Hypoxia-induced glucose-6-phosphate dehydrogenase overexpression and -activation in pulmonary artery smooth muscle cells: implication in pulmonary hypertension.

Severe pulmonary hypertension is a debilitating disease with an alarmingly low 5-yr life expectancy. Hypoxia, one of the causes of pulmonary hypertension, elicits constriction and remodeling of the pulmonary arteries. We now know that pulmonary arterial remodeling is a consequence of hyperplasia and hypertrophy of pulmonary artery smooth muscle (PASM), endothelial, myofibroblast, and stem cells. However, our knowledge about the mechanisms that cause these cells to proliferate and hypertrophy in response to hypoxic stimuli is still incomplete, and, hence, the treatment for severe pulmonary arterial hypertension is inadequate. Here we demonstrate that the activity and expression of glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme of the pentose phosphate pathway, are increased in hypoxic PASM cells and in lungs of chronic hypoxic rats. G6PD overexpression and -activation is stimulated by H2O2. Increased G6PD activity contributes to PASM cell proliferation by increasing Sp1 and hypoxia-inducible factor 1α (HIF-1α), which directs the cells to synthesize less contractile (myocardin and SM22α) and more proliferative (cyclin A and phospho-histone H3) proteins. G6PD inhibition with dehydroepiandrosterone increased myocardin expression in remodeled pulmonary arteries of moderate and severe pulmonary hypertensive rats. These observations suggest that altered glucose metabolism and G6PD overactivation play a key role in switching the PASM cells from the contractile to synthetic phenotype by increasing Sp1 and HIF-1α, which suppresses myocardin, a key cofactor that maintains smooth muscle cell in contractile state, and increasing hypoxia-induced PASM cell growth, and hence contribute to pulmonary arterial remodeling and pathogenesis of pulmonary hypertension.

Contractile protein expression is upregulated by reactive oxygen species in aorta of Goto-Kakizaki rat.

Although it is known that blood vessels undergo remodeling in type 2 diabetes (T2D), the signaling pathways that underlie the structural and functional changes seen in diabetic arteries remain unclear. Our objective was to determine whether the remodeling in type 2 diabetic Goto-Kakizaki (GK) rats is evoked by elevated reactive oxygen species (ROS). Our results show that aortas from GK rats produced greater force (P < 0.05) in response to stimulation with KCl and U46619 than aortas from Wistar rats. Associated with these changes, aortic expression of contractile proteins (measured as an index of remodeling) and the microRNA (miR-145), which act to upregulate transcription of contractile protein genes, was twofold higher (P < 0.05) in GK than Wistar (age-matched control) rats, and there was a corresponding increase in ROS and decrease in nitric oxide signaling. Oral administration of the antioxidant Tempol (1 mmol/l) to Wistar and GK rats reduced (P < 0.05) myocardin and calponin expression. Tempol (1 mmol/l) decreased expression of miR-145 in Wistar and GK rat aorta. To elucidate the mechanism through which ROS increases miR-145, we measured their levels in freshly isolated aorta and cultured aortic smooth muscle cells incubated for 12 h in the presence of H2O2 (300 µmol/l). H2O2 increased expression of miR-145, and there were corresponding nuclear increases in myocardin, a miR-145 target protein. Intriguingly, H2O2-induced expression of miR-145 was decreased by U0126 (10 µmol/l), a MEK1/2 inhibitor, and myocardin was decreased by anti-miR-145 (50 nmol/l) and U0126 (10 µmol/l). Our novel findings demonstrate that ROS evokes vascular wall remodeling and dysfunction by enhancing expression of contractile proteins in T2D.

Glucose-6-phosphate dehydrogenase and NADPH redox regulates cardiac myocyte L-type calcium channel activity and myocardial contractile function.

We recently demonstrated that a 17-ketosteroid, epiandrosterone, attenuates L-type Ca(2+) currents (I(Ca-L)) in cardiac myocytes and inhibits myocardial contractility. Because 17-ketosteroids are known to inhibit glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme in the pentose phosphate pathway, and to reduce intracellular NADPH levels, we hypothesized that inhibition of G6PD could be a novel signaling mechanism which inhibit I(Ca-L) and, therefore, cardiac contractile function. We tested this idea by examining myocardial function in isolated hearts and Ca(2+) channel activity in isolated cardiac myocytes. Myocardial function was tested in Langendorff perfused hearts and I(Ca-L) were recorded in the whole-cell patch configuration by applying double pulses from a holding potential of -80 mV and then normalized to the peak amplitudes of control currents. 6-Aminonicotinamide, a competitive inhibitor of G6PD, increased pCO(2) and decreased pH. Additionally, 6-aminonicotinamide inhibited G6PD activity, reduced NADPH levels, attenuated peak I(Ca-L) amplitudes, and decreased left ventricular developed pressure and ±dp/dt. Finally, dialyzing NADPH into cells from the patch pipette solution attenuated the suppression of I(Ca-L) by 6-aminonicotinamide. Likewise, in G6PD-deficient mice, G6PD insufficiency in the heart decreased GSH-to-GSSG ratio, superoxide, cholesterol and acetyl CoA. In these mice, M-mode echocardiographic findings showed increased diastolic volume and end-diastolic diameter without changes in the fraction shortening. Taken together, these findings suggest that inhibiting G6PD activity and reducing NADPH levels alters metabolism and leads to inhibition of L-type Ca(2+) channel activity. Notably, this pathway may be involved in modulating myocardial contractility under physiological and pathophysiological conditions during which the pentose phosphate pathway-derived NADPH redox is modulated (e.g., ischemia-reperfusion and heart failure).

