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Identification of Sirt3-regulated acetylation sites in human cells. (A) Generation of Sirt3 knockdown and overexpression U2OS cells. U2OS cells were either transfected with a human Sirt3 encoding cDNA or with a Sirt3-specific shRNA. To induce expression of Sirt3-shRNA, cells were treated with doxycycline for the indicated time periods. Expression of Sirt3 was analyzed by immunostaining using anti-Sirt3 antibody. (B) Distribution of identified acetylation sites between mitochondrial or non-mitochondrial cellular compartments. (C) Increased acetylation of mitochondrial acetylation in Sirt3 knockdown cells. Logarithmized SILAC ratios of acetylated peptides from mitochondrial and non-mitochondrial proteins were plotted. These data shows the upwardly shifted distribution of SILAC ratios of mitochondrial acetylation sites in Sirt3 knockdown cells. (D) Gene Ontology enrichment analysis of Sirt3-regulated proteins. Proteins with increased acetylation in Sirt3 knockdown cells showed significant enrichment of mitochondrial associated GO terms. doi:10.1371/journal.pone.0050545.g003
Source publication
Lysine acetylation is a posttranslational modification that is dynamically regulated by the activity of acetyltransferases and deacetylases. The human and mouse genomes encode 18 different lysine deacetylases (KDACs) which are key regulators of many cellular processes. Identifying substrates of KDACs and pinpointing the regulated acetylation sites...
Contexts in source publication
Context 1
... these cells we either increased cellular Sirt3 levels by retroviral overexpression of human Sirt3, or reduced its expression using an inducible shRNA- based knockdown approach. Overexpression and conditional knockdown of Sirt3 was confirmed at the protein level by Western blotting ( Figure 3A). Sirt3 overexpressing cells were grown in 'light' SILAC media whereas Sirt3 knockdown cells were cultured in 'heavy' SILAC media and acetylation analysis was performed as described above ( Figure 1A). ...
Context 2
... overexpressing cells were grown in 'light' SILAC media whereas Sirt3 knockdown cells were cultured in 'heavy' SILAC media and acetylation analysis was performed as described above ( Figure 1A). Using this approach, we identified over 3,000 acetylation sites in human U2OS cells, of which about 23% were present on mitochondrial proteins ( Figure 3B, Table S2). In agreement with the data obtained from Sirt3 knockout MEFs, acetylation of mitochondrial sites was significantly increased in comparison to non-mitochondrial acetylation sites ( Figure 3C). ...
Context 3
... this approach, we identified over 3,000 acetylation sites in human U2OS cells, of which about 23% were present on mitochondrial proteins ( Figure 3B, Table S2). In agreement with the data obtained from Sirt3 knockout MEFs, acetylation of mitochondrial sites was significantly increased in comparison to non-mitochondrial acetylation sites ( Figure 3C). Furthermore, analysis of proteins with increased acetylation in Sirt3 deficient cells revealed that mitochondria associated GO terms were enriched among Sirt3-regulated proteins ( Figure 3D). ...
Context 4
... agreement with the data obtained from Sirt3 knockout MEFs, acetylation of mitochondrial sites was significantly increased in comparison to non-mitochondrial acetylation sites ( Figure 3C). Furthermore, analysis of proteins with increased acetylation in Sirt3 deficient cells revealed that mitochondria associated GO terms were enriched among Sirt3-regulated proteins ( Figure 3D). ...
Citations
... SIRT3 is a deacetylase that regulates most mitochondrial lysine acetylation, including tricarboxylic acid cycle-associated dehydrogenase, forkhead box protein O3a (FOXO3a), superoxide dismutase, etc [22][23] . ...
Background
Our previous studies have confirmed that Honokiol exerts protective effect on cardiomyocytes and cardiac tissue against ATO-induced cardiotoxicity, which is attributed to inhibiting cardiomyocyte apoptosis via reduced oxidative stress.
Objectives
This research aimed to explore the entire mechanism by which Honokiol provides protection against arsenic trioxide-induced cardiotoxicity.
Materials and methods
129S1/SvImJ male wild type (WT) mice and SIRT3-knockout (SIRT3−/−) mice with 129S1 background were applied to verify the pathway of Honokiol on myocardial toxicity of arsenic trioxide; MitoSox, carboxy-DCFDA and Amplex Red were used to detect oxidative stress; ferroptosis-associated markers (MDA, GSH, tissue iron, GPX4 and 4-HNE expression) were tested by test kit or immunohistochemistry; autophagic flux was analyzed by western blot.
