Browsing by Subject "Acetylation"

SIRT3 is an NAD+-dependent mitochondrial protein deacetylase purported to influence cellular and systemic metabolism through modulation of the mitochondrial acetylproteome. Fuel-stimulated insulin secretion from pancreatic islets involves mitochondrial metabolism and might be susceptible to SIRT3-mediated effects. To investigate this idea, we used CRISPR/Cas9 technology to obtain complete SIRT3 knockout in the INS-1 832/13 insulinoma cell line. In the context of this SIRT3 knockout cell line, we re-expressed wild-type SIRT3, β-Galactosidase, or one of three enzymatically inactive mutant forms of SIRT3 to generate lines representing a wide range of SIRT3 expression and mitochondrial protein deacetylase activity. We performed large-scale acetylproteome profiling by mass spectrometry on the different lines, and observed wide-spread, SIRT3-dependent changes in acetylation of enzymes involved in fatty acid oxidation, the TCA cycle, and the electron transport chain. Remarkably, despite these broad changes, the cell lines had indistinguishable insulin secretion responses to glucose or pyruvate, and exhibited no differences in function or viability in response to metabolic or ER stress-inducing agents. Moreover, metabolomic profiling revealed that, when compared to SIRT3-null cell lines, expression of wild-type SIRT3 does not result in appreciable changes in a host of organic acid, amino acid or fatty acid-derived acylcarnitine metabolites during glucose stimulation.

We also studied mice with global SIRT3 knockout (KO) fed a standard chow (STD) or high-fat/high-sucrose (HFHS) diet. Importantly, we performed these studies in the C57Bl/6J background in which we replaced the mutant allele of nicotinamide nucleotide transhydrogenase (NNT) present in the “J” substrain, with the wild-type allele in order to restore endogenous NNT function. SIRT3 KO and wild-type (WT) mice fed a STD diet exhibited no differences in insulin secretion during oral or IP glucose tolerance tests, and the function of islets isolated from these mice was indistinguishable in islet perifusion studies conducted with a broad array of secretagogues. Only when chronically fed a HFHS diet did SIRT3 KO animals exhibit a modest impairment in insulin secretion, but without an effect on glycemic control. Our broad conclusion is that major changes in mitochondrial protein acetylation in response to manipulation of SIRT3 are not sufficient to cause changes in islet function or metabolism. However, under conditions of chronic nutritional stress (feeding of a HFHS diet for 12 weeks), a negative effect on function appears, suggesting that islets are more susceptible to nutrition-induced factors (oxidative stress, local cytokine production, etc.) when SIRT3 is absent. Further studies will be required to identify factors that may interact with SIRT3 deficiency and mitochondrial protein hyperacetylation to increase the risk of -cell dysfunction.

Background

Methods and results

Conclusion

Heart failure is a worldwide public health problem with substantial clinical burden and economic costs. In the progression into failure, the heart undergoes dramatic alterations in mitochondrial fuel metabolism and bioenergetics. As such, there is considerable interest in the delineation of regulatory events involved in the metabolic dysfunction of heart failure. Previous collaborative work identified three metabolic signatures associated with early stage heart failure: 1) accumulation of acylcarnitine metabolites; 2) mitochondrial hyperacetylation; and 3) elevated ketone catabolism. The goal of this dissertation was to explore the role of these metabolic signatures in the pathogenesis of heart failure.

Tissue accumulation of acylcarnitine metabolites is characteristic of mitochondrial dysfunction and indicative of incomplete β-oxidation. This occurs when a large portion of the fatty acids (i.e., acyl groups) within the mitochondria are not fully catabolized and the resulting intermediates are transferred to carnitine esters, enabling the traversal of biological membranes and departure from the mitochondrial matrix.

