Regulation of TSC2 lysosome translocation and mitochondrial turnover by TSC2 acetylation status.

Sirtuin1 (SIRT1) activity decreases the tuberous sclerosis complex 2 (TSC2) lysine acetylation status, inhibiting the mechanistic target of rapamycin complex 1 (mTORC1) signalling and concomitantly, activating autophagy. This study analyzes the role of TSC2 acetylation levels in its translocation to the lysosome and the mitochondrial turnover in both mouse embryonic fibroblast (MEF) and in mouse insulinoma cells (MIN6) as a model of pancreatic ß cells. Resveratrol (RESV), an activator of SIRT1 activity, promotes TSC2 deacetylation and its translocation to the lysosome, inhibiting mTORC1 activity. An improvement in mitochondrial turnover was also observed in cells treated with RESV, associated with an increase in the fissioned mitochondria, positive autophagic and mitophagic fluxes and an enhancement of mitochondrial biogenesis. This study proves that TSC2 in its deacetylated form is essential for regulating mTORC1 signalling and the maintenance of the mitochondrial quality control, which is involved in the homeostasis of pancreatic beta cells and prevents from several metabolic disorders such as Type 2 Diabetes Mellitus.

Targeting pancreatic beta cell death in type 2 diabetes by polyphenols.

Diabetes is a very complex disease which is characterized by the appearance of insulin resistance that is primarily compensated by an increase in pancreatic beta cell mass, generating hyperinsulinemia. After time, pancreatic beta cells die by apoptosis appearing in the second phase of the disease, and characterized by hypoinsulinemia. There are multiple conditions that can alter pancreatic beta cell homeostasis and viability, being the most relevant ones; ER stress, cytotoxicity by amylin, mTORC1 hyperactivity, oxidative stress, mitochondrial dysfunction, inflammation and alterations in autophagy/mitophagy flux. In addition, the possible effects that different polyphenols could exert in the modulation of these mechanisms and regulating pancreatic beta cell viability are analyzed. It is necessary a profound analysis and understanding of all the possible mechanisms involved in the control and maintenance of pancreatic beta cell viability to develop more accurate and target treatments for controlling beta cell homeostasis and preventing or even reversing type 2 diabetes mellitus.

Biological Actions and Molecular Mechanisms of Sambucus nigra L. in Neurodegeneration: A Cell Culture Approach.

Sambucus nigra flowers (elderflower) have been widely used in traditional medicine for the relief of early symptoms of common cold. Its chemical composition mainly consists of polyphenolic compounds such as flavonoids, hydroxycinnamic acids, and triterpenes. Although the antioxidant properties of polyphenols are well known, the aim of this study is to assess the antioxidant and protective potentials of Sambucus nigra flowers in the human neuroblastoma (SH-SY5Y) cell line using different in vitro approaches. The antioxidant capacity is first evaluated by the oxygen radical absorbance capacity (ORAC) and the free radical scavenging activity (DPPH) methods. Cell viability is assessed by the crystal violet method; furthermore, the intracellular ROS formation (DCFH-DA method) is determined, together with the effect on the cell antioxidant defenses: reduced glutathione (GSH) and antioxidant enzyme activities (GPx, GR). On the other hand, mTORC1 hyperactivation and autophagy blockage have been associated with an increase in the formation of protein aggregates, this promoting the transference and expansion of neurodegenerative diseases. Then, the ability of Sambucus nigra flowers in the regulation of mTORC1 signaling activity and the reduction in oxidative stress through the activation of autophagy/mitophagy flux is also examined. In this regard, search for different molecules with a potential inhibitory effect on mTORC1 activation could have multiple positive effects either in the molecular pathogenic events and/or in the progression of several diseases including neurodegenerative ones.

Dietary Polyphenols in Metabolic and Neurodegenerative Diseases: Molecular Targets in Autophagy and Biological Effects.