Results
SIRT3 knockout abolishes protective effects of HKL in ATO-induced myocardial injury and hypertrophy; Honokiol protects myocardium from ATO-induced oxidative stress through SIRT3/SOD2 pathway; Honokiol inhibited ATO-induced ferroptosis via the SIRT3 pathway in myocardial tissues; Honokiol improve autophagic flux in cardiomyocytes exposed to ATO treatment. Autophagic flux plays a critical role in protective effect of Honokiol on ATO-induced ferroptosis. The present study confirmed that Honokiol inhibits oxidative stress injury and ferroptosis in cardiomyocytes by promoting the autophagic flux in cardiomyocytes through the activation of SIRT3.
... Sirt-3 is most often described as a mitochondrial deacetylase that plays a pivotal role in regulating mitochondrial function and maintaining cellular energy balance. Proteomic analyses have identified hundreds of (de)acetylation sites regulated by Sirt-3 [69], and focused studies are still revealing new Sirt-3 targets. Given the large number of cellular processes in which Sirt-3 is involved, it is likely that it serves as a mediator in fine tuning the cell's metabolic status and not as an on/off switch. ...
Sirt-3 is an important regulator of mitochondrial function and cellular energy homeostasis, whose function is associated with aging and various pathologies such as Alzheimer’s disease, Parkinson’s disease, cardiovascular diseases, and cancers. Many of these conditions show differences in incidence, onset, and progression between the sexes. In search of hormone-independent, sex-specific roles of Sirt-3, we performed mRNA sequencing in male and female Sirt-3 WT and KO mouse embryonic fibroblasts (MEFs). The aim of this study was to investigate the sex-specific cellular responses to the loss of Sirt-3. By comparing WT and KO MEF of both sexes, the differences in global gene expression patterns as well as in metabolic and stress responses associated with the loss of Sirt-3 have been elucidated. Significant differences in the activities of basal metabolic pathways were found both between genotypes and between sexes. In-depth pathway analysis of metabolic pathways revealed several important sex-specific phenomena. Male cells mount an adaptive Hif-1a response, shifting their metabolism toward glycolysis and energy production from fatty acids. Furthermore, the loss of Sirt-3 in male MEFs leads to mitochondrial and endoplasmic reticulum stress. Since Sirt-3 knock-out is permanent, male cells are forced to function in a state of persistent oxidative and metabolic stress. Female MEFs are able to at least partially compensate for the loss of Sirt-3 by a higher expression of antioxidant enzymes. The activation of neither Hif-1a, mitochondrial stress response, nor oxidative stress response was observed in female cells lacking Sirt-3. These findings emphasize the sex-specific role of Sirt-3, which should be considered in future research.
... First, SIRT3 can deacetylate some key enzymes in the mitochondria involved in respiratory chain such as NADH dehydrogenase ubiquinone subcomplex subunit 9 (NDUF9A), succinate dehydrogenase A (SDHA), pyruvate dehydrogenase (PDH), ATP5A1 and other ATP synthase subunits to improve mitochondrial respiration efficiency and maintain ATP production. 31,[64][65][66][67] The data showed that, SDHA and ATP5A1 were highly acetylated in the TAC group, while NADPH could significantly reverse this phenotype. These results indicate that NADPH may enhance ATPase activity and increase ATP synthesis through SIRT3-mediated deacetylation of SDHA and ATP5A1. ...
Background
Therapies are urgently required to ameliorate pathological cardiac hypertrophy and enhance cardiac function in heart failure. Our preliminary experiments have demonstrated that exogenous NADPH exhibits a positive inotropic effect on isolated heart. This study aims to investigate the positive inotropic effects of NADPH in pathological cardiac hypertrophy and heart failure, as well as the underlying mechanisms involved.
Methods
Endogenous plasma NADPH contents were determined in patients with chronic heart failure and control adults. The positive inotropic effects of NADPH were investigated in isolated toad heart or rat heart. The effects of NADPH were investigated in isoproterenol (ISO)–induced cardiac hypertrophy or transverse aortic constriction (TAC)–induced heart failure. The underlying mechanisms of NADPH were studied using SIRT3 knockout mice, echocardiography, Western blotting, transmission electron microscopy, and immunoprecipitation.
