Sirtuin 6 inhibition protects against glucocorticoid-induced skeletal muscle atrophy by regulating IGF/PI3K/AKT signaling.

Chronic activation of stress hormones such as glucocorticoids leads to skeletal muscle wasting in mammals. However, the molecular events that mediate glucocorticoid-induced muscle wasting are not well understood. Here, we show that SIRT6, a chromatin-associated deacetylase indirectly regulates glucocorticoid-induced muscle wasting by modulating IGF/PI3K/AKT signaling. Our results show that SIRT6 levels are increased during glucocorticoid-induced reduction of myotube size and during skeletal muscle atrophy in mice. Notably, overexpression of SIRT6 spontaneously decreases the size of primary myotubes in a cell-autonomous manner. On the other hand, SIRT6 depletion increases the diameter of myotubes and protects them against glucocorticoid-induced reduction in myotube size, which is associated with enhanced protein synthesis and repression of atrogenes. In line with this, we find that muscle-specific SIRT6 deficient mice are resistant to glucocorticoid-induced muscle wasting. Mechanistically, we find that SIRT6 deficiency hyperactivates IGF/PI3K/AKT signaling through c-Jun transcription factor-mediated increase in IGF2 expression. The increased activation, in turn, leads to nuclear exclusion and transcriptional repression of the FoxO transcription factor, a key activator of muscle atrophy. Further, we find that pharmacological inhibition of SIRT6 protects against glucocorticoid-induced muscle wasting in mice by regulating IGF/PI3K/AKT signaling implicating the role of SIRT6 in glucocorticoid-induced muscle atrophy.

Maternal obesogenic diet combined with postnatal exposure to high-fat diet induces metabolic alterations in offspring.

Maternal obesity has been shown to impact the offspring health during childhood and adult life. This study aimed to evaluate whether maternal obesity combined with postnatal exposure to an obesogenic diet could induce metabolic alterations in offspring. Female CD1 mice were fed a control diet (CD, 11.1% of energy from fat) or with a high-fat diet (HFD, 44.3% of energy from fat) for 3 months. After weaning, pups born from control and obese mothers were fed with CD or HFD for 3 months. Both mothers and offspring were weighted weekly and several blood metabolic parameters levels were evaluated. Here, we present evidence that the offspring from mothers exposed to a HFD showed increased acetylation levels of histone 3 on lysine 9 (H3K9) in the liver at postnatal Day 1, whereas the levels of acetylation of H4K16, dimethylation of H3K27, and trimethylation of H3K9 showed no change. We also observed a higher perinatal weight and increased blood cholesterol levels when compared to the offspring on postnatal Day 1 born from CD-fed mothers. When mice born from obese mothers were fed with HFD, we observed that they gained more weight, presented higher blood cholesterol levels, and abdominal adipose tissue than mice born to the same mothers but fed with CD. Collectively, our results point toward maternal obesity and HFD consumption as a risk factor for epigenetic changes in the liver of the offspring, higher perinatal weight, increased weight gain, and altered blood cholesterol levels.

Epigenetic control of early neurodegenerative events in diabetic retinopathy by the histone deacetylase SIRT6.

Diabetic retinopathy (DR) is one of the common complications associated with diabetes mellitus and the leading cause of blindness worldwide. Recent research has demonstrated that DR is not only a microvascular disease but may be a result of neurodegenerative processes. Moreover, glucose-induced neuron and glial cell damage may occur shortly after the onset of diabetes which makes the disease hard to diagnose at early stages. SIRT6, a NAD-dependent sirtuin deacylase, modulates aging, energy metabolism, and neurodegeneration. In previous studies we showed that SIRT6 deficiency causes major retinal transmission defects, changes in the expression of glycolytic genes, and elevated levels of apoptosis. Given the importance of glucose availability for retinal function and the critical role of SIRT6 in modulating glycolysis, we aimed to analyze SIRT6 participation in the molecular machinery that regulates the development of experimental DR. Using non-obese diabetic mice, we determined by western blot that 2 weeks after the onset of the disease, high glucose concentrations induced retinal increase in a neovascularization promoting factor (vascular endothelial growth factor, VEGF), and the loss of a neuroprotective factor (brain-derived neurotrophic factor, BDNF) associated with reduced levels of SIRT6 and increased acetylation levels of its substrates (H3K9 and H3K56) suggesting a deregulation of key neural factors. Noteworthy, retinas from CNS conditionally deleted SIRT6 mice showed a resemblance to diabetic retinas exhibiting lower protein levels of BDNF factor and increased protein levels of VEGF. Moreover, cultured Müller glial cells subjected to high glucose concentrations exhibited decreased levels of SIRT6 and increased levels of H3K56 acetylation. In addition, the increment of VEGF levels induced by high glucose was reverted by the over-expression of SIRT6 in this cell type. Accordingly, siRNA experiments showed that, when SIRT6 was silenced, VEGF levels increased. Our findings suggest that epigenetically regulated neurodegenerative events may occur at an early diabetic stage prior to the characteristic proliferative and vascular changes observed at a later diabetic stage.

