Antiapoptosis and Mitochondrial Effect of Pioglitazone Preconditioning in the Ischemic/reperfused Heart of Rat

Purpose  Pioglitazone, used clinically in the treatment of type 2 diabetes mellitus, has been implicated as a regulator of cellular inflammatory and ischemic responses. The present study examined whether pioglitazone could inhibit cadiocyte apoptosis and reduce mitochondrial ultrastructure injury and membrane potential loss in the ischemic/reperfused heart of the rat. Furthermore, we investigated whether the protective effect of pioglitazone was related to opening of the mitochondrial_ATP-sensitive potassium channels. Methods  Adult male Sprague–Dawley rats were subjected to 30 min of ischemia followed by 4 h of reperfusion. At 24 h before ischemia, rats were randomized to receive 0.9% saline, 5-hydroxydecanoate (5-HD, 10 mg kg⁻¹, i.v.) plus pioglitazone (3 mg kg⁻¹, i.v.) or pioglitazone (3 mg kg⁻¹, i.v.). One group served as sham control. We investigated mitochondrial structure, apoptosis rate and Bcl-2, Bax and Caspase-3 proteins by immunohistochemistry staining. RT-PCR was used to determine the expression of P38MAPKmRNA and JNKmRNA. Western blotting was used to measure the expression of P38MAPK, JNK and NFκB P65. A second group of rats were randomly divided into sham-operated, ischemia/reperfusion (I/R), pioglitazone treatment, 5-HD + pioglitazone and 5-HD groups and the size of myocardial infarction was determined. Primary cultured cardiomyocytes of neonatal Sprague–Dawley rats were divided into control, hypoxia reoxygenation, different concentrations of pioglitazone and 5-HD + pioglitazone groups. JC-1 staining flowcytometry was used to examine mitochondrial membrane potential (ΔΨm). Results  Pioglitazone decreased mitochondrial ultrastructural damage compared to I/R, and reduced infarct size from 34.93 ± 5.55% (I/R) to 20.24 ± 3.93% (P < 0.05). Compared with the I/R group, the apoptosis rate and positive cell index (PCI) of Bax and Caspase-3 proteins in the pioglitazone group were significantly decreased (P < 0.05), while the PCI of Bcl-2 protein was increased (P < 0.05). There was no significant difference between the I/R and 5-HD + pioglitazone groups. Compared with the sham-operated group, the expression of P38MAPK mRNA, JNK mRNA and protein of P38MAPK, JNK and NFκB P65 in I/R was increased (P < 0.05). Pioglitazone did inhibit the increase in expressions vs I/R (P < 0.05). The rate of loss ΔΨm cells in the pioglitazone group was significantly lower than in the hypoxia reoxygenation group, while the addition of 5-HD inhibited the effect of pioglitazone. Conclusion  Pioglitazone inhibited cadiocyte apoptosis and reduced mitochondrial ultrastructure injury and membrane potential loss in the ischemic/reperfused heart of rat. These protective effects of pioglitazone may be related to opening mitochondrial_ATP-sensitive potassium channels.     

Effects of pioglitazone and metformin on NEFA-induced insulin resistance in type 2 diabetes

Aims/hypothesis  We sought to determine whether pioglitazone and metformin alter NEFA-induced insulin resistance in type 2 diabetes and, if so, the mechanism whereby this is effected. Methods  Euglycaemic–hyperinsulinaemic clamps (glucose ∼5.3 mmol/l, insulin ∼200 pmol/l) were performed in the presence of Intralipid–heparin (IL/H) or glycerol before and after 4 months of treatment with pioglitazone (n = 11) or metformin (n = 9) in diabetic participants. Hormone secretion was inhibited with somatostatin in all participants. Results  Pioglitazone increased insulin-stimulated glucose disappearance (p < 0.01) and increased insulin-induced suppression of glucose production (p < 0.01), gluconeogenesis (p < 0.05) and glycogenolysis (p < 0.05) during IL/H. However, glucose disappearance remained lower (p < 0.05) whereas glucose production (p < 0.01), gluconeogenesis (p < 0.05) and glycogenolysis (p < 0.05) were higher on the IL/H study day than on the glycerol study day, indicating persistence of NEFA-induced insulin resistance. Metformin increased (p < 0.001) glucose disappearance during IL/H to rates present during glycerol treatment, indicating protection against NEFA-induced insulin resistance in extrahepatic tissues. However, glucose production and gluconeogenesis (but not glycogenolysis) were higher (p < 0.01) during IL/H than during glycerol treatment with metformin, indicating persistence of NEFA-induced hepatic insulin resistance. Conclusions/interpretation  We conclude that pioglitazone improves both the hepatic and the extrahepatic action of insulin but does not prevent NEFA-induced insulin resistance. In contrast, whereas metformin prevents NEFA-induced extrahepatic insulin resistance, it does not protect against NEFA-induced hepatic insulin resistance. B. R. Landau died after the completion of this study.     

