Julien Auquier

Inhibition of the mTOR/p70S6K pathway is not involved in the insulin-sensitizing effect of AMPK on cardiac glucose uptake

The AMP-activated protein kinase (AMPK) is known to increase cardiac insulin sensitivity on glucose uptake. AMPK also inhibits the mammalian target of rapamycin (mTOR)/p70 ribosomal S6 kinase (p70S6K) pathway. Once activated by insulin, mTOR/p70S6K phosphorylates insulin receptor substrate-1 (IRS-1) on serine residues, resulting in its inhibition and reduction of insulin signaling. AMPK was postulated to act on insulin by inhibiting this mTOR/p70S6K-mediated negative feedback loop. We tested this hypothesis in cardiomyocytes. The stimulation of glucose uptake by AMPK activators and insulin correlated with AMPK and protein kinase B (PKB/Akt) activation, respectively. Both treatments induced the phosphorylation of Akt substrate 160 (AS160) known to control glucose uptake. Together, insulin and AMPK activators acted synergistically to induce PKB/Akt overactivation, AS160 overphosphorylation, and glucose uptake overstimulation. This correlated with p70S6K inhibition and with a decrease in serine phosphorylation of IRS-1, indicating the inhibition of the negative feedback loop. We used the mTOR inhibitor rapamycin to confirm these results. Mimicking AMPK activators in the presence of insulin, rapamycin inhibited p70S6K and reduced IRS-1 phosphorylation on serine, resulting in the overphosphorylation of PKB/Akt and AS160. However, rapamycin did not enhance the insulin-induced stimulation of glucose uptake. In conclusion, although the insulin-sensitizing effect of AMPK on PKB/Akt is explained by the inhibition of the insulin-induced negative feedback loop, its effect on glucose uptake is independent of this mechanism. This disconnection revealed that the PKB/Akt/AS160 pathway does not seem to be the rate-limiting step in the control of glucose uptake under insulin treatment.

Catalase expression in MCF-7 breast cancer cells is mainly controlled by PI3K/Akt/mTor signaling pathway.

Catalase is an antioxidant enzyme that catalyzes mainly the transformation of hydrogen peroxide into water and oxygen. Although catalase is frequently down-regulated in tumors the underlying mechanism remains unclear. Few transcription factors have been reported to directly bind the human catalase promoter. Among them FoxO3a has been proposed as a positive regulator of catalase expression. Therefore, we decided to study the role of the transcription factor FoxO3a and the phosphatidylinositol-3 kinase (PI3K) signaling pathway, which regulates FoxO3a, in the expression of catalase. To this end, we developed an experimental model of mammary breast MCF-7 cancer cells that acquire resistance to oxidative stress, the so-called Resox cells, in which catalase is overexpressed as compared with MCF-7 parental cell line. In Resox cells, Akt expression is decreased but its phosphorylation is enhanced when compared with MCF-7 cells. A similar profile is observed for FoxO3a, with less total protein but more phosphorylated FoxO3a in Resox cells, correlating with its higher Akt activity. The modulation of FoxO3a expression by knockdown and overexpression strategies did not affect catalase expression, neither in MCF-7 nor in Resox cells. Inhibition of PI3K and mTOR by LY295002 and rapamycin, respectively, decreases the phosphorylation of downstream targets (i.e. GSK3β and p70S6K) and leads to an increase of catalase expression only in MCF-7 but not in Resox cells. In conclusion, FoxO3a does not appear to play a critical role in the regulation of catalase expression in both cancer cells. Only MCF-7 cells are sensitive and dependent on PI3K/Akt/mTOR signaling.

Metabolism and acetylation contribute to leucine-mediated inhibition of cardiac glucose uptake

High plasma leucine levels strongly correlate with type 2 diabetes. Studies of muscle cells have suggested that leucine alters the insulin response for glucose transport by activating an insulin-negative feedback loop driven by the mammalian target of rapamycin/p70 ribosomal S6 kinase (mTOR/p70S6K) pathway. Here, we examined the molecular mechanism involved in leucines action on cardiac glucose uptake. Leucine was indeed able to curb glucose uptake after insulin stimulation in both cultured cardiomyocytes and perfused hearts. Although leucine activated mTOR/p70S6K, the mTOR inhibitor rapamycin did not prevent leucines inhibitory action on glucose uptake, ruling out the contribution of the insulin-negative feedback loop. α-Ketoisocaproate, the first metabolite of leucine catabolism, mimicked leucines effect on glucose uptake. Incubation of cardiomyocytes with [13C]leucine ascertained its metabolism to ketone bodies (KBs), which had a similar negative impact on insulin-stimulated glucose transport. Both leucine and KBs reduced glucose uptake by affecting translocation of glucose transporter 4 (GLUT4) to the plasma membrane. Finally, we found that leucine elevated the global protein acetylation level. Pharmacological inhibition of lysine acetyltransferases counteracted this increase in protein acetylation and prevented leucines inhibitory action on both glucose uptake and GLUT4 translocation. Taken together, these results indicate that leucine metabolism into KBs contributes to inhibition of cardiac glucose uptake by hampering the translocation of GLUT4-containing vesicles via acetylation. They offer new insights into the establishment of insulin resistance in the heart.NEW & NOTEWORTHY Catabolism of the branched-chain amino acid leucine into ketone bodies efficiently inhibits cardiac glucose uptake through decreased translocation of glucose transporter 4 to the plasma membrane. Leucine increases protein acetylation. Pharmacological inhibition of acetylation reverses leucines action, suggesting acetylation involvement in this phenomenon.Listen to this articles corresponding podcast at http://ajpheart.podbean.com/e/leucine-metabolism-inhibits-cardiac-glucose-uptake/.

