Sirt1 Mediates Vitamin D Deficiency-Driven Gluconeogenesis in the Liver via mTorc2/Akt Signaling.

As an active form of vitamin D (VD), 1,25-dihydroxyvitamin D (1,25(OH)2D3) is involved in the development of many metabolic diseases, such as diabetes, autoimmune diseases, and tumours. While prospective epidemiological studies have consistently implicated VD deficiency in the regulation of glucose metabolism and insulin sensitivity, the specific mechanism remains unclear. Here, we generated 1α(OH)ase-null mice (targeted ablation of the 25-hydroxyvitamin D 1α hydroxylase enzyme) and found that these mice developed hepatic glucose overproduction, glucose intolerance, and hepatic insulin resistance accompanied by reduced Sirtuin 1 (Sirt1) expression. The chromatin immunoprecipitation (ChIP) and a luciferase reporter assay revealed that 1,25(OH)2D3-activated VD receptor (VDR) directly interacted with one VD response element (VDRE) in the Sirt1 promoter to upregulate Sirt1 transcription, triggering a cascade of serine/threonine kinase (AKT) phosphorylation at S473 and FOXO1 phosphorylation at S256. This phosphorylation cascade reduced the expression of gluconeogenic genes, eventually attenuating glucose overproduction in the liver. In addition, a signaling pathway was found to modulate gluconeogenesis involving VDR, Sirt1, Rictor (a component of mTOR complex 2 [mTorc2]), AKT, and FOXO1, and Sirt1 and FOXO1 were identified as key modulators of dysregulated gluconeogenesis due to VD deficiency.

Norcantharidin counteracts acquired everolimus resistance in renal cell carcinoma by dual inhibition of mammalian target of rapamycin complex 1 and complex 2 pathways in Vitro.

Everolimus, an oral mammalian target of rapamycin complex 1 (mTORC1) inhibitor, presents a therapeutic option in metastatic renal cell carcinoma (RCC) patients who were intolerant to, or previously failed, immune- and vascular endothelial growth factor-targeted therapies. However, the onset of drug resistance limits its clinical use. One possible mechanism underpinning the resistance is that inhibiting mTORC1 by everolimus results in mTORC2-dependent activation of v-Akt murine thymoma viral oncogene (AKT) and upregulation of hypoxia-inducible transcription factors (HIF). Norcantharidin (NCTD) is a demethylated derivative of cantharidin with antitumor properties which is an active ingredient of the traditional Chinese medicine Mylabris. In this study, everolimus-resistant RCC cells (786-O-R) obtained by chronic everolimus treatment revealed higher level of HIF2α and over-activated mTORC2 pathway and NCTD inhibits cell proliferation in both everolimus-resistant and -sensitive RCC cells by arresting cell cycle in G0/G1 phase and reducing cell cycle-related proteins of C-Myc and cyclin D. Furthermore, NCTD shows synergistic anticancer effects combined with everolimus in everolimus-resistant 786-O-R cells. Mechanically, NCTD repressed both mTORC1 and mTORC2 signaling pathways as well as downstream molecular signaling pathways, such as p-4EBP1, p-AKT, HIF1α and HIF2α. Our findings provide sound evidence that combination of NCTD and everolimus is a potential therapeutic strategy for treating RCC and overcoming everolimus resistance by dual inhibition of mTORC1 and mTORC2.

Simvastatin preconditioning confers neuroprotection against hypoxia-ischemia induced brain damage in neonatal rats via autophagy and silent information regulator 1 (SIRT1) activation.

Previous studies have shown that simvastatin (Sim) has neuroprotective effects in a neonatal model of hypoxia-ischemia (HI)-induced brain injury when administered before but not after HI, pointing to the preconditioning (PC)-like effects of the statin. The present study aimed to gain more insight into the PC-like effect of Sim by studying the role of autophagy and its modulation by mTOR and SIRT1 in neuroprotection. Sim potentiated the autophagy response induced by neonatal HI, as shown by the increased expression of both microtubule-associated protein 1 light chain 3 (LC3) and beclin 1, increased monodansylcadaverine (MDC) labeling, and reduced expression of p62. The autophagy inhibitor 3-methyladenine (3MA) completely blocked the neuroprotective effect of Sim. Two hours after HI, there was a reduction in the activity of mTORC1 and a concomitant increase in that of mTORC2. Sim preconditioning further decreased the activity of mTORC1, but did not affect that of mTORC2. However, 24 h after injury, mTORC2 activity was significantly preserved in Sim-treated rats. Sim preconditioning also prevented the depletion of SIRT1 induced by HI, an effect that was completely blocked by 3MA. These data show that Sim preconditioning may modulate autophagy and survival pathways by affecting mTORC1, mTORC2, and SIRT1 activities. This study provides further preclinical evidence of the PC-like effect of statins in brain tissue, supporting their beneficial effects in improving stroke outcome after prophylactic treatments.

Discovery of Small-Molecule Selective mTORC1 Inhibitors via Direct Inhibition of Glucose Transporters.

The mechanistic target of rapamycin (mTOR) is a central regulator of cellular metabolic processes. Dysregulation of this kinase complex can result in a variety of human diseases. Rapamycin and its analogs target mTORC1 directly; however, chronic treatment in certain cell types and in vivo results in the inhibition of both mTORC1 and mTORC2. We have developed a high-throughput cell-based screen for the detection of phosphorylated forms of the mTORC1 (4E-BP1, S6K1) and mTORC2 (Akt) substrates and have identified and characterized a chemical scaffold that demonstrates a profile consistent with the selective inhibition of mTORC1. Stable isotope labeling of amino acids in cell culture-based proteomic target identification revealed that class I glucose transporters were the primary target for these compounds yielding potent inhibition of glucose uptake and, as a result, selective inhibition of mTORC1. The link between the glucose uptake and selective mTORC1 inhibition are discussed in the context of a yet-to-be discovered glucose sensor.

Role of the mTOR signalling pathway in salivary gland development.

Development of the salivary gland is characterized by extensive branching morphogenesis. Although various molecules have been implicated in salivary gland development, the role of the mammalian target of rapamycin (mTOR) signalling pathway, including both mTOR complexes 1 and 2 (mTORC1 and 2), in salivary gland development is unknown. Here, we examined protein expression levels related to the mTOR signalling pathway using an ex vivo submandibular salivary gland (SMG) organ culture. We showed that branching buds in the salivary glands were substantially decreased and phosphorylation of mTORC1 signalling pathway related proteins (mTOR, p70 ribosomal protein S6 kinase 1 and eukaryotic initiation factor 4E-binding protein 1) was inhibited by rapamycin (an mTOR inhibitor). In addition, AKT, which is an upstream protein kinase of mTORC1 and is downstream of mTORC2, is inhibited by LY294002 (a phosphatidylinositol 3-kinase inhibitor), but not by rapamycin. Moreover, rapamycin-treated ICR neonatal mice exhibited a reduction in both body weight and salivary glands compared with vehicle-treated neonatal mice. The present data indicate that the mTOR signalling pathway, including both mTORC1 and mTORC2, plays a critical role in salivary gland development both in ex vivo SMG organ culture and ICR neonatal mice in vivo.