Adiponectin receptor genes: mutation screening in syndromes of insulin resistance and association studies for type 2 diabetes and metabolic traits in UK populations

Aims/hypothesis Adiponectin is an adipokine with insulin-sensitising and anti-atherogenic properties. Several reports suggest that genetic variants in the adiponectin gene are associated with circulating levels of adiponectin, insulin sensitivity and type 2 diabetes risk. Recently two receptors for adiponectin have been cloned. Genetic studies have yielded conflicting results on the role of these genes and type 2 diabetes predisposition. In this study we aimed to evaluate the potential role of genetic variation in these genes in syndromes of severe insulin resistance, type 2 diabetes and in related metabolic traits in UK Europid populations. Materials and methods Exons and splice junctions of the adiponectin receptor 1 and 2 genes (ADIPOR1; ADIPOR2) were sequenced in patients from our severe insulin resistance cohort (n=129). Subsequently, 24 polymorphisms were tested for association with type 2 diabetes in population-based type 2 diabetes case–control studies (n=2,127) and with quantitative traits in a population-based longitudinal study (n=1,721). Results No missense or nonsense mutations in ADIPOR1 and ADIPOR2 were detected in the cohort of patients with severe insulin resistance. None of the 24 polymorphisms (allele frequency 2.3–48.3%) tested was associated with type 2 diabetes in the case–control study. Similarly, none of the polymorphisms was associated with fasting plasma insulin, fasting and 2-h post-load plasma glucose, 30-min insulin increment or BMI. Conclusions/interpretation Genetic variation in ADIPOR1 and ADIPOR2 is not a major cause of extreme insulin resistance in humans, nor does it contribute in a significant manner to type 2 diabetes risk and related traits in UK Europid populations. Electronic supplementary material The online version of this article () contains supplementary material, which is accessible to authorised users.     

Genes involved in mitochondrial biogenesis/function are induced in response to bilio-pancreatic diversion in morbidly obese individuals with normal glucose tolerance but not in type 2 diabetic patients

Aims/hypothesis  The mechanisms allowing normalisation of insulin sensitivity and reversal of type 2 diabetes after bilio-pancreatic diversion (BPD) have not been elucidated. We studied whether the expression of genes relevant to mitochondrial biogenesis/function is induced in response to BPD and whether the response differs between morbidly obese patients with normal glucose tolerance (NGT) and patients with type 2 diabetes. Methods  The effect of stable weight reduction after BPD on metabolic variables and expression of nuclear genes encoding for mitochondrial proteins or regulators of mitochondrial function was investigated in skeletal muscle. Insulin sensitivity was assessed by euglycaemic–hyperinsulinaemic clamp and substrate oxidation by indirect calorimetry. Results  Both NGT and type 2 diabetic patients showed a net improvement of insulin sensitivity, with the latter also showing blood glucose normalisation. NGT patients had a large increase in glucose oxidation and substantial reduction in lipid oxidation. In contrast, type 2 diabetic patients had a blunted response to BPD in terms of glucose oxidation. NGT patients showed increased expression of genes encoding mitofusin 2, porin or citrate synthase; no significant changes were detected in diabetic patients. The expression of genes regulating mitochondrial activity (PGC-1β [also known as PPARGC1B], PGC-1α [also known as PPARGC1A], PPARδ [also known as PPARD], SIRT1) was induced only in NGT patients. Conclusions/interpretation  These findings indicate that weight loss after BPD exerts a beneficial effect on insulin sensitivity via mechanisms that are independent of the expression of genes involved in mitochondrial biogenesis/activity. Furthermore, the observation that gene expression is not altered with weight loss in type 2 diabetic patients while it is induced in NGT patients suggests a heritable component. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorised users. G. Mingrone and A. Zorzano share senior authorship.     