Findings
The endogenous NADPH content in the blood of patients and animals with pathological cardiac hypertrophy or heart failure was significantly reduced compared with age-sex matched control subjects. Exogenous NADPH showed positive inotropic effects on the isolated normal and failing hearts, while antagonism of ATP receptor partially abolished the positive inotropic effect of NADPH. Exogenous NADPH administration significantly reduced heart weight indices, and improved cardiac function in the mice with pathological cardiac hypertrophy or heart failure. NADPH increased SIRT3 expression and activity, deacetylated target proteins, improved mitochondrial function and facilitated ATP production in the hypertrophic myocardium. Importantly, inhibition of SIRT3 abolished the positive inotropic effect of NADPH, and the anti-heart failure effect of NADPH was significantly reduced in the SIRT3 Knockout mice.
Interpretation
Exogenous NADPH shows positive inotropic effect and improves energy metabolism via SIRT3 in pathological cardiac hypertrophy and heart failure. NADPH thus may be one of the potential candidates for the treatment of pathological cardiac hypertrophy or heart failure.
Funding
This work was supported by grants from the 10.13039/501100001809National Natural Science Foundation of China (No. 81973315, 82173811, 81730092), Natural Science Foundation of Jiangsu Higher Education (20KJA310008), Jiangsu Key Laboratory of Neuropsychiatric Diseases (BM2013003) and the Priority Academic Program Development of the Jiangsu Higher Education Institutes (PAPD).
... 6 A twofold increase in >100 acetylation sites in mitochondrial proteins was reported in SIRT3 knockout (SIRT3-KO) cells. 64 Furthermore, >90% of the SIRT3 interactome was found to be acetylated, 59 although only $50% of the mitochondrial proteome has been reported to be acetylated. Investigating the involvement of SIRT3 in mitochondrial biology, Rardin and colleagues discovered the SIRT3-regulated acetylome in the mitochondria of the liver. ...
Sirtuin 3 (SIRT3), a mitochondrial deacetylase expressed preferentially in high-metabolic-demand tissues including the brain, requires NAD+ as a cofactor for catalytic activity. It regulates various processes such as energy homeostasis, redox balance, mitochondrial quality control, mitochondrial unfolded protein response (UPRmt), biogenesis, dynamics and mitophagy by altering protein acetylation status. Reduced SIRT3 expression or activity causes hyperacetylation of hundreds of mitochondrial proteins, which has been linked with neurological abnormalities, neuro-excitotoxicity and neuronal cell death. A body of evidence has suggested, SIRT3 activation as a potential therapeutic modality for age-related brain abnormalities and neurodegenerative disorders.
... We next asked whether mTOR-driven AS in CDS also affects proteome-wide PTMs [45][46][47][48]. We cataloged the unique peptide sequences belonging to each AS isoform and compared them to peptide sequences in published PTM proteomics [43,[49][50][51][52][53][54]. In this proteome-wide search, we found that exon skipping could abolish existing sites for four interrogated PTMs in select genes and could also create new PTM sites in other genes, demonstrating that mTOR-modulated AS serves as a molecular scaffold for PTMs in functional proteomics ( Figure 4D). ...
The mammalian target of rapamycin (mTOR) pathway is crucial in energy metabolism and cell proliferation. Previously, we reported transcriptome-wide 3′-untranslated region (UTR) shortening by alternative polyadenylation upon mTOR activation and its impact on the proteome. Here, we further interrogated the mTOR-activated transcriptome and found that hyperactivation of mTOR promotes transcriptome-wide exon skipping/exclusion, producing short isoform transcripts from genes. This widespread exon skipping confers multifarious regulations in the mTOR-controlled functional proteomics: AS in coding regions widely affects the protein length and functional domains. They also alter the half-life of proteins and affect the regulatory post-translational modifications. Among the RNA processing factors differentially regulated by mTOR signaling, we found that SRSF3 mechanistically facilitates exon skipping in the mTOR-activated transcriptome. This study reveals a role of mTOR in AS regulation and demonstrates that widespread AS is a multifaceted modulator of the mTOR-regulated functional proteome.
