Lonidamine extends lifespan of adult Caenorhabditis elegans by increasing the formation of mitochondrial reactive oxygen species.

Compounds that delay aging in model organisms may be of significant interest to antiaging medicine, since these substances potentially provide pharmaceutical approaches to promote healthy lifespan in humans. The aim of the study was to test whether pharmaceutical concentrations of the glycolytic inhibitor lonidamine are capable of extending lifespan in a nematodal model organism for aging processes, the roundworm Caenorhabditis elegans. Several hundreds of adult C. elegans roundworms were maintained on agar plates and fed E. coli strain OP50 bacteria. Lonidamine was applied to test whether it may promote longevity by quantifying survival in the presence and absence of the compound. In addition, several biochemical and metabolic assays were performed with nematodes exposed to lonidamine. Lonidamine significantly extends both median and maximum lifespan of C. elegans when applied at a concentration of 5 micromolar by 8% each. Moreover, the compound increases paraquat stress resistance, and promotes mitochondrial respiration, culminating in increased formation of reactive oxygen species (ROS). Extension of lifespan requires activation of pmk-1, an orthologue of p38 MAP kinase, and is abolished by co-application of an antioxidant, indicating that increased ROS formation is required for the extension of lifespan by lonidamine. Consistent with the concept of mitohormesis, lonidamine is capable of promoting longevity in a pmk-1 sensitive manner by increasing formation of ROS.

Genetic population structure, fitness variation and the importance of population history in remnant populations of the endangered plant Silene chlorantha (Willd.) Ehrh. (Caryophyllaceae).

Habitat fragmentation can lead to a decline of genetic diversity, a potential risk for the survival of natural populations. Fragmented populations can become highly differentiated due to reduced gene flow and genetic drift. A decline in number of individuals can result in lower reproductive fitness due to inbreeding effects. We investigated genetic variation within and between 11 populations of the rare and endangered plant Silene chlorantha in northeastern Germany to support conservation strategies. Genetic diversity was evaluated using AFLP techniques and the results were correlated to fitness traits. Fitness evaluation in nature and in a common garden approach was conducted. Our analysis revealed population differentiation was high and within population genetic diversity was intermediate. A clear population structure was supported by a Bayesian approach, AMOVA and neighbour-joining analysis. No correlation between genetic and geographic distance was found. Our results indicate that patterns of population differentiation were mainly caused by temporal and/or spatial isolation and genetic drift. The fitness evaluation revealed that pollinator limitation and habitat quality seem, at present, to be more important to reproductive fitness than genetic diversity by itself. Populations of S. chlorantha with low genetic diversity have the potential to increase in individual number if habitat conditions improve. This was detected in a single large population in the investigation area, which was formerly affected by bottleneck effects.

The phytochemical glaucarubinone promotes mitochondrial metabolism, reduces body fat, and extends lifespan of Caenorhabditis elegans.

Naturally occurring compounds that promote energy expenditure and delay aging in model organisms may be of significant interest, since these substances potentially provide pharmaceutical approaches to tackle obesity and promote healthy lifespan in humans. We aimed to test whether pharmaceutical concentrations of glaucarubinone, a cytotoxic and antimalarial quassinoid known from different species of the plant family Simaroubaceae, are capable of affecting metabolism and/or extending lifespan in a nematodal model organism for aging processes, the roundworm Caenorhabditis elegans. Adult C. elegans roundworms, maintained on agar plates, were fed with E. coli strain OP50 bacteria, and glaucarubinone was applied to the agar to test (i) whether it alters respiration rates and mitochondrial activity, (ii) whether it affects body fat content, and (iii) whether it may promote longevity by quantifying survival in the presence and absence of the compound. We have found that glaucarubinone induces oxygen consumption and reduces body fat content of C. elegans. Moreover and consistent with the concept of mitohormesis, glaucarubinone extends C. elegans lifespan when applied at a concentration of 1 or 10 nanomolar. Taken together, glaucarubinone is capable of reducing body fat and promoting longevity in C. elegans, tentatively suggesting that this compound may promote metabolic health and lifespan in mammals and possibly humans.