NIA0004Unsaturated fatty acids diminish the detrimental effects of saturated fatty acids on insulin signalling in human muscle by decreasing de novo ceramide production

Insulin resistance in skeletal muscle is associated with impaired oxidative capacity and reduced size, number and function of mitochondria. Insulin‐resistant individuals have lower adiponectin concentrations, a characteristic predating the development of type‐2 diabetes (T2D). The aim of this study was to test the potential role of adiponectin in mitochondrial bioenergetics in individuals predisposed to develop T2D, in adiponectin KO mice and in primary muscle cell culture. Individuals with a family history of T2D displayed lower plasma adiponectin concentration (P = 0.03), reduced PGC1β (P = 0.04) and mtFAM (P = 0.03) mRNA, lower mitochondrial content (P = 0.006), citrate synthase (P = 0.02) and (‐HAD (P = 0.03) activity, suggesting defective mitochondrial bioenergetics in skeletal muscle. In addition, AdipoR1 was the principle correlate of mitochondrial content (r² = 0.81), suggesting an important role in mitochondrial biogenesis. Knock out of adiponectin in mice was associated with low PGC1α and PPARδ mRNA (both, P < 0.05), reduced mitochondrial content (P < 0.05) and COX II activity (P < 0.05) and markers for type‐1 oxidative fibers in skeletal muscle. This suggests that mitochondrial function is dependent on circulating adiponectin. In primary cultures of human myotubes, treatment with adiponectin induced AMPKK/ AMPK activity, PGC1α protein, mitochondrial biogenesis (P < 0.05), palmitate oxidation (P < 0.05), citrate synthase (P < 0.05) and SOD activity (P < 0.05), and reduced mitochondrial membrane potential and the production of ROS (P < 0.05). The inhibition of adiponectin receptor expression by siRNA or of AMPK by a pharmacological agent blunted the induction in mitochondrial function. Our findings indicate a novel pathway in skeletal muscle by which adiponectin increase mitochondrial number and function and exerts an insulin sensitizing effect.     

Pioglitazone does not enhance the effectiveness of lifestyle modification in preventing conversion of impaired glucose tolerance to diabetes in Asian Indians: results of the Indian Diabetes Prevention Programme-2 (IDPP-2)

Aims/hypothesis  The objective of this prevention programme was to study whether combining pioglitazone with lifestyle modification would enhance the efficacy of lifestyle modification in preventing type 2 diabetes in Asian Indians with impaired glucose tolerance. Methods  In a community-based, placebo-controlled 3 year prospective study, 407 participants with impaired glucose tolerance (mean age 45.3 ± 6.2 years, mean BMI 25.9 ± 3.3 kg/m²) were sequentially grouped to receive either: lifestyle modification plus pioglitazone, 30 mg (n = 204) or lifestyle modification plus placebo (n = 203). The participants and investigators were blinded to the assignment. The primary outcome was development of diabetes. Results  At baseline, both groups had similar demographic, anthropometric and biochemical characteristics. At year 3, the response rate was 90.2%. The cumulative incidence of diabetes was 29.8% with pioglitazone and 31.6% with placebo (unadjusted HR 1.084 [95% CI 0.753–1.560], p = 0.665). Normoglycaemia was achieved in 40.9% and 32.3% of participants receiving pioglitazone and placebo, respectively (p = 0.109). In pioglitazone group, two deaths and two non-fatal hospitalisations occurred due to cardiac problems; in the placebo group there were two occurrences of cardiac disease. Conclusions/interpretation  Despite good adherence to lifestyle modification and drug therapy, no additional effect of pioglitazone was seen above that achieved with placebo. The effectiveness of the intervention in both groups was comparable with that of lifestyle modification alone, as reported from the Indian Diabetes Prevention Programme-1. The results are at variance with studies that showed significant relative risk reduction in conversion to diabetes with pioglitazone in Americans with IGT. An ethnicity-related difference in the action of pioglitazone in non-diabetic participants may be one explanation. Trial registration: ClinicalTrials.gov NCT00276497 Funding: This study was funded by the India Diabetes Research Foundation     

