Protein hypoacylation induced by Sirt5 overexpression has minimal metabolic effect in mice.

Sirtuins are a family of evolutionary conserved enzymes that dynamically regulate cellular physiology. Mammals have 7 sirtuins, which are located in different cellular compartments. Sirt5, a sirtuin isoform located in multiple subcellular sites, is involved in regulating a diverse range of cellular and metabolic processes through the removal of a range of acyl-lysine modifications on target proteins. Loss of Sirt5 leads to hyper-malonylation and hyper-succinylation of both mitochondrial and extra-mitochondrial proteins, influencing oxidative phosphorylation, the TCA cycle and glycolysis. However despite these findings, the effect of Sirt5 overexpression on metabolism remains poorly investigated. Here we report that overexpression of Sirt5 has minimal effect on mitochondrial metabolism and overall physiology in mice, despite inducing widespread decreases in protein acylation. Our data confirms the role of Sirt5 as an important demalonylase and desuccinylase enzyme in vivo, but questions the relevance of physiological changes in protein acylation levels in the regulation of cellular metabolism.

Regulation of glucose homeostasis and insulin action by ceramide acyl-chain length: A beneficial role for very long-chain sphingolipid species.

In a recent study, we showed that in response to high fat feeding C57BL/6, 129X1, DBA/2 and FVB/N mice all developed glucose intolerance, while BALB/c mice displayed minimal deterioration in glucose tolerance and insulin action. Lipidomic analysis of livers across these five strains has revealed marked strain-specific differences in ceramide (Cer) and sphingomyelin (SM) species with high-fat feeding; with increases in C16-C22 (long-chain) and reductions in C>22 (very long-chain) Cer and SM species observed in the four strains that developed HFD-induced glucose intolerance. Intriguingly, the opposite pattern was observed in sphingolipid species in BALB/c mice. These strain-specific changes in sphingolipid acylation closely correlated with ceramide synthase 2 (CerS2) protein content and activity, with reduced CerS2 levels/activity observed in glucose intolerant strains and increased content in BALB/c mice. Overexpression of CerS2 in primary mouse hepatocytes induced a specific elevation in very long-chain Cer, but despite the overall increase in ceramide abundance, there was a substantial improvement in insulin signal transduction, as well as decreased ER stress and gluconeogenic markers. Overall our findings suggest that very long-chain sphingolipid species exhibit a protective role against the development of glucose intolerance and hepatic insulin resistance.

Cardiac and skeletal muscles show molecularly distinct responses to cancer cachexia.

Cancer cachexia is a systemic, paraneoplastic syndrome seen in patients with advanced cancer. There is growing interest in the altered muscle pathophysiology experienced by cachectic patients. This study reports the microarray analysis of gene expression in cardiac and skeletal muscle in the colon 26 (C26) carcinoma mouse model of cancer cachexia. A total of 268 genes were found to be differentially expressed in cardiac muscle tissue, compared with nontumor-bearing controls. This was fewer than the 1,533 genes that changed in cachectic skeletal muscle. In addition to different numbers of genes changing, different cellular functions were seen to change in each tissue. The cachectic heart showed signs of inflammation, similar to cachectic skeletal muscle, but did not show the upregulation of ubiquitin-dependent protein catabolic processes or downregulation of genes involved in cellular energetics and muscle regeneration that characterizes skeletal muscle cachexia. Quantitative PCR was used to investigate a subset of inflammatory genes in the cardiac and skeletal muscle of independent cachectic samples; this revealed that B4galt1, C1s, Serpina3n, and Vsig4 were significantly upregulated in cardiac tissue, whereas C1s and Serpina3n were significantly upregulated in skeletal tissue. Our skeletal muscle microarray results were also compared with those from three published microarray studies and found to be consistent in terms of the genes differentially expressed and the functional processes affected. Our study highlights that skeletal and cardiac muscles are affected differently in the C26 mouse model of cachexia and that therapeutic strategies cannot assume that both muscle types will show a similar response.

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.

Proteomic profiling of skeletal and cardiac muscle in cancer cachexia: alterations in sarcomeric and mitochondrial protein expression.

