Types of dairy foods and risk of fragility fracture in the prospective Nurses' Health Study cohort.

BACKGROUND: Fragility fractures present enormous health challenges for women. Dairy products provide many bone-beneficial nutrients, such as calcium and vitamin D. Individual dairy foods may exert different effects on bone health. OBJECTIVES: The aim of this study was to investigate the associations between total dairy, yogurt, milk, and cheese and fragility fracture risk among females in the prospective Nurses' Health Study (NHS) conducted in the United States. METHODS: In the current analysis, 103,003 females with mean age of 48 y were followed from 1980-2004. Proportional hazards models were used to estimate risk of first fracture (of the wrist, hip, or vertebrae) by intakes of dairy foods (total dairy, milk, yogurt, or cheese) obtained from a food frequency questionnaire. Fractures that were caused by high-trauma events were not included. We relied on self-reported data for wrist and hip fractures whereas for vertebral fractures, medical records were used to confirm cases. RESULTS: A total of 5495 incident fracture cases were documented during follow-up. After controlling for relevant confounding variables, consumption of ≥2 servings/d of total dairy (compared with <1 serving/d) was associated with lower fracture risk (hazard ratio [HR]: 0.74; 95% confidence interval [CI]: 0.61, 0.89). More than 2 servings of milk per day (compared with <1 serving/d) were associated with a lower fracture risk (HR: 0.85; 95% CI: 0.77, 0.94). Intakes of calcium, vitamin D, and protein from nondairy sources did not modify the effects of total dairy or milk on fracture risk. There was no association between yogurt intake and fracture risk. Intake of cheese (≥1 servings/d compared with <1 serving/wk) was weakly associated with lower fracture risk (HR: 0.89; 95% CI: 0.79, 0.99). CONCLUSIONS: Higher total dairy, milk, and cheese intakes are associated with lower risks of fracture in females in the NHS.

Dairy Foods, Weight Change, and Risk of Obesity During the Menopausal Transition.

BACKGROUND: Weight gain during the menopausal transition is common. Dairy consumption may impact weight change during this critical period, and different dairy foods may have different effects. OBJECTIVES: This study aimed to investigate the associations of different types of dairy foods with weight gain and risk of obesity in perimenopausal women from the Nurses' Health Study II cohort. METHODS: The examination at menopause was selected as the exam closest to the reported age at menopause. Weight change during 12 y surrounding menopause was derived from self-reported weight data for 3 exams before and 3 after menopause. The mean age of the first weight measure was 45.8 y and the average BMI was 25.0 kg/m2. Dairy food intakes were estimated as mean intakes over the same 12 y. Generalized linear models were used to assess the association between dairy foods and annualized weight change. Cox proportional hazard models were used to estimate the adjusted relative risks for becoming obese over 12 y surrounding menopause. RESULTS: In longitudinal analyses, those with the highest yogurt intakes had the lowest weight gain at every exam. This was not the case for other forms of dairy. After adjusting for potential covariates, those consuming ≥2.0 servings/wk of yogurt (compared with <1.0 serving/month) had a 31% (RR: 0.69; 95% CI: 0.64, 0.74) lower risk of obesity. The highest total dairy intake (≥2.0 servings/d compared with <1.0) was associated with only a 12% (RR: 0.88; 95% CI: 0.82, 0.95) reduction in obesity risk. Higher activity levels and alternative healthy eating index scores were independently associated with statistically significant reductions in risk of obesity, but higher intakes of yogurt strengthened these beneficial associations. CONCLUSION: Yogurt intake was associated with less weight gain and lower obesity risk in women during the menopausal transition.

Dairy Food Intakes, Postpartum Weight Retention, and Risk of Obesity.

