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

Shc proteins play a role in energy metabolism through interaction with the insulin receptor. The aim of this study was to determine whether Shc proteins influence liver glycolysis and gluconeogenesis under both fed and fasted states. Decreased glycolytic and increased gluconeogenic and transamination enzyme activities were observed in ShcKO versus WT mice. Levels of key regulatory metabolites, such as fructose-2,6-bisphosphate, matched the activity of metabolic pathways. Protein levels of glycolytic and gluconeogenic enzymes were not different. pAMPK protein levels increased with fasting and were higher in ShcKO versus WT mice. Therefore, Shc proteins play a role in shifting the metabolism from glucose oxidation to gluconeogenesis and lipid catabolism and should be considered as regulators of fuel selection. Fuel selection and utilization could play a critical role in healthy aging. Characterization of metabolic events in ShcKO mice would help to elucidate how metabolism is influenced by these proteins

Shc depletion stimulates brown fat activity in vivo and in vitro.

Adipose tissue is an important metabolic organ that integrates a wide array of homeostatic processes and is crucial for whole-body insulin sensitivity and energy metabolism. Brown adipose tissue (BAT) is a key thermogenic tissue with a well-established role in energy expenditure. BAT dissipates energy and protects against both hypothermia and obesity. Thus, BAT stimulation therapy is a rational strategy for the looming pandemic of obesity, whose consequences and comorbidities have a huge impact on the aged. Shc-deficient mice (ShcKO) were previously shown to be lean, insulin sensitive, and resistant to high-fat diet and obesity. We investigated the contribution of BAT to this phenotype. Insulin-dependent BAT glucose uptake was higher in ShcKO mice. Primary ShcKO BAT cells exhibited increased mitochondrial respiration; increased expression of several mitochondrial and lipid-oxidative enzymes was observed in ShcKO BAT. Levels of brown fat-specific markers of differentiation, UCP1, PRDM16, ELOVL3, and Cox8b, were higher in ShcKO BAT. In vitro, Shc knockdown in BAT cell line increased insulin sensitivity and metabolic activity. In vivo, pharmacological stimulation of ShcKO BAT resulted in higher energy expenditure. Conversely, pharmacological inhibition of BAT abolished the improved metabolic parameters, that is the increased insulin sensitivity and glucose tolerance of ShcKO mice. Similarly, in vitro Shc knockdown in BAT cell lines increased their expression of UCP1 and metabolic activity. These data suggest increased BAT activity significantly contributes to the improved metabolic phenotype of ShcKO mice

Complex I-Associated Hydrogen Peroxide Production Is Decreased and Electron Transport Chain Enzyme Activities Are Altered in n-3 Enriched fat-1 Mice

The polyunsaturated nature of n-3 fatty acids makes them prone to oxidative damage. However, it is not clear if n-3 fatty acids are simply a passive site for oxidative attack or if they also modulate mitochondrial reactive oxygen species (ROS) production. The present study used fat-1 transgenic mice, that are capable of synthesizing n-3 fatty acids, to investigate the influence of increases in n-3 fatty acids and resultant decreases in the n-6∶n-3 ratio on liver mitochondrial H2O2 production and electron transport chain (ETC) activity. There was an increase in n-3 fatty acids and a decrease in the n-6∶n-3 ratio in liver mitochondria from the fat-1 compared to control mice. This change was largely due to alterations in the fatty acid composition of phosphatidylcholine and phosphatidylethanolamine, with only a small percentage of fatty acids in cardiolipin being altered in the fat-1 animals. The lipid changes in the fat-1 mice were associated with a decrease (p<0.05) in the activity of ETC complex I and increases (p<0.05) in the activities of complexes III and IV. Mitochondrial H2O2 production with either succinate or succinate/glutamate/malate substrates was also decreased (p<0.05) in the fat-1 mice. This change in H2O2 production was due to a decrease in ROS production from ETC complex I in the fat-1 animals. These results indicate that the fatty acid changes in fat-1 liver mitochondria may at least partially oppose oxidative stress by limiting ROS production from ETC complex I

Key glycolytic enzyme activities of skeletal muscle are decreased under fed and fasted states in mice with knocked down levels of Shc proteins.

Shc proteins interact with the insulin receptor, indicating a role in regulating glycolysis. To investigate this idea, the activities of key glycolytic regulatory enzymes and metabolites levels were measured in skeletal muscle from mice with low levels of Shc proteins (ShcKO) and wild-type (WT) controls. The activities of hexokinase, phosphofructokinase-1 and pyruvate kinase were decreased in ShcKO versus WT mice under both fed and fasted conditions. Increased alanine transaminase and branched-chain amino acid transaminase activities were also observed in ShcKO mice under both fed and fasting conditions. Protein expression of glycolytic enzymes was unchanged in the ShcKO and WT mice, indicating that decreased activities were not due to changes in their transcription. Changes in metabolite levels were consistent with the observed changes in enzyme activities. In particular, the levels of fructose-2,6-bisphosphate, a potent activator of phosphofructokinase-1, were consistently decreased in the ShcKO mice. Furthermore, the levels of lactate (inhibitor of hexokinase and phosphofructokinase-1) and citrate (inhibitor of phosphofructokinase-1 and pyruvate kinase) were increased in fed and fasted ShcKO versus WT mice. Pyruvate dehydrogenase activity was lower in ShcKO versus WT mice under fed conditions, and showed inhibition under fasting conditions in both ShcKO and WT mice, with ShcKO mice showing less inhibition than the WT mice. Pyruvate dehydrogenase kinase 4 levels were unchanged under fed conditions but were lower in the ShcKO mice under fasting conditions. These studies indicate that decreased levels of Shc proteins in skeletal muscle lead to a decreased glycolytic capacity in both fed and fasted states

Calorie restriction influences key metabolic enzyme activities and markers of oxidative damage in distinct mouse liver mitochondrial sub-populations

AIMS: The purpose of the study was to establish if enzyme activities from key metabolic pathways and levels of markers of oxidative damage to proteins and lipids differed between distinct liver mitochondrial sub-populations, and which specific sub-populations contributed to these differences. MAIN METHODS: Male C57BL/6J mice were fed non-purified diet for one month then separated into two groups, control and calorie-restricted (CR). The two groups were fed semi-purified diet (AIN93G), with the CR group receiving 40% less calories than controls. After two months, enzyme activities and markers of oxidative damage in mitochondria were determined. KEY FINDINGS: In all mitochondrial sub-populations, Enzyme activities and markers of oxidative damage, from control and CR groups, showed a pattern of M1 > M3 > M10. Higher acyl-CoA dehydrogenase (β-oxidation) and β-hydroxybutryate dehydrogenase (ketogenesis) activities and lower carbonyl and TBARS levels were observed in M1 and M3 fractions from CR mice. ETC enzyme activities did not show a consistent pattern. In the Krebs cycle, citrate synthase and aconitase activities decreased while succinate dehydrogenase and malate dehydrogenase activities increased in the M1 mitochondria from the CR versus control mice. SIGNIFICANCE: CR does not produce uniform changes in enzyme activities or markers of oxidative damage in mitochondrial sub-populations, with changes occurring primarily in the heavy mitochondrial populations. Centrifugation at 10,000 g to isolate mitochondria likely dilutes the mitochondrial populations which show the greatest response to CR. Use of lower centrifugal force (3,000 g or lower) may be beneficial for some studies