Immp2l Enhances the Structure and Function of Mitochondrial Gpd2 Dehydrogenase.

'Inner mitochondrial membrane peptidase 2 like' (IMMP2L) is a nuclear-encoded mitochondrial peptidase that has been conserved through evolutionary history, as has its target enzyme, 'mitochondrial glycerol phosphate dehydrogenase 2' (GPD2). IMMP2L is known to cleave the mitochondrial transit peptide from GPD2 and another nuclear-encoded mitochondrial respiratory-related protein, cytochrome C1 (CYC1). However, it is not known whether IMMP2L peptidase activates or alters the activity or respiratory-related functions of GPD2 or CYC1. Previous investigations found compelling evidence of behavioural change in the Immp2lKD-/- KO mouse, and in this study, EchoMRI analysis found that the organs of the Immp2lKD-/- KO mouse were smaller and that the KO mouse had significantly less lean mass and overall body weight compared with wildtype littermates (p < 0.05). Moreover, all organs analysed from the Immp2lKD-/- KO had lower relative levels of mitochondrial reactive oxygen species (mitoROS). The kidneys of the Immp2lKD-/- KO mouse displayed the greatest decrease in mitoROS levels that were over 50% less compared with wildtype litter mates. Mitochondrial respiration was also lowest in the kidney of the Immp2lKD-/- KO mouse compared with other tissues when using succinate as the respiratory substrate, whereas respiration was similar to the wildtype when glutamate was used as the substrate. When glycerol-3-phosphate (G3P) was used as the substrate for Gpd2, we observed ~20% and ~7% respective decreases in respiration in female and male Immp2lKD-/- KO mice over time. Together, these findings indicate that the respiratory-related functions of mGpd2 and Cyc1 have been compromised to different degrees in different tissues and genders of the Immp2lKD-/- KO mouse. Structural analyses using AlphaFold2-Multimer further predicted that the interaction between Cyc1 and mitochondrial-encoded cytochrome b (Cyb) in Complex III had been altered, as had the homodimeric structure of the mGpd2 enzyme within the inner mitochondrial membrane of the Immp2lKD-/- KO mouse. mGpd2 functions as an integral component of the glycerol phosphate shuttle (GPS), which positively regulates both mitochondrial respiration and glycolysis. Interestingly, we found that nonmitochondrial respiration (NMR) was also dramatically lowered in the Immp2lKD-/- KO mouse. Primary mouse embryonic fibroblast (MEF) cell lines derived from the Immp2lKD-/- KO mouse displayed a ~27% decrease in total respiration, comprising a ~50% decrease in NMR and a ~12% decrease in total mitochondrial respiration, where the latter was consistent with the cumulative decreases in substrate-specific mediated mitochondrial respiration reported here. This study is the first to report the role of Immp2l in enhancing Gpd2 structure and function, mitochondrial respiration, nonmitochondrial respiration, organ size and homeostasis.

SIRT2 transgenic over-expression does not impact lifespan in mice.

The NAD+ -dependent deacylase family of sirtuin enzymes have been implicated in biological ageing, late-life health and overall lifespan, though of these members, a role for sirtuin-2 (SIRT2) is less clear. Transgenic overexpression of SIRT2 in the BubR1 hypomorph model of progeria can rescue many aspects of health and increase overall lifespan, due to a specific interaction between SIRT2 and BubR1 that improves the stability of this protein. It is less clear whether SIRT2 is relevant to biological ageing outside of a model where BubR1 is under-expressed. Here, we sought to test whether SIRT2 over-expression would impact the overall health and lifespan of mice on a nonprogeroid, wild-type background. While we previously found that SIRT2 transgenic overexpression prolonged female fertility, here, we did not observe any additional impact on health or lifespan, which was measured in both male and female mice on standard chow diets, and in males challenged with a high-fat diet. At the biochemical level, NMR studies revealed an increase in total levels of a number of metabolites in the brain of SIRT2-Tg animals, pointing to a potential impact in cell composition; however, this did not translate into functional differences. Overall, we conclude that strategies to enhance SIRT2 protein levels may not lead to increased longevity.

Antioxidant Behavioural Phenotype in the Immp2l Gene Knock-Out Mouse.