Excessive postpartum weight retention puts women at risk for health problems. This study aimed to investigate the effects of dairy foods on weight retention and risk of obesity in postpartum women in the Nurses' Health Study II. Weight was reported every 2 years. We identified the pre-pregnancy and postpartum exams that were approximately 2 years before and after the birth year. Dairy consumption was averaged during these 4 years. Linear models were used to assess postpartum weight retention. Multivariable models were used to estimate risk of obesity. Women with higher yogurt (≥2 servings/week vs. <1 serving/month) intakes had 0.61 pounds less postpartum weight retention. Consuming ≥ 5 cheese servings/week was associated with 0.63 pounds less weight retention than the lowest intake. Among sedentary women, only yogurt intake was associated with lower risk of postpartum obesity (RR: 0.84; 95% CI: 0.71−1.00), though of borderline statistical significance. Among women with less healthy diets, yogurt consumption was also associated with lower postpartum obesity risk (RR: 0.70; 95% CI: 0.57−0.85). In sum, higher yogurt and cheese intakes were associated with less postpartum weight retention and among higher risk women (sedentary or lower diet quality) greater yogurt intake was associated with lower risks of postpartum obesity.

What Regulates Basal Insulin Secretion and Causes Hyperinsulinemia?

We hypothesize that basal hyperinsulinemia is synergistically mediated by an interplay between increased oxidative stress and excess lipid in the form of reactive oxygen species (ROS) and long-chain acyl-CoA esters (LC-CoA). In addition, ROS production may increase in response to inflammatory cytokines and certain exogenous environmental toxins that mislead ß-cells into perceiving nutrient excess when none exists. Thus, basal hyperinsulinemia is envisioned as an adaptation to sustained real or perceived nutrient excess that only manifests as a disease when the excess demand can no longer be met by an overworked ß-cell. In this article we will present a testable hypothetical mechanism to explain the role of lipids and ROS in basal hyperinsulinemia and how they differ from glucose-stimulated insulin secretion (GSIS). The model centers on redox regulation, via ROS, and S-acylation-mediated trafficking via LC-CoA. These pathways are well established in neural systems but not ß-cells. During GSIS, these signals rise and fall in an oscillatory pattern, together with the other well-established signals derived from glucose metabolism; however, their precise roles have not been defined. We propose that failure to either increase or decrease ROS or LC-CoA appropriately will disturb ß-cell function.

The Redox Communication Network as a Regulator of Metabolism.

Key tissues are dysfunctional in obesity, diabetes, cardiovascular disease, fatty liver and other metabolic diseases. Focus has centered on individual organs as though each was isolated. Attention has been paid to insulin resistance as the key relevant pathosis, particularly insulin receptor signaling. However, many tissues play important roles in synergistically regulating metabolic homeostasis and should be considered part of a network. Our approach identifies redox as an acute regulator of the greater metabolic network. Redox reactions involve the transfer of electrons between two molecules and in this work refer to commonly shared molecules, reflective of energy state, that can readily lose electrons to increase or gain electrons to decrease the oxidation state of molecules including NAD(P), NAD(P)H, and thiols. Metabolism alters such redox molecules to impact metabolic function in many tissues, thus, responding to anabolic and catabolic stimuli appropriately and synergistically. It is also important to consider environmental factors that have arisen or increased in recent decades as putative modifiers of redox and reactive oxygen species (ROS) and thus metabolic state. ROS are highly reactive, controlled by the thiol redox state and influence the function of thousands of proteins. Lactate (L) and pyruvate (P) in cells are present in a ratio of about 10 reflective of the cytosolic NADH to NAD ratio. Equilibrium is maintained in cells because lactate dehydrogenase is highly expressed and near equilibrium. The major source of circulating lactate and pyruvate is muscle, although other tissues also contribute. Acetoacetate (A) is produced primarily by liver mitochondria where ß-hydroxybutyrate dehydrogenase is highly expressed, and maintains a ratio of ß-hydroxybutyrate (ß) to A of about 2, reflective of the mitochondrial NADH to NAD ratio. All four metabolites as well as the thiols, cysteine and glutathione, are transported into and out of cells, due to high expression of relevant transporters. Our model supports regulation of all collaborating metabolic organs through changes in circulating redox metabolites, regardless of whether change was initiated exogenously or by a single organ. Validation of these predictions suggests novel ways to understand function by monitoring and impacting redox state.

