Mechanisms and Consequences of GLO1 Attenuation in Obese Skeletal Muscle: Exploring a Potential Role for SIRT1-Mediated GLO1 Stability

Abstract

The obesity pandemic is persistent with global rates of obesity exceeding 13%. Obesity results in the development of insulin resistance which through a myriad of mechanisms increases the risk for secondary disease development such as type 2 diabetes (T2D). Glyoxalase I (GLO1) is the rate-limiting enzyme for detoxification of the reactive dicarbonyl methylglyoxal which is formed in proportion to glycolytic flux and rapidly modifies and damages proteins, and DNA. Loss of GLO1 drives the development of insulin resistance (IR) and T2D in preclinical models. Skeletal muscle is critical for whole body insulin stimulated glucose uptake and plays a major role in T2D development. We previously demonstrated that muscle from patients with T2D possess lower levels GLO1 protein which was correlated with BMI and clamp-derived insulin sensitivity. Our preliminary data demonstrated GLO1 is attenuated in muscle from individuals with obesity prior to T2D development. However, the mechanisms, and consequences of attenuated muscle GLO1 are unknown. Recent in vitro evidence suggests that GLO1 is acetylated under obesogenic conditions, marking it for ubiquitination and degradation although the factors regulating GLO1 acetylation were not identified. SIRT1 is an NAD+-dependent deacetylase that promotes oxidative metabolism, is augmented by exercise, and is purported to be attenuated with obesity. Therefore, we tested the hypothesis that loss of SIRT1 would promote GLO1 acetylation thereby destabilizing and attenuating GLO1 protein. We also set out to determine if acute exercise could rescue GLO1 protein in individuals with obesity, potentially by promoting SIRT1 and GLO1 deacetylation. Lastly, we employed human immortalized myotubes to directly test the proposed mechanisms of GLO1 acetylation and determine the proteomic consequences of GLO1 knock down in muscle. We collected muscle biopsies from 15 lean healthy (LH) individuals and 5 individuals with obesity (OB) before, 30 minutes, and 3 hours after treadmill exercise at 80% of participants’ VO2Max. Baseline GLO1 (p < 0.01) and SIRT1 (p < 0.05) proteins were lower in OB muscle and GLO1 protein was correlated to SIRT1 protein (Rho = 0.639, p < 0.01). Knock down of GLO1 in human immortalized myotubes upregulated ubiquitin proteasome, and apoptosis pathways while downregulating glycolytic proteins and proteins involved in sarcomere organization. Acute exercise provoked a trend in increased SIRT1 protein 30 minutes after exercise in the OB group (post-hoc pre – 30 min. p = 0.13) along with a trend for decreased GLO1 acetylation (p = 0.09). However, GLO1 protein abundance and activity were not affected by exercise in OB muscle. Studies in human immortalized myotubes aimed at depleting or rescuing SIRT1 activity by modulating cellular NAD+ or by directly knocking down SIRT1 did not alter GLO1 acetylation, protein, mRNA or activity. Lastly, a screen of other factors that regulate protein acetylation revealed differential effects of specific deacetylases (SIRT2) and acetyltransferases (GCN5 and P300) on GLO1 protein and activity. Collectively these data are the first to demonstrate lower GLO1 protein abundance in muscle from individuals with obesity and to describe the proteomic consequences of GLO1 attenuation in human muscle. We also excluded SIRT1 as a regulator of GLO1 while demonstrating the potential for more nuanced regulation of GLO1 by acetylation. These data establish GLO1 as a novel target for the prevention and treatment of obesity-related maladaptive phenotypes in muscle. Future work is still needed to uncover targetable mechanisms underpinning GLO1 attenuation with obesity in muscle.