Protection of pancreatic INS-1 β-cells from glucose- and fructose-induced cell death by inhibiting mitochondrial permeability transition with cyclosporin A or metformin

Hyperglycemia is detrimental to β-cell viability, playing a major role in the progression of β-cell loss in diabetes mellitus. The permeability transition pore (PTP) is a mitochondrial channel involved in cell death. Recent evidence suggests that PTP inhibitors prevent hyperglycemia-induced cell death in human endothelial cells. In this work, we have examined the involvement of PTP opening in INS-1 cell death induced by high levels of glucose or fructose. PTP regulation was studied by measuring the calcium retention capacity in permeabilized INS-1 cells and by confocal microscopy in intact INS-1 cells. Cell death was analyzed by flow cytometry. We first reported that metformin and cyclosporin A (CsA) prevented Ca2+-induced PTP opening in permeabilized and intact INS-1 cells. We then showed that incubation of INS-1 cells in the presence of 30 mM glucose or 2.5 mM fructose induced PTP opening and led to cell death. As both metformin and CsA prevented glucose- and fructose- induced PTP opening, and hampered glucose- and fructose- induced cell death, we conclude that PTP opening is involved in high glucose- and high fructose- induced INS-1 cell death. We therefore suggest that preventing PTP opening might be a new approach to preserve β-cell viability

Non-Invasive In Vivo Imaging of Calcium Signaling in Mice

Rapid and transient elevations of Ca2+ within cellular microdomains play a critical role in the regulation of many signal transduction pathways. Described here is a genetic approach for non-invasive detection of localized Ca2+ concentration ([Ca2+]) rises in live animals using bioluminescence imaging (BLI). Transgenic mice conditionally expressing the Ca2+-sensitive bioluminescent reporter GFP-aequorin targeted to the mitochondrial matrix were studied in several experimental paradigms. Rapid [Ca2+] rises inside the mitochondrial matrix could be readily detected during single-twitch muscle contractions. Whole body patterns of [Ca2+] were monitored in freely moving mice and during epileptic seizures. Furthermore, variations in mitochondrial [Ca2+] correlated to behavioral components of the sleep/wake cycle were observed during prolonged whole body recordings of newborn mice. This non-invasive imaging technique opens new avenues for the analysis of Ca2+ signaling whenever whole body information in freely moving animals is desired, in particular during behavioral and developmental studies

Substrate cycling between de novo lipogenesis and lipid oxidation: a thermogenic mechanism against skeletal muscle lipotoxicity and glucolipotoxicity

Life is a combustion, but how the major fuel substrates that sustain human life compete and interact with each other for combustion has been at the epicenter of research into the pathogenesis of insulin resistance ever since Randle proposed a 'glucose-fatty acid cycle' in 1963. Since then, several features of a mutual interaction that is characterized by both reciprocality and dependency between glucose and lipid metabolism have been unravelled, namely: 1. the inhibitory effects of elevated concentrations of fatty acids on glucose oxidation (via inactivation of mitochondrial pyruvate dehydrogenase or via desensitization of insulin-mediated glucose transport), 2. the inhibitory effects of elevated concentrations of glucose on fatty acid oxidation (via malonyl-CoA regulation of fatty acid entry into the mitochondria), and more recently 3. the stimulatory effects of elevated concentrations of glucose on de novo lipogenesis, that is, synthesis of lipids from glucose (via SREBP1c regulation of glycolytic and lipogenic enzymes). This paper first revisits the physiological significance of these mutual interactions between glucose and lipids in skeletal muscle pertaining to both blood glucose and intramyocellular lipid homeostasis. It then concentrates upon emerging evidence, from calorimetric studies investigating the direct effect of leptin on thermogenesis in intact skeletal muscle, of yet another feature of the mutual interaction between glucose and lipid oxidation: that of substrate cycling between de novo lipogenesis and lipid oxidation. It is proposed that this energy-dissipating substrate cycling that links glucose and lipid metabolism to thermogenesis could function as a 'fine-tuning' mechanism that regulates intramyocellular lipid homeostasis, and hence contributes to the protection of skeletal muscle against lipotoxicity

