Reductions in liver enzymes are associated with anti-hyperglycaemic and anti-obesity effects of tofogliflozin in people with type 2 diabetes: Post-hoc analyses.

AIMS: How the pathology of type 2 diabetes (T2D), including hyperglycaemia and obesity, affects liver enzymes has not been clinically demonstrated. Thus, we compared time courses of gamma-glutamyltransferase (GGT) and alanine aminotransferase (ALT) with those of fasting plasma glucose (FPG) and body weight (BW) during treatment with the SGLT2 inhibitor tofogliflozin for T2D. MATERIALS AND METHODS: We post-hoc analysed preexisting data on 1046 people with T2D administered tofogliflozin or placebo for 24 weeks in four tofogliflozin studies. First, time courses of percent changes in variables during the intervention were analysed using a mixed effect model to explore the similarity of the time courses and to evaluate time-treatment interactions. Second, clinical factors related to the percent changes in GGT and ALT were clarified using multivariate analyses. RESULTS: GGT levels and FPG values rapidly and significantly decreased via tofogliflozin as early as week 4, with decreases maintained until week 24. Conversely, BW and ALT decreased progressively until week 24. Time courses of FPG (p = .365, time-treatment interaction) and GGT (p = .510) reductions were parallel between tofogliflozin and placebo from weeks 4 to 24, while BW and ALT reductions (p < .001, respectively) were not. Reductions in GGT at week 24 were associated with reductions in FPG and BW at week 24, whereas ALT reductions were only associated with reductions in BW. CONCLUSIONS: Reductions in GGT and ALT were associated with the anti-hyperglycaemic and anti-obesity effects of tofogliflozin, respectively, in people with T2D. Therefore, GGT and ALT may be surrogate markers for hyperglycaemia and obesity in T2D.

Pravastatin-induced proangiogenic effects depend upon extracellular FGF-2.

The HMG-CoA reductase inhibitors (statins) have been shown to exert several protective effects on the vasculature that are unrelated to changes in the cholesterol profile, and to induce angiogenesis. The proangiogenic effect exerted by statins has been attributed to the activation of the PI3K/Akt pathway in endothelial cells; however, it is unclear how statins activate this pathway. Pravastatin-mediated activation of Akt and MAPK occurs rapidly (within 10 min.) and at low doses (10 nM). Here, we hypothesized that FGF-2 contributes to the proangiogenic effect of statins. We found that pravastatin, a hydrophilic statin, induced phosphorylation of the FGF receptor (FGFR) in human umbilical vein endothelial cells. SU5402, an inhibitor of FGFR, abolished pravastatin-induced PI3K/Akt and MAPK activity. Likewise, anti-FGF-2 function-blocking antibodies inhibited Akt and MAPK activity. Moreover, depletion of extracellular FGF-2 by heparin prevented pravastatin-induced phosphorylation of Akt and MAPK. Treatment with FGF-2 antibody inhibited pravastatin-enhanced endothelial cell proliferation, migration and tube formation. These observations indicate that pravastatin exerts proangiogenic effects in endothelial cells depending upon the extracellular FGF-2.

Heat shock cognate protein 70 is essential for Akt signaling in endothelial function.

OBJECTIVE: Heat shock protein 70s (Hsp70s) are molecular chaperones that protect cells from damage in response to various stress stimuli. However, the functions and mechanisms in endothelial cells (ECs) have not been examined. Herein, we investigate the role of Hsp70s, including heat shock cognate protein 70 (Hsc70), which is constitutively expressed in nonstressed cells (ie, ECs). METHODS AND RESULTS: The Hsp70 inhibitor, KNK437, significantly decreased vascular endothelial growth factor (VEGF)-induced cell migration and tube formation in vitro. KNK437 inhibited the phosphorylation of VEGF-induced Akt and endothelial nitric oxide synthase (eNOS) in human umbilical vein endothelial cells. In a mouse hind limb model of vascular insufficiency, intramuscular inhibition of Hsp70s attenuated collateral and capillary vessel formation. Silencing the Hsc70 gene by short interfering RNA abolished VEGF-induced Akt phosphorylation and VEGF-stimulated human umbilical vein endothelial cell migration and tube formation. As the molecular mechanisms, Hsc70 knockdown reduced the expression of phosphatidylinositol 3-kinase. CONCLUSIONS: Collectively, Hsc70 plays a significant role in ECs via the phosphatidylinositol 3-kinase/Akt pathway. Hsc70 may provide the basis for the development of new therapeutic strategies for angiogenesis.

Pravastatin accelerates ischemia-induced angiogenesis through AMP-activated protein kinase.

Statins exert pleiotropic effects on the cardiovascular system, in part through an increase in nitric oxide (NO) bioavailability. In this study, we examined the role of pravastatin in ischemia-induced angiogenesis. Unilateral hindlimb ischemia was surgically induced in C57BL/6J mice. Phosphorylation of AMP-activated protein kinase (AMPK), acetyl-CoA carboxylase (ACC) and endothelial NO synthase (eNOS) was increased in ischemic tissues. Furthermore, mice treated with pravastatin showed higher increases in phosphorylation than did untreated mice. Laser Doppler analysis has shown that pravastatin treatment accelerates the development of collateral vessels and angiogenesis in response to hindlimb ischemia. Capillary density in the ischemic hindlimb was also increased by pravastatin treatment. An in vitro study on human umbilical vein endothelial cells (HUVECs) revealed that pravastatin increased the phosphorylation of AMPK. Pravastatin-induced phosphorylation of eNOS, one of the downstreams of AMPK, was inhibited by compound C, an AMPK antagonist. The increased migration and tube formation of HUVECs by pravastatin were significantly blocked by compound C treatment. The accelerated angiogenesis by pravastatin after hindlimb ischemia was significantly reduced after treatment with compound C. Thus, ischemia induced AMPK phosphorylation in vivo. Furthermore, pravastatin could also activate AMPK in vivo and in vitro. Such phosphorylation results in eNOS activation and angiogenesis, which provide a novel explanation for one of the pleiotropic effects of statins that is beneficial for angiogenesis.

Pharmacogenomics of cardiovascular pharmacology: molecular network analysis in pleiotropic effects of statin -- an experimental elucidation of the pharmacologic action from protein-protein interaction analysis.

The ebb and flow of cellular life depends largely on signaling pathways and networks, which are regulated by specific protein-protein interactions. These interactions often involve assembly of large signaling complexes containing many different protein kinases, protein phosphatases, their substrates, and scaffold proteins. Identification of protein complexes is the key to understanding cellular functions. One of the techniques used for the isolation of protein complexes is the affinity purification system. Inhibitors of 3-hydroxyl-3-methyglutaryl coenzyme A (HMG-CoA) reductase (i.e., statins) exert cholesterol-independent vasoprotective effects that are mediated, in part, through the activation of Akt. However, the molecular mechanism remains unknown. To elucidate the molecular mechanisms of the pleioptropic effects of statins, we searched for the binding molecule of Akt1 by using a combined mass spectrometry and affinity purification strategy. By this technique, we identified the protein-protein interactions of 23 proteins from statin-treated rat aortic endothelial cells (rAECs). Our results suggest that this approach is very effective and statin activates many Akt down-stream targets, not only endothelial nitric oxide synthase (eNOS). The methodology presented here would provide a new tool for chemical proteomics in medicinal science.