0325 : Catabolism of leucine in the heart inhibits glucose transport

Branched-chain amino acids like leucine induce insulin resistance in muscle and adipose tissues. The mechanism explaining leucine action involves mTOR/p70S6K signaling. This pathway is activated by leucine and is implicated in the stimulation of an insulin negative feedback loop. Knowing that insulin-resistance participates in diabetic cardiomyopathy, we were interested in studying leucine action in cardiomyocytes. Primary cultured adult rat cardiomyocytes were pretreated with different concentrations of leucine (1 to 10mM) during different periods of time (up to 20h) before being exposed to insulin (3x10-9M, 30min). Insulin increased glucose transport. This correlated with the increase of PKB and AS160 phosphorylation, both known to regulate GLUT4 translocation to the plasma membrane allowing glucose uptake. 1h pre-incubation with leucine stimulated mTOR/p70S6K pathway. This is accompanied by a decrease in PKB and AS160 phosphorylation but, surprisingly, insulin-stimulated glucose uptake was preserved. On the other hand, a longer incubation (14h) with leucine induced a drastic decrease in glucose transport. The mTOR/p70S6K inhibitor rapamycin did not prevent this inhibition. The non-metabolized leucine analog BCH had no effect on the insulin-induced glucose uptake. By contrast, intermediates of leucine catabolism, alpha-ketoisocaproate and ketone bodies inhibited glucose uptake similarly to leucine. This inhibition is clearly independent of insulin signaling because leucine also inhibited basal glucose transport and glucose uptake stimulated by the insulin-unrelated pathway involving AMPK. The leucine-mediated inhibition of glucose transport resulted from the inhibition of GLUT4 translocation. The exact molecular mechanism downstream leucine’s metabolites and responsible for the inhibition of GLUT4 translocation and glucose uptake is under investigation. Leucine catabolism reduces cardiac glucose transport independently of insulin signaling by an undefined mechanism.

0341: AMPK exerts an insulin-sensitizing effect on cardiac glucose uptake by multiple molecular mechanisms including cytoskeleton reorganization

Background Insulin-resistant cardiomyocytes are characterized by a decreased ability of insulin to stimulate glucose uptake. We have previously shown that the activation of AMPK by metformin or phenformin restores insulin-sensitivity in insulin-resistant cardiomyocytes. The aim of our present work is to understand by which molecular mechanisms AMPK exerts its insulin sensitizing effect. In this study we focused on the mTOR/p70S6K pathway and on cytoskeleton reorganization. mTOR/p70S6K, which is known to be inhibited by AMPK, is able to reduce insulin signaling via a negative feedback loop involving serine phosphorylation of IRS-1. On the other hand, cytoskeleton reorganization, which is a known target of AMPK, is responsible for the translocation of the glucose transporter GLUT4 to the plasma membrane. Methods Adult rat cardiomyocytes were primary cultured and treated with different agents including insulin, AMPK activator (phenformin), mTOR inhibitor rapamycin and/or actin cytoskeleton inhibitor latrunculin B. Glucose uptake was assessed by detritiation of 2- 3 H-glucose. Results First, we tested if rapamycin was able to mimic AMPK activators. Similarly to phenformin, rapamycin increased the insulin-dependent phosphorylation of Akt involved in the regulation of glucose uptake. Despite the ability of rapamycin to induce this Akt over-phosphorylation, rapamycin was not able to restore the insulin-dependent stimulation of glucose uptake like phenformin did. On the other hand, latrunculin B abolished the insulin-sensitizing action of phenformin on glucose uptake, in insulin-sensitive as well as in insulinresistant cells. Conclusions actin cytoskeleton reorganization but not mTOR/p70S6K, is involved in the insulin-sensitizing effect of AMPK on cardiac glucose uptake. The role played by Small G proteins, known to be involved in the regulation of actin cytoskeleton is under investigation.