Long-term effects of early life stress exposure: Role of epigenetic mechanisms.

Stress is an adaptive response to demands of the environment and thus essential for survival. Exposure to stress during the first years of life has been shown to have profound effects on the growth and development of an adult individual. There are evidences demonstrating that stressful experiences during gestation or in early life can lead to enhanced susceptibility to mental disorders. Early-life stress triggers hypothalamic-pituitary-adrenocortical (HPA) axis activation and the associated neurochemical reactions following glucocorticoid release are accompanied by a rapid physiological response. An excessive response may affect the developing brain resulting in neurobehavioral and neurochemical changes later in life. This article reviews the data from experimental studies aimed to investigate hormonal, functional, molecular and epigenetic mechanisms involved in the stress response during early-life programming. We think these studies might prove useful for the identification of novel pharmacological targets for more effective treatments of mental disorders.

Identification of and Molecular Basis for SIRT6 Loss-of-Function Point Mutations in Cancer.

Chromatin factors have emerged as the most frequently dysregulated family of proteins in cancer. We have previously identified the histone deacetylase SIRT6 as a key tumor suppressor, yet whether point mutations are selected for in cancer remains unclear. In this manuscript, we characterized naturally occurring patient-derived SIRT6 mutations. Strikingly, all the mutations significantly affected either stability or catalytic activity of SIRT6, indicating that these mutations were selected for in these tumors. Further, the mutant proteins failed to rescue sirt6 knockout (SIRT6 KO) cells, as measured by the levels of histone acetylation at glycolytic genes and their inability to rescue the tumorigenic potential of these cells. Notably, the main activity affected in the mutants was histone deacetylation rather than demyristoylation, pointing to the former as the main tumor-suppressive function for SIRT6. Our results identified cancer-associated point mutations in SIRT6, cementing its function as a tumor suppressor in human cancer.

The histone deacetylase SIRT6 controls embryonic stem cell fate via TET-mediated production of 5-hydroxymethylcytosine.

How embryonic stem cells (ESCs) commit to specific cell lineages and yield all cell types of a fully formed organism remains a major question. ESC differentiation is accompanied by large-scale histone and DNA modifications, but the relations between these epigenetic categories are not understood. Here we demonstrate the interplay between the histone deacetylase sirtuin 6 (SIRT6) and the ten-eleven translocation enzymes (TETs). SIRT6 targets acetylated histone H3 at Lys 9 and 56 (H3K9ac and H3K56ac), while TETs convert 5-methylcytosine into 5-hydroxymethylcytosine (5hmC). ESCs derived from Sirt6 knockout (S6KO) mice are skewed towards neuroectoderm development. This phenotype involves derepression of OCT4, SOX2 and NANOG, which causes an upregulation of TET-dependent production of 5hmC. Genome-wide analysis revealed neural genes marked with 5hmC in S6KO ESCs, thereby implicating TET enzymes in the neuroectoderm-skewed differentiation phenotype. We demonstrate that SIRT6 functions as a chromatin regulator safeguarding the balance between pluripotency and differentiation through Tet-mediated production of 5hmC.

SIRT6 is required for normal retinal function.