The effect of moderate alcohol consumption on adiponectin oligomers and muscle oxidative capacity: a human intervention study

Aims/hypothesis The aim of this study was to investigate whether moderate alcohol consumption increases plasma high molecular weight (HMW) adiponectin and/or muscle oxidative capacity. Materials and methods Eleven lean (BMI 18–25 kg/m²) and eight overweight (BMI ≥27 kg/m²) men consumed 100 ml whisky (∼32 g alcohol) or water daily for 4 weeks in a randomised, controlled, crossover trial. After each treatment period, muscle biopsies and fasting blood samples were collected. Results Adiponectin concentrations increased (p < 0.001) by 12.5% after 4 weeks of moderate alcohol consumption. Moderate alcohol consumption tended to increase HMW adiponectin by 57% (p = 0.07) and medium molecular weight adiponectin by 12.5% (p = 0.07), but not low molecular weight (LMW) adiponectin. Skeletal muscle citrate synthase, cytochrome c oxidase and β-3-hydroxyacyl coenzyme A dehydrogenase (β-HAD) activity were not changed after moderate alcohol consumption, but an interaction between alcohol consumption and BMI was observed for cytochrome c oxidase (p = 0.072) and citrate synthase (p = 0.102) activity. Among lean men, moderate alcohol consumption tended to increase cytochrome c oxidase (p = 0.08) and citrate synthase activity (p = 0.12) by 23 and 26%, respectively, but not among overweight men. In particular, plasma HMW adiponectin correlated positively with activities of skeletal muscle citrate synthase (r = 0.64, p = 0.009), cytochrome c oxidase (p = 0.59, p = 0.009) and β-HAD (r = 0.46, p = 0.056), while such correlation was not present for LMW adiponectin. Whole-body insulin sensitivity and intramyocellular triacylglycerol content were not affected by moderate alcohol consumption. Conclusions/interpretation Moderate alcohol consumption increases adiponectin concentrations, and in particular HMW adiponectin. Concentrations of HMW adiponectin in particular were positively associated with skeletal muscle oxidative capacity.     

Serum leptin, adiponectin, and resistin concentration in colorectal adenoma and carcinoma (CC) patients

Introduction  Leptin, adiponectin, and resistin are the proteins secreted by adipocytes, which affects the metabolism. While the role of leptin in colon carcinogenesis is documented, the effect of adiponectin and resistin remains unclear. It has been indicated that while leptin may potentiate the cancer cells growth, adiponectin and resistin may act oppositely. Aim  The aim of this study is to determine the concentration of leptin, adiponectin, and resistin in patients with adenomatous polyps and colorectal cancer. Methods  The serum concentration investigated adipohormones had been measured with ELISA in 37 patients with colorectal adenomas, 36 with colorectal cancer (CC) and in 25 controls with no colorectal pathology. Endoscopically removed polyps and CC biopsies had been evaluated with histopathology. Mean BMI value was calculated for all patients. Results  Among 37 adenomas, 25 revealed high-grade dysplasia (HGD) and 12 low-grade dysplasia (LGD). All cases of CC were adenocarcinomas. No difference in the level of investigated adipohormones in serum between patients with HGD and LGD polyps was observed. The serum concentration of leptin and adiponectin in CC patients was lower than in patients with adenomas (p < 0.05; p < 0.05, respectively) as well as in controls (p < 0.01; p < 0.05, respectively). The concentration of resistin in CC was not significantly different in the adenoma group (p > 0.05) but higher than in controls (p < 0.05). There was a correlation between adiponectin and leptin serum concentration (r = 0.61). Conclusion  We conclude that serum concentration of adiponectin and resistin may play an important role in colon carcinogenesis. We also assume that leptin may possibly have the prognostic value useful in clinical practice and its concentration is independent of BMI value.     