Mitochondrial dysfunction is strongly associated with autism spectrum disorder (ASD) and the Inner mitochondrial membrane protein 2-like (IMMP2L) gene is linked to autism inheritance. However, the biological basis of this linkage is unknown notwithstanding independent reports of oxidative stress in association with both IMMP2L and ASD. To better understand IMMP2L's association with behaviour, we developed the Immp2lKD knockout (KO) mouse model which is devoid of Immp2l peptidase activity. Immp2lKD -/- KO mice do not display any of the core behavioural symptoms of ASD, albeit homozygous Immp2lKD -/- KO mice do display increased auditory stimulus-driven instrumental behaviour and increased amphetamine-induced locomotion. Due to reports of increased ROS and oxidative stress phenotypes in an earlier truncated Immp2l mouse model resulting from an intragenic deletion within Immp2l, we tested whether high doses of the synthetic mitochondrial targeted antioxidant (MitoQ) could reverse or moderate the behavioural changes in Immp2lKD -/- KO mice. To our surprise, we observed that ROS levels were not increased but significantly lowered in our new Immp2lKD -/- KO mice and that these mice had no oxidative stress-associated phenotypes and were fully fertile with no age-related ataxia or neurodegeneration as ascertained using electron microscopy. Furthermore, the antioxidant MitoQ had no effect on the increased amphetamine-induced locomotion of these mice. Together, these findings indicate that the behavioural changes in Immp2lKD -/- KO mice are associated with an antioxidant-like phenotype with lowered and not increased levels of ROS and no oxidative stress-related phenotypes. This suggested that treatments with antioxidants are unlikely to be effective in treating behaviours directly resulting from the loss of Immp2l/IMMP2L activity, while any behavioural deficits that maybe associated with IMMP2L intragenic deletion-associated truncations have yet to be determined.

Metabolic Profiling of Mice with Deletion of the Orphan G Protein-Coupled Receptor, GPR37L1.

Understanding the neurogenic causes of obesity may reveal novel drug targets to counter the obesity crisis and associated sequelae. Here, we investigate whether the deletion of GPR37L1, an astrocyte-specific orphan G protein-coupled receptor, affects whole-body energy homeostasis in mice. We subjected male Gpr37l1-/- mice and littermate wildtype (Gpr37l1+/+, C57BL/6J background) controls to either 12 weeks of high-fat diet (HFD) or chow feeding, or to 1 year of chow diet, with body composition quantified by EchoMRI, glucose handling by glucose tolerance test and metabolic rate by indirect calorimetry. Following an HFD, Gpr37l1-/- mice had similar glucose handling, body weight and fat mass compared with wildtype controls. Interestingly, we observed a significantly elevated respiratory exchange ratio in HFD- and chow-fed Gpr37l1-/- mice during daylight hours. After 1 year of chow feeding, we again saw no differences in glucose and insulin tolerance or body weight between genotypes, nor in energy expenditure or respiratory exchange ratio. However, there was significantly lower fat mass accumulation, and higher ambulatory activity in the Gpr37l1-/- mice during night hours. Overall, these results indicate that while GPR37L1 may play a minor role in whole-body metabolism, it is not a viable clinical target for the treatment of obesity.

A selective inhibitor of ceramide synthase 1 reveals a novel role in fat metabolism.

Specific forms of the lipid ceramide, synthesized by the ceramide synthase enzyme family, are believed to regulate metabolic physiology. Genetic mouse models have established C16 ceramide as a driver of insulin resistance in liver and adipose tissue. C18 ceramide, synthesized by ceramide synthase 1 (CerS1), is abundant in skeletal muscle and suggested to promote insulin resistance in humans. We herein describe the first isoform-specific ceramide synthase inhibitor, P053, which inhibits CerS1 with nanomolar potency. Lipidomic profiling shows that P053 is highly selective for CerS1. Daily P053 administration to mice fed a high-fat diet (HFD) increases fatty acid oxidation in skeletal muscle and impedes increases in muscle triglycerides and adiposity, but does not protect against HFD-induced insulin resistance. Our inhibitor therefore allowed us to define a role for CerS1 as an endogenous inhibitor of mitochondrial fatty acid oxidation in muscle and regulator of whole-body adiposity.