Type 2 diabetes mellitus (T2DM) is a metabolic disorder in which pancreatic insulin secretion does not meet the demands of insulin sensitivity [1, 2]. Over a period of time, consistently elevated levels of blood glucose and free fatty acids lead to glucolipotoxicity- mediated pancreatic beta cell dysfunction [3, 4]. It is now accepted that elevated glucose levels are required to mediate the lipotoxic effects, including inhibition of glucose-stimulated insulin secretion (GSIS), impaired insulin gene expression and apoptosis [4–8].
GSIS involves both glucose oxidation-coupled ATP production and the anaplerotic/cataplerotic pathway-mediated generation of coupling factors that trigger and amplify insulin secretion, respectively [9, 10]. Briefly, glucose uptake initiates metabolic pathways in which glucose is first converted to pyruvate mediated by glucokinase, and then to oxaloacetate by pyruvate carboxylase. Mitochondrial oxaloacetate generates citrate, a cataplerotic signal, which is transported to the cytosol and then broken down into acetyl-CoA initiating fatty acid synthesis. Acetyl-CoA is subsequently converted to malonyl-CoA, the concomitant step in fatty acid synthesis. In pancreatic beta cells, malonyl-CoA inhibits carnitine-palmitoyl transferase-1 (CPT-1) blocking fatty acid oxidation and resulting in the buildup of long-chain acyl-CoA esters (LC-CoA) in the cytosol . Long chain-CoA is thought to be a potential modulator of insulin secretion stimulating insulin granule docking and exocytosis [11, 12]. Glucose metabolism also raises the cytosolic ATP/ADP ratio, which inhibits the ATP-sensitive potassium channel (KATP) resulting in plasma membrane depolarization. In response to this, voltage-gated calcium channels open, causing an influx of extracellular calcium and exocytosis of insulin granules .
Another well-known role of glucose is augmenting insulin secretion by promoting phospholipase-C (PLC)-mediated hydrolysis of phosphatidylinositol 4, 5-biphosphate (PIP2) into diacylglycerol (DAG) and inositol triphosphate (IP3) . The DAG generated, in turn, activates protein kinase C (PKC), which is known to maintain insulin exocytosis [15, 16], while IP3 mobilizes calcium from endoplasmic reticulum stores. The PLC pathway is also known to upregulate cAMP levels in beta cells, which show glucose-mediated oscillations that correlate with insulin secretion [17, 18].
Further, glucose is known to increase insulin content through insulin gene transcription mediated by PDX1 and MAFa . Under normal conditions, the synthesized insulin is held in readily releasable pools which are transported to the plasma membrane by the small GTPase, Rab27a and the SNARE complex for acute calcium-mediated release [20, 21].
Chronic hyperglycemia (glucotoxicity) and hyperlipidemia (lipotoxicity) have been known to impair beta cell function [22, 23], and glucolipotoxicity has been defined as ‘the deleterious effects of elevated glucose and fatty acids on pancreatic beta cell-function and mass’ . Studies by Kashyap et al. in human subjects have shown that the ability of the beta cell to increase insulin secretion in response to fatty acids is a component that may predispose to T2DM . In accordance with this, animal models for T2DM show a glucolipotoxicity-mediated dysfunction in multiple cellular processes involved in insulin secretion [26–27 and references therein]. In vitro studies have been an important source of information to understand the molecular basis of glucolipotoxicity. For example, fatty acid-mediated inhibition of insulin gene transcription, which was identified in vitro, has been recapitulated in vivo. However, a known limitation of the in vitro studies in this area of research has been the varying concentrations of fatty acid used .
Here, we used specific concentrations of glucose and palmitate to study the effects of in vitro chronic glucolipotoxic conditions on intracellular signaling pathways and cellular processes that mediate glucose responsiveness and insulin secretion. We confirmed metabolic stress in pancreatic islets under these conditions using known stress markers. We found that chronic glucolipotoxicity impaired glucose and fat uptake/metabolism in rat pancreatic cells resulting in lower cellular ATP along with mitochondrial number and activity. In agreement with this, IP3 levels were also reduced as was the calcium mobilized by the IP3 receptor and the L-type voltage gated calcium channels. Finally, we found that chronic glucolipotoxicity significantly decreased insulin secretion by reducing both insulin gene expression and granule docking to the plasma membrane in pancreatic islets. Thus, our results present the first integrated view of glucolipotoxicity in vitro linking known and novel signaling events to reduced glucose sensitivity and insulin secretion.