The retina is one of the major energy consuming tissues within the body. In this context, synaptic transmission between light-excited rod and cone photoreceptors and downstream ON-bipolar neurons is a highly demanding energy consuming process. Sirtuin 6 (SIRT6), a NAD-dependent deacylase, plays a key role in regulating glucose metabolism. In this study, we demonstrate that SIRT6 is highly expressed in the retina, controlling levels of histone H3K9 and H3K56 acetylation. Notably, despite apparent normal histology, SIRT6 deficiency caused major retinal transmission defects concomitant to changes in expression of glycolytic genes and glutamate receptors, as well as elevated levels of apoptosis in inner retina cells. Our results identify SIRT6 as a critical modulator of retinal function, likely through its effects on chromatin.

Assaying chromatin sirtuins.

Most of the sirtuins' nuclear substrates identified so far are histones or other chromatin-associated proteins and, thus, it is of special relevance the development of good biochemical techniques to analyze the biology of these proteins in the context of chromatin. Here, we describe several of the chromatin-based techniques to identify sirtuins' substrates, including a chromatin immunoprecipitation (ChIP) protocol, an acid-extraction protocol, and a nucleosomal immunoprecipitation protocol to analyze putative sirtuin chromatin interactors.

SIRT6 recruits SNF2H to DNA break sites, preventing genomic instability through chromatin remodeling.

DNA damage is linked to multiple human diseases, such as cancer, neurodegeneration, and aging. Little is known about the role of chromatin accessibility in DNA repair. Here, we find that the deacetylase sirtuin 6 (SIRT6) is one of the earliest factors recruited to double-strand breaks (DSBs). SIRT6 recruits the chromatin remodeler SNF2H to DSBs and focally deacetylates histone H3K56. Lack of SIRT6 and SNF2H impairs chromatin remodeling, increasing sensitivity to genotoxic damage and recruitment of downstream factors such as 53BP1 and breast cancer 1 (BRCA1). Remarkably, SIRT6-deficient mice exhibit lower levels of chromatin-associated SNF2H in specific tissues, a phenotype accompanied by DNA damage. We demonstrate that SIRT6 is critical for recruitment of a chromatin remodeler as an early step in the DNA damage response, indicating that proper unfolding of chromatin plays a rate-limiting role. We present a unique crosstalk between a histone modifier and a chromatin remodeler, regulating a coordinated response to prevent DNA damage.

The histone deacetylase SIRT6 is a tumor suppressor that controls cancer metabolism.

Reprogramming of cellular metabolism is a key event during tumorigenesis. Despite being known for decades (Warburg effect), the molecular mechanisms regulating this switch remained unexplored. Here, we identify SIRT6 as a tumor suppressor that regulates aerobic glycolysis in cancer cells. Importantly, loss of SIRT6 leads to tumor formation without activation of known oncogenes, whereas transformed SIRT6-deficient cells display increased glycolysis and tumor growth, suggesting that SIRT6 plays a role in both establishment and maintenance of cancer. By using a conditional SIRT6 allele, we show that SIRT6 deletion in vivo increases the number, size, and aggressiveness of tumors. SIRT6 also functions as a regulator of ribosome metabolism by corepressing MYC transcriptional activity. Lastly, Sirt6 is selectively downregulated in several human cancers, and expression levels of SIRT6 predict prognosis and tumor-free survival rates, highlighting SIRT6 as a critical modulator of cancer metabolism. Our studies reveal SIRT6 to be a potent tumor suppressor acting to suppress cancer metabolism.

Ischemic tolerance protects the rat retina from glaucomatous damage.