Metformin-treated patients with type 2 diabetes have normal mitochondrial complex I respiration

Aims/hypothesis  

The glucose-lowering drug metformin has been shown to inhibit complex I of the mitochondrial electron transport chain in skeletal muscle. To investigate this effect in vivo we studied skeletal muscle mitochondrial respiratory capacity and content from patients with type 2 diabetes treated with metformin (n = 14) or sulfonylurea (n = 8) and healthy control (n = 18) participants.     

Association analysis of Notch pathway signalling genes in diabetic nephropathy

Aims/hypothesis  

Several studies have provided compelling evidence implicating the Notch signalling pathway in diabetic nephropathy. Co-regulation of Notch signalling pathway genes with GREM1 has recently been demonstrated and several genes involved in the Notch pathway are differentially expressed in kidney biopsies from individuals with diabetic nephropathy. We assessed single-nucleotide polymorphisms (SNPs; n = 42) in four of these key genes (JAG1, HES1, NOTCH3 and ADAM10) for association with diabetic nephropathy using a case–control design.     

Polymorphisms in the gene encoding adiponectin receptor 1 are associated with insulin resistance and high liver fat

Aims/hypothesis The adipokine adiponectin has insulin-sensitising, anti-atherogenic and anti-inflammatory properties. Recently, the genes for mouse and human adiponectin receptor-1 (ADIPOR1) and -2 (ADIPOR2) have been cloned. The aim of this study was to investigate whether genetic variants of the genes encoding ADIPOR1 and ADIPOR2 play a role in human metabolism.Materials and methods We screened ADIPOR1 and ADIPOR2 for polymorphisms and determined their association with glucose metabolism, lipid metabolism, an atherogenic lipid profile and inflammatory markers in 502 non-diabetic subjects. A subgroup participated in a longitudinal study; these subjects received diet counselling and increased their physical activity.Results We identified six variants of ADIPOR1 and seven variants of ADIPOR2. A single-nucleotide polymorphism (SNP) in the putative promoter region 8503 bp upstream of the translational start codon (–8503 G/A) of ADIPOR1 (frequency of allele A=0.31) was in almost complete linkage disequilibrium with another SNP (–1927 T/C) in intron 1. Subjects carrying the –8503 A and –1927 C alleles had lower insulin sensitivity, as estimated from a 75 g OGTT (p=0.04) and determined during a euglycaemic clamp (n=295, p=0.04); they also had higher HbA₁c levels (p=0.02) and, although the difference was not statistically significant, higher liver fat (n=85, determined by proton magnetic resonance spectroscopy, p=0.056) (all p values are adjusted for age, sex and percentage of body fat). In the longitudinal study (n=45), the –8503 A and –1927 C alleles were associated with lower insulin sensitivity (p=0.03) and higher liver fat (p=0.02) at follow-up compared with the –8503 G and –1927 T alleles, independently of basal measurements, sex and baseline and follow-up percentage of body fat.Conclusions/interpretation The present findings suggest that the –8503 G/A SNP in the promoter or the –1927 T/C SNP in intron 1 of ADIPOR1 may affect insulin sensitivity and liver fat in humans.Electronic Supplementary Material Supplementary material is available for this article at .     

Effects of peroxisome proliferator-activated receptor (PPAR)-α and PPAR-γ agonists on glucose and lipid metabolism in patients with type 2 diabetes mellitus