Mitochondrial function as a determinant of life span

Average human life expectancy has progressively increased over many decades largely due to improvements in nutrition, vaccination, antimicrobial agents, and effective treatment/prevention of cardiovascular disease, cancer, etc. Maximal life span, in contrast, has changed very little. Caloric restriction (CR) increases maximal life span in many species, in concert with improvements in mitochondrial function. These effects have yet to be demonstrated in humans, and the duration and level of CR required to extend life span in animals is not realistic in humans. Physical activity (voluntary exercise) continues to hold much promise for increasing healthy life expectancy in humans, but remains to show any impact to increase maximal life span. However, longevity in Caenorhabditis elegans is related to activity levels, possibly through maintenance of mitochondrial function throughout the life span. In humans, we reported a progressive decline in muscle mitochondrial DNA abundance and protein synthesis with age. Other investigators also noted age-related declines in muscle mitochondrial function, which are related to peak oxygen uptake. Long-term aerobic exercise largely prevented age-related declines in mitochondrial DNA abundance and function in humans and may increase spontaneous activity levels in mice. Notwithstanding, the impact of aerobic exercise and activity levels on maximal life span is uncertain. It is proposed that age-related declines in mitochondrial content and function not only affect physical function, but also play a major role in regulation of life span. Regular aerobic exercise and prevention of adiposity by healthy diet may increase healthy life expectancy and prolong life span through beneficial effects at the level of the mitochondrion

Fish oil normalizes plasma glucose levels and improves liver carbohydrate metabolism in rats fed a sucrose-rich diet

A sucrose-rich diet (SRD) induces insulin resistance and dyslipidemia with impaired hepatic glucose production and gluconeogenesis, accompanied by altered post-receptor insulin signaling steps. The aim of this study was to examine the effectiveness of fish oil (FO) to reverse or improve the impaired hepatic glucose metabolism once installed in rats fed 8 months a SRD. In the liver of rats fed SRD in which FO replaced corn-oil during the last 2 months, as dietary fat, several key enzyme activities and metabolites involved in glucose metabolisms (phosphorylation, glycolysis, gluconeogenesis and oxidative and non oxidative glucose pathway) were measured. The protein mass levels of IRS-1 and ap85 PI-3K at basal conditions were also analyzed. FO improved the altered activities of some enzymes involved in the glycolytic and oxidative pathways observed in the liver of SRD fed rats but was unable to restore the impaired capacity of glucose phosphorylation. Moreover, FO reversed the increase in PEPCK and G-6-Pase and reduced the G-6-Pase/GK ratio. Glycogen concentration and GSa activity returned to levels similar to those observed in the liver of the control-fed rats. Besides, FO did not modify the altered protein mass levels of IRS-1 and αp85 PI-3K. Finally, dietary FO was effective in reversing or improving the impaired activities of several key enzymes of hepatic carbohydrate metabolism contributing, at least in part, to the normalization of plasma glucose levels in the SRD-fed rats. However, these positive effects of FO were not observed under basal conditions in the early steps of insulin signaling transduction. © 2011 AOCS.Fil: Hein, Gustavo Juan. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas; ArgentinaFil: Chicco, Adriana Graciela. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas; ArgentinaFil: Lombardo, Yolanda B.. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas; Argentin

Respostas metabólicas à suplementação com frutose em exercício de força de membros inferiores Metabolic responses to fructose supplementation in strength exercise of lower limbs