... In mitochondria, SIRT3 plays a pivotal role in regulating the activity of fatty acid oxidation enzymes by deacetylation [49]. SIRT3 was induced by RES treatment, which increases fatty acid oxidation and ameliorates fat accumulation in HFD-induced obese mice [50]. ...
Introduction: Despite decades of research, obesity and its related medical complications remain a major health concern globally. Therefore, novel therapeutic strategies are needed to combat obesity and its numerous debilitating complications. Resveratrol (RES) has a potential therapeutic effect in obesity and diabetes by improving oxidative metabolism and insulin signaling. Background and Objectives: The aim of this study was to investigate the effect of RES treatment on weight loss and glucose and fatty acid metabolism. Methods: Obesity was induced in 24 mice by exposure to a high-fat diet (HFD) for 8 weeks. Mice were randomly assigned to one group of either: group 1: control, non-treated low-fat diet (LFD) for 12 weeks (n = 8), group 2: non-treated high-fat diet (HFD) for 12 weeks (n = 8), group 3: RES-treated HFD (HFD + RES) (n = 8), or group 4: RES-treated and switched to LFD (HFD-LFD + RES) (n = 8). HFD + RES mice were first fed an HFD for 8 weeks followed by 4 weeks of RES. The HFD-LFD + RES group was first fed an HFD for 8 weeks and then treated with RES and switched to an LFD for 4 weeks. Results: After 12 weeks, group 2 mice had significantly higher body weights compared to group 1 (23.71 ± 1.95 vs. 47.83 ± 2.27; p < 0.05). Group 4 had a significant decrease in body weight and improvement in glucose tolerance compared to mice in group 2 (71.3 ± 1.17 vs. 46.1 ± 1.82 and 40.9 ± 1.75, respectively; p < 0.05). Skeletal muscles expression of SIRT1, SIRT3, and PGC1α were induced in group 3 and 4 mice compared to group 2 (p < 0.01), with no changes in AMP-activated protein kinase expression levels. Furthermore, combination of RES and diet ameliorated skeletal muscle intermediate lipid accumulation and significantly improved insulin sensitivity and secretion. Conclusions: The results of this study suggest a synergistic beneficial effect of LFD and RES to lower body weight and enhance glucose and fatty acid metabolism.
... Future studies could probe the roles that these modified TFAM forms could play in mitochondrial replication processivity as well as transcription processivity through these "roadblocks" in the presence of transcription elongation factor that clamps POLRMT on DNA. Additionally, TFAM has been missing as a target of the mitochondrial deacetylase, SIRT3, in all studies except one, in which K154 was identified as the only target (43)(44)(45). Determining whether or not other residues on TFAM are regulatable via sirtuin-catalyzed deacetylation would help to solidify the acetylated sites as more consequential to TFAM regulation within the mitochondria. ...
Mitochondrial transcription factor A (TFAM) plays important roles in mitochondrial DNA (mtDNA) compaction, transcription initiation, and in the regulation of processes like transcription and replication processivity. It is possible that TFAM is locally regulated within the mitochondrial matrix via such mechanisms like phosphorylation by protein kinase A (PKA) and non-enzymatic acetylation by acetyl-CoA. Here we demonstrate that DNA-bound TFAM is less susceptible to these modifications. We confirmed using electrophoretic mobility shift assays that phosphorylated or acetylated TFAM compacted circular double-stranded DNA just as well as unmodified TFAM and provide an in-depth analysis of acetylated sites on TFAM. We show that both modifications of TFAM increase the processivity of mitochondrial RNA polymerase during transcription through TFAM-imposed barriers on DNA, but that TFAM bearing either modification retains its full activity in transcription initiation. We conclude that TFAM phosphorylation by PKA and non-enzymatic acetylation by acetyl-CoA are unlikely to occur at the mtDNA and that modified free TFAM retains its vital functionalities like compaction and transcription initiation while enhancing transcription processivity.
... Finally, the substrates from the SILAC studies of the dif ferential protein acetylation between the SIRT3 KO and the wt murine embryonic fibroblasts and U2OS cells with SIRT3 upregulated by retroviral overexpression com pared to the cells with shRNA silenced SIRT3 were included. The differential threshold was set to the 2 fold acetylation change for the ratios SIRT3KO/wt and SIRT3KO/SIRT3 overexpression [32]. The final list of SIRT3 substrates used for the analysis contained 407 pro teins (Table S2 in the Supplement). ...