Elamipretide Promotes Mitophagosome Formation and Prevents Its Reduction Induced by Nutrient Excess in INS1 ß-cells.

Elamipretide is a tetrapeptide that restores defects in mitochondrial function, binds to cardiolipin, and is being tested in clinical trials for mitochondria-related diseases. However, whether elamipretide modulates mitochondrial quality control and dynamics, processes essential to preserve mitochondrial function, is unclear. Thus, we tested the effects of elamipretide on mitochondrial morphology, mitophagosome formation, and their early disruption induced by excess nutrients in INS1 ß-cells. Elamipretide treatment was sufficient to increase engulfment of mitochondria into autophagosomes in control INS1 ß-cells, without inducing widespread changes in mitochondrial morphology or membrane potential. In an early pathogenic context mimicked by short-term exposure to nutrient excess, elamipretide treatment prevented both mitochondrial fragmentation and defects in the engulfment of mitochondria into autophagosomes. On the other hand, elamipretide did not prevent lysosomal defects induced by nutrient excess. Accordingly, elamipretide treatment did not entail benefits on pathogenic p62 and LC3II accumulation or on insulin secretory function. In conclusion, our data show that elamipretide selectively stimulates the engulfment of mitochondria into autophagosomes and prevents its defects induced by nutrient excess. Thus, we propose that improved selectivity of mitochondrial quality control processes might contribute to the benefits stemming from elamipretide treatments in other disease models.

BET proteins in abnormal metabolism, inflammation, and the breast cancer microenvironment.

Obesity and its associated pathology Type 2 diabetes are two chronic metabolic and inflammatory diseases that promote breast cancer progression, metastasis, and poor outcomes. Emerging critical opinion considers unresolved inflammation and abnormal metabolism separately from obesity; settings where they do not co-occur can inform disease mechanism. In breast cancer, the tumor microenvironment is often infiltrated with T effector and T regulatory cells programmed by metabolic signaling. The pathways by which tumor cells evade immune surveillance, immune therapies, and take advantage of antitumor immunity are poorly understood, but likely depend on metabolic inflammation in the microenvironment. Immune functions are abnormal in metabolic disease, and lessons learned from preclinical studies in lean and metabolically normal environments may not translate to patients with obesity and metabolic disease. This problem is made more urgent by the rising incidence of breast cancer among women who are not obese but who have metabolic disease and associated inflammation, a phenotype common in Asia. The somatic BET proteins, comprising BRD2, BRD3, and BRD4, are new critical regulators of metabolism, coactivate transcription of genes that encode proinflammatory cytokines in immune cell subsets infiltrating the microenvironment, and could be important targets in breast cancer immunotherapy. These transcriptional coregulators are well known to regulate tumor cell progression, but only recently identified as critical for metabolism, metastasis, and expression of immune checkpoint molecules. We consider interrelationships among metabolism, inflammation, and breast cancer aggressiveness relevant to the emerging threat of breast cancer among women with metabolic disease, but without obesity.

Type 1 diabetes alters lipid handling and metabolism in human fibroblasts and peripheral blood mononuclear cells.