Glaucoma is a leading cause of acquired blindness which may involve an ischemic-like insult to retinal ganglion cells and optic nerve head. We investigated the effect of a weekly application of brief ischemia pulses (ischemic conditioning) on the rat retinal damage induced by experimental glaucoma. Glaucoma was induced by weekly injections of chondroitin sulfate (CS) in the rat eye anterior chamber. Retinal ischemia was induced by increasing intraocular pressure to 120 mmHg for 5 min; this maneuver started after 6 weekly injections of vehicle or CS and was weekly repeated in one eye, while the contralateral eye was submitted to a sham procedure. Glaucoma was evaluated in terms of: i) intraocular pressure (IOP), ii) retinal function (electroretinogram (ERG)), iii) visual pathway function (visual evoked potentials, (VEPs)) iv) histology of the retina and optic nerve head. Retinal thiobarbituric acid substances levels were assessed as an index of lipid peroxidation. Ischemic conditioning significantly preserved ERG, VEPs, as well as retinal and optic nerve head structure from glaucomatous damage, without changes in IOP. Moreover, ischemia pulses abrogated the increase in lipid peroxidation induced by experimental glaucoma. These results indicate that induction of ischemic tolerance could constitute a fertile avenue for the development of new therapeutic strategies in glaucoma treatment.

Circadian variations of prostaglandin E2 and F2 alpha release in the golden hamster retina.

Circadian variations of prostaglandin E2 and F2alpha release were examined in the golden hamster retina. Both parameters showed significant diurnal variations with maximal values at midnight. When hamsters were placed under constant darkness for 48 h, the differences in prostaglandin release between subjective mid-day and subjective midnight persisted. Western blot analysis showed that cyclooxygenase (COX)-1 levels were significantly higher at midnight than at mid-day, and at subjective midnight than at subjective mid-day, whereas no changes in COX-2 levels were observed among these time points. Immunohistochemical studies indicated the presence of COX-1 and COX-2 in the inner (but not outer) retina. Circadian variations of retinal prostaglandin release were also assessed in suprachiasmatic nuclei (SCN)-lesioned animals. Significant differences in retinal prostaglandin release between subjective mid-day and subjective midnight were observed in SCN-lesioned animals. These results indicate that hamster retinal prostaglandin release is regulated by a retinal circadian clock independent from the SCN. Thus, the present results suggest that the prostaglandin/COX-1 system could be a retinal clock output or part of the retinal clock mechanism.

Regulation of hippocampal gene expression is conserved in two species subjected to different stressors and antidepressant treatments.

BACKGROUND: Chronic stress has significant effects on hippocampal structure and function. We have previously identified nerve growth factor (NGF), membrane glycoprotein 6a (M6a), the guanine nucleotide binding protein (G protein) alpha q polypeptide (GNAQ), and CDC-like kinase 1 (CLK-1) as genes regulated by psychosocial stress and clomipramine treatment in the hippocampus of tree shrews. These genes encode proteins involved in neurite outgrowth. METHODS: To analyze whether regulation of the above-mentioned genes is conserved between different species, stressors, and antidepressant drugs, we subjected mice to repeated restraint stress and tianeptine treatment and measured hippocampal messenger RNA (mRNA) levels by real time reverse transcription polymerase chain reaction (RT-PCR). RESULTS: Chronically stressed mice displayed a reduction in transcript levels for NGF, M6a, GNAQ, and CLK-1. In addition, other genes implicated in neuronal plasticity, such as brain-derived neurotrophic factor (BDNF), cyclic adenosine monophosphate (cAMP) response element binding protein (CREB), protein kinase C (PKC), neural cell adhesion molecule (NCAM), and synapsin I were downregulated in stressed mice. Tianeptine treatment reversed the stress effects for the genes analyzed. Alterations in gene expression were dependent on the duration of the stress treatment and, in some cases, were only observed in male mice. CONCLUSIONS: These results suggest that genes involved in neurite remodeling are one of the main targets for regulation by chronic stress. The finding that this regulation is conserved in different stress models and antidepressant treatments highlights the biological relevance of the genes analyzed and suggests that they might be involved in stress-related disorders.

Acute and chronic stress exert opposing effects on antibody responses associated with changes in stress hormone regulation of T-lymphocyte reactivity.

Here we show that stress exerts a differential effect on T-cell-dependent antibody production. IgG production is augmented after acute stress and impaired in a chronic situation. We found catecholamines and corticosterone levels were increased in acute situations although they were not modified after prolonged stress conditions. However, lymphocyte sensitivity to corticosterone and catecholamines was altered under stress conditions. These results point out the role of the adrenal's hormones as mediators of the differential effects of stress on the immune response providing the basis for a functional significance of stress hormone receptors on lymphocytes.