Abstract
The biochemical mechanisms underlying glucose-stimulated insulin secretion from pancreatic beta-cells are not completely understood. To identify metabolic disturbances in beta-cells that impair glucose-stimulated insulin secretion, we compared two INS-1-derived clonal beta-cell lines, which are glucose-responsive (832/13 cells) or glucose-unresponsive (832/2 cells). To this end, we analyzed a number of parameters in glycolytic and mitochondrial metabolism, including mRNA expression of genes involved in cellular energy metabolism. We found that despite a marked impairment of glucose-stimulated insulin secretion, 832/2 cells exhibited a higher rate of glycolysis. Still, no glucose-induced increases in respiratory rate, ATP production, or respiratory chain complex I, III, and IV activities were seen in the 832/2 cells. Instead, 832/2 cells, which expressed lactate dehydrogenase A, released lactate regardless of ambient glucose concentrations. In contrast, the glucose-responsive 832/13 line lacked lactate dehydrogenase and did not produce lactate. Accordingly, in 832/2 cells mRNA expression of genes for glycolytic enzymes were up-regulated, whereas mitochondria-related genes were down-regulated. This could account for a Warburg-like effect in the 832/2 cell clone, lacking in 832/13 cells as well as primary beta-cells. In human islets, mRNA expression of genes such as lactate dehydrogenase A and hexokinase I correlated positively with HbA(1c) levels, reflecting perturbed long term glucose homeostasis, whereas that of Slc2a2 (glucose transporter 2) correlated negatively with HbA(1c) and thus better metabolic control. We conclude that tight metabolic regulation enhancing mitochondrial metabolism and restricting glycolysis in 832/13 cells is required for clonal beta-cells to secrete insulin robustly in response to glucose. Moreover, a similar expression pattern of genes controlling glycolytic and mitochondrial metabolism in clonal beta-cells and human islets was observed, suggesting that a similar prioritization of mitochondrial metabolism is required in healthy human beta-cells. The 832 beta-cell lines may be helpful tools to resolve metabolic perturbations occurring in Type 2 diabetes.
Highlights
Pyruvate carboxylase (PC), catalyzing the carboxylation of pyruvate to oxaloacetate, is a key regulator of cellular anaplerosis, providing a two-carbon net addition to the tricarboxylic acid cycle, thereby replenishing the cycle with intermediates
To gain further insight into the mechanisms controlling insulin secretion, we have examined two clonal cell lines derived from the INS-1 cell line, which was initially established from cells isolated from an x-ray-induced rat transplantable insulinoma [9]
We found that the glucose-responsive 832/13 cells exhibited a tight regulation of mitochondrial metabolism in response to changes in ambient glucose; this regulation was lost in the glucose-unresponsive 832/2 cell line, which instead favored glycolytic metabolism and production of lactate
Summary
Reagents—All of the reagents and solutions were obtained from Sigma unless otherwise indicated. Lactate released from cells incubated at 2.8 and 16.7 mM glucose was measured in supernatants, using a colorimetric lactate assay kit (Biovision, Mountain View, CA). Total RNA (100 –200 ng) was processed following the GeneChip expression 3Ј-amplification reagents one-cycle cDNA synthesis kit instructions (Affymetrix Inc., Santa Clara, CA) to produce double-stranded cDNA. This was used as a template to generate biotin-targeted cRNA following the manufacturer’s specifications. Protein Measurement—Protein content was determined in the obtained cell lysates by the bicinchoninic acid protein assay (Thermo), measuring the absorbance at 590 nm and comparing it with a bovine serum albumin standard curve. Differences between mean values were considered significant when p Ͻ 0.05
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