Prevalence of injection-meal interval usage and its association with variables of metabolic control in patients with Type 1 and Type 2 diabetes.

OBJECTIVE: Regular human insulin is usually recommended with an injection-meal interval. It is not known how many patients follow these recommendations and, of those who do, the injection-meal interval remains incompletely studied. We investigated the injection-meal interval in patients with Type 1 and Type 2 diabetes and the association with metabolic control in routine care. METHODS: Four hundred and seventy-one consecutive patients with Type 1 or Type 2 diabetes were interviewed to determine their injection-meal interval in a university outpatient clinic setting in Germany in 2006. Four hundred and thirty-three interviews were suitable for analysis (143 Type 1 diabetes, 290 Type 2 diabetes). HbA(1c) was Diabetes Control and Complications Trial adjusted. RESULTS: Among those with Type 1 diabetes, 27% 'always', 27% 'sometimes' and 46% 'never' used an injection-meal interval. Forty-three per cent of patients with Type 2 diabetes always used an injection-meal interval, 12% sometimes and 45% never. Among patients with Type 1 diabetes, there was no difference in HbA(1c) between those who always used an injection-meal interval (n=39, age 58 years, duration of diabetes 21.1 years, BMI 28.7 kg/m², HbA(1c) 7.50%/58 mmol/mol) compared with those who never used an injection-meal interval (n=66, age 47.3 years, duration of diabetes 17.4 years, BMI 27.3 kg/m², HbA(1c) 7.55%/59 mmol/mol). Among patients with Type 2 diabetes, HbA(1c) in those who always used an injection-meal interval (n = 124, age 65 years, duration of diabetes 13.8 years, BMI 32.6 kg/m², HbA(1c) 7.31%/56 mmol/mol) is 0.27% lower compared with those who never used an injection-meal interval (n=130, age 64.3 years, duration of diabetes 16 years, BMI 32.8 kg/m², HbA(1c) 7.58%/59 mmol/mol). CONCLUSION: Nearly half of insulin-treated patients do not use an injection-meal interval. We found no significant association between adherence to injection-meal interval and HbA(1c) in patients with Type 1 diabetes, but a slightly lower HbA(1c) in patients with Type 2 diabetes who always use an injection-meal interval.

Differential effects of resveratrol and SRT1720 on lifespan of adult Caenorhabditis elegans.

Resveratrol and SRT1720 have been shown to act as sirtuin activators that may ameliorate type 2 diabetes and metabolic diseases in mice. Moreover, resveratrol extends lifespan in model organisms like C. elegans, N. FURZERI, and possibly D. melanogaster. The aim of the study was to test whether pharmacological concentrations of resveratrol and SRT1720 are capable of extending lifespan in a nematodal model organism for aging processes, the roundworm Caenorhabditis elegans. Several hundreds of adult C. ELEGANS roundworms were maintained on agar plates and fed E. COLI strain OP50 bacteria. Resveratrol (5 micromolar, 500 nanomolar) or SRT1720 (1 micromolar, 100 nanomolar) was applied to the agar to test whether they may promote longevity by quantifying survival in the presence and absence of the respective compounds. At a dose of 5 micromolar, which is pharmacologically relevant and 20 times lower than previously published concentrations, resveratrol significantly extends C. elegans lifespan by 3.6% (mean lifespan) and 3.4% (maximum lifespan). By unexpected contrast, SRT1720, which was previously proposed to be several hundred times more active than resveratrol, did not extend lifespan at none of the concentrations tested. Thus, in the model organisms C. elegans, resveratrol is capable of promoting longevity at a concentration that pharmacologically relevant and 20 times lower than previously published doses. The sirtuin activator SRT1720 did not extend lifespan, suggesting that in C. elegans, some relevant effects of resveratrol cannot be mimicked by SRT1720.

Physical activity does not influence the effect of antioxidant supplementation at nutritional doses on the incidence of impaired fasting glucose: a 7.5 year post-hoc analysis from the SU.VI.MAX study.