Triggers of the autoimmune response that leads to type 1 diabetes (T1D) remain poorly understood. A possibility is that parallel changes in both T cells and target cells provoke autoimmune attack. We previously documented greater Ca2+ transients in fibroblasts from T1D subjects than non-T1D after exposure to fatty acids (FA) and tumor necrosis factor α (TNFα). These data indicate that metabolic and signal transduction defects present in T1D can be elicited ex vivo in isolated cells. Changes that precede T1D, including inflammation, may activate atypical responses in people that are genetically predisposed to T1D. To identify such cellular differences in T1D, we quantified a panel of metabolic responses in fibroblasts and peripheral blood cells (PBMCs) from age-matched T1D and non-T1D subjects, as models for non-immune and immune cells, respectively. Fibroblasts from T1D subjects accumulated more lipid, had higher LC-CoA levels and converted more FA to CO2, with less mitochondrial proton leak in response to oleate alone or with TNFα, using the latter as a model of inflammation. T1D-PBMCs contained and also accumulated more lipid following FA exposure. In addition, they formed more peroxidized lipid than controls following FA exposure. We conclude that both immune and non-immune cells in T1D subjects differ from controls in terms of responses to FA and TNFα. Our results suggest a differential sensitivity to inflammatory insults and FA that may precede and contribute to T1D by priming both immune cells and their targets for autoimmune reactions.

Direct Stimulation of Islet Insulin Secretion by Glycolytic and Mitochondrial Metabolites in KCl-Depolarized Islets.

We have previously demonstrated that islet depolarization with 70 mM KCl opens Cx36 hemichannels and allows diffusion of small metabolites and cofactors through the ß-cell plasma membrane. We have investigated in this islet "permeabilized" model whether glycolytic and citric acid cycle intermediates stimulate insulin secretion and how it correlates with ATP production (islet content plus extracellular nucleotide accumulation). Glycolytic intermediates (10 mM) stimulated insulin secretion and ATP production similarly. However, they showed differential sensitivities to respiratory chain or enzyme inhibitors. Pyruvate showed a lower secretory capacity and less ATP production than phosphoenolpyruvate, implicating an important role for glycolytic generation of ATP. ATP production by glucose-6-phosphate was not sensitive to a pyruvate kinase inhibitor that effectively suppressed the phosphoenolpyruvate-induced secretory response and islet ATP rise. Strong suppression of both insulin secretion and ATP production induced by glucose-6-phosphate was caused by 10 µM antimycin A, implicating an important role for the glycerophosphate shuttle in transferring reducing equivalents to the mitochondria. Five citric acid cycle intermediates were investigated for their secretory and ATP production capacity (succinate, fumarate, malate, isocitrate and α-ketoglutarate at 5 mM, together with ADP and/or NADP+ to feed the NADPH re-oxidation cycles). The magnitude of the secretory response was very similar among the different mitochondrial metabolites but α-ketoglutarate showed a more sustained second phase of secretion. Gabaculine (1 mM, a GABA-transaminase inhibitor) suppressed the second phase of secretion and the ATP-production stimulated by α-ketoglutarate, supporting a role for the GABA shuttle in the control of glucose-induced insulin secretion. None of the other citric acid intermediates essayed showed any suppression of both insulin secretion or ATP-production by the presence of gabaculine. We propose that endogenous GABA metabolism in the "GABA-shunt" facilitates ATP production in the citric acid cycle for an optimal insulin secretion.

BET Bromodomain Proteins Brd2, Brd3 and Brd4 Selectively Regulate Metabolic Pathways in the Pancreatic ß-Cell.

Displacement of Bromodomain and Extra-Terminal (BET) proteins from chromatin has promise for cancer and inflammatory disease treatments, but roles of BET proteins in metabolic disease remain unexplored. Small molecule BET inhibitors, such as JQ1, block BET protein binding to acetylated lysines, but lack selectivity within the BET family (Brd2, Brd3, Brd4, Brdt), making it difficult to disentangle contributions of each family member to transcriptional and cellular outcomes. Here, we demonstrate multiple improvements in pancreatic ß-cells upon BET inhibition with JQ1 or BET-specific siRNAs. JQ1 (50-400 nM) increases insulin secretion from INS-1 cells in a concentration dependent manner. JQ1 increases insulin content in INS-1 cells, accounting for increased secretion, in both rat and human islets. Higher concentrations of JQ1 decrease intracellular triglyceride stores in INS-1 cells, a result of increased fatty acid oxidation. Specific inhibition of both Brd2 and Brd4 enhances insulin transcription, leading to increased insulin content. Inhibition of Brd2 alone increases fatty acid oxidation. Overlapping yet discrete roles for individual BET proteins in metabolic regulation suggest new isoform-selective BET inhibitors may be useful to treat insulin resistant/diabetic patients. Results imply that cancer and diseases of chronic inflammation or disordered metabolism are related through shared chromatin regulatory mechanisms.

