Most differentiated cells convert glucose to pyruvate in the cytosol through glycolysis, followed by pyruvate oxidation in the mitochondria. These processes are linked by the Mitochondrial Pyruvate Carrier (MPC), which is required for efficient mitochondrial pyruvate uptake. In contrast, proliferative cells, including many cancer and stem cells, perform glycolysis robustly but limit fractional mitochondrial pyruvate oxidation. We sought to understand the role this transition from glycolysis to pyruvate oxidation plays in stem cell maintenance and differentiation. Loss of the MPC in Lgr5-EGFP positive stem cells, or treatment of intestinal organoids with an MPC inhibitor, increases proliferation and expands the stem cell compartment. Similarly, genetic deletion of the MPC in Drosophila intestinal stem cells also increases proliferation, whereas MPC overexpression suppresses stem cell proliferation. These data demonstrate that limiting mitochondrial pyruvate metabolism is necessary and sufficient to maintain the proliferation of intestinal stem cells.

Vascular dysfunction that accompanies obesity and insulin resistance may be mediated by lipid metabolites. We sought to determine if vascular ceramide leads to arterial dysfunction and to elucidate the underlying mechanisms. Pharmacological inhibition of de novo ceramide synthesis, using the Ser palmitoyl transferase inhibitor myriocin, and heterozygous deletion of dihydroceramide desaturase prevented vascular dysfunction and hypertension in mice after high-fat feeding. These findings were recapitulated in isolated arteries in vitro, confirming that ceramide impairs endothelium-dependent vasorelaxation in a tissue-autonomous manner. Studies in endothelial cells reveal that de novo ceramide biosynthesis induced protein phosphatase 2A (PP2A) association directly with the endothelial nitric oxide synthase (eNOS)/Akt/Hsp90 complex that was concurrent with decreased basal and agonist-stimulated eNOS phosphorylation. PP2A attenuates eNOS phosphorylation by preventing phosphorylation of the pool of Akt that colocalizes with eNOS and by dephosphorylating eNOS. Ceramide decreased the association between PP2A and the predominantly cytosolic inhibitor 2 of PP2A. We conclude that ceramide mediates obesity-related vascular dysfunction by a mechanism that involves PP2A-mediated disruption of the eNOS/Akt/Hsp90 signaling complex. These results provide important insight into a pathway that represents a novel target for reversing obesity-related vascular dysfunction.

Abstract-Impaired insulin signaling via phosphatidylinositol 3-kinase/Akt to endothelial nitric oxide synthase (eNOS) in the vasculature has been postulated to lead to arterial dysfunction and hypertension in obesity and other insulin resistant states. To investigate this, we compared insulin signaling in the vasculature, endothelial function, and systemic blood pressure in mice fed a high-fat (HF) diet to mice with genetic ablation of insulin receptors in all vascular tissues (TTr-IR Ϫ/Ϫ ) or mice with genetic ablation of Akt1 (Akt1Ϫ/Ϫ). HF mice developed obesity, impaired glucose tolerance, and elevated free fatty acids that was associated with endothelial dysfunction and hypertension. Basal and insulin-mediated phosphorylation of extracellular signal-regulated kinase 1/2 and Akt in the vasculature was preserved, but basal and insulin-stimulated eNOS phosphorylation was abolished in vessels from HF versus lean mice. In contrast, basal vascular eNOS phosphorylation, endothelial function, and blood pressure were normal despite absent insulinmediated eNOS phosphorylation in TTr-IR Ϫ/Ϫ mice and absent insulin-mediated eNOS phosphorylation via Akt1 in Akt1Ϫ/Ϫ mice. In cultured endothelial cells, 6 hours of incubation with palmitate attenuated basal and insulinstimulated eNOS phosphorylation and NO production despite normal activation of extracellular signal-regulated kinase 1/2 and Akt. Moreover, incubation of isolated arteries with palmitate impaired endothelium-dependent but not vascular smooth muscle function. Collectively, these results indicate that lower arterial eNOS phosphorylation, hypertension, and vascular dysfunction following HF feeding do not result from defective upstream signaling via Akt, but from free fatty acid-mediated impairment of eNOS phosphorylation. Key Words: arterial insulin signaling Ⅲ hypertension Ⅲ endothelial dysfunction Ⅲ mice Ⅲ diabetes S timulation of insulin receptors in the vasculature leads to increased activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway and the mitogen-activated protein (MAP) kinase (eg, extracellular signaling-regulated kinase [ERK]1/2) pathway. [1][2][3][4] Insulin receptor (IR)-mediated stimulation of PI3K/Akt leads to endothelial nitric oxide (NO) synthase (eNOS) phosphorylation, NO production, and vasorelaxation. 4 -7 Insulin-mediated activation of ERK1/2 leads to endothelin (ET)-1 production, inhibition of eNOS phosphorylation, and subsequent vasocontraction. 1,4,8 Evidence from several experimental models of insulin resistance reveals impaired insulin-mediated PI3K/Aktdependent signaling in the vasculature, whereas ERK1/2 pathways are preserved or even augmented. 2,8,9 Collectively, these observations have led to the hypothesis that an imbalance in vascular insulin-mediated signaling can precipitate cardiovascular complications including endothelial dysfunction and hypertension. 1,4 Recently, it was shown that insulinmediated Akt phosphorylation was preserved, but NOmediated vasorelaxation was blunted, in arteries from obese, glucose ...