Supplementation with high doses of antioxidant vitamins prevents the insulin-sensitizing effects of physical exercise. However, little is known whether antioxidant supplementation affects the incidence of impaired fasting glucose (IFG). Data from 8938 subjects included in a randomized controlled trial on supplementation with antioxidants vitamins and trace elements at nutritional doses (SU.VI.MAX) were used to examine the effects of antioxidants on incident IFG after 7.5 years of follow-up, with and without stratification for daily physical exercise. The odds-ratio (95% CI) for developing an IFG among study participants receiving antioxidant supplementation was 1.34 (0.90-1.97) (p=0.33), in comparison to placebo. This risk did not vary significantly according to physical activity level (p for homogeneity=0.10). Supplementation with trace elements and antioxidants at nutritional doses apparently does not affect the incidence of IFG irrespective of self-reported physical exercise habits.

Cannabinoid type 1 receptor blockade induces transdifferentiation towards a brown fat phenotype in white adipocytes.

AIM: The endocannabinoid (EC) system is a major component in the control of energy homeostasis. It mediates a positive energy balance via central and peripheral pathways. Blockade of the cannabinoid type 1 receptor induces weight reduction and improves cardiovascular risk factors in overweight patients. Cannabinoid receptor type 1 (CB1R)-deficient mice are resistant to diet-induced obesity. The mechanisms responsible for these effects remain only partially elucidated. We hypothesized peripheral effects via direct modulation of adipocyte function to be an integral part of EC action on energy metabolism and insulin sensitivity. METHODS: SV40 immortalized murine white and brown adipocytes were used for all experiments. We investigated the effect of CB1R blockade by stimulating the cells acutely and chronically with rimonabant, a selective antagonist for the CB1R, or by knocking down the receptor with small interfering RNA (siRNA). Changes in thermogenic mRNA and protein expression as well as mitochondrial biogenesis and function were assessed by real-time RT-PCR, immunoblotting, fluorescent staining techniques, electron microscopy and by measuring oxygen consumption. RESULTS: Acute and chronic blockade of the CB1R with the selective antagonist rimonabant or by siRNA in murine white adipocytes strongly induced the thermogenic uncoupling protein-1 (UCP-1). UCP-1 expression was increased in a time- and dose-dependent manner both at the RNA and protein level. Furthermore, this effect was paralleled by enhanced peroxisome proliferator-activated receptor gamma coactivator 1alpha (PGC-1alpha) expression. In accordance with these findings, AMP-activated protein kinase (AMPK) phosphorylation was also increased after rimonabant treatment. Mitochondria-specific fluorescent staining demonstrated an augmentation in the number of mitochondria. This was confirmed by electron microscopy images. Moreover, rimonabant treatment enhanced the cytochrome c oxidase activity and increased cellular oxygen consumption. CONCLUSIONS: Taken together, our data demonstrate that inhibition of peripheral CB1R action in adipocytes directly promotes transdifferentiation of white adipocytes into a mitochondria-rich, thermogenic brown fat phenotype. Enhanced thermogenesis and insulin sensitivity may represent a peripheral mechanism contributing to weight loss and improved glucose homeostasis in rimonabant-treated patients.

A beta cell-specific knockout of hormone-sensitive lipase in mice results in hyperglycaemia and disruption of exocytosis.

AIMS/HYPOTHESIS: The enzyme hormone-sensitive lipase (HSL) is produced and is active in pancreatic beta cells. Because lipids are known to play a crucial role in normal control of insulin release and in the deterioration of beta cell function, as observed in type 2 diabetes, actions of HSL in beta cells may be critical. This notion has been addressed in different lines of HSL knockout mice with contradictory results. METHODS: To resolve this, we created a transgenic mouse lacking HSL specifically in beta cells, and characterised this model with regard to glucose metabolism and insulin secretion, using both in vivo and in vitro methods. RESULTS: We found that fasting basal plasma glucose levels were significantly elevated in mice lacking HSL in beta cells. An IVGTT at 12 weeks revealed a blunting of the initial insulin response to glucose with delayed elimination of the sugar. Additionally, arginine-stimulated insulin secretion was markedly diminished in vivo. Investigation of the exocytotic response in single HSL-deficient beta cells showed an impaired response to depolarisation of the plasma membrane. Beta cell mass and islet insulin content were increased, suggesting a compensatory mechanism, by which beta cells lacking HSL strive to maintain normoglycaemia. CONCLUSIONS/INTERPRETATION: Based on these results, we suggest that HSL, which is located in close proximity of the secretory granules, may serve as provider of a lipid-derived signal essential for normal insulin secretion.