Phosphoinositide signalling in type 2 diabetes: a ß-cell perspective.

Type 2 diabetes is a complex disease. It results from a failure of the body to maintain energy homoeostasis. Multicellular organisms have evolved complex strategies to preserve a relatively stable internal nutrient environment, despite fluctuations in external nutrient availability. This complex strategy involves the co-ordinated responses of multiple organs to promote storage or mobilization of energy sources according to the availability of nutrients and cellular bioenergetics needs. The endocrine pancreas plays a central role in these processes by secreting insulin and glucagon. When this co-ordinated effort fails, hyperglycaemia and hyperlipidaemia develops, characterizing a state of metabolic imbalance and ultimately overt diabetes. Although diabetes is most likely a collection of diseases, scientists are starting to identify genetic components and environmental triggers. Genome-wide association studies revealed that by and large, gene variants associated with type 2 diabetes are implicated in pancreatic ß-cell function, suggesting that the ß-cell may be the weakest link in the chain of events that results in diabetes. Thus, it is critical to understand how environmental cues affect the ß-cell. Phosphoinositides are important 'decoders' of environmental cues. As such, these lipids have been implicated in cellular responses to a wide range of growth factors, hormones, stress agents, nutrients and metabolites. Here we will review some of the well-established and potential new roles for phosphoinositides in ß-cell function/dysfunction and discuss how our knowledge of phosphoinositide signalling could aid in the identification of potential strategies for treating or preventing type 2 diabetes.

Inhibition of Monoacylglycerol Lipase Activity Decreases Glucose-Stimulated Insulin Secretion in INS-1 (832/13) Cells and Rat Islets.

Lipid signals derived from lipolysis and membrane phospholipids play an important role in glucose-stimulated insulin secretion (GSIS), though the exact secondary signals remain unclear. Previous reports have documented a stimulatory role of exogenously added mono-acyl-glycerol (MAG) on insulin secretion from cultured ß-cells and islets. In this report we have determined effects of increasing intracellular MAG in the ß-cell by inhibiting mono-acyl-glycerol lipase (MGL) activity, which catalyzes the final step in triacylglycerol breakdown, namely the hydrolysis of MAG to glycerol and free fatty acid (FA). To determine the role of MGL in GSIS, we used three different pharmacological agents (JZL184, MJN110 and URB602). All three inhibited GSIS and depolarization-induced insulin secretion in INS-1 (832/13). JZL184 significantly inhibited both GSIS and depolarization-induced insulin secretion in rat islets. JZL184 significantly decreased lipolysis and increased both mono- and diacyglycerol species in INS-1 cells. Analysis of the kinetics of GSIS showed that inhibition was greater during the sustained phase of secretion. A similar pattern was observed in the response of Ca2+ to glucose and depolarization but to a lesser degree suggesting that altered Ca2+ handling alone could not explain the reduction in insulin secretion. In addition, a significant reduction in long chain-CoA (LC-CoA) was observed in INS-1 cells at both basal and stimulatory glucose following inhibition of MGL. Our data implicate an important role for MGL in insulin secretion.

Inborn Errors of Long-Chain Fatty Acid ß-Oxidation Link Neural Stem Cell Self-Renewal to Autism.

