain fatty acids acetate and propionate as their ligands. The objective of this study was to investigate the involvement of GPCR41, GPCR43, and their ligands in the process of adipogenesis. We measured the levels of GPCR41 and GPCR43 mRNA in both adipose and other tissues of the mouse. GRP43 mRNA expression was higher in four types of adipose tissue than in other tissues, whereas GPCR41 mRNA was not detected in any adipose tissues. A high level of GPCR43 expression was found in isolated adipocytes, but expression level was very low in stromal-vascular cells. Expression of GPCR43 was up-regulated in adipose tissues of mice fed a high-fat diet compared with those fed a normal-fat diet. GPCR43 mRNA could not be detected in confluent and undifferentiated 3T3-L1 adipocytes; however, the levels rose with time after the initiation of differentiation. GPCR41 expression was not detected in confluent and differentiated adipocytes. Acetate and propionate treatments increased lipids present as multiple droplets in 3T3-L1 adipocytes. Propionate significantly elevated the level of GPCR43 expression during adipose differentiation, with up-regulation of PPAR-gamma2. Small interfering RNA mediated a reduction of GPCR43 mRNA in 3T3-L1 cells and blocked the process of adipocyte differentiation. In addition, both acetate and propionate inhibited isoproterenol-induced lipolysis in a dose-dependent manner. We conclude that acetate and propionate short chain fatty acids may have important physiological roles in adipogenesis through GPCR43, but not through GPCR41.     

Differential expression of a novel seven transmembrane domain protein in epididymal fat from aged and diabetic mice.

To identify novel seven transmembrane domain proteins from 3T3-L1 adipocytes, we used PCR to amplify 3T3-L1 adipocyte complementary DNA (cDNA) with primers homologous to the N- and C-termini of pancreatic glucagon-like peptide-1 (GLP-1) receptor. We screened a cDNA library prepared from fully differentiated 3T3-L1 adipocytes using a 500-bp cDNA PCR product probe. Herein describes the isolation and characterization of a 1.6-kb cDNA clone that encodes a novel 298-amino acid protein that we termed TPRA40 (transmembrane domain protein of 40 kDa regulated in adipocytes). TPRA40 has seven putative transmembrane domains and shows little homology with the known GLP-1 receptor or with other G protein-coupled receptors. The levels of TPRA40 mRNA and protein were higher in 3T3-L1 adipocytes than in 3T3-L1 fibroblasts. TPRA40 is present in a number of mouse and human tissues. Interestingly, TPRA40 mRNA levels were significantly increased by 2- to 3-fold in epididymal fat of 24-month-old mice vs. young controls as well as in db/db and ob/ob mice vs. nondiabetic control littermates. No difference in TPRA40 mRNA levels was observed in brain, heart, skeletal muscle, liver, or kidney. Furthermore, no difference in TPRA40 expression was detected in brown fat of ob/ob mice when compared with age-matched controls. Taken together, these data suggest that TPRA40 represents a novel membrane-associated protein whose expression in white adipose tissue is altered with aging and type 2 diabetes.     

Depot-specific regulation of perilipin by rosiglitazone in a diabetic animal model

Treatment with rosiglitazone, a potent peroxisome proliferator-activated receptor (PPAR) gamma agonist, results in lipid storage coupled with reduced release of free fatty acids into the circulation. Many studies have reported that PPAR-gamma agonists increase subcutaneous adiposity but have no effect on visceral fat mass. Perilipin, a family of phosphoproteins that coat intracellular lipid droplets in adipocytes, is essential for enlargement of lipid droplets. Recently, a functional PPAR-responsive element was identified within the murine perilipin gene. We hypothesized that the depot-specific regulation of perilipin by rosiglitazone may be associated with the fat-redistribution and insulin-sensitizing effects of rosiglitazone. After 6 weeks of rosiglitazone treatment in Otusuka Long-Evans Tokushima Fatty rats, an animal model of type 2 diabetes mellitus, we measured changes in adiposity, triglyceride content in liver and muscle, morphology of the pancreas, and perilipin messenger RNA and protein expression in adipose tissue. Rosiglitazone increased subcutaneous adiposity, decreased triglyceride content of liver and muscle, decreased plasma free fatty acids (2107 +/- 507 micromol/L in the placebo group vs 824 +/- 148 micromol/L in the rosiglitazone group; P < .05), and improved insulin resistance. The islets of placebo-treated rats showed hypertrophy and destruction, whereas the islets of rosiglitazone-treated rats showed hypertrophy, but the islet architecture remained intact. Perilipin messenger RNA and protein expression increased in subcutaneous fat, but did not change in visceral fat, after rosiglitazone treatment. In 3T3-L1 cells, rosiglitazone pretreatment decreased lipolysis and increased perilipin protein. In conclusion, increased perilipin expression in subcutaneous fat after rosiglitazone treatment is likely to be a mediator of reduced lipolysis, resulting in lipid storage in subcutaneous fat, fat redistribution, and insulin sensitization.     