A cell-based high-throughput assay system reveals modulation of oxidative and nonoxidative glucose metabolism due to commonly used organic solvents.

A 96-well format screening system was generated to quantify changes in nonoxidative glucose metabolism and oxidative pyruvate metabolism. D-Glucose uptake from the supernatant media was quantified by the glucose oxidase method, and L-lactate production of cells was quantified by the lactate dehydrogenase method applied on supernatant media. Mitochondrial membrane potential was quantified using tetramethylrhodamine methyl ester (TMRM) fluorescence, and reactive oxygen species (ROS) formation was determined by quantification of dihydrodichlorofluorescein fluorescence. Adenosine triphosphate (ATP) content of myocytes was determined using the luciferin reaction, and cellular respiration was quantified using commercially available, precoated microtiter plates. These six assays were used to determine the putative influence of organic solvents, namely dimethyl sulfoxide (DMSO), ethanol, methanol, and N-methylpyrrolidone (NMP) at concentrations of 0.01, 0.1, 1.0, and 5.0% (vol/vol), respectively, on glucose and pyruvate metabolism after 4 and 24 hours. In summary, all solvents induced significant changes in regard to one or several of the parameters evaluated, affecting cellular glucose uptake, glycolysis, mitochondrial metabolism, or oxidative phosphorylation. Accordingly, this comprehensive HTS evaluation should enable researchers to choose specific organic solvents on a rational basis to avoid nonspecific effects in cultured cells and tissue culture based experimental setups.

Alterations of pancreatic beta-cell mass and islet number due to Ins2-controlled expression of Cre recombinase: RIP-Cre revisited; part 2.

Tissue-specific disruption of genes by targeted expression of Cre recombinase in insulin-producing cells has been widely used to explore pathways involved in regulation of pancreatic beta-cell mass. One particular line of transgenic mice [B6.Cg-Tg(Ins2-cre)25Mgn/J], commonly called RIP-Cre, in which the expression of Cre recombinase is controlled by a short fragment of the rat insulin II gene promoter has been used on at least 20 genes in at least 27 studies. In the majority of these studies (15 out of 27) inactivation of the gene of interest was associated with alterations in islet architecture, islet mass, or pancreatic insulin content. We have tested the hypothesis that genomic integration or expression of Cre recombinase alone causes alterations of beta-cell mass by quantifying islet number and mass in RIP-Cre mice. We have observed a significant hypoplasia of beta-cells in young RIP-Cre mice, and a significant hyperplasia of islets in older RIP-Cre animals. These findings suggest that glucose intolerance and impaired insulin secretion previously described for younger RIP-Cre mice might be caused by transgene-associated islet hypoplasia, and that hyperplasia in older mice might reflect a compensatory response to transgene-related glucose intolerance.

Adiponectin oligomers in human serum during acute and chronic exercise: relation to lipid metabolism and insulin sensitivity.

Beneficial effects of physical exercise include improved insulin sensitivity, which may be affected by a modulated release of adiponectin, which is exclusively synthesized in white adipose tissue and mediates insulin sensitivity. Adiponectin circulates in three different oligomers, which also have a distinct biological function. We therefore aimed to investigate the distribution of adiponectin oligomers in human serum in relation to physical activity. Thirty-eight lean and healthy individuals were investigated. Seven healthy women and 8 healthy men volunteered to investigate the effect of chronic exercise, at 3 different time points with different training intensities. These individuals were all highly trained and were compared to a control group with low physical activity (n = 15). For studying acute exercise effects, 8 healthy men participated in a bicycle test. Adiponectin was determined by ELISA, oligomers were detected by non-denaturating western blot. Total adiponectin and oligomers were unchanged by acute exercise. LDL cholesterol was significantly lower in the chronic exercise group (p = 0.03). Total adiponectin levels and oligomers were not different between these two groups and were unaltered by different training intensities. However, total adiponectin and specifically HMW oligomers correlated with HDL cholesterol (r = 0.459; p = 0.009). We conclude that acute and chronic exercise does not directly affect circulating adiponectin or oligomer distribution in lean and healthy individuals. Whether such regulation is relevant in individuals with a metabolic disorder remains to be determined. However, our data suggest that adiponectin oligomers have distinct physiological functions IN VIVO, and specifically HMW adiponectin is closely correlated with HDL cholesterol.