Polyphenols represent a group of secondary metabolites of plants which have been analyzed as potent regulators of multiple biological processes, including cell proliferation, apoptosis, and autophagy, among others. These natural compounds exhibit beneficial effects and protection against inflammation, oxidative stress, and related injuries including metabolic diseases, such as cardiovascular damage, obesity and diabetes, and neurodegeneration. This review aims to summarize the mechanisms of action of polyphenols in relation to the activation of autophagy, stimulation of mitochondrial function and antioxidant defenses, attenuation of oxidative stress, and reduction in cell apoptosis, which may be responsible of the health promoting properties of these compounds.

Overexpression of Mitochondrial IF1 Prevents Metastatic Disease of Colorectal Cancer by Enhancing Anoikis and Tumor Infiltration of NK Cells.

Increasing evidences show that the ATPase Inhibitory Factor 1 (IF1), the physiological inhibitor of the ATP synthase, is overexpressed in a large number of carcinomas contributing to metabolic reprogramming and cancer progression. Herein, we show that in contrast to the findings in other carcinomas, the overexpression of IF1 in a cohort of colorectal carcinomas (CRC) predicts less chances of disease recurrence, IF1 being an independent predictor of survival. Bioinformatic and gene expression analyses of the transcriptome of colon cancer cells with differential expression of IF1 indicate that cells overexpressing IF1 display a less aggressive behavior than IF1 silenced (shIF1) cells. Proteomic and functional in vitro migration and invasion assays confirmed the higher tumorigenic potential of shIF1 cells. Moreover, shIF1 cells have increased in vivo metastatic potential. The higher metastatic potential of shIF1 cells relies on increased cFLIP-mediated resistance to undergo anoikis after cell detachment. Furthermore, tumor spheroids of shIF1 cells have an increased ability to escape from immune surveillance by NK cells. Altogether, the results reveal that the overexpression of IF1 acts as a tumor suppressor in CRC with an important anti-metastatic role, thus supporting IF1 as a potential therapeutic target in CRC.

Changes in the Turnover of the Cellular Proteome during Metabolic Reprogramming: A Role for mtROS in Proteostasis.

The role played by protein turnover in metabolic reprogramming is unknown. Herein, using a SILAC approach, we have studied the changes in the half-life of 266 proteins of energy metabolism and of translation during the metabolic switch induced by the prolyl hydroxylases inhibitor dimethyloxalylglycine (DMOG). DMOG induces HIF-1α expression and triggers the activation of glycolysis and the concurrent inhibition of mitochondrial respiration in colon cancer cells. Changes in the activity of energy provision pathways correlated with increased turnover rates of glycolytic enzymes and the stabilization of mitochondrial proteins. Moreover, reprogramming also stabilized the proteins of translation. The partial DMOG-mediated arrest of the synthesis of mitochondrial and translation proteins results from the inhibition of the mTORC1/p70SK/S6 signaling pathway. In contrast, DMOG stimulated the synthesis of glycolytic enzymes, emphasizing the opposite and differential regulation of the two pathways of energy provision. Addition of MitoQ, a mitochondrial reactive oxygen species (mtROS) scavenger, stabilized the turnover of cellular proteins similarly as when protein degradation is inhibited with leupeptin, a serine-protease inhibitor. Overall, the results show that the higher the activity of a pathway the lower is the half-life of the proteins involved and suggest a role for mtROS in cellular proteostasis. Data are available via ProteomeXchange with identifier PXD013482.

MTORC1 Regulates both General Autophagy and Mitophagy Induction after Oxidative Phosphorylation Uncoupling.

Mechanistic target of rapamycin complex 1 (MTORC1) is a critical negative regulator of general autophagy. We hypothesized that MTORC1 may specifically regulate autophagic clearance of damaged mitochondria. To test this, we used cells lacking tuberous sclerosis complex 2 (TSC2-/- cells), which show constitutive MTORC1 activation. TSC2-/- cells show MTORC1-dependent impaired autophagic flux after chemical uncoupling of mitochondria, increased mitochondrial-protein aging, and accumulation of p62/SQSTM1-positive mitochondria. Mitochondrial autophagy (mitophagy) was also deficient in cells lacking TSC2, associated with altered expression of PTEN-induced putative kinase 1 (PINK1) and PARK2 translocation to uncoupled mitochondria, all of which were recovered by MTORC1 inhibition or expression of constitutively active forkhead box protein O1 (FoxO1). These data prove the necessity of intact MTORC1 signaling to regulate two synergistic processes required for clearance of damaged mitochondria: (i) general autophagy initiation and (ii) PINK1/PARK2-mediated selective targeting of uncoupled mitochondria to the autophagic machinery.

