Abstract
Chronic high glucose (HG) inflicts glucotoxicity on vulnerable cell types such as pancreatic β cells and contributes to insulin resistance and impaired insulin secretion in diabetic patients. To identify HG-induced cellular aberrations that are candidate mediators of glucotoxicity in pancreatic β cells, we analyzed gene expression in ERoSHK6, a mouse insulin-secreting cell line after chronic HG exposure (six-day exposure to 33.3 mM glucose). Chronic HG exposure which reduced glucose-stimulated insulin secretion (GSIS) increased transcript levels of 185 genes that clustered primarily in 5 processes namely cellular growth and proliferation; cell death; cellular assembly and organization; cell morphology; and cell-to-cell signaling and interaction. The former two were validated by increased apoptosis of ERoSHK6 cells after chronic HG exposure and reaffirmed the vulnerability of β cells to glucotoxicity. The three remaining processes were partially substantiated by changes in cellular morphology and structure, and instigated an investigation of the cytoskeleton and cell-cell adhesion. These studies revealed a depolymerized actin cytoskeleton that lacked actin stress fibers anchored at vinculin-containing focal adhesion sites as well as loss of E-cadherin-mediated cell-cell adherence after exposure to chronic HG, and were concomitant with constitutive ERK1/2 phosphorylation that was refractory to serum and glucose deprivation. Although inhibition of ERK phosphorylation by PD98059 promoted actin polymerization, it increased apoptosis and GSIS impairment. These findings suggest that ERK phosphorylation is a proximate regulator of cellular processes targeted by chronic HG-induced gene expression and that dynamic actin polymerization and depolymerization is important in β cell survival and function. Therefore, chronic HG alters gene expression and signal transduction to predispose the cytoskeleton towards apoptosis and GSIS impairment.
Highlights
Diabetes mellitus is an epidemic metabolic disease with mild early symptomatic manifestations that as a result of poor disease management, often lead to severe complications such as cardiovascular disease, limb amputation, diabetic retinopathy, renal failure, diabetic neuropathy
ERoSHK6 cells, the cells were cultured in INS medium containing either low glucose (LG) or high glucose (HG)
Relative to ERoSHKLG, basal insulin secretion of ERoSHKHG when assayed in the presence of 2.8 mM glucose was increased by 43.70% (P,0.001) while its glucose stimulated insulin secretion (GSIS) as assayed in the presence of 16.7 mM glucose was reduced by 36.33% in ERoSHKHG (P,0.005) (Fig. 1)
Summary
Diabetes mellitus is an epidemic metabolic disease with mild early symptomatic manifestations that as a result of poor disease management, often lead to severe complications such as cardiovascular disease, limb amputation, diabetic retinopathy, renal failure, diabetic neuropathy (http://www.who.int/topics/ diabetes_mellitus/en/; accessed August 2011). Excessive glucose uptake has been shown to induce biochemical changes such as increased flux through the polyol pathway, enhanced production of advanced glycation end products (AGEs), activation of PKC, and increased activity of the hexosoamine pathway [2]. These biochemical changes have the potential to increase reactive oxygen species production and to modify proteins and lipids that could result in global cellular damage and severe phenotypic alterations such as mitochondria dysfunction [7], endoplasmic reticulum stress [8], increased intracellular calcium and cell death [9]. Since b cell dysfunction has been shown to be central to the development and progression of T2D [18], timely interventional therapies to preserve the important b cell function of GSIS and viability are likely to improve the prognosis of the disease [19,20]
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