Glc-6-PD and PKG contribute to hypoxia-induced decrease in smooth muscle cell contractile phenotype proteins in pulmonary artery.

Persistent hypoxic pulmonary vasoconstriction (HPV) plays a significant role in the pathogenesis of pulmonary hypertension, which is an emerging clinical problem around the world. We recently showed that hypoxia-induced activation of glucose-6-phosphate dehydrogenase (Glc-6-PD) in pulmonary artery smooth muscle links metabolic changes within smooth muscle cells to HPV and that inhibition of Glc-6PD reduces acute HPV. Here, we demonstrate that exposing pulmonary arterial rings to hypoxia (20-30 Torr) for 12 h in vitro significantly (P < 0.05) reduces (by 30-50%) SM22α and smooth muscle myosin heavy chain expression and evokes HPV. Glc-6-PD activity was also elevated in hypoxic pulmonary arteries. Inhibition of Glc-6-PD activity prevented the hypoxia-induced reduction in SM22α expression and inhibited HPV by 80-90% (P < 0.05). Furthermore, Glc-6-PD and protein kinase G (PKG) formed a complex in pulmonary artery, and Glc-6-PD inhibition increased PKG-mediated phosphorylation of VASP (p-VASP). In turn, increasing PKG activity upregulated SM22α expression and attenuated HPV evoked by Glc-6-PD inhibition. Increasing passive tension (from 0.8 to 3.0 g) in hypoxic arteries for 12 h reduced Glc-6-PD, increased p-VASP and SM22α levels, and inhibited HPV. The present findings indicate that increases in Glc-6-PD activity influence PKG activity and smooth muscle cell phenotype proteins, all of which affect pulmonary artery contractility and remodeling.

Effects of laparoscopic Roux-en-Y gastric bypass on glucose-6 phosphate dehydrogenase activity in obese type 2 diabetics.

BACKGROUND: Glucose-6-phosphate dehydrogenase (G6PD) is the rate-limiting enzyme of the pentose phosphate pathway that provides the majority of NADPH required for lipid biosynthesis. G6PD overexpression has been implicated in insulin resistance, hyperlipidemia, and increased oxidative stress in animals. This study examines G6PD expression in obese diabetic and nondiabetic subjects pre- and post-laparoscopic Roux-en-Y gastric bypass (LRYGB). METHODS: Patients undergoing LRYGB were recruited for the IRB-approved study and placed in either the diabetic (n = 11) or nondiabetic group (n = 16) (diabetic, HbA1c > 6.5%; nondiabetic, HbA1c < 6.0%). Blood samples were collected at baseline and throughout the first 3 postoperative months. Liver, adipose, and omental samples were taken during surgery. Results are expressed as mean ± SEM and were compared statistically using the Mann-Whitney test. RESULTS: The two groups were not significantly different at baseline except for fasting glucose and HbA1c. G6PD activity (nm/min/mg protein) was significantly higher in red blood cells (RBCs) (3.12 ± 1.39 vs. 0.67 ± 0.14) and liver (17.23 ± 2.40 vs. 9.74 ± 2.18) in diabetics compared to nondiabetics. There was good correlation between increased liver G6PD activity and the severity of diabetes as measured by HbA1c (r (2) = 0.525) and fasting glucose (r (2) = 0.542). No significant difference was found in the adipose or omental G6PD expression. Both groups experienced a significant increase in G6PD blood activity shortly following surgery (1 week) followed by a reduction 3 months after surgery. CONCLUSION: These results are the first ever seen in human subjects and demonstrate increased G6PD activity in diabetics compared to nondiabetics. These results suggest a correlation between G6PD activity and the severity of type 2 diabetes. The early increases in G6PD activity after LRYGB were unexpected and longer follow-up is needed to determine the effects of LRYGB on G6PD activity.