Inborn errors of metabolism (IEMs) occur with high incidence in human populations. Especially prevalent among these are inborn deficiencies in fatty acid ß-oxidation (FAO), which are clinically associated with developmental neuropsychiatric disorders, including autism. We now report that neural stem cell (NSC)-autonomous insufficiencies in the activity of TMLHE (an autism risk factor that supports long-chain FAO by catalyzing carnitine biosynthesis), of CPT1A (an enzyme required for long-chain FAO transport into mitochondria), or of fatty acid mobilization from lipid droplets reduced NSC pools in the mouse embryonic neocortex. Lineage tracing experiments demonstrated that reduced flux through the FAO pathway potentiated NSC symmetric differentiating divisions at the expense of self-renewing stem cell division modes. The collective data reveal a key role for FAO in controlling NSC-to-IPC transition in the mammalian embryonic brain and suggest NSC self renewal as a cellular mechanism underlying the association between IEMs and autism.

KCl -Permeabilized Pancreatic Islets: An Experimental Model to Explore the Messenger Role of ATP in the Mechanism of Insulin Secretion.

Our previous work has demonstrated that islet depolarization with KCl opens connexin36 hemichannels in ß-cells of mouse pancreatic islets allowing the exchange of small metabolites with the extracellular medium. In this study, the opening of these hemichannels has been further characterized in rat islets and INS-1 cells. Taking advantage of hemicannels'opening, the uptake of extracellular ATP and its effect on insulin release were investigated. 70 mM KCl stimulated light emission by luciferin in dispersed rat islets cells transduced with the fire-fly luciferase gene: it was suppressed by 20 mM glucose and 50 µM mefloquine, a specific connexin36 inhibitor. Extracellular ATP was taken up or released by islets depolarized with 70 mM KCl at 5 mM glucose, depending on the external ATP concentration. 1 mM ATP restored the loss of ATP induced by the depolarization itself. ATP concentrations above 5 mM increased islet ATP content and the ATP/ADP ratio. No ATP uptake occurred in non-depolarized or KCl-depolarized islets simultaneously incubated with 50 µM mefloquine or 20 mM glucose. Extracellular ATP potentiated the secretory response induced by 70 mM KCl at 5 mM glucose in perifused rat islets: 5 mM ATP triggered a second phase of insulin release after the initial peak triggered by KCl-depolarization itself; at 10 mM, it increased both the initial, KCl-dependent, peak and stimulated a greater second phase of secretion than at 5 mM. These stimulatory effects of extracellular ATP were almost completely suppressed by 50 µM mefloquine. The magnitude of the second phase of insulin release due to 5 mM extracellular ATP was decreased by addition of 5 mM ADP (extracellular ATP/ADP ratio = 1). ATP acts independently of KATP channels closure and its intracellular concentration and its ATP/ADP ratio seems to regulate the magnitude of both the first (triggering) and second (amplifying) phases of glucose-induced insulin secretion.

Chronic Exposure to Excess Nutrients Left-shifts the Concentration Dependence of Glucose-stimulated Insulin Secretion in Pancreatic ß-Cells.

Hyperinsulinemia (HI) is elevated plasma insulin at basal glucose. Impaired glucose tolerance is associated with HI, although the exact cause and effect relationship remains poorly defined. We tested the hypothesis that HI can result from an intrinsic response of the ß-cell to chronic exposure to excess nutrients, involving a shift in the concentration dependence of glucose-stimulated insulin secretion. INS-1 (832/13) cells were cultured in either a physiological (4 mm) or high (11 mm) glucose concentration with or without concomitant exposure to oleate. Isolated rat islets were also cultured with or without oleate. A clear hypersensitivity to submaximal glucose concentrations was evident in INS-1 cells cultured in excess nutrients such that the 25% of maximal (S0.25) glucose-stimulated insulin secretion was significantly reduced in cells cultured in 11 mm glucose (S0.25 = 3.5 mm) and 4 mm glucose with oleate (S0.25 = 4.5 mm) compared with 4 mm glucose alone (S0.25 = 5.7 mm). The magnitude of the left shift was linearly correlated with intracellular lipid stores in INS-1 cells (r(2) = 0.97). We observed no significant differences in the dose responses for glucose stimulation of respiration, NAD(P)H autofluorescence, or Ca(2+) responses between left- and right-shifted ß-cells. However, a left shift in the sensitivity of exocytosis to Ca(2+) was documented in permeabilized INS-1 cells cultured in 11 versus 4 mm glucose (S0.25 = 1.1 and 1.7 µm, respectively). Our results suggest that the sensitivity of exocytosis to triggering is modulated by a lipid component, the levels of which are influenced by the culture nutrient environment.