Regular insulin secretory oscillations despite impaired ATP synthesis in Friedreich Ataxia patients.

Friedreich Ataxia is an inherited disorder caused by decreased expression of a mitochondrial protein called frataxin. Deficiency of this protein causes reduced biogenesis of iron-sulfur clusters, and subsequently impaired synthesis and replenishment of ATP IN VIVO. Basal secretion of insulin occurs in an oscillating manner presumably triggered by ATP-dependent feedback inhibition of glycolytic flux. Hence, individuals with reduced ATP synthesis rates should possibly exhibit impaired insulin secretory oscillations if these were solely dependent on ATP. In the present study Friedreich Ataxia patients with a presumptive impairment of ATP synthesis in pancreatic beta-cells were evaluated for regularity of basal secretory oscillations of insulin. Healthy siblings were employed as controls. In conflict with the initial hypothesis, no differences in regards to oscillation patterns were observed between patients and controls. Supported by EX VIVO evidence, these findings tentatively suggest that pulsatile insulin secretion might not be exclusively dependent on ATP feedback inhibition in humans.

Impact of cereal fibre on glucose-regulating factors.

AIMS/HYPOTHESIS: Insoluble dietary fibre intake is associated, by unknown mechanisms, with a reduced risk of type 2 diabetes. We investigated whether a short-term dietary intervention with purified insoluble fibres influences acute and delayed responses of glucose, insulin, glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide 1. METHODS: Fourteen healthy women with NGT were studied for 300 min on six to eight occasions. Subjects consumed three matched portions of control (C) or fibre-enriched bread (10.4-10.6 g/portion; wheat fibre [WF], oat fibre [OF], and, in a substudy [n=9], resistant starch [RS]) followed by control (C-C, C-WF, C-OF, C-RS) on subsequent days. RESULTS: Fibre enrichment accelerated the early insulin response (fibrextime interaction p=0.026 for WF, p<0.001 for OF, p=0.126 for RS; time of maximal concentration [T(max)], C 57.9+/-5.9, WF 49.3+/-2.5 [p=0.086], OF 46.1+/-2.9 [p=0.026], RS 46.7+/-5.8 min [p=0.029]). It was also associated with an earlier postprandial GIP response after OF (T(max), C 83.6+/-7.2, WF 70.7+/-6.0 [p=0.054], OF 64.3+/-6.9 [p=0.022], RS 60.0+/-5.0 [p>0.15]). Increased fibre intake for 24 h was further associated with a reduced postprandial glucose response on the following day subsequent to ingestion of a control meal (AUC(C-C) 4,140+/-401, AUC(C-WF) 2,850+/-331 [p=0.007], AUC(C-OF) 2,830+/-277 [p=0.011]), with no difference in maximal concentration and T(max) of glucose responses. No differences in insulin responses were observed 24 h after the fibre-enriched diets compared with control (p>0.15). Colonic fermentation was increased only on study days C-OF (p=0.017) and C-RS (p=0.016). CONCLUSIONS/INTERPRETATION: The consumption of highly purified insoluble dietary fibres accelerated the acute GIP and insulin response and was further associated with enhanced postprandial carbohydrate handling the following day upon ingestion of a control meal.

Ghrelin is not suppressed in hyperglycemic clamps by gastric inhibitory polypeptide and arginine.

