Amino Acid Restriction Triggers Angiogenesis via GCN2/ATF4 Regulation of VEGF and H2S Production.

Angiogenesis, the formation of new blood vessels by endothelial cells (ECs), is an adaptive response to oxygen/nutrient deprivation orchestrated by vascular endothelial growth factor (VEGF) upon ischemia or exercise. Hypoxia is the best-understood trigger of VEGF expression via the transcription factor HIF1α. Nutrient deprivation is inseparable from hypoxia during ischemia, yet its role in angiogenesis is poorly characterized. Here, we identified sulfur amino acid restriction as a proangiogenic trigger, promoting increased VEGF expression, migration and sprouting in ECs in vitro, and increased capillary density in mouse skeletal muscle in vivo via the GCN2/ATF4 amino acid starvation response pathway independent of hypoxia or HIF1α. We also identified a requirement for cystathionine-γ-lyase in VEGF-dependent angiogenesis via increased hydrogen sulfide (H2S) production. H2S mediated its proangiogenic effects in part by inhibiting mitochondrial electron transport and oxidative phosphorylation, resulting in increased glucose uptake and glycolytic ATP production.

Author Correction: Glycerol phosphate shuttle enzyme GPD2 regulates macrophage inflammatory responses.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Glycerol phosphate shuttle enzyme GPD2 regulates macrophage inflammatory responses.

Macrophages are activated during microbial infection to coordinate inflammatory responses and host defense. Here we find that in macrophages activated by bacterial lipopolysaccharide (LPS), mitochondrial glycerol 3-phosphate dehydrogenase (GPD2) regulates glucose oxidation to drive inflammatory responses. GPD2, a component of the glycerol phosphate shuttle, boosts glucose oxidation to fuel the production of acetyl coenzyme A, acetylation of histones and induction of genes encoding inflammatory mediators. While acute exposure to LPS drives macrophage activation, prolonged exposure to LPS triggers tolerance to LPS, where macrophages induce immunosuppression to limit the detrimental effects of sustained inflammation. The shift in the inflammatory response is modulated by GPD2, which coordinates a shutdown of oxidative metabolism; this limits the availability of acetyl coenzyme A for histone acetylation at genes encoding inflammatory mediators and thus contributes to the suppression of inflammatory responses. Therefore, GPD2 and the glycerol phosphate shuttle integrate the extent of microbial stimulation with glucose oxidation to balance the beneficial and detrimental effects of the inflammatory response.

Optimizing preclinical models of ageing for translation to clinical trials.

Preclinical models have been the backbone of translational research for more than a century. Rats and mice are critical models in the preliminary stages of drug testing, both for determining efficacy and ruling out potential human-relevant toxicities. Historically, most preclinical pharmacological studies have used young, relatively healthy, inbred male models in highly controlled environments. In the field of geriatric pharmacology, there is a growing focus on the importance of using more appropriate preclinical models both in the testing of therapeutics commonly used in older populations, and in the evaluation of potential geroprotective drug candidates. Here we provide a commentary on optimizing preclinical models of ageing for translation to clinical trials. We will discuss approaches to modelling clinically relevant contexts such as age, sex, genetic diversity, exposures and environment, as well as measures of clinically relevant outcomes such as frailty and healthspan. We will identify the strengths and limitations of these approaches and areas for improvement. We will also briefly cover new preclinical models that move beyond rodents. We hope this commentary will be a springboard for larger discussions on optimizing preclinical ageing models for testing therapeutics.

AI-Predicted mTOR Inhibitor Reduces Cancer Cell Proliferation and Extends the Lifespan of C. elegans.

The mechanistic target of rapamycin (mTOR) kinase is one of the top drug targets for promoting health and lifespan extension. Besides rapamycin, only a few other mTOR inhibitors have been developed and shown to be capable of slowing aging. We used machine learning to predict novel small molecules targeting mTOR. We selected one small molecule, TKA001, based on in silico predictions of a high on-target probability, low toxicity, favorable physicochemical properties, and preferable ADMET profile. We modeled TKA001 binding in silico by molecular docking and molecular dynamics. TKA001 potently inhibits both TOR complex 1 and 2 signaling in vitro. Furthermore, TKA001 inhibits human cancer cell proliferation in vitro and extends the lifespan of Caenorhabditis elegans, suggesting that TKA001 is able to slow aging in vivo.

Preoperative Protein or Methionine Restriction Preserves Wound Healing and Reduces Hyperglycemia.

