p46Shc Inhibits Thiolase and Lipid Oxidation in Mitochondria

Abstract

Although the p46Shc isoform has been known to be mitochondrially localized for 11 years, its function in mitochondria has been a mystery. We confirmed p46Shc to be mitochondrially localized and showed that the major mitochondrial partner of p46Shc is the lipid oxidation enzyme 3-ketoacylCoA thiolase ACAA2, to which p46Shc binds directly and with a strong affinity. Increasing p46Shc expression inhibits, and decreasing p46Shc stimulates enzymatic activity of thiolase in vitro. Thus, we suggest p46Shc to be a negative mitochondrial thiolase activity regulator, and reduction of p46Shc expression activates thiolase. This is the first demonstration of a protein that directly binds and controls thiolase activity. Thiolase was thought previously only to be regulated by metabolite balance and steadystate flux control. Thiolase is the last enzyme of the mitochondrial fatty acid beta-oxidation spiral, and thus is important for energy metabolism. Mice with reduction of p46Shc are lean, resist obesity, have higher lipid oxidation capacity, and increased thiolase activity. The thiolase-p46Shc connection shown here in vitro and in organello may be an important underlying mechanism explaining the metabolic phenotype of Shcdepleted mice in vivo.Shc proteins have three isoforms: p46, p52, and p66, and have major effects on metabolism (1-4). p52Shc contacts the insulin receptor and regulates the signaling between the IRS1 and Ras pathway (5). In 2004, it was demonstrated that the major mitochondrial Shc isoform is p46Shc (6), but since that time the mitochondrial partner and physiological function of p46Shc has been a mystery. Here we show the mitochondrial partner and function of p46Shc, and suggest how this could contribute to the obesity-resistance observed in ShcKO mice.There are three isoforms at the mammalian Shc locus, the highly expressed isoforms p46Shc and p52Shc, and the minor p66Shc. All three Shc isoforms are derived from a single DNA locus. First, two mRNA are produced: p66Shc and p52/46Shc by means of trans-splicing. The p66Shc mRNA has start codons for all three Shc isoforms. The p52/46Shc-mRNA does not have a start codon for p66Shc and only produces p52Shc and p46Shc (7). For this reason, knockdowns of either p66Shc, or all three Shc isoforms together have been achieved to date. There are two mouse models of Shc depletion produced to date: ShcL, also known as p66ShcKO developed by the Tom Prolla group (ShcProlla, Ref. 4), and Shc mice developed by the Pelicci group (ShcP or ShcPelicci, also known as p66Shc (Ϫ/Ϫ, Refs. 3,4,8,9). Names in literature for these two models can be described by these definitions: ShcL, ShcProlla; ShcKO, ShcP ϭ ShcPelicci ϭ p66Shc(Ϫ/Ϫ).Briefly, ShcL or ShcProlla mice have a deletion of only the minor p66Shc isoform and have no health benefits: ShcL mice are not lean, do not resist weight gain on high fat diets (HFD), 2 and are not longer-lived on HFD, and do not have improved insulin sensitivity (4). Thus p66Shc deletion by itself does not result in health benefits.By contrast, ShcKO have...

Nonphagocytic Activation of NOX2 Is Implicated in Progressive Nonalcoholic Steatohepatitis During Aging

“…We saw that p46Shc was also induced in aging, which raises the question regarding its role in NASH. In fact, p46Shc dysregulated mitochondrial β-oxidation by inhibiting 3-ketoacyl-CoA thiolase, (23) thereby increasing the FFA load in hepatocytes. More work would be required to decipher the respective roles of p46 and p52Shc isoforms in NASH.…”

Mice with low levels of Shc proteins display reduced glycolytic and increased gluconeogenic activities in liver

“…However, similar to skeletal muscle [8] , Shc-related changes in enzyme activity do not appear to be due to alterations in gene expression since there were no differences in the protein levels of glycolytic or gluconeogenic enzymes between ShcKO and WT mice. We have recently reported that the activity of thiolase was decreased through direct binding with p46Shc [10] and it is possible that the activities of other enzymes could be altered through binding with Shc proteins. Additional studies are needed to determine if Shc proteins bind and directly influence the activities of any enzymes in the glycolysis or gluconeogenesis pathways.…”

“…The activities of fatty acid β-oxidation enzymes in liver and skeletal muscle were also increased in ShcKO compared to wild-type mice in the fasted state, and the activities of ketogenic enzymes were increased in liver from ShcKO versus wild-type mice [9] . Moreover, we have reported recently that the β-oxidation enzyme 3-ketoacyl-CoA thiolase was directly influenced by p46Shc through binding that resulted in decreased activity and that lower levels of p46Shc resulted in increased activity of the enzyme [10] . The results of our current studies are consistent with the idea that Shc proteins may play a role in regulating substrate oxidation.…”

The biochemistry of feed efficiency, energy metabolism, and mitochondrial function, an animal and molecular approach

“…In both liver and muscle, the activities of β-hydroxybutyrate dehydrogenase and acetoacetyl-CoA thiolase were increased (P < 0.05) in both the fed and fasted states in the ShcKO compared to WT mice. Overall, these results indicate that skeletal muscle from the ShcKO animals has an increased capacity for oxidizing both fatty acids and ketone bodies Tomilov et al, 2016).…”

“…These results indicate that Shc proteins may influence the fuels used for energy but they do not produce a net change in overall capacity for energy metabolism in the mitochondria. Shc proteins may play an important role in energy metabolism and, particularly, a decrease in Shc levels leads to an increased capacity for β-oxidation, ketone body metabolism, amino acid catabolism and gluconeogenesis under fasting conditions Tomilov et al, 2016). Decreases in Shc proteins may play an important role in transitioning the animal from a fed to a fasted state.…”