Differential Mitochondrial Adaptation in Primary Vascular Smooth Muscle Cells from a Diabetic Rat Model.

Amy C Keller,Jane E B Reusch,Dwight J Klemm,Matthew W Miller,Chelsea A Connon,P Mason Mcclatchey,Leslie A Knaub,Kate E Geary,Lori A Walker,Ron Bouchard

doi:10.1155/2016/8524267

Abstract

Diabetes affects more than 330 million people worldwide and causes elevated cardiovascular disease risk. Mitochondria are critical for vascular function, generate cellular reactive oxygen species (ROS), and are perturbed by diabetes, representing a novel target for therapeutics. We hypothesized that adaptive mitochondrial plasticity in response to nutrient stress would be impaired in diabetes cellular physiology via a nitric oxide synthase- (NOS-) mediated decrease in mitochondrial function. Primary smooth muscle cells (SMCs) from aorta of the nonobese, insulin resistant rat diabetes model Goto-Kakizaki (GK) and the Wistar control rat were exposed to high glucose (25 mM). At baseline, significantly greater nitric oxide evolution, ROS production, and respiratory control ratio (RCR) were observed in GK SMCs. Upon exposure to high glucose, expression of phosphorylated eNOS, uncoupled respiration, and expression of mitochondrial complexes I, II, III, and V were significantly decreased in GK SMCs (p < 0.05). Mitochondrial superoxide increased with high glucose in Wistar SMCs (p < 0.05) with no change in the GK beyond elevated baseline concentrations. Baseline comparisons show persistent metabolic perturbations in a diabetes phenotype. Overall, nutrient stress in GK SMCs caused a persistent decline in eNOS and mitochondrial function and disrupted mitochondrial plasticity, illustrating eNOS and mitochondria as potential therapeutic targets.

Highlights

Diabetes imparts staggering social and economic costs worldwide
Collagenase, ethylenediaminetetraacetic acid (EDTA), ethylene glycol tetra-acetic acid (EGTA), sodium pyrophosphate, sodium orthovanadate, sodium fluoride, okadaic acid, 1% protease inhibitor cocktail, dithiothreitol, magnesium chloride, Klactobionate, taurine, potassium phosphate, HEPES, digitonin, pyruvate, malic acid, glutamic acid, adenosine diphosphate (ADP), succinic acid, oligomycin, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), antibody to β-actin, phenylephrine and acetylcholine, trypsin inhibitor, and cytochrome c were procured from SigmaAldrich (MO, USA)
In the representative blot pictured, dimer Endothelial nitric oxide synthase GK (eNOS) is visible at 8.09% higher concentrations in aorta from the 18-week-old GK as compared to the Wistar rats (Figure 1(b)), and 40% higher concentrations of the monomer of eNOS are seen in GK aorta (Figure 1(b))

Summary

Introduction

Diabetes imparts staggering social and economic costs worldwide. It is known that people with diabetes have a 3–5-fold higher risk of cardiovascular disease (CVD) than the nondiabetic population [1]. Vascular remodeling, characterized by endothelial dysfunction and vascular stiffness and seen in the context of diabetes, hyperglycemia, and elevated oxidative stress, heralds CVD onset [2, 3]. Elucidating early cellular mechanistic pathology is critical to understanding disease progression. Mitochondria have recently emerged as therapeutic targets in chronic diseases. They are critical signaling hubs in vascular processes such as the endothelial regulation of vasomotion, calcium signaling associated with vascular relaxation, smooth muscle cell proliferation, and apoptosis [4,5,6]. Excess cellular ROS in the vasculature may originate from dysfunctional mitochondria, and this excess ROS precedes vascular inflammation, vascular stiffness, and SMCs apoptosis [7,8,9,10]. Excess mitochondrial ROS is associated with characteristics of type 2 diabetes such as hyperglycemia, decreased antioxidant defense, and insulin resistance [11,12,13,14,15]

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