BACKGROUND: Dietary restriction (DR), defined as reduced nutrient intake without malnutrition, is associated with longevity extension, improved glucose metabolism, and increased stress resistance, but also poor wound healing. Short-term preoperative DR followed by a return to normal feeding after surgery results in improved surgical outcomes in preclinical models. However, the effect of preoperative DR on wound healing and perioperative glucose homeostasis is currently unknown. Here, we tested the effects of two different preoperative DR regimens-protein restriction (PR) and methionine restriction (MR)-on wound healing and perioperative glucose homeostasis using an established murine model of wound healing in both nondiabetic and diabetic mice. MATERIALS AND METHODS: Surgical outcomes were tested using the McFarlane flap in nondiabetic and streptozotocin-induced diabetic mice. Short-term dietary preconditioning included 1 wk of PR or MR diet (1-2 wk) versus an isocaloric complete diet before surgery; all mice were returned to a complete diet postoperatively. Outcome measures of flap wound recovery included skin viability and laser Doppler imaging of flap perfusion and assessment of CD45+ cell infiltration. Glucose homeostasis was assessed by glucose tolerance testing and by perioperative glucose levels in the diabetic cohort. RESULTS: No significant differences were observed in percentage of viable skin, perfusion, or immune cell infiltration at 7-10 d after surgery in PR or MR mice compared with controls in healthy or diabetic mice. Preoperative glucose tolerance and postoperative glucose levels were however significantly improved by both PR and MR in diabetic mice. CONCLUSIONS: Short-term dietary preconditioning with PR or MR did not impair wound healing in nondiabetic or diabetic mice. However, both regimens reduced preoperative hyperglycemia in diabetic mice. Thus, brief preoperative dietary manipulations stand as strategies to potentially improve perioperative hyperglycemia with no deleterious effects on wound healing in mice.

The mitochondrial DNA common deletion as a potential biomarker of cancer-associated fibroblasts from skin basal and squamous cell carcinomas.

Cancer-associated fibroblasts (CAFs) are components of the tumor microenvironment and represent appealing therapeutic targets for translational studies. Conventional protein-based biomarkers for CAFs have been reported to be limited in their specificity, rendering difficult the identification of CAFs from normal fibroblasts (NFs) in clinical samples and dampening the development of CAF-targeted therapies to treat cancer. In this study, we propose the mitochondrial RNA and the mitochondrial DNA (mtDNA) common deletion (CD) as novel indicators of CAF identity. We found that cancer-activation correlated with decreased levels of the mtDNA CD, a condition not due to altered mitochondria count or cellular redox state, but potentially linked to the generalized overexpression of mtDNA maintenance genes in CAFs. Decreased mtDNA CD content in CAFs was associated with moderate to strong overexpression of mtDNA-encoded genes and to slightly improved mitochondrial function. We identified similar patterns of upregulation of mtDNA-encoded genes in independent single-cell RNA seq data obtained from squamous cell carcinoma (SCC) patients. By using the identified nucleic acids-based indicators, identification of CAFs from NFs could be improved, leading to potential therapeutic benefits in advancing translational and clinical studies.

Sulfur Amino Acid Restriction Enhances Exercise Capacity in Mice by Boosting Fat Oxidation in Muscle.

Dietary restriction of the sulfur-containing amino acids methionine and cysteine (SAAR) improves body composition, enhances insulin sensitivity, and extends lifespan; benefits seen also with endurance exercise. Yet, the impact of SAAR on skeletal muscle remains largely unexplored. Here we demonstrate that one week of SAAR in sedentary, young, male mice increases endurance exercise capacity. Indirect calorimetry showed that SAAR increased lipid oxidation at rest and delayed the onset of carbohydrate utilization during exercise. Transcriptomic analysis revealed increased expression of genes involved in fatty acid catabolism especially in glycolytic muscle following SAAR. These findings were functionally supported by increased fatty acid circulatory turnover flux and muscle ß-oxidation. Reducing lipid uptake from circulation through endothelial cell (EC)-specific CD36 deletion attenuated the running phenotype. Mechanistically, VEGF-signaling inhibition prevented exercise increases following SAAR, without affecting angiogenesis, implicating noncanonical VEGF signaling and EC CD36-dependent fatty acid transport in regulating exercise capacity by influencing muscle substrate availability.

Dietary isoleucine content defines the metabolic and molecular response to a Western diet.