Nϵ-acetylation in the mitochondrial matrix is a non-enzymatic, post-translational modification (PTM) that spontaneously arises from the relatively basic pH and abundance of acetyl-CoA. Accumulation of this PTM has been observed in other tissues and disease states with evidence suggesting it impairs mitochondrial metabolism and causes dysfunction. However, convincing studies are lacking to establish a direct causal connection between dysfunction and acetylation. To address this shortcoming, a novel assay platform for the comprehensive assessment of mitochondrial bioenergetic transduction was developed and validated. Next, we generated and validated a novel mouse model of cardiac mitochondrial hyperacetylation and utilized the bioenergetic assay platform to test the hypothesis that it causes metabolic perturbations. Surprisingly, these hyperacetylated mitochondria exhibited almost no deficits in mitochondrial oxidative metabolism. To determine if hyperacetylation causes mitochondrial dysfunction in vivo under pathologic stimuli, the mouse model and littermate controls were subjected to transaortic constriction, a surgical method to induce pressure-overload heart failure. The hyperacetylated animals did not exhibit enhanced sensitivity toward cardiac dysfunction relative controls. With these results, we concluded that mitochondrial hyperacetylation does not contribute to the pathogenesis of heart failure.

Elevated ketone catabolism was observed in early stage failing hearts. Through a series of murine and canine heart failure models, ketone catabolism was shown to be adaptive in response to pathological stress. Additionally, the mitochondrial bioenergetic assay platform was applied to cardiac mitochondria under substrate limited-conditions. These results indicate that ketone catabolism improves bioenergetic efficiency under constraints which mimic the failing heart. With these results, we conclude ketone catabolism is an important metabolic defense in response to the dysfunction of the failing heart.

Identifying metabolic vulnerabilities of cancer cells remains a subject of investigation for the identification of potential metabolically based therapies for cancer. It is well known that proliferating cells become largely dependent on glucose and glutamine for their growth. Interestingly, we find that lipid oxidizing genes are consistently downregulated across a wide variety of cancers while lipid synthesizing genes are elevated. This indicates that lipid oxidation may be refractory to cancer cell growth. Studies have shown beneficial effects of carbohydrate restriction in various forms in the treatment of cancer. For example, the use of ketogenic diets, which contain high levels of fat and protein with very low levels of carbohydrates, have shown efficacy in decreasing tumor growth in glioma, colon, prostate, and gastric cancers. A major challenge facing the use of these diets in cancer therapy is that the mechanism by which they show efficacy in cancer remains unclear. By using octanoate, the most well-known ketogenic fatty acid, we are able to drive fatty acid oxidation and ketogenesis to study these processes in proliferating cells. We found that supplementation of octanoate into complete culture medium causes a dramatic, dose-dependent and reversible suppression of proliferation across numerous cell lines and significant changes to anabolic cellular metabolism. Importantly, we have found that ketone production from octanoate had no effect on cell proliferation but that the overall cellular response to the lipid causes inhibition of cell growth.

Nutrients and metabolites are sensed by the cell at many levels and the cellular response to metabolites is critical to proliferation and survival of a cancer cell. One way in which the cell responds to glucose, the major fuel source in cancer cells, is by increasing histone acetylation to promote gene expression. Wellen et al., found that upon glucose addition there was a specific gene expression pattern characterized by the upregulation of genes involved in glucose metabolism. In this way the cell promotes glucose-derived fatty acid synthesis, a rate-limiting process for cancer cell proliferation. This is one way in which the metabolic response to nutrients is integrated into cellular signaling and the epigenome. Remarkably, we have found that lipids can promote a feed-forward mechanism of lipid metabolism by inducing histone acetylation and increasing gene expression of lipid metabolizing genes. We find that upon treatment of cells with octanoate there is an inhibition of both glucose and glutamine metabolism and that octanoate-derived carbon becomes the major fuel source in the cell. We then found that histones were hyperacetylated after octanoate treatment and remarkably, close to 90% of the carbon on histones was octanoate derived. In addition, octanoate is a weak HDAC inhibitor which further promotes octanoate-derived acetyl-CoA being deposited onto histones. A gene array from octanoate treated samples finds that fatty acid metabolism is the top pathway in our gene ontology analysis. This provides evidence that the cell responds to nutrient sources in a specific manner depending on the nature of the carbon source. Finally, we find the most negatively regulated pathway upon octanoate treatment is DNA replication. Consistently, we find that octanoate causes an accumulation of cells in G1 phase of the cell cycle and induction of apoptosis.

Here we describe a mechanism for how fatty acids are sensed and how they communicate with the nucleus to alter gene expression. We show that the cell responds to lipids via a coordinated response to promote lipid metabolism and induce histone acetylation. This feed-forward mechanism of lipid metabolism consists of a reprograming of anabolic metabolism, and promotion of gene expression changes culminating in inhibition of cell growth and apoptosis.