... The SIRT3 substrates used for data mining originated from the analysis of the mitochondrial fractions [30,31], where the mitochondr ial proteins are overrepresented. The exception is the SILAC study [32] (Table S1), which used the whole cell lysis step before the LC MS/MS identification. The SIRT3 substrate list obtained from this study contains multiple histone proteins (Fig. 1b), while their occur rence in the lists of the substrates from other sources [30,31] is limited. ...
... Cluster V includes several remarkable SIRT3 sub strates with interesting molecular functions located in the nucleoplasm such as LMNA (lamin A), LMNB, and HIF 1A, p53, and PRKDC, which are highlighted fur ther. The SIRT3 substrate LMNA was identified by the SILAC method in the U2OS cells overexpressing SIRT3 versus the KO cells, while LMNB was found by the iden tical technique in the SIRT3 KO MEF compared to the wt cells (Table S1 in the Supplement) [32]. Interestingly, similar to the aging related functions of SIRT3, muta tions of its substrate, LMNA, cause the syndrome of pre mature aging called Hutchinson-Gilford progeria syn drome [71]. ...
Белок сиртуин 3 (SIRT3) представляет собой лизиндеацетилазу, играющую важную роль в поддержании целостности митохондрий, являющихся уязвимой мишенью при многих заболеваниях. Интересно, что клеточное старение может быть обращено только лишь путём суперэкспрессии SIRT3, что вызывает много вопросов о роли SIRT3 в молекулярных механизмах борьбы со старением. Анализ функционирования SIRT3 мы провели на основе имеющихся данных по взаимодействию 407 субстратов этого белка. Результаты изучения многообразия путей и прогнозирования функций генов подтвердили роль SIRT3 в первичном метаболизме и производстве ATP митохондриями. Кроме того, SIRT3, предположительно, задействован в термогенезе, при развитии дегенеративных заболеваний головного мозга, таких как болезнь Альцгеймера, болезнь Паркинсона, болезнь Хантингтона, а также неалкогольной жировой болезни печени. Приоритизация белков в узлах исследуемого пути продемонстрировала, что субъединицы комплекса I дыхательной цепи митохондрий (MRC) являются основными регуляторными точками во всей сети взаимодействий. Дополнительными приоритетными узлами оказались субъединица сукцинатдегидрогеназы B (SDHB) комплекса II и ATP5F1 комплекса V MRC. Проведенный анализ подтверждает существование NADH/NAD+-зависимой регуляторной петли обратной связи между SIRT3, комплексом I MRC и ацетил-КоА-синтетазами, а также наличие ядерных субстратов SIRT3. Малоисследованные функции субстратов SIRT3, таких как LMNA и LMNB, HIF‑1α, p53, DNA‑PK и PARK7, отмечены как перспективные для дальнейших научных исследований. SIRT3 действует как репрессор BACE1 через SIRT3-LKB1-AMPK-CREB-PGC1A-PPARG-BACE1 (SIRT3-BACE1), функции которого наилучшим образом соответствуют механизмам циркадного ритма. Формируется новая рабочая гипотеза терапевтической мишени для лечения болезни Альцгеймера. Также обозначены другие важные пути терапевтических вмешательств, ассоциированные с активностью SIRT3.
... Finally, the substrates from the SILAC studies of the dif ferential protein acetylation between the SIRT3 KO and the wt murine embryonic fibroblasts and U2OS cells with SIRT3 upregulated by retroviral overexpression com pared to the cells with shRNA silenced SIRT3 were included. The differential threshold was set to the 2 fold acetylation change for the ratios SIRT3KO/wt and SIRT3KO/SIRT3 overexpression [32]. The final list of SIRT3 substrates used for the analysis contained 407 pro teins (Table S2 in the Supplement). ...
... The SIRT3 substrates used for data mining originated from the analysis of the mitochondrial fractions [30,31], where the mitochondr ial proteins are overrepresented. The exception is the SILAC study [32] (Table S1), which used the whole cell lysis step before the LC MS/MS identification. The SIRT3 substrate list obtained from this study contains multiple histone proteins (Fig. 1b), while their occur rence in the lists of the substrates from other sources [30,31] is limited. ...
