SUMMARY Type 2 diabetes Mellitus (T2DM) and aging are characterized by insulin resistance, lower mitochondrial density and function and increased production of reactive oxygen species (ROS). In lower organisms continuous remodeling critically maintains the function and life cycle of mitochondria, in part by the protease pcp1 (PARL ortholog). We therefore examined whether variation in PARL protein content is associated with mitochondrial abnormalities and insulin resistance. Relative to healthy, young individuals (23±1y), PARL mRNA and mitochondrial mass were both reduced in elderly subjects (64.4±1.2 y; 51% and 44% respectively) and in subjects with T2DM (51.8±3 y; 31% and 41% respectively; all p<0.05). Muscle knock-down of PARL in mice resulted in lower mitochondrial content (−31±3%, p<0.05), lower OPA1 and PGC1α protein levels and impaired insulin signaling. Furthermore, mitochondrial cristae were malformed and resulted in elevated in vivo oxidative stress. Adenoviral suppression of PARL protein in healthy myotubes lowered mitochondrial mass (−33±8%), insulin stimulated glycogen synthesis (−33±9%) and increased ROS production (2-fold) (all p<0.05). We propose that lower PARL expression may contribute to the mitochondrial abnormalities seen in aging and T2DM.

Aims/hypothesis: This study aimed to identify genes that are expressed in skeletal muscle, encode proteins with functional significance in mitochondria, and are associated with type 2 diabetes. Methods: We screened for differentially expressed genes in skeletal muscle of Psammomys obesus (Israeli sand rats), and prioritised these on the basis of genomic localisation and bioinformatics analysis for proteins with likely mitochondrial functions. Results: We identified a mitochondrial intramembrane protease, known as presenilins-associated rhomboid-like protein (PSARL) that is associated with insulin resistance and type 2 diabetes. Expression of PSARL was reduced in skeletal muscle of diabetic Psammomys obesus, and restored after exercise training to successfully treat the diabetes. PSARL gene expression in human skeletal muscle was correlated with insulin sensitivity as assessed by glucose disposal during a hyperinsulinaemic-euglycaemic clamp. In 1,031 human subjects, an amino acid substitution (Leu262Val) in PSARL was associated with increased plasma insulin concentration, a key risk factor for diabetes. Furthermore, this variant interacted strongly with age to affect insulin levels, acounting for 5% of the variation in plasma insulin in elderly subjects. Conclusions/interpretation: Variation in PSARL sequence and/or expression may be an important new risk factor for type 2 diabetes and other components of the metabolic syndrome.

Drugs that recapitulate aspects of the exercise adaptive response have the potential to provide better treatment for diseases associated with physical inactivity. We previously observed reduced skeletal muscle class IIa HDAC (histone deacetylase) transcriptional repressive activity during exercise. Here, we find that exercise-like adaptations are induced by skeletal muscle expression of class IIa HDAC mutants that cannot form a corepressor complex. Adaptations include increased metabolic gene expression, mitochondrial capacity, and lipid oxidation. An existing HDAC inhibitor, Scriptaid, had similar phenotypic effects through disruption of the class IIa HDAC corepressor complex. Acute Scriptaid administration to mice increased the expression of metabolic genes, which required an intact class IIa HDAC corepressor complex. Chronic Scriptaid administration increased exercise capacity, whole-body energy expenditure and lipid oxidation, and reduced fasting blood lipids and glucose. Therefore, compounds that disrupt class IIa HDAC function could be used to enhance metabolic health in chronic diseases driven by physical inactivity.

To identify genes involved in the central regulation of energy balance, we compared hypothalamic mRNA from lean and obese Psammomys obesus, a polygenic model of obesity, using differential display PCR. One mRNA transcript was observed to be elevated in obese, and obese diabetic, P. obesus compared with lean animals and was subsequently found to be increased 4-fold in the hypothalamus of lethal yellow agouti (A(y)/a) mice, a murine model of obesity and diabetes. Intracerebroventricular infusion of antisense oligonucleotide targeted to this transcript selectively suppressed its hypothalamic mRNA levels and resulted in loss of body weight in both P. obesus and Sprague Dawley rats. Reductions in body weight were mediated by profoundly reduced food intake without a concomitant reduction in metabolic rate. Yeast two-hybrid screening, and confirmation in mammalian cells by bioluminescence resonance energy transfer analysis, demonstrated that the protein it encodes interacts with endophilins, mediators of synaptic vesicle recycling and receptor endocytosis in the brain. We therefore named this transcript Src homology 3-domain growth factor receptor-bound 2-like (endophilin) interacting protein 1 (SGIP1). SGIP1 encodes a large proline-rich protein that is expressed predominantly in the brain and is highly conserved between species. Together these data suggest that SGIP1 is an important and novel member of the group of neuronal molecules required for the regulation of energy homeostasis.

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Given their involvement in processes necessary for life, mitochondrial damage and subsequent dysfunction can lead to a wide range of human diseases. Previous studies of both animal models and humans have suggested that PARL, presenilins-associated rhomboid-like protein, is a key regulator of mitochondrial integrity and function, and plays a role in cellular apoptosis. As a surrogate measure of mitochondrial integrity, we previously measured mitochondrial content in a Caucasian population consisting of large extended pedigrees, with results highlighting a substantial genetic component to this trait. To assess the influence of variation in the PARL gene on mitochondrial content, we resequenced 6.5kb of the gene, identifying 16 SNPs and genotyped these in 1,031 of these Caucasian individuals, distributed across 162 families. Statistical genetic analysis revealed that one promoter variant, T-191C, exhibited significant effects (after correction for multiple testing) on mitochondrial content levels. Comparison of the transcription factor binding characteristics of the T-191C promoter SNP by EMSA indicates preferential binding of nuclear factors to the T allele, suggesting functional variation in PARL expression. These results suggest that genetic variation within PARL influences mitochondrial abundance and integrity.

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