The amino acid composition of the diet has recently emerged as a critical regulator of metabolic health. Consumption of the branched-chain amino acid isoleucine is positively correlated with body mass index in humans, and reducing dietary levels of isoleucine rapidly improves the metabolic health of diet-induced obese male C57BL/6J mice. However, it is unknown how sex, strain, and dietary isoleucine intake may interact to impact the response to a Western Diet (WD). Here, we find that although the magnitude of the effect varies by sex and strain, reducing dietary levels of isoleucine protects C57BL/6J and DBA/2J mice of both sexes from the deleterious metabolic effects of a WD, while increasing dietary levels of isoleucine impairs aspects of metabolic health. Despite broadly positive responses across all sexes and strains to reduced isoleucine, the molecular response of each sex and strain is highly distinctive. Using a multi-omics approach, we identify a core sex- and strain- independent molecular response to dietary isoleucine, and identify mega-clusters of differentially expressed hepatic genes, metabolites, and lipids associated with each phenotype. Intriguingly, the metabolic effects of reduced isoleucine in mice are not associated with FGF21 - and we find that in humans plasma FGF21 levels are likewise not associated with dietary levels of isoleucine. Finally, we find that foods contain a range of isoleucine levels, and that consumption of dietary isoleucine is lower in humans with healthy eating habits. Our results demonstrate that the dietary level of isoleucine is critical in the metabolic and molecular response to a WD, and suggest that lowering dietary levels of isoleucine may be an innovative and translatable strategy to protect from the negative metabolic consequences of a WD.

Short-term hypercaloric carbohydrate loading increases surgical stress resilience by inducing FGF21.

Dietary restriction promotes resistance to surgical stress in multiple organisms. Counterintuitively, current medical protocols recommend short-term carbohydrate-rich drinks (carbohydrate loading) prior to surgery, part of a multimodal perioperative care pathway designed to enhance surgical recovery. Despite widespread clinical use, preclinical and mechanistic studies on carbohydrate loading in surgical contexts are lacking. Here we demonstrate in ad libitum-fed mice that liquid carbohydrate loading for one week drives reductions in solid food intake, while nearly doubling total caloric intake. Similarly, in humans, simple carbohydrate intake is inversely correlated with dietary protein intake. Carbohydrate loading-induced protein dilution increases expression of hepatic fibroblast growth factor 21 (FGF21) independent of caloric intake, resulting in protection in two models of surgical stress: renal and hepatic ischemia-reperfusion injury. The protection is consistent across male, female, and aged mice. In vivo, amino acid add-back or genetic FGF21 deletion blocks carbohydrate loading-mediated protection from ischemia-reperfusion injury. Finally, carbohydrate loading induction of FGF21 is associated with the induction of the canonical integrated stress response (ATF3/4, NF-kB), and oxidative metabolism (PPARγ). Together, these data support carbohydrate loading drinks prior to surgery and reveal an essential role of protein dilution via FGF21.

TIME-seq reduces time and cost of DNA methylation measurement for epigenetic clock construction.

Epigenetic 'clocks' based on DNA methylation have emerged as the most robust and widely used aging biomarkers, but conventional methods for applying them are expensive and laborious. Here we develop tagmentation-based indexing for methylation sequencing (TIME-seq), a highly multiplexed and scalable method for low-cost epigenetic clocks. Using TIME-seq, we applied multi-tissue and tissue-specific epigenetic clocks in over 1,800 mouse DNA samples from eight tissue and cell types. We show that TIME-seq clocks are accurate and robust, enriched for polycomb repressive complex 2-regulated loci, and benchmark favorably against conventional methods despite being up to 100-fold less expensive. Using dietary treatments and gene therapy, we find that TIME-seq clocks reflect diverse interventions in multiple tissues. Finally, we develop an economical human blood clock (R > 0.96, median error = 3.39 years) in 1,056 demographically representative individuals. These methods will enable more efficient epigenetic clock measurement in larger-scale human and animal studies.

Improved resilience and proteostasis mediate longevity upon DAF-2 degradation in old age.

Little is known about the possibility of reversing age-related biological changes when they have already occurred. To explore this, we have characterized the effects of reducing insulin/IGF-1 signaling (IIS) during old age. Reduction of IIS throughout life slows age-related decline in diverse species, most strikingly in the nematode Caenorhabditis elegans. Here we show that even at advanced ages, auxin-induced degradation of DAF-2 in single tissues, including neurons and the intestine, is still able to markedly increase C. elegans lifespan. We describe how reversibility varies among senescent changes. While senescent pathologies that develop in mid-life were not reversed, there was a rejuvenation of the proteostasis network, manifesting as a restoration of the capacity to eliminate otherwise intractable protein aggregates that accumulate with age. Moreover, resistance to several stressors was restored. These results support several new conclusions. (1) Loss of resilience is not solely a consequence of pathologies that develop in earlier life. (2) Restoration of proteostasis and resilience by inhibiting IIS is a plausible cause of the increase in lifespan. And (3), most interestingly, some aspects of the age-related transition from resilience to frailty can be reversed to a certain extent. This raises the possibility that the effect of IIS and related pathways on resilience and frailty during aging in higher animals might possess some degree of reversibility.