... Cluster V includes several remarkable SIRT3 sub strates with interesting molecular functions located in the nucleoplasm such as LMNA (lamin A), LMNB, and HIF 1A, p53, and PRKDC, which are highlighted fur ther. The SIRT3 substrate LMNA was identified by the SILAC method in the U2OS cells overexpressing SIRT3 versus the KO cells, while LMNB was found by the iden tical technique in the SIRT3 KO MEF compared to the wt cells (Table S1 in the Supplement) [32]. Interestingly, similar to the aging related functions of SIRT3, muta tions of its substrate, LMNA, cause the syndrome of pre mature aging called Hutchinson-Gilford progeria syn drome [71]. ...
Abstract—SIRT3 is a protein lysine deacetylase with a prominent role in the maintenance of mitochondrial integrity, which is a vulnerable target in many diseases. Intriguingly, cellular aging is reversible just by SIRT3 overexpression, which raises many questions about the role of SIRT3 in the molecular anti-aging mechanisms. Therefore, functions of SIRT3 were analyzed through the interaction network of 407 substrates collected by data mining. Results of the pathway enrichment and gene function prediction confirmed functions in the primary metabolism and mitochondrial ATP production. However, it also suggested involvement in thermogenesis, brain-related neurodegenerative diseases Alzheimer’s (AD), Parkinson’s, Huntington’s disease (HD), and non-alcoholic fatty liver disease. The protein node prioritization analysis identified subunits of the complex I of the mitochondrial respiratory chain (MRC) as the nodes with the main regulatory effect within the entire interaction network. Additional high-ranked nodes were succinate dehydrogenase subunit B (SDHB), complex II, and ATP5F1, complex V of MRC. The analysis supports existence of the NADH/NAD+ driven regulatory feedback loop between SIRT3, complex I (MRC), and acetyl-CoA synthetases, and existence of the nuclear substrates of SIRT3. Unexplored functions of SIRT3 substrates such as LMNA and LMNB; HIF-1a, p53, DNA-PK, and PARK7 are highlighted for further scientific advances. SIRT3 acts as a repressor of BACE1 through the SIRT3-LKB1-AMPK-CREB-PGC1A-PPARG-BACE1 (SIRT3-BACE1), which functions are fitted the best by the Circadian Clock pathway. It forms a new working hypothesis as the therapeutical target for AD treatment. Other important pathways linked to SIRT3 activity are highlighted for therapeutical interventions.
... Thus, the hyperacetylation of mitochondrial proteins will be further accelerated due to SIRT3 inhibition. As a result, the activity of the pyruvate dehydrogenase complex, Electron Transport Chain respiratory complex I, and TCA cycle enzymes including citrate synthase, isocitrate dehydrogenase 2, α-ketoglutarate dehydrogenase and malate dehydrogenase will be reduced [92,96,97]. ...
... The mitochondrial ROS levels are determined by the rates of superoxide production and of its detoxification. Ethanol consumption results in hyperacetylation of mitochondrial proteins that has an inhibitory effect on the activity of some of the detoxification enzymes [92,96,97]. The hyperacetylation of SOD2 leads to increased levels of ROS due to reduced rate of superoxide detoxification by this enzyme [104,105]. ...
Alcohol abuse and dependence in humans causes an extreme shift in metabolism for which the human brain is not evolutionarily prepared. Oxidation of ethanol and acetaldehyde are not regulated, making ethanol a dominating metabolic substrate that prevents the activity of enzymes from oxidizing their usual endogenous substrates. The enzymes required to oxidize ethanol across the variety of affected tissues all produce acetaldehyde which is then converted to acetate by aldehyde dehydrogenases (ALDHs). ALDHs are NAD+-dependent enzymes, and mitochondrial ALDH2 is likely the primary contributor to ethanol-derived acetaldehyde clearance in cells. Metabolism of alcohol has several adverse effects on mitochondria including increased free radical levels, hyperacetylation of mitochondrial proteins, and excessive mitochondrial fragmentation. This review discusses the role of astrocytic and neuronal mitochondria in ethanol metabolism that contributes to the acute and chronic changes in mitochondrial function and morphology, that might promote tolerance, dependence and withdrawal. We also propose potential modes of therapeutic intervention to reduce the toxicity of chronic alcohol consumption.
