Mitofusins Mfn1 and Mfn2 Are Required to Preserve Glucose- but Not Incretin-Stimulated ß-Cell Connectivity and Insulin Secretion.

Mitochondrial glucose metabolism is essential for stimulated insulin release from pancreatic ß-cells. Whether mitofusin gene expression, and hence, mitochondrial network integrity, is important for glucose or incretin signaling has not previously been explored. Here, we generated mice with ß-cell-selective, adult-restricted deletion knock-out (dKO) of the mitofusin genes Mfn1 and Mfn2 (ßMfn1/2 dKO). ßMfn1/2-dKO mice displayed elevated fed and fasted glycemia and a more than fivefold decrease in plasma insulin. Mitochondrial length, glucose-induced polarization, ATP synthesis, and cytosolic and mitochondrial Ca2+ increases were all reduced in dKO islets. In contrast, oral glucose tolerance was more modestly affected in ßMfn1/2-dKO mice, and glucagon-like peptide 1 or glucose-dependent insulinotropic peptide receptor agonists largely corrected defective glucose-stimulated insulin secretion through enhanced EPAC-dependent signaling. Correspondingly, cAMP increases in the cytosol, as measured with an Epac-camps-based sensor, were exaggerated in dKO mice. Mitochondrial fusion and fission cycles are thus essential in the ß-cell to maintain normal glucose, but not incretin, sensing. These findings broaden our understanding of the roles of mitofusins in ß-cells, the potential contributions of altered mitochondrial dynamics to diabetes development, and the impact of incretins on this process.

High-Throughput Image Analysis of Lipid-Droplet-Bound Mitochondria.

Changes to mitochondrial architecture are associated with various adaptive and pathogenic processes. However, quantification of changes to mitochondrial structures is limited by the yet unmet challenge of defining the borders of each individual mitochondrion within an image. Here, we describe a novel method for segmenting primary brown adipocyte (BA) mitochondria images. We describe a granular approach to quantifying subcellular structures, particularly mitochondria in close proximity to lipid droplets: peridroplet mitochondria. In addition, we lay out a novel machine-learning-based mitochondrial segmentation method that eliminates the bias of manual mitochondrial segmentation and improves object recognition compared to conventional thresholding analyses. By applying these methods, we discovered a significant difference between cytosolic and peridroplet BA mitochondrial H2O2 production and validated the machine-learning algorithm in BA via norepinephrine-induced mitochondrial fragmentation and comparing manual analyses to the automated analysis. This approach provides a high-throughput analysis protocol to quantify ratiometric probes in subpopulations of mitochondria in adipocytes.

Mitochondrial Proton Leak Regulated by Cyclophilin D Elevates Insulin Secretion in Islets at Nonstimulatory Glucose Levels.

Fasting hyperinsulinemia precedes the development of type 2 diabetes. However, it is unclear whether fasting insulin hypersecretion is a primary driver of insulin resistance or a consequence of the progressive increase in fasting glycemia induced by insulin resistance in the prediabetic state. Herein, we have discovered a mechanism that specifically regulates non-glucose-stimulated insulin secretion (NGSIS) in pancreatic islets that is activated by nonesterified free fatty acids, the major fuel used by ß-cells during fasting. We show that the mitochondrial permeability transition pore regulator cyclophilin D (CypD) promotes NGSIS, but not glucose-stimulated insulin secretion, by increasing mitochondrial proton leak. Islets from prediabetic obese mice show significantly higher CypD-dependent proton leak and NGSIS compared with lean mice. Proton leak-mediated NGSIS is conserved in human islets and is stimulated by exposure to nonesterified free fatty acids at concentrations observed in obese subjects. Mechanistically, proton leak activates islet NGSIS independently of mitochondrial ATP synthesis but ultimately requires closure of the KATP channel. In summary, we have described a novel nonesterified free fatty acid-stimulated pathway that selectively drives pancreatic islet NGSIS, which may be therapeutically exploited as an alternative way to halt fasting hyperinsulinemia and the progression of type 2 diabetes.

Cell culture models of fatty acid overload: Problems and solutions.

High plasma levels of fatty acids occur in a variety of metabolic diseases. Cellular effects of fatty acid overload resulting in negative cellular responses (lipotoxicity) are often studied in vitro, in an attempt to understand mechanisms involved in these diseases. Fatty acids are poorly soluble, and thus usually studied when complexed to albumins such as bovine serum albumin (BSA). The conjugation of fatty acids to albumin requires care pertaining to preparation of the solutions, effective free fatty acid concentrations, use of different fatty acid species, types of BSA, appropriate controls and ensuring cellular fatty acid uptake. This review discusses lipotoxicity models, the potential problems encountered when using these cellular models, as well as practical solutions for difficulties encountered.

Weight, blood pressure, and dietary benefits after 12 months of a Web-based Nutrition Education Program (DASH for health): longitudinal observational study.

BACKGROUND: The dietary habits of Americans are creating serious health concerns, including obesity, hypertension, diabetes, cardiovascular disease, and even some types of cancer. While considerable attention has been focused on calorie reduction and weight loss, approaches are needed that will not only help the population reduce calorie intake but also consume the type of healthy, well-balanced diet that would prevent this array of medical complications. OBJECTIVE: To design an Internet-based nutrition education program and to explore its effect on weight, blood pressure, and eating habits after 12 months of participation. METHODS: We designed the DASH for Health program to provide weekly articles about healthy nutrition via the Internet. Dietary advice was based on the DASH diet (Dietary Approaches to Stop Hypertension). The program was offered as a free benefit to the employees of EMC Corporation, and 2834 employees and spouses enrolled. Enrollees voluntarily entered information about themselves on the website (food intake), and we used these self-entered data to determine if the program had any effect. Analyses were based upon the change in weight, blood pressure, and food intake between the baseline period (before the DASH program began) and the 12th month. To be included in an outcome, a subject had to have provided both a baseline and 12th-month entry. RESULTS: After 12 months, 735 of 2834 original enrollees (26%) were still actively using the program. For subjects who were overweight/obese (body mass index > 25; n = 151), weight change at 12 months was -4.2 lbs (95% CI: -2.2, -6.2; P < .001). For subjects with hypertension or prehypertension at baseline (n = 62), systolic blood pressure fell 6.8 mmHg at 12 months (CI: -2.6, -11.0; P < .001; n = 62). Diastolic pressure fell 2.1 mmHg (P = .16). Based upon self-entered food surveys, enrollees (n = 181) at 12 months were eating significantly more fruits, more vegetables, and fewer grain products. They also reduced consumption of carbonated beverages. Enrollees who had visited the website more often tended to have greater blood pressure and weight loss effect, suggesting that use of the DASH for Health program was at least partially responsible for the benefits we observed. CONCLUSIONS: We have found that continued use of a nutrition education program delivered totally via the Internet, with no person-to-person contact with health professionals, is associated with significant weight loss, blood pressure lowering, and dietary improvements after 12 months. Effective programs like DASH for Health, delivered via the Internet, can provide benefit to large numbers of subjects at low cost and may help address the nutritional public health crisis.