It has been reported that increased expression of UCP-2 in the vasculature may prevent the development of atherosclerosis in patients with increased production of reactive oxygen species, as in the diabetes, obesity or hypertension. Thus, a greater understanding in the modulation of UCP-2 could improve the atherosclerotic process. However, the effect of TNF-α or insulin modulating UCP-2 in the vascular wall is completely unknown. In this context, we propose to study new molecular mechanisms that help to explain whether the moderate hyperinsulinemia or lowering TNF-α levels might have a protective role against vascular damage mediated by UCP-2 expression levels.

Type 2 diabetes results from a combination of insulin resistance and impaired insulin secretion. To directly address the effects of hepatic insulin resistance in adult animals, we developed an inducible liver-specific insulin receptor knockout mouse (iLIRKO).

Recent reports suggested frequent occurrence of cancer associated somatic mutations within regulatory elements of the genome. Based on initial exome sequencing of 21 melanomas, we report frequent somatic mutations in skin cancers in a bidirectional promoter of diphthamide biosynthesis 3 (DPH3) and oxidoreductase NAD-binding domain containing 1 (OXNAD1) genes. The UV-signature mutations occurred at sites adjacent and within a binding motif for E-twenty six/ternary complex factors (Ets/TCF), at -8 and -9 bp from DPH3 transcription start site. Follow up screening of 586 different skin lesions showed that the DPH3 promoter mutations were present in melanocytic nevi (2/114; 2%), melanoma (30/304; 10%), basal cell carcinoma of skin (BCC; 57/137; 42%) and squamous cell carcinoma of skin (SCC; 12/31; 39%). Reporter assays carried out in one melanoma cell line for DPH3 and OXNAD1 orientations showed statistically significant increased promoter activity due to -8/-9CC > TT tandem mutations; although, no effect of the mutations on DPH3 and OXNAD1 transcription in tumors was observed. The results from this study show occurrence of frequent somatic non-coding mutations adjacent to a pre-existing binding site for Ets transcription factors within the directional promoter of DPH3 and OXNAD1 genes in three major skin cancers. The detected mutations displayed typical UV signature; however, the functionality of the mutations remains to be determined.

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.

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.

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.

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.

Energy sensing is indispensable to balance anabolic and catabolic processes for the maintenance of cell viability. Pancreatic β cells are especially relevant because of their involvement in the coordination of insulin secretion when glucose concentration arises in the local milieu. In this work, we uncover the increased susceptibility of pancreatic β cells to cell death in response to different energy stressors. Upon glucose decline, from 25 to 5 mM, caused by stimulation with either 2-deoxyglucose or metformin, only pancreatic β cells showed an increase in cell death. Very interestingly, when we transfected either mouse insulinoma cell or human embryo kidney cells with a phospho-mutant form of B cell lymphoma 2 associated agonist of cell death at serine 155 (BAD S155D), an increase in the pro-survival factor B cell lymphoma 2 was detected in pancreatic β cells and not in human embryonic kidney cells in the presence of the energetic stressors. This data suggests that the protective capacity of this mutant form is only present in cells that present glucokinase. In contrast, upon hyperactivation of mechanistic target of rapamycin complex 1 signaling by knocking-down tuberous sclerosis complex protein, we observed increased susceptibility to cell death in response to energy stress in both pancreatic and non-pancreatic β cells. Therefore, mechanistic target of rapamycin complex 1 signaling presents a dual effect on cell viability. On the one hand, a chronic inhibition of mechanistic target of rapamycin complex 1 activity in response to the energy status is deleterious for pancreatic β cells, being attenuated by the overexpression of B cell lymphoma 2 associated agonist of cell death S155D. On the other hand, mechanistic target of rapamycin complex 1 hyperactivity provokes a susceptibility to energetic stress-induced cell death. Taken together, these results may open potential implications for the use of glucokinase activators or mechanistic target of rapamycin complex 1 modulators for the maintenance of pancreatic β cells for longer periods of time avoiding its loss in different pathologies such as type 2 diabetes mellitus.

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.

Hutchinson-Gilford Progeria Syndrome is an ultrarare disease which is characterized by an accelerated senescence phenotype with deleterious consequences to people suffering this pathology. The production of an abnormal protein derived from lamin A, called progerin, presents a farnesylated domain, which is not eliminated by the causal mutation of the disease, and accumulates in the interior of the nucleus, provoking a disruption of nuclear membrane, chromatin organization and an altered gene expression. The mutation in these patients occurs in a single nucleotide change, which creates a de novo splicing site, producing a shorter version of the protein. Apart from this mutation, an alteration in the metalloproteinase Zmpste24, involved in the maturation of lamin A, causing a similar alteration than in progeria. However, in this case, patients accumulate a protein, called prelamin A, which generates similar alterations in the nucleus than progerin. The reduction of prelamin A protein levels facilitates the recovery of the phenotype in different mice models of the disease, reducing the aging process. Different strategies have been studied for eliminating this toxic protein. Here, we report that immortalization of primary cells derived from the Zmpste24 KO mice, facilitates prelamin A degradation by different mechanisms, being essential, the enhancing proliferative capacity that the immortalized cells present. Then, these data suggest that using different treatments for increasing proliferative capacity of these cells, potentially could have a beneficial effect, facilitating prelamin A toxicity.