Sex differences in pharmacological interventions and their effects on lifespan and healthspan outcomes: a systematic review.

With an increasing aging population, the burden of age-related diseases magnifies. To alleviate this burden, geroprotection has been an area of intense research focus with the development of pharmacological interventions that target lifespan and/or healthspan. However, there are often sex differences, with compounds mostly tested in male animals. Given the importance of considering both sexes in preclinical research, this neglects potential benefits for the female population, as interventions tested in both sexes often show clear sexual dimorphisms in their biological responses. To further understand the prevalence of sex differences in pharmacological geroprotective intervention studies, we performed a systematic review of the literature according to the PRISMA guidelines. Seventy-two studies met our inclusion criteria and were classified into one of five subclasses: FDA-repurposed drugs, novel small molecules, probiotics, traditional Chinese medicine, and antioxidants, vitamins, or other dietary supplements. Interventions were analyzed for their effects on median and maximal lifespan and healthspan markers, including frailty, muscle function and coordination, cognitive function and learning, metabolism, and cancer. With our systematic review, we found that twenty-two out of sixty-four compounds tested were able to prolong both lifespan and healthspan measures. Focusing on the use of female and male mice, and on comparing their outcomes, we found that 40% of studies only used male mice or did not clarify the sex. Notably, of the 36% of pharmacologic interventions that did use both male and female mice, 73% of these studies showed sex-specific outcomes on healthspan and/or lifespan. These data highlight the importance of studying both sexes in the search for geroprotectors, as the biology of aging is not the same in male and female mice. Systematic Review Registration: [website], identifier [registration number].

Cystathionine-γ-lyase overexpression modulates oxidized nicotinamide adenine dinucleotide biosynthesis and enhances neovascularization.

Objective: Hydrogen sulfide is a proangiogenic gas produced primarily by the transsulfuration enzyme cystathionine-γ-lyase (CGL). CGL-dependent hydrogen sulfide production is required for neovascularization in models of peripheral arterial disease. However, the benefits of increasing endogenous CGL and its mechanism of action have not yet been elucidated. Methods: Male whole body CGL-overexpressing transgenic (CGLTg) mice and wild-type (WT) littermates (C57BL/6J) were subjected to the hindlimb ischemia model (age, 10-12 weeks). Functional recovery was assessed via the treadmill exercise endurance test. Leg perfusion was measured by laser Doppler imaging and vascular endothelial-cadherin immunostaining. To examine the angiogenic potential, aortic ring sprouting assay and postnatal mouse retinal vasculature development studies were performed. Finally, comparative metabolomics analysis, oxidized/reduced nicotinamide adenine dinucleotide (NAD+/NADH) analysis, and quantitative real-time polymerase chain reaction were performed on CGLWT and CGLTg gastrocnemius muscle. Results: The restoration of blood flow occurred more rapidly in CGLTg mice. Compared with the CGLWT mice, the median ± standard deviation running distance and time were increased for the CGLTg mice after femoral artery ligation (159 ± 53 m vs 291 ± 74 m [P < .005] and 17 ± 4 minutes vs 27 ± 5 minutes [P < .05], respectively). Consistently, in the CGLTg ischemic gastrocnemius muscle, the capillary density was increased fourfold (0.05 ± 0.02 vs 0.20 ± 0.12; P < .005). Ex vivo, the endothelial cell (EC) sprouting length was increased in aorta isolated from CGLTg mice, especially when cultured in VEGFA (vascular endothelial growth factor A)-only media (63 ± 2 pixels vs 146 ± 52 pixels; P < .05). Metabolomics analysis demonstrated a higher level of niacinamide, a precursor of NAD+/NADH in the muscle of CGLTg mice (61.4 × 106 ± 5.9 × 106 vs 72.4 ± 7.7 × 106 area under the curve; P < .05). Similarly, the NAD+ salvage pathway gene expression was increased in CGLTg gastrocnemius muscle. Finally, CGL overexpression or supplementation with the NAD+ precursor nicotinamide mononucleotide improved EC migration in vitro (wound closure: control, 35% ± 9%; CGL, 55% ± 11%; nicotinamide mononucleotide, 42% ± 13%; P < .05). Conclusions: Our results have demonstrated that CGL overexpression improves the neovascularization of skeletal muscle on hindlimb ischemia. These effects correlated with changes in the NAD pathway, which improved EC migration.

