Hyperglycemia decreases mitochondrial function: The regulatory role of mitochondrial biogenesis
Introduction
Diabetes mellitus is a metabolic disorder characterized by hyperglycemia and insufficiency of secretion or receptor insensitivity to endogenous insulin. The number of adults with clinical diagnosis of diabetes has been increasing dramatically worldwide. It has been estimated that the number of adults affected by diabetes in the world will grow from 135 million in 1995 to 300 million in the year 2025 (King et al., 1998). While exogenous insulin and other medications can control many aspects of diabetes, assorted complications affecting the vascular system, kidney, and peripheral nerves are common and extremely costly in terms of longevity and quality of life.
It is well established that hyperglycemia elicits an increase in reactive oxygen species (ROS) production, due to increased input of reducing equivalents into the mitochondrial electron transport chain (Nishikawa et al., 2000a, Brownlee, 2001). ROS overproduction is the trigger of the pathways responsible for hyperglycemia-induced cell damage: (1) increased polyol pathway flux; (2) increased advanced glycation end product (AGE) formation; (3) activation of protein kinase C (PKC) isoforms; and (4) increased hexosamine pathway flux (Nishikawa et al., 2000b, Brownlee, 2001, Robertson, 2004).
Diabetes-associated alterations in mitochondrial phenotype have been widely described (Rolo and Palmeira, 2006). In animal models of diabetes, distinct mitochondrial alterations as a function of age have been observed, indicating differential adaptation mechanisms to counteract high glucose levels (Palmeira et al., 1999, Ferreira et al., 1999, Ferreira et al., 2003). Since mitochondrial number and function require both nuclear and mitochondrial-encoded genes, coordinated mechanisms exist to regulate the two genomes and determine the overall oxidative capacity (Kelly and Scarpulla, 2004). Adaptive responses of mitochondrial function to diabetic stress may reflect changes in mitochondrial gene expression induced by hyperglycemia. Growing evidence indicates that several transcriptional changes in diabetes are associated with impaired mitochondrial function and altered glucose and fatty acid metabolism, characteristics of diabetes mellitus. Reduced expression of oxidative phosphorylation genes has been observed in type 2 diabetes (Mootha et al., 2003), accompanied by decreased expression of peroxisomal proliferators activator receptor γ coactivator-α (PGC-1α) in prediabetic and diabetic muscle (Patti et al., 2003). PGC-1α is an integrator of the molecular regulatory circuit involved in the transcriptional control of cellular energy metabolism, including mitochondrial biogenesis, hepatic gluconeogenesis, and fatty acid β-oxidation (Puigserver and Spiegelman, 2003). Recent work by Yu and collaborators has shown that exposure to high glucose conditions leads to dynamic changes in mitochondrial morphology due to prolonged ROS overproduction (Yu et al., 2006). However, the relationship between ROS production and mitochondrial biogenesis is still unclear.
Based on the indication of modulation of gene expression and alteration of mitochondrial function by hyperglycemia, we hypothesize that hyperglycemia-induced increased ROS production is the trigger for decreased mitochondrial biogenesis. We demonstrated that prolonged hyperglycemia-induced ROS overproduction causes a decrease in mitochondrial copy number in HepG2 cells. We also found that this decrease in mitochondrial biogenesis is coincident to a decrease in TFAM transcripts and results in a loss of respiratory efficiency. Liver being the primary organ involved in blood glucose metabolism, decreased mitochondrial biogenesis and subsequent impairment of oxidative metabolism will establish a vicious cycle of metabolic alterations implicated in diabetes pathogenesis.
Section snippets
Cell culture
HepG2/C3A cells obtained from American Type Culture Collection (ATCC, Rockville, MD) were cultured in Eagle's minimum essential medium (MEM) (with 2 mM l-glutamine and Earl's BSS adjusted to contain 1.5 g/l sodium bicarbonate, 0.1 mM nonessential amino acids, and 1 mM sodium pyruvate (Sigma, St. Louis, MO)), supplemented with 10% fetal bovine serum. Cells were plated on 25- or 75-cm2 tissue culture flasks and grown in a 5% CO2 incubator at 37 °C with saturating humidity and growth media changes
Hyperglycemia induces an increase in PAI-1 expression without changes in cell viability
We examined cell viability in HepG2 cells exposed to hyperglycemic conditions (30 mM glucose) for 48 h, 96 h and 7 days. No differences were observed in ethidium homodimer-1 and calcein-AM fluorescence when compared with cells cultured in 5.5 mM glucose (data not shown). All cell cultures were greater than 85% viable with all treatments and at all time points. To confirm that extended exposure of HepG2 cells to high glucose conditions was mimicking untreated diabetic conditions, PAI-1
Discussion
Our data demonstrate that long-term exposure of HepG2 cells to high glucose conditions leads to loss of respiratory capacity and decreased mitochondrial biogenesis, the two of which may be causally related.
ROS production via the mitochondrial respiratory chain has been shown to be the causal link between high glucose and the main pathways responsible for hyperglycemic damage. The prevailing hypothesis is that hyperglycemia-induced increase in electron transfer donors (NADH and FADH2) increases
Acknowledgments
We would like to acknowledge LaRae Peterson for fluorescence microscopy. This work was in part supported by HL 58016 and HL 72175 grants from National Institute of Health. C.M. Palmeira is recipient of a fellowship from Science and Technology Foundation (SFRH/BSAB/349/2003). A. P. Rolo is a recipient of fellowship from Science and Technology Foundation (SFRH/BPD/26514/2006).
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