UNLABELLED: Systemic ghrelin concentration falls rapidly after nutrient ingestion in vivo. The effect incretins on ghrelin secretion in humans remains unclear. We quantified circulating ghrelin concentrations under hyperglycemic conditions combined with infusion of gastric inhibitory polypeptide (GIP) and arginine. METHODS: Eight healthy volunteers were studied with a hyperglycemic clamp followed by addition of GIP (2 pmol.kg(-1).min(-1), 60-115 min) and an arginine-bolus and -infusion (10 mg.kg(-1).min(-1), 90-115 min). RESULTS: Hyperglycemia alone increased circulating insulin concentrations (p<0.01), and decreased ghrelin concentrations to 89.8% of basal (p=0.208). GIP-infusion resulted in circulating insulin concentration of 1109+/-942 pmol/l (p<0.02) and no further decrease of ghrelin (86.2% of baseline, p=0.050). Under arginine- and GIP-infusion together, insulin concentrations increased progressively to 3005+/-1604 pmol/l (p<0.01) without further decreasing in ghrelin concentrations (98.9% of baseline, p=0.575). CONCLUSIONS: Hyperglycemic hyperinsulinemia and further increases of hyperinsulinemia to supraphysiological and high supraphysiological concentrations under GIP- and arginine-infusion do not significantly decrease ghrelin concentrations in healthy subjects. Moreover, there is no dose-dependent suppression of ghrelin by insulin in the hyperglycemic condition. Neither GIP nor arginine affected ghrelin release.

Post-prandial decrease of human plasma ghrelin in the absence of insulin.

Ghrelin is the most powerful orexigenic hormone in mammalian physiology. Ghrelin plasma concentrations increase prior to meal onset, but decrease post-prandially. We and others reported previously that insulin reduces circulating ghrelin levels and might therefore be a driving force for post-prandial suppression of ghrelin. To test the influence of insulin on post-prandial ghrelin regulation, a patient with Type I diabetes with complete insulin deficiency received a low glycemic index meal and subsequently an additional high glycemic index meal in the absence of insulin substitution. Subsequently, a sc injection of 0.08 IU Lispro insulin per kg body weight was given. Results were compared to those of a healthy control subject matched for sex, age and body mass index, which was undergoing the same test series (without Lispro bolus) in the presence of endogenous post-prandial insulin secretion. A substantial decrease of plasma ghrelin levels was observed in the insulin-deficient patient following low glycemic index carbohydrate load (27% plasma ghrelin decrease). The subsequent exposure to a high glycemic index meal resulted in a slight additional reduction of ghrelin levels (32% from baseline), while Lispro bolus did not induce further changes in circulating ghrelin (27% of baseline at termination). This post-prandial response was comparable to that of the healthy control subject (33% reduction after the first meal, 40% after the second meal). These data tentatively suggest that post-prandial secretion of ghrelin is not exclusively regulated by plasma insulin or plasma glucose but may depend on other metabolic factors yet to be identified.

The Pro115Gln polymorphism within the PPAR gamma2 gene has no epidemiological impact on morbid obesity.

The peroxisome-proliferator-activated receptor gamma2 (PPAR gamma2) is a transcriptional key regulator of adipocyte differentiation. PPAR gamma2 can be inactivated by phosphorylation of a serine residue at position 114. A point mutation leading to an amino acid exchange at position 115 (Pro115Gln) was shown to preclude serine phosphorylation and to consecutively accelerate adipocyte differentiation emphasizing the pathophysiological relevance of this mutation. So far, four markedly obese heterozygote carriers of the Pro115Gln mutation (body mass index 37.9-47.3 kgxm (-2)) have been identified in a circumscribed study population. In order to evaluate the epidemiological relevance of the Pro115Gln mutation in morbid obesity we screened the DNA of all subjects with a body mass index greater than 35 kgxm (-2) who had participated in a nationwide German epidemiological field survey. There was no homozygote or heterozygote carrier of the Pro115Gln polymorphism among them. We conclude that the Pro115Gln polymorphism within the PPAR gamma2 gene has no relevant epidemiological impact on morbid obesity in Germany. It needs further investigation whether this polymorphism might play a role in related metabolic disorders.