Aims/hypothesis The aim of the study was to examine the effects of pioglitazone (PIO), a peroxisome proliferator-activated receptor (PPAR)-γ agonist, and fenofibrate (FENO), a PPAR-α agonist, as monotherapy and in combination on glucose and lipid metabolism. Subjects and methods Fifteen type 2 diabetic patients received FENO (n = 8) or PIO (n = 7) for 3 months, followed by the addition of the other agent for 3 months in an open-label study. Subjects received a 4 h hyperinsulinaemic–euglycaemic clamp and a hepatic fat content measurement at 0, 3 and 6 months. Results Following PIO, fasting plasma glucose (FPG) (p < 0.05) and HbA_1c (p < 0.01) decreased, while plasma adiponectin (AD) (5.5 ± 0.9 to 13.8 ± 3.5 μg/ml [SEM], p < 0.03) and the rate of insulin-stimulated total-body glucose disposal (R _d) (23.8 ± 3.8 to 40.5 ± 4.4 μmol kg⁻¹ min⁻¹, p < 0.005) increased. After FENO, FPG, HbA_1c, AD and R _d did not change. PIO reduced fasting NEFA (784 ± 53 to 546 ± 43 μmol/l, p < 0.05), triacylglycerol (2.12 ± 0.28 to 1.61 ± 0.22 mmol/l, p < 0.05) and hepatic fat content (20.4 ± 4.8 to 10.2 ± 2.5%, p < 0.02). Following FENO, fasting NEFA and hepatic fat content did not change, while triacylglycerol decreased (2.20 ± 0.14 to 1.59 ± 0.13 mmol/l, p < 0.01). Addition of FENO to PIO had no effect on R _d, FPG, HbA_1c, NEFA, hepatic fat content or AD, but triacylglycerol decreased (1.61 ± 0.22 to 1.00 ± 0.15 mmol/l, p < 0.05). Addition of PIO to FENO increased R _d (24.9 ± 4.4 to 36.1 ± 2.2 μmol kg⁻¹ min⁻¹, p < 0.005) and AD (4.1 ± 0.8 to 13.1 ± 2.5 μg/ml, p < 0.005) and reduced FPG (p < 0.05), HbA_1c (p < 0.05), NEFA (p < 0.01), hepatic fat content (18.3 ± 3.1 to 13.5 ± 2.1%, p < 0.03) and triacylglycerol (1.59 ± 0.13 to 0.96 ± 0.9 mmol/l, p < 0.01). Muscle adenosine 5′-monophosphate-activated protein kinase (AMPK) activity did not change following FENO; following the addition of PIO, muscle AMPK activity increased significantly (phosphorylated AMPK:total AMPK ratio 1.2 ± 0.2 to 2.2 ± 0.3, p < 0.01). Conclusions/interpretation We conclude that PPAR-α therapy has no effect on NEFA or glucose metabolism and that addition of a PPAR-α agonist to a PPAR-γ agent causes a further decrease in plasma triacylglycerol, but has no effect on NEFA or glucose metabolism.     

Absence of an association between the polymorphisms in the genes encoding adiponectin receptors and type 2 diabetes

Aims/hypothesis Secreted by adipocytes, adiponectin is a hormone that acts as an antidiabetic and anti-atherogenic adipokine. We recently cloned the genes encoding two adiponectin receptors (ADIPOR1 and ADIPOR2). The aim of this study was to examine whether ADIPOR1 and/or ADIPOR2 play a major role in genetic susceptibility to insulin resistance or type 2 diabetes in the Japanese population.Methods By direct sequencing and a search of public databases, we identified single nucleotide polymorphisms (SNPs) in ADIPOR1 and ADIPOR2, and investigated whether these SNPs are associated with insulin resistance and type 2 diabetes in the Japanese population.Results The linkage disequilibrium (LD) in the chromosomal region of ADIPOR1 was almost completely preserved, whereas the LD in ADIPOR2 was less well preserved. None of the SNPs in ADIPOR1 or ADIPOR2 were significantly associated with insulin resistance or type 2 diabetes. No differences in ADIPOR1 or ADIPOR2 haplotype frequencies were observed between type 2 diabetic and non-diabetic subjects.Conclusions/interpretation Genetic variations in ADIPOR1 or ADIPOR2 are unlikely to lead to a common genetic predisposition to insulin resistance or type 2 diabetes in the Japanese population.Electronic supplementary material Supplementary material is available for this article at .     

Mitochondrial cytochrome <Emphasis Type="Italic">c</Emphasis> release: a factor to consider in mitochondrial disease?