A frutose, por seu metabolismo independente da insulina, realiza significativas alterações no metabolismo hepático, promovendo um entorno metabólico favorável ao metabolismo tanto da glicose como dos lipídios, durante o exercício. Essa condição tem sido bastante estudada em exercício de endurance; no entanto, nenhum estudo sobre a suplementação com frutose no exercício de força (EF) foi encontrado. O objetivo do presente estudo foi avaliar os efeitos agudos da adição de frutose a um suplemento de glicose sobre o metabolismo de lipídios em EF. Vinte homens treinados ingeriram suplemento de glicose (G) ou glicose mais frutose (G+F), 15 minutos antes de realizar exercício de força (10 séries de 10 repetições). Os sujeitos foram testados em ordem randômica em um desenho cruzado e com uma semana de intervalo em duas condições experimentais: EF+(G) e EF+(G+F). A análise dos resultados mostrou que os valores de triglicérides durante o exercício foram maiores (p < 0,05) quando os sujeitos foram suplementados com G+F do que quando suplementados apenas com G. Ao final do exercício, os valores de ácidos graxos livres foram maiores quando os sujeitos foram suplementados G+F (p < 0,05). A glicemia foi menor durante o exercício e maior na recuperação (p < 0,05) para essa condição. O comportamento da insulina não diferiu entre os experimentos durante o exercício de força (p > 0,05), mas foi maior em G+F que em G (p < 0,05) durante a recuperação. A percepção subjetiva de esforço (PSE) foi menor (p < 0,05) para a suplementação com G+F do que com G. Em conclusão, a suplementação com G+F afeta positivamente o metabolismo de lipídios durante o exercício de força e favorece seu metabolismo imediatamente após o esforço, proporcionando condição metabólica que reflete em uma condição que afeta favoravelmente a PSE.<br>Due to its insulin-independent metabolism, fructose promotes significant changes in liver metabolism, promoting a metabolic surrounding favorable to the glucose as well as lipids metabolism during the exercise. This condition has been widely studied in endurance exercises; however, none study about fructose supplementation in strength exercise (SE) was found. This study aimed to assess the acute effects of the fructose addition to a glucose supplement on lipid metabolism in strength exercise. Twenty trained male subjects ingested a glucose (G) or glucose plus fuctose (G+F) supplement, 15 minutes before practicing a strength exercise (10 sets of 10 repetitions). The subjects were tested randomly in a crossover design and with a week of pause in two experimental conditions: SE+(G) and SE+(G+F). The analysis of the results showed that values of triglycerides during the exercise were higher (p < 0.05) when the subjects were supplemented with G+F than when they were supplemented only with G. By the end of the exercise the values of free fatty acid were higher when in G+F (p < 0.05). Glycemia was lower during the exercise and higher in the recovery (p < 0.05) in this condition. Insulin values did not differ among the experiments during strength exercises (p > 0.05), but they were higher in G+F than in G (p < 0.05) during recovery. Perceived exertion (PE) was lower (p < 0.05) in G+F than in G. It can be concluded that the G+F supplementation positively affects the lipid metabolism during the strength exercise and favors its metabolism immediately after the effort, promoting a metabolic condition that reflects on a condition that favorably affects the PE

Skeletal muscle oxidative capacity in rats fed high-fat diet.

To investigate whether young rats respond to high-fat feeding through changes in energy efficiency and fuel partitioning at the level of skeletal muscle, to avoid obesity development. In addition, to establish whether the two mitochondrial subpopulations, which exist in skeletal muscle, ie subsarcolemmal and intermyofibrillar, are differently affected by high-fat feeding. DESIGN: Weaning rats were fed a low-fat or a high-fat diet for 15 days. MEASUREMENTS: Energy balance and lipid partitioning in the whole animal. State 3 and state 4 oxygen consumption rates in whole skeletal muscle homogenate. State 3 and state 4 oxygen consumption rates, membrane potential and uncoupling effect of palmitate in subsarcolemmal and intermyofibrillar mitochondria from skeletal muscle. RESULTS: Rats fed a high-fat diet showed an increased whole body lipid utilization. Skeletal muscle NAD-linked and lipid oxidative capacity significantly increased at the whole-tissue level, due to an increase in lipid oxidative capacity in subsarcolemmal and intermyofibrillar mitochondria and in NAD-linked activity only in intermyofibrillar ones. In addition, rats fed a high-fat diet showed an increase in the uncoupling effect of palmitate in both the mitochondrial populations. CONCLUSIONS: In young rats fed a high-fat diet, skeletal muscle contributes to enhanced whole body lipid oxidation through an increased mitochondrial capacity to use lipids as metabolic fuels, associated with a decrease in energy couplin