BACKGROUND: Cancer cachexia is observed in more than 50% of advanced cancer patients, and impairs quality of life and prognosis. A variety of pathways are likely to be dysregulated. Hence, a broad-spectrum understanding of the disease process is best achieved by a discovery based approach such as proteomics. RESULTS: More than 300 proteins were identified with > 95% confidence in correct sequence identification, of which 5-10% were significantly differentially expressed in cachectic tissues (p-value of 0.05; 27 proteins from gastrocnemius, 34 proteins from soleus and 24 proteins from heart). The two most pronounced functional groups being sarcomeric proteins (mostly upregulated across all three muscle types) and energy/metabolism proteins (mostly downregulated across all muscle types). Electron microscopy revealed disintegration of the sarcomere and morphological aberrations of mitochondria in the cardiac muscle of colon 26 (C26) carcinoma mice. MATERIALS AND METHODS: The colon 26 (C26) carcinoma mouse model of cachexia was used to analyse soleus, gastrocnemius and cardiac muscles using two 8-plex iTRAQ proteomic experiments and tandem mass spectrometry (LCMSMS). Differentially expressed proteomic lists for protein clustering and enrichment of biological processes, molecular pathways, and disease related pathways were analysed using bioinformatics. Cardiac muscle ultrastructure was explored by electron microscopy. CONCLUSIONS: Morphological and proteomic analyses suggested molecular events associated with disintegrated sarcomeric structure with increased dissolution of Z-disc and M-line proteins. Altered mitochondrial morphology, in combination with the reduced expression of proteins regulating substrate and energy metabolism, suggest that muscle cells are likely to be undergoing a state of energy crisis which ultimately results in cancer-induced cachexia.

Niclosamide reduces glucagon sensitivity via hepatic PKA inhibition in obese mice: Implications for glucose metabolism improvements in type 2 diabetes.

Type 2 diabetes (T2D) is a global pandemic. Currently, the drugs used to treat T2D improve hyperglycemic symptom of the disease but the underlying mechanism causing the high blood glucose levels have not been fully resolved. Recently published data showed that salt form of niclosamide improved glucose metabolism in high fat fed mice via mitochondrial uncoupling. However, based on our previous work we hypothesised that niclosamide might also improve glucose metabolism via inhibition of the glucagon signalling in liver in vivo. In this study, mice were fed either a chow or high fat diet containing two different formulations of niclosamide (niclosamide ethanolamine salt - NENS or niclosamide - Nic) for 10 weeks. We identified both forms of niclosamide significantly improved whole body glucose metabolism without altering total body weight or body composition, energy expenditure or insulin secretion or sensitivity. Our study provides evidence that inhibition of the glucagon signalling pathway contributes to the beneficial effects of niclosamide (NENS or Nic) on whole body glucose metabolism. In conclusion, our results suggest that the niclosamide could be a useful adjunctive therapeutic strategy to treat T2D, as hepatic glucose output is elevated in people with T2D and current drugs do not redress this adequately.

The role of mitochondrial sirtuins in health and disease.

Mitochondria play a critical role in energy production, cell signalling and cell survival. Defects in mitochondrial function contribute to the ageing process and ageing-related disorders such as metabolic disease, cancer, and neurodegeneration. The sirtuin family of deacylase enzymes have a variety of subcellular localisations and have been found to remove a growing list of post-translational acyl modifications from target proteins. SIRT3, SIRT4, and SIRT5 are found primarily located in the mitochondria, and are involved in many of the key processes of this organelle. SIRT3 has been the subject of intense research and is primarily a deacetylase thought to function as a mitochondrial fidelity protein, with roles in mitochondrial substrate metabolism, protection against oxidative stress, and cell survival pathways. Less is known about the functional targets of SIRT4, which has deacetylase, ADP-ribosylase, and a newly-described lipoamidase function, although key roles in lipid and glutamine metabolism have been reported. SIRT5 modulates a host of newly-discovered acyl modifications including succinylation, malonylation, and glutarylation in both mitochondrial and extra-mitochondrial compartments, however the functional significance of SIRT5 in the regulation of many of its proposed target proteins remains to be discovered. Because of their influence on a broad range of pathways, SIRT3, SIRT4, and SIRT5 are implicated in a range of disease-states including metabolic disease such as diabetes, neurodegenerative diseases, cancer, and ageing-related disorders such as hearing-loss and cardiac dysfunction. We review the current knowledge on the function of the three mitochondrial sirtuins, their role in disease, and the current outstanding questions in the field.