Brd2 gene disruption causes "metabolically healthy" obesity: epigenetic and chromatin-based mechanisms that uncouple obesity from type 2 diabetes.

Disturbed body energy balance can lead to obesity and obesity-driven diseases such as Type 2 diabetes, which have reached an epidemic level. Evidence indicates that obesity-induced inflammation is a major cause of insulin resistance and Type 2 diabetes. Environmental factors, such as nutrients, affect body energy balance through epigenetic or chromatin-based mechanisms. As a bromodomain and external domain family transcription regulator, Brd2 regulates expression of many genes through interpretation of chromatin codes and participates in the regulation of body energy balance and immune function. In the severely obese state, Brd2 knockdown in mice prevented obesity-induced inflammatory responses, protected animals from insulin resistance, glucose intolerance and pancreatic beta cell dysfunction, and thus uncoupled obesity from diabetes. Brd2 provides an important model for investigation of the function of transcription regulators and the development of obesity and diabetes; it also provides a possible, innovative target to treat obesity and diabetes through modulation of the function of a chromatin code reader.

Reactive oxygen species stimulate insulin secretion in rat pancreatic islets: studies using mono-oleoyl-glycerol.

Chronic exposure (24-72 hrs) of pancreatic islets to elevated glucose and fatty acid leads to glucolipoxicity characterized by basal insulin hypersecretion and impaired glucose-stimulated insulin secretion (GSIS). Our aim was to determine the mechanism for basal hypersecretion of insulin. We used mono-oleoyl-glycerol (MOG) as a tool to rapidly increase lipids in isolated rat pancreatic ß-cells and in the clonal pancreatic ß-cell line INS-1 832/13. MOG (25-400 µM) stimulated basal insulin secretion from ß-cells in a concentration dependent manner without increasing intracellular Ca(2+) or O(2) consumption. Like GSIS, MOG increased NAD(P)H and reactive oxygen species (ROS). The mitochondrial reductant ß-hydroxybutyrate (ß-OHB) also increased the redox state and ROS production, while ROS scavengers abrogated secretion. Diazoxide (0.4 mM) did not prevent the stimulatory effect of MOG, confirming that the effect was independent of the K(ATP)-dependent pathway of secretion. MOG was metabolized to glycerol and long-chain acyl-CoA (LC-CoA), whereas, acute oleate did not similarly increase LC-CoA. Inhibition of diacylglycerol kinase (DGK) did not mimic the effect of MOG on insulin secretion, indicating that MOG did not act primarily by inhibiting DGK. Inhibition of acyl-CoA synthetase (ACS) reduced the stimulatory effect of MOG on basal insulin secretion by 30% indicating a role for LC-CoA. These data suggest that basal insulin secretion is stimulated by increased ROS production, due to an increase in the mitochondrial redox state independent of the established components of GSIS.

Respiration in adipocytes is inhibited by reactive oxygen species.