The intracellular signalling kinases Akt/protein kinase B (Akt), protein kinase A (PKA) and adenosine monophosphate-activated protein kinase (AMPK) are phosphorylated in response to increased mechanical force or perfusion rate in cultured endothelial cells or isolated blood vessels. All three kinases phosphorylate endothelial nitric oxide synthase (eNOS) on serine (S) 1177, while Akt and PKA additionally phosphorylate eNOS on S617 and S635 respectively. Although these kinases might contribute to subsequent activation of eNOS during dynamic exercise, the specific mediators of exercise-induced eNOS phosphorylation and activation in vivo are unknown. We determined the impact of 50 min of treadmill running on the phosphorylation of Akt, AMPK, cyclic adenosine monophosphate response element binding protein (CREB -a target of PKA) and eNOS (S 1177, 635 and 617 and threonine (T) 495) in the presence or absence of pharmacological inhibition of PI3 kinase (PI3K) and Akt signalling using wortmannin. Compared to arteries from sedentary mice, eNOS enzyme activity was greater in vessels from treadmill-running animals and was associated with increased phosphorylation of Akt (S473), CREB (S133), AMPK (T172), and eNOS at S1177 and S617 but not at S635 or T495. These data suggest that Akt signalling is a major mediator of eNOS activation. To confirm this, treadmill-running was performed in the presence of vehicle (DMSO) or PI3K inhibition. Compared to results from sedentary mice, vascular Akt phosphorylation and eNOS phosphorylation at S617 during treadmill-running were prevented by wortmannin but not vehicle treatment, whereas exercise-related increases in AMPK and CREB phosphorylation were similar between groups. Arterial eNOS phosphorylation at S1177 increased during exercise after wortmannin treatment relative to values obtained from sedentary animals, but the elevation was blunted by ∼50% compared to results from vehicle-treated mice. These findings indicate that Akt and AMPK contribute importantly to vascular eNOS S1177 phosphorylation during treadmill-running, and that AMPK is sufficient to activate p-eNOS S1177 in the presence of PI3K inhibition. Frictional forces along the surface of the endothelium exerted by flowing blood are potent stimuli for endothelial nitric oxide (NO) production (Jo et al. 1997). These forces are proportional to the product of blood viscosity and blood velocity at the endothelial wall and collectively have been termed 'endothelial shear stress' . Endothelial surfaces possess mechanoreceptors that detect shear stress and data concerning the pathways responsible for phosphorylation and activation of endothelial NO synthase (eNOS) are emerging (