br Materials and Methods br Results br
Materials and Methods
Discussion Mitochondrion not only generates energy but also produces ROS under physiological conditions. However, overnutrition causes mitochondrial congestion and elicits excessive ROS generation, leading to insulin resistance (Muoio, 2014, Houstis et al., 2006). Our findings reveal that RGSE can modulate key enzymes for the oxidation of glucose and fatty Vitamin B12 under conditions of lipid overload, thus attenuates mitochondrial oxidative stress and improves myocardial insulin signaling. There is a crosstalk between competing substrates described as Randle cycle. Metabolic intermediates arising from fat catabolism act as negative regulators of glucose oxidation and vice versa, by regulating the key enzymes controlling fuel metabolism. PDH is a key enzyme mediating pyruvate oxidation in mitochondria, linking glycolysis with glucose oxidation. CPT1 is located on the outer membrane of mitochondria and acts as the rate-limiting enzyme for mitochondrial fatty acid uptake and β-oxidation. We found that PA stimulation decreased PDH activity while increased CPT1 expression, which was blocked by mildronate (a carnitine acyltransferase inhibitor), a partial inhibitor of β-oxidation and able to improve insulin signaling (Aguer et al., 2015). In addition, we observed a similar effect of PDKs inhibitor DCA. DCA can activate the activity of PDH by dephosphorylation and thus enhance glucose oxidation. These data suggested that reduction of FAO or promotion of glucose oxidation can restore substrate metabolic homeostasis in the setting of lipid overload. HFD-induced increased FAO has been demonstrated in heart and skeletal muscle (Carley and Severson, 2005, Henstridge et al., 2015, Koves et al., 2008, Ussher et al., 2009). We found that HFD induced an elevation of blood FFA (Control), with a significant increase in expression for p-PDH and CPT1 in heart, which were counteracted by DCA and metformin. In previous work, we have also observed that another β-oxidation inhibitor trimetazidine effectively reduced myocardial p-PDH expression in HFD-fed mice (Li et al., 2017). These results, together with others, demonstrated that PDH activity and CPT1 expression acting as sensitive targets in response to modulators of glucose oxidation or FAO. RGSE displayed prominent effects as DCA did, indicative of its role in modulation of fuel catabolism under conditions of lipid overload. A large body of evidence suggests that long-term overload of lipids leads to metabolic insensitivity and inflexibility, including the insensitivity to insulin (Mazumder et al., 2004, Muoio, 2014). Herein, it is further demonstrated in cardiomyocytes because exposure to PA interfered insulin-mediated Akt/AS160/Glut4 signaling, leading to a decrease in glucose uptake and subsequent glucose utilization, characterized by decreased mitochondrial binding HK-II, elevated p-PDH and reduction of ATP content. HK-II is a predominant isoform in insulin-sensitive tissues including heart, catalyzing phosphorylation of glucose during glycolysis. It can translocate to outer membrane of mitochondria and bind to voltage-dependent anion channel 1, which provides facilitation of coupling between glycolysis and oxidative phosphorylation (Roberts and Miyamoto, 2015). Impairment of insulin sensitivity in heart was also observed in HFD-fed mice, evidenced by suppressed response to an acute insulin bolus. RGSE can correct these alterations in vitro or in vivo, suggesting the beneficial role of RGSE as a metabolic modulator in improvement of insulin sensitivity. Of note, the protective effect of RGSE in vivo might partially attribute to a low level of blood FFA, because this regulation would decrease myocardial fatty acid availability and mitochondrial flux. RGSE ameliorated glucose intolerance in HFD-fed mice, indicating its potential to prevent systemic insulin resistance. It is somewhat interesting to find that AMPK activator metformin decreased the CPT1 expression in the setting of PA stimulation or HFD feeding. It is known that AMPK promotes phosphorylation of acetyl-CoA carboxylase and increase CPT1 activity, thus enhance FAO. We observed that metformin, DCA, mildronate and RGSE were all able to prevent the loss of phosphorylation of AMPK (Suppl. Fig. 3) and block the elevation of CPT1 expression. Consistently, Roberts and colleagues observed that the wild type super-healing Murphy Roths Large mouse displayed decreased p-AMPK level but increased β-oxidation in heart (Roberts et al., 2015). The evidence suggests that AMPK activation in cardiomyocytes prefers to promote glucose utilization but not FAO in some specific circumstances.