Resveratrol long-term treatment differentiates INS-1E beta-cell towards improved glucose response and insulin secretion.

The clonal INS-1E beta-cell line has proven to be instrumental for numerous studies investigating the mechanisms of glucose-stimulated insulin secretion. The composition of its culture medium has not changed over the years, although some compounds have been recently highlighted for their effects on tissue differentiation. The present study investigated the effects of long-term treatment of INS-1E cells with 1 µM resveratrol on glucose-stimulated insulin secretion, testing an extended glucose dose response. The data demonstrate that chronic exposure to low-dose resveratrol expands the range of the glucose dose response of INS-1E cells beyond 15 mM glucose. We also assessed whether such beneficial effects could be retained after resveratrol withdrawal from the culture medium. This was not the case as INS-1E cells deprived of resveratrol returned to the phenotype of naïve cells, i.e., exhibiting a plateau phase at 15 mM glucose. Of note, although resveratrol has antioxidant properties, it cannot substitute for ß-mercaptoethanol normally present in the medium of INS-1E cells as a reducing agent. In conclusion, the addition of resveratrol as a standard component of the culture medium of INS-1E cells improves glucose-stimulated insulin secretion.

Silencing of the mitochondrial NADH shuttle component aspartate-glutamate carrier AGC1/Aralar1 in INS-1E cells and rat islets.

Transfer of reducing equivalents between cytosolic compartments and the mitochondrial matrix is mediated by NADH shuttles. Among these, the malate-aspartate shuttle has been proposed to play a major role in beta-cells for the control of glucose-stimulated insulin secretion. AGC1 or Aralar1 (aspartate-glutamate carrier 1) is a key component of the malate-aspartate shuttle. Overexpression of AGC1 increases the capacity of the malate-aspartate shuttle, resulting in enhanced metabolism-secretion coupling, both in INS-1E cells and rat islets. In the present study, knockdown of AGC1 was achieved in the same beta-cell models, using adenovirus-mediated delivery of shRNA (small-hairpin RNA). Compared with control INS-1E cells, down-regulation of AGC1 blunted NADH formation (-57%; P<0.05), increased lactate production (+16%; P<0.001) and inhibited glucose oxidation (-22%; P<0.01). This correlated with a reduced secretory response at 15 mM glucose (-25%; P<0.05), while insulin release was unchanged at intermediate 7.5 mM and basal 2.5 mM glucose. In isolated rat islets, efficient AGC1 knockdown did not alter insulin exocytosis evoked by 16.7 mM glucose. However, 4 mM amino-oxyacetate, commonly used to block transaminases of the malate-aspartate shuttle, inhibited glucose-stimulated insulin secretion to similar extents in INS-1E cells (-66%; P<0.01) and rat islets (-56%; P<0.01). These results show that down-regulation of the key component of the malate-aspartate shuttle AGC1 reduced glucose-induced oxidative metabolism and insulin secretion in INS-1E cells, whereas similar AGC1 knockdown in rat islets did not affect their secretory response.

The antiepileptic drug topiramate preserves metabolism-secretion coupling in insulin secreting cells chronically exposed to the fatty acid oleate.

Topiramate (Topamax), primarily prescribed against epilepsy, was reported to reduce body weight and to ameliorate glycemic control in obese patients with diabetes. In rodent models of obesity and diabetes, topiramate treatment counteracts hyperglycemia and increases insulin levels upon glucose tolerance test. These observations suggest that topiramate might exert direct action on insulin secreting cells, in particular regarding obesity associated beta-cell dysfunction. In this study, INS-1E beta-cells were exposed for 3 days to the fatty acid oleate (0.4mM) and concomitantly treated with therapeutic concentrations of topiramate before measurements of insulin secretion and metabolic parameters. In healthy cells, topiramate had no acute or chronic effects on insulin release. Exposure of INS-1E cells to oleate for 3 days increased insulin release at basal 2.5mM glucose and blunted the response to stimulatory glucose concentration (15mM). Such lipotoxic effects were associated with impaired mitochondrial function, as evidenced by partial loss of resting mitochondrial membrane potential and reduced hyperpolarization in response to glucose. Oil-red-O staining and triglyceride measurements revealed lipid accumulation in oleate treated cells. Topiramate treatment counteracted oleate-induced lipid load and partially protected against mitochondrial membrane dysfunction. In particular, topiramate restored glucose stimulated insulin secretion, essentially by maintaining low insulin release at basal glucose. Topiramate increased expression of the nutrient sensor PPARalpha and of the mitochondrial fatty acid carrier CPT-1, correlating with enhancement of beta-oxidation rate. The data demonstrate that a drug originally used as mood stabilizer exerts a direct action on beta-cells, protecting against lipid-induced dysfunction.

Glucose sensitivity and metabolism-secretion coupling studied during two-year continuous culture in INS-1E insulinoma cells.

Rat insulinoma-derived INS-1 cells constitute a widely used beta-cell surrogate. However, due to their nonclonal nature, INS-1 cells are heterogeneous and are not stable over extended culture periods. We have isolated clonal INS-1E cells from parental INS-1 based on both their insulin content and their secretory responses to glucose. Here we describe the stable differentiated INS-1E beta-cell phenotype over 116 passages (no. 27-142) representing a 2.2-yr continuous follow-up. INS-1E cells can be safely cultured and used within passages 40-100 with average insulin contents of 2.30 +/- 0.11 microg/million cells. Glucose-induced insulin secretion was dose-related and similar to rat islet responses. Secretion saturated with a 6.2-fold increase at 15 mm glucose, showing a 50% effective concentration of 10.4 mm. Secretory responses to amino acids and sulfonylurea were similar to those of islets. Moreover, INS-1E cells retained the amplifying pathway, as judged by glucose-evoked augmentation of insulin release in a depolarized state. Regarding metabolic parameters, INS-1E cells exhibited glucose dose-dependent elevations of NAD(P)H, cytosolic Ca(2+), and mitochondrial Ca(2+) levels. In contrast, mitochondrial membrane potential, ATP levels, and cell membrane potential were all fully activated by 7.5 mm glucose. Using the perforated patch clamp technique, 7.5 and 15 mm glucose elicited electrical activity to a similar degree. A K(ATP) current was identified in whole cell voltage clamp using diazoxide and tolbutamide. As in native beta-cells, tolbutamide induced electrical activity, indicating that the K(ATP)conductance is important in setting the resting potential. Therefore, INS-1E cells represent a stable and valuable beta-cell model.