It is a desirable goal to stimulate fuel oxidation in adipocytes and shift the balance toward less fuel storage and more burning. To understand this regulatory process, respiration was measured in primary rat adipocytes, mitochondria, and fat-fed mice. Maximum O(2) consumption, in vitro, was determined with a chemical uncoupler of oxidative phosphorylation (carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP)). The adenosine triphosphate/adenosine diphosphate (ATP/ADP) ratio was measured by luminescence. Mitochondria were localized by confocal microscopy with MitoTracker Green and their membrane potential (Delta psi(M)) measured using tetramethylrhodamine ethyl ester perchlorate (TMRE). The effect of N-acetylcysteine (NAC) on respiration and body composition in vivo was assessed in mice. Addition of FCCP collapsed Delta psi(M) and decreased the ATP/ADP ratio. However, we demonstrated the same rate of adipocyte O(2) consumption in the absence or presence of fuels and FCCP. Respiration was only stimulated when reactive oxygen species (ROS) were scavenged by pyruvate or NAC: other fuels or fuel combinations had little effect. Importantly, the ROS scavenging role of pyruvate was not affected by rotenone, an inhibitor of mitochondrial complex I. In addition, mice that consumed NAC exhibited increased O(2) consumption and decreased body fat in vivo. These studies suggest for the first time that adipocyte O(2) consumption may be inhibited by ROS, because pyruvate and NAC stimulated respiration. ROS inhibition of O(2) consumption may explain the difficulty to identify effective strategies to increase fat burning in adipocytes. Stimulating fuel oxidation in adipocytes by decreasing ROS may provide a novel means to shift the balance from fuel storage to fuel burning.

The CB1 antagonist rimonabant decreases insulin hypersecretion in rat pancreatic islets.

Type 2 diabetes and obesity are characterized by elevated nocturnal circulating free fatty acids, elevated basal insulin secretion, and blunted glucose-stimulated insulin secretion (GSIS). The CB1 receptor antagonist, Rimonabant, has been shown to improve glucose tolerance and insulin sensitivity in vivo but its direct effect on islets has been unclear. Islets from lean littermates and obese Zucker (ZF) and Zucker Diabetic Fatty (ZDF) rats were incubated for 24 h in vitro and exposed to 11 mmol/l glucose and 0.3 mmol/l palmitate (GL) with or without Rimonabant. Insulin secretion was determined at basal (3 mmol/l) or stimulatory (15 mmol/l) glucose concentrations. As expected, basal secretion was significantly elevated in islets from obese or GL-treated lean rats whereas the fold increase in GSIS was diminished. Rimonabant decreased basal hypersecretion in islets from obese rats and GL-treated lean rats without decreasing the fold increase in GSIS. However, it decreased GSIS in islets from lean rats without affecting basal secretion. These findings indicate that Rimonabant has direct effects on islets to reduce insulin secretion when secretion is elevated above normal levels by diet or in obesity. In contrast, it appears to decrease stimulated secretion in islets from lean animals but not in obese or GL-exposed islets.

Fission and selective fusion govern mitochondrial segregation and elimination by autophagy.

Accumulation of depolarized mitochondria within beta-cells has been associated with oxidative damage and development of diabetes. To determine the source and fate of depolarized mitochondria, individual mitochondria were photolabeled and tracked through fusion and fission. Mitochondria were found to go through frequent cycles of fusion and fission in a 'kiss and run' pattern. Fission events often generated uneven daughter units: one daughter exhibited increased membrane potential (delta psi(m)) and a high probability of subsequent fusion, while the other had decreased membrane potential and a reduced probability for a fusion event. Together, this pattern generated a subpopulation of non-fusing mitochondria that were found to have reduced delta psi(m) and decreased levels of the fusion protein OPA1. Inhibition of the fission machinery through DRP1(K38A) or FIS1 RNAi decreased mitochondrial autophagy and resulted in the accumulation of oxidized mitochondrial proteins, reduced respiration and impaired insulin secretion. Pulse chase and arrest of autophagy at the pre-proteolysis stage reveal that before autophagy mitochondria lose delta psi(m) and OPA1, and that overexpression of OPA1 decreases mitochondrial autophagy. Together, these findings suggest that fission followed by selective fusion segregates dysfunctional mitochondria and permits their removal by autophagy.