TSC2 N-terminal lysine acetylation status affects to its stability modulating mTORC1 signaling and autophagy.

There is a growing evidence of the role of protein acetylation in different processes controlling metabolism. Sirtuins (histone deacetylases nicotinamide adenine dinucleotide-dependent) activate autophagy playing a protective role in cell homeostasis. This study analyzes tuberous sclerosis complex (TSC2) lysine acetylation, in the regulation of mTORC1 signaling activation, autophagy and cell proliferation. Nicotinamide 5mM (a concentration commonly used to inhibit SIRT1), increased TSC2 acetylation in its N-terminal domain, and concomitantly with an augment in its ubiquitination protein status, leading to mTORC1 activation and cell proliferation. In contrast, resveratrol (RESV), an activator of sirtuins deacetylation activity, avoided TSC2 acetylation, inhibiting mTORC1 signaling and promoting autophagy. Moreover, TSC2 in its deacetylated state was prevented from ubiquitination. Using MEF Sirt1 +/+ and Sirt1 -/- cells or a SIRT1 inhibitor (EX527) in MIN6 cells, TSC2 was hyperacetylated and neither NAM nor RESV were capable to modulate mTORC1 signaling. Then, silencing Tsc2 in MIN6 or in MEF Tsc2-/- cells, the effects of SIRT1 modulation by NAM or RESV on mTORC1 signaling were abolished. We also observed that two TSC2 lysine mutants in its N-terminal domain, derived from TSC patients, differentially modulate mTORC1 signaling. TSC2 K599M variant presented a lower mTORC1 activity. However, with K106Q mutant, there was an activation of mTORC1 signaling at the basal state as well as in response to NAM. This study provides, for the first time, a relationship between TSC2 lysine acetylation status and its stability, representing a novel mechanism for regulating mTORC1 pathway.

Pancreatic ß-cell failure mediated by mTORC1 hyperactivity and autophagic impairment.

Hyperactivation of the mammalian target of rapamycin complex 1 (mTORC1) in ß-cells is usually found as a consequence of increased metabolic load. Although it plays an essential role in ß-cell compensatory mechanisms, mTORC1 negatively regulates autophagy. Using a mouse model with ß-cell-specific deletion of Tsc2 (ßTsc2(-/-)) and, consequently, mTORC1 hyperactivation, we focused on the role that chronic mTORC1 hyperactivation might have on ß-cell failure. mTORC1 hyperactivation drove an early increase in ß-cell mass that later declined, triggering hyperglycemia. Apoptosis and endoplasmic reticulum stress markers were found in islets of older ßTsc2(-/-) mice as well as accumulation of p62/SQSTM1 and an impaired autophagic response. Mitochondrial mass was increased in ß-cells of ßTsc2(-/-) mice, but mitophagy was also impaired under these circumstances. We provide evidence of ß-cell autophagy impairment as a link between mTORC1 hyperactivation and mitochondrial dysfunction that probably contributes to ß-cell failure.

Concerted expression of the thermogenic and bioenergetic mitochondrial protein machinery in brown adipose tissue.

Brown adipose tissue (BAT) is specialized in non-shivering thermogenesis through the expression of the mitochondrial uncoupling protein-1 (UCP1). In this paper, we describe the relationship between UCP1 and proteins involved in ATP synthesis. By the use of BATIRKO mice, which have enhanced UCP1 expression in BAT, an increase in ATP synthase as well as in ubiquinol cytochrome c reductase levels was observed. Alterations in mitochondrial mass or variations in ATP levels were not observed in BAT of these mice. In addition, using a protocol of brown adipocyte differentiation, the concerted expression of UCP1 with ATP synthase was found. These two scenarios revealed that increases in the uncoupling machinery of brown adypocites must be concomitantly followed by an enhancement of proteins involved in ATP synthesis. These concerted changes reflect the need to maintain ATP production in an essentially uncoupling cell type.