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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. ©, MINECO (SAF2013-41945-R and SAF2016-75916-R), CIBERER-ISCIII, and Fundación Ramón Areces, Spain, We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI).

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. In the present review, we report the main biological effects in relationship to autophagy regulation in response to different dietary polyphenols and its impact on metabolic and neurodegenerative diseases

Human islet amyloid polypeptide (hIAPP), or amylin, has the tendency to aggregate into insoluble amyloid fibrils, a typical feature of islets from type 2 diabetes individuals. Thus, we investigated comparatively the impact of hIAPP on key pathways involved in pancreatic beta survival. INS1E-hIAPP cells present a hyperactivation of MTORC1 and an inhibition of autophagy signaling, those cells showing an increase in cell size. Resveratrol, a MTORC1 inhibitor, can reverse TSC2 degradation that occurs in INS1E-hIAPP cells and diminished MTORC1 hyperactivation with concomitant autophagy stimulation. At the same time, a blockade in mitophagy was found in INS1E-hIAPP cells, as compared with control or INS1E-rIAPP cells. Consistently, human amylin overexpression generates a basal induction of nitrotyrosine levels and polyubiquitinated aggregates. Failure of the protein degradation machinery finally results in an accumulation of damaged and fissioned mitochondria, ROS production, and increased susceptibility to endoplasmic reticulum (ER)-stress-induced apoptosis. Overall, hIAPP overexpression in INS1E cells induced MTORC1 activation and mitophagy inhibition, favoring a pro-fission scenario of damaged mitochondria, these cells turn out to be more susceptible to the ER-stress-induced apoptosis and malfunction.

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.

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.

It is unknown how the lack of insulin receptor (IR)/insulinlike growth factor I receptor (IGFIR) in a tissue-specific manner affects brown fat development and mitochondrial integrity and function, as well as its effect on the redistribution of the adipose organ and the metabolic status. To address this important issue, we developed IR/IGFIR double-knockout (DKO) in a brown adipose tissue–specific manner. Lack of those receptors caused severe brown fat atrophy, enhanced beige cell clusters in inguinal fat; loss of mitochondrial mass; mitochondrial damage related to cristae disruption; and the loss of proteins involved in autophagosome formation, mitophagy, mitochondrial quality control, and dynamics and thermogenesis. More important, DKO mice showed an impaired thermogenesis upon cold exposure, based on a failure in the mitochondrial fission mechanisms and a much lower uncoupling protein 1 transcription rate and content. As a result, DKO mice under normal conditions showed an obesity susceptibility, revealed by increased body fat mass and insulin resistance. Upon consumption of a high-fat diet, DKO mice displayed frank obesity, as shown by increased body weight, increased adiposity, insulin resistance, hyperinsulinemia, and hypertriglyceridemia, all consistent with a metabolic syndrome. Collectively, our data suggest a cause-and-effect relationship between failure in brown fat thermogenesis and increased adiposity and obesity.

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., Ministerio de Ciencia, Innovación y Universidades (SAF2013-41945-R, SAF2016-75916-R and SAF2016-75452-R), CIBERER-ISCIII (CB06/07/0017) and Fundación Ramón Areces, Spain

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.

Brown fat is a thermogenic tissue that generates heat to maintain body temperature in cold environments and dissipate excess energy in response to overfeeding. We have addressed the role of the IGFIR in the brown fat development and function. Mice lacking IGFIR exhibited normal brown adipose tissue/body weight in knockout (KO) vs control mice. However, lack of IGFIR decreased uncoupling protein 1 expression in interscapular brown fat and beige cells in inguinal fat. More importantly, the lack of IGFIR resulted in an impaired cold acclimation. No differences in the total fat volume were found in the KO vs control mice. Epididymal fat showed larger adipocytes but with a lower number of adipocytes in KO vs control mice at age 12 months. In addition, KO mice showed a sustained moderate hyperinsulinemia and hypertriglyceridemia upon time and hepatic insulin insensitivity associated with lipid accumulation, with the outcome of a global insulin resistance. In addition, we found that the expression of uncoupling protein 3 in the skeletal muscle was decreased and its expression was increased in the heart in parallel with the expression of beta-2 adrenergic receptors. Upon nonobesogenic high-fat diet, we found a severe insulin resistance in the liver and in the skeletal muscle, but unchanged insulin sensitivity in the heart. In conclusion, our data suggest that IGFIR it is not an essential growth factor in the brown fat development in the presence of the IR and very high plasma levels of IGF-I, but it is indispensable for full brown fat functionality.

Hyperactivation of the mammalian target of rapamycin complex 1 (mTORC1) in β-cells is usually found as a consequence of increased metabolic load. Although it plays an essential role in β-cell compensatory mechanisms, mTORC1 negatively regulates autophagy. Using a mouse model with β-cell–specific deletion of Tsc2 (βTsc2−/−) and, consequently, mTORC1 hyperactivation, we focused on the role that chronic mTORC1 hyperactivation might have on β-cell failure. mTORC1 hyperactivation drove an early increase in β-cell mass that later declined, triggering hyperglycemia. Apoptosis and endoplasmic reticulum stress markers were found in islets of older βTsc2−/− mice as well as accumulation of p62/SQSTM1 and an impaired autophagic response. Mitochondrial mass was increased in β-cells of βTsc2−/− mice, but mitophagy was also impaired under these circumstances. We provide evidence of β-cell autophagy impairment as a link between mTORC1 hyperactivation and mitochondrial dysfunction that probably contributes to β-cell failure.

Autophagy is a highly regulated homoeostatic process involved in the lysosomal degradation of damaged cell organelles and proteins. This process is considered an important pro-survival mechanism under diverse stress conditions. A diabetic milieu is known to hamper osteoblast viability and function. In the present study, we explored the putative protective role of autophagy in osteoblastic cells exposed to an HG (high glucose) medium. HG was found to increase protein oxidation and triggered autophagy by a mechanism dependent on reactive oxygen species overproduction in osteoblastic MC3T3-E1 cells. MC3T3-E1 cell survival was impaired by HG and worsened by chemical or genetic inhibition of autophagy. These findings were mimicked by H2O2-induced oxidative stress in these cells. Autophagy impairment led to both defective mitochondrial morphology and decreased bioenergetic machinery and inhibited further osteoblast differentiation in MC3T3-E1 cells upon exposure to HG. These novel findings indicate that autophagy is an essential mechanism to maintain osteoblast viability and function in an HG environment.

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.