Summary  The pathogenesis of mitochondrial disorders has largely focused on the impairment of cellular energy metabolism. However, mitochondrial dysfunction has also been implicated as a factor in the initiation of apoptosis due to the translocation of cytochrome c, from mitochondria to the cytosol, and the subsequent cleavage of pro-caspase 3. In this study, we determined the cytochrome c content of cytosols (skeletal muscle) prepared from 22 patients with evidence of compromised mitochondrial electron transport chain enzyme activity and 26 disease controls. The cytochrome c content of the mitochondrial electron transport chain-deficient group was found to be significantly (p < 0.02) elevated when compared with the control group (63.7 ± 15.5 versus 27.7 ± 2.5 ng/mg protein). Furthermore, a relationship between the cytosolic cytochrome c content of skeletal muscle and complex I and complex IV activities was demonstrated. Such data raise the possibility that mitochondrial cytochrome c release may be a feature of mitochondrial disorders, particularly for those patients with marked deficiencies of respiratory chain enzymes. Whether initiation of apoptosis occurs as a direct consequence of this cytochrome c release has not been fully evaluated here. However, for one patient with the greatest documented cytosolic cytochrome c content, caspase 3 could be demonstrated in the cytosolic preparation. Further work is required in order to establish whether a relationship also exists between caspase 3 formation and the magnitude of respiratory chain deficiency. Competing interests: None declared     

Impact of the peroxisome proliferator activated receptor-γ coactivator-1β (<Emphasis Type="Italic">PGC-1β</Emphasis>) Ala203Pro polymorphism on in vivo metabolism, <Emphasis Type="Italic">PGC-1β</Emphasis> expression and fibre type composition in human skeletal muscle

Aims/hypothesis Peroxisome proliferator activated receptor-γ coactivator-1β (PGC-1β, also known as PPARGC1B) expression is reduced in skeletal muscle from patients with type 2 diabetes mellitus and in elderly subjects. Ala203Pro, a common variant in the PGC-1β gene is associated with obesity. The aim of this study was to investigate whether the PGC-1β Ala203Pro polymorphism influences the age-related decline in skeletal muscle PGC-1β expression, in vivo metabolism and markers for muscle fibre type composition. Materials and methods The PGC-1β Ala203Pro polymorphism was genotyped in 110 young (age 28.0 ± 1.9 years) and 86 elderly (age 62.4 ± 2.0 years) twins and related to muscle PGC-1β expression, in vivo metabolism and markers for fibre type composition. Results Insulin-stimulated non-oxidative glucose metabolism (NOGM; p = 0.025) and glycolytic flux rate (GF; p = 0.026) were reduced in young Ala/Ala carriers compared with carriers of a 203Pro allele. In addition, a regression analysis, correcting for covariates, showed that the PGC-1β 203Pro allele was positively related to insulin-stimulated NOGM and GF in the young twins. While muscle expression of PGC-1β was reduced in elderly compared with young carriers of the Ala/Ala genotype (p ≤ 0.001), there was no significant age-related decline in PGC-1β expression in carriers of the 203Pro allele (p ≥ 0.4). However, a regression analysis, correcting for covariates, showed that only age was significantly related to muscle PGC-1β expression. Finally, PGC-1β expression correlated positively with markers for oxidative fibres in human muscle. Conclusions/interpretation This study suggests that young carriers of a PGC-1β 203Pro allele have enhanced insulin-stimulated glucose metabolism and may be protected against an age-related decline in PGC-1β expression in muscle. C. Ling and L. Wegner contributed equally to this study.     

Curcumin ameliorates impaired insulin/IGF signalling and memory deficit in a streptozotocin-treated rat model

Increased serum insulin levels and reduced peripheral insulin activities seen in insulin resistance syndrome are associated with age-dependent cognitive impairment and Sporadic Alzheimer’s Disease (SAD), suggesting a disturbance in the insulin signalling system in the brain and possibly being one of the causes of dementia. Therefore, the streptozotocin (STZ)-induced animal may be an appropriate model for the investigation of SAD and related dementia. This study was designed to investigate the beneficial effect of Curcumin (CUR), a neuroprotective agent, on intracerebroventricular (ICV) STZ-induced cognitive impairment in rats. For this purpose, adult male Wistar rats were bilaterally ICV injected with STZ (3 mg/kg). An artificial cerebrospinal fluid (aCSF) was given to the control group (SHAM) instead of STZ on days 1 and 3. Learning and memory performance were assessed using the “passive avoidance task” and the “Morris water maze test”. After confirmation of acquisition impairment with these tests, the STZ group was divided into two subgroups: STZ + vehicle (Vh) and STZ + CUR. The rats in the SHAM and STZ + Vh groups were administered intraperitoneally with 0.5 ml Vh and the rats in the STZ + CUR group were treated intraperitoneally with CUR (300 mg kg⁻¹ day⁻¹ in Vh) for 10 days starting from the 25th day after STZ injection. The Morris water maze test was reapplied on the 35th day after STZ injection and all of the rats were sacrificed on day 36 for quantitation of IGF-1 and for histopathological evaluation. Rats in the STZ + CUR group were found to have a higher performance in cognitive tests than rats in the STZ + Vh group (P < 0.01). In parallel with the cognitive tests, IGF-1 levels were decreased in all of the STZ-injected groups (1.78 ± 0.34) compared to the SHAM group (3.46 ± 0.41). In contrast, CUR treatment significantly increased IGF-1 levels (P < 0.001). The degree of neuronal loss decreased after CUR treatment compared to the SHAM group (P < 0.02). These results clearly indicate that CUR treatment is effective in reducing the cognitive impairment caused by STZ in rats, and may be a potential therapeutic agent for altering neurodegeneration in SAD.     