氯化钴诱导低氧上调3T3-L1脂肪细胞单核细胞趋化蛋白1的表达

研究氯化钴体外模拟低氧对小鼠3T3-L1脂肪细胞单核细胞趋化蛋白1表达的影响.体外培养3T3-L1前脂肪细胞,并将其诱导分化为成熟脂肪细胞,油红O染色鉴定脂肪细胞分化程度和脂质积聚情况.氯化钴化学模拟脂肪细胞低氧环境,采用实时荧光定量PCR与蛋白免疫印迹法检测低氧诱导因子-1α的mRNA和蛋白水平;采用实时荧光定量PCR与酶联免疫吸附法检测单核细胞趋化蛋白1的mRNA和蛋白水平,并对两者的蛋白水平进行相关性分析.氯化钴处理分化成熟的3T3-L1脂肪细胞后,低氧诱导因子-1α在mRNA及蛋白水平均显著上调;同时发现单核细胞趋化蛋白1 mRNA的表达水平和培养液中单核细胞趋化蛋白1蛋白的分泌水平均升高,且低氧诱导因子-1α与单核细胞趋化蛋白1蛋白水平呈现线性相关( r=0.864,P＜0.01).氯化钴诱导低氧上调3T3-L1脂肪细胞单核细胞趋化蛋白1的表达,参与脂肪组织慢性低度炎症的发生.     

未知功能基因JAZF1过表达对3T3-L1脂肪细胞糖脂代谢的影响

目的 观察JAZF1基因(Juxtaposed with another zincfinger gene 1)过表达对3T3-L1脂肪细胞糖脂代谢相关基因的影响.方法 采用实时荧光定量PCR(RT-QPCR)法检测JAZF1 mRNA在健康C57BL/6J小鼠多种组织表达分布情况;构建JAZF1真核表达载体并瞬时转染3T3-L1细胞,RT-QPCR法检测JAZF1、糖脂代谢相关基因mRNA的表达;用Western印迹法测定各组细胞JAZF1蛋白水平;油红O染色比色法检测细胞内脂质积聚的变化.结果 转染48 h后,JAZF1转染组脂肪细胞中,JAZF1 mRNA及蛋白表达明显高于阴性对照和空载组,激素敏感脂肪酶(HSL)mRNA表达水平明显增加(P<0.05),脂肪酸合成酶(FAS)、乙酰辅酶A羧化酶(ACC)、类固醇调节元件结合蛋白1(SREBP1)mRNA相对表达量明显降低(均P<0.01),脂肪细胞甘油三酯酶(ATGL)、葡萄糖转运子1(GLUT1)、GLUT4 mRNA表达并无明显改变;油红O染色显示JAZF1转染组细胞内脂质积聚明显降低(P<0.05).结论 JAZFl在C57BL/6J小鼠多种组织均有表达,提示其可能在维持正常生理功能中起着一定作用.3T3-L1细胞过表达JAZF1可减少脂质合成、增加脂解,并可明显改善脂质积聚,可能是肥胖和糖尿病治疗的一个潜在靶点.     

Effects of cevoglitazar, a dual PPARα/γ agonist, on ectopic fat deposition in fatty Zucker rats

Aim:  By acting as both insulin sensitizers and lipid-lowering agents, dual-acting peroxisome proliferator-activated receptors α/γ (PPARα/γ) agonists may be used to improve glucose tolerance in type 2 diabetic patients without inducing adiposity and body weight gain. Here, in an animal model of obesity and insulin resistance, the metabolic response to cevoglitazar, a dual PPARα/γ, was characterized using a combination of in vivo and ex vivo magnetic resonance methodologies and compared to treatment effects of fenofibrate, a PPARα agonist, and pioglitazone, a PPARγ agonist.
Methods:  Four groups of fatty Zucker rats: (i) Vehicle; (ii) fenofibrate 150 mg/kg; (iii) pioglitazone 30 mg/kg; and (iv) cevoglitazar 5 mg/kg were investigated before and after treatment. Animals were fed a fat-enriched (54% kcal fat) diet for 6 weeks, 2 weeks high of fat–exposure alone followed by a 4-week dosing period.
Results and conclusions:  Cevoglitazar was as effective as pioglitazone at improving glucose tolerance. However, unlike pioglitazone, both fenofibrate and cevoglitazar reduced BW gain and adiposity, independent of food intake. All three treatment regimens normalized intramyocellular lipids. Metabolic profiling showed that in the muscle cevoglitazar improves the lipid profile via both PPARα- and PPARγ-mediated mechanisms. Pioglitazone reduced hepatic lipid accumulation, while cevoglitazar and fenofibrate reduced hepatic lipid concentration below baseline levels (p < 0.05). Metabolic profiling showed that in the liver, cevoglitazar functions largely through PPARα agonism resulting in increased β-oxidation. Cevoglitazar only induced small changes to the lipid composition of visceral fat. In subcutaneous fat, however, cevoglitazar induced changes similar to those observed with fenofibrate suggesting export of fatty acids from this depot.