Supraphysiological hyperinsulinaemia is necessary to stimulate skeletal muscle protein anabolism in older adults: evidence of a true age-related insulin resistance of muscle protein metabolism

Aims/hypothesis  The physiological increase in muscle protein anabolism induced by insulin is blunted in healthy, glucose-tolerant older adults. We hypothesised that the age-related defect in muscle protein anabolism is a true insulin resistance state and can be overridden by supraphysiological hyperinsulinaemia. Methods  We used dye dilution, stable isotopic and immunoblotting techniques to measure leg blood flow, muscle protein synthesis, protein kinase B/mammalian target of rapamycin (Akt/mTOR) signalling, and amino acid kinetics in 14 healthy, glucose-tolerant older volunteers at baseline, and during an insulin infusion at postprandial (PD, 0.15 mU min⁻¹ 100 ml⁻¹) or supraphysiologically high (HD, 0.30 mU min⁻¹ 100 ml⁻¹) doses. Results  Leg blood flow, muscle protein synthesis, and Akt/mTOR signalling were not different at baseline. During hyperinsulinaemia, leg blood flow (p < 0.01) and muscle protein synthesis increased in the HD group only (PD [%/h]: from 0.063 ± 0.006 to 0.060 ± 0.005; HD [%/h]: from 0.061 ± 0.007 to 0.098 ± 0.007; p < 0.01). Muscle Akt phosphorylation increased in both groups, but the increase tended to be greater in the HD group (p = 0.07). The level of p70 ribosomal S6 kinase 1 (S6K1) phosphorylation increased in the HD group only (p < 0.05). Net amino acid balance across the leg improved in both groups, but a net anabolic effect was observed only in the HD group (p < 0.05). Conclusions/interpretation  We conclude that supraphysiological hyperinsulinaemia is necessary to stimulate muscle protein synthesis and anabolic signalling in healthy older individuals, suggesting the existence of a true age-related insulin resistance of muscle protein metabolism.     

PPARγ ligands, rosiglitazone and pioglitazone, inhibit bFGF- and VEGF-mediated angiogenesis

Objective  To study the effect of peroxisome proliferator-activated receptor-gamma (PPARγ) agonists, pioglitazone and rosiglitazone, on vascular endothelial growth factor (VEGF)- and basic fibroblast growth factor (bFGF)-induced angiogenesis and on endothelial cell migration. Methods  Chick chorioallantoic membrane (CAM) model was used to evaluate the efficacy of pioglitazone and rosiglitazone on VEGF- and bFGF-induced angiogenesis. In addition, the effect of pioglitazone and rosiglitazone on endothelial cell migration was evaluated using 8 mm pore filter to a feeder layer containing vitronectin as chemoattractant. Results  Pioglitazone and rosiglitazone inhibited the pro-angiogenic effects of bFGF and VEGF in the CAM model significantly (P < 0.001) to the same extent. Endothelial cell migration was also inhibited by both pioglitazone and rosiglitazone (P < 0.001). Conclusions  These results suggest that PPARγ ligands, pioglitazone and rosiglitazone, in addition to their important regulatory role in adipogenesis and inflammation, possess anti-angiogenic properties. Thus, PPARγ ligands may be useful in the treatment of diabetic retinopathy, macular degeneration, and other ocular disorders and may lower the risk to develop cancer in diabetic patients.