Short-term Pre-operative Methionine Restriction Induces Browning of Perivascular Adipose Tissue and Improves Vein Graft Remodeling in Mice.

Short-term preoperative methionine restriction (MetR) shows promise as a translatable strategy to modulate the body's response to surgical injury. Its application, however, to improve post-interventional vascular remodeling remains underexplored. Here, we find that MetR protects from arterial intimal hyperplasia in a focal stenosis model and adverse vascular remodeling after vein graft surgery. RNA sequencing reveals that MetR enhances the brown adipose tissue phenotype in arterial perivascular adipose tissue (PVAT) and induces it in venous PVAT. Specifically, PPAR-α was highly upregulated in PVAT-adipocytes. Furthermore, MetR dampens the post-operative pro-inflammatory response to surgery in PVAT-macrophages in vivo and in vitro . This study shows for the first time that the detrimental effects of dysfunctional PVAT on vascular remodeling can be reversed by MetR, and identifies pathways involved in browning of PVAT. Furthermore, we demonstrate the potential of short-term pre-operative MetR as a simple intervention to ameliorate vascular remodeling after vascular surgery.

Short-Term Ketogenic Diet Induces a Molecular Response That Is Distinct From Dietary Protein Restriction.

There is increasing interest in utilizing short-term dietary interventions in the contexts of cancer, surgical stress and metabolic disease. These short-term diets may be more feasible than extended interventions and may be designed to complement existing therapies. In particular, the high-fat, low-carbohydrate ketogenic diet (KD), traditionally used to treat epilepsy, has gained popularity as a potential strategy for weight loss and improved metabolic health. In mice, long-term KD improves insulin sensitivity and may extend lifespan and healthspan. Dietary protein restriction (PR) causes increased energy expenditure, weight loss and improved glucose homeostasis. Since KD is inherently a low-protein diet (10% of calories from protein vs. >18% in control diet), here we evaluated the potential for mechanistic overlap between PR and KD via activation of a PR response. Mice were fed control, protein-free (PF), or one of four ketogenic diets with varying protein content for 8 days. PF and KD both decreased body weight, fat mass, and liver weights, and reduced fasting glucose and insulin levels, compared to mice fed the control diet. However, PF-fed animals had significantly improved insulin tolerance compared to KD. Furthermore, contrary to the PF-fed mice, mice fed ketogenic diets containing more than 5% of energy from protein did not increase hepatic Fgf21 or brown adipose Ucp1 expression. Interestingly, mice fed KD lacking protein demonstrated greater elevations in hepatic Fgf21 than mice fed a low-fat PF diet. To further elucidate potential mechanistic differences between PF and KD and the interplay between dietary protein and carbohydrate restriction, we conducted RNA-seq analysis on livers from mice fed each of the six diets and identified distinct gene sets which respond to dietary protein content, dietary fat content, and ketogenesis. We conclude that KD with 10% of energy from protein does not induce a protein restriction response, and that the overlapping metabolic benefits of KD and PF diets may occur via distinct underlying mechanisms.

Sodium thiosulfate acts as a hydrogen sulfide mimetic to prevent intimal hyperplasia via inhibition of tubulin polymerisation.

BACKGROUND: Intimal hyperplasia (IH) remains a major limitation in the long-term success of any type of revascularisation. IH is due to vascular smooth muscle cell (VSMC) dedifferentiation, proliferation and migration. The gasotransmitter Hydrogen Sulfide (H2S), mainly produced in blood vessels by the enzyme cystathionine- γ-lyase (CSE), inhibits IH in pre-clinical models. However, there is currently no H2S donor available to treat patients. Here we used sodium thiosulfate (STS), a clinically-approved source of sulfur, to limit IH. METHODS: Low density lipoprotein receptor deleted (LDLR-/-), WT or Cse-deleted (Cse-/-) male mice randomly treated with 4 g/L STS in the water bottle were submitted to focal carotid artery stenosis to induce IH. Human vein segments were maintained in culture for 7 days to induce IH. Further in vitro studies were conducted in primary human vascular smooth muscle cells (VSMCs). FINDINGS: STS inhibited IH in WT mice, as well as in LDLR-/- and Cse-/- mice, and in human vein segments. STS inhibited cell proliferation in the carotid artery wall and in human vein segments. STS increased polysulfides in vivo and protein persulfidation in vitro, which correlated with microtubule depolymerisation, cell cycle arrest and reduced VSMC migration and proliferation. INTERPRETATION: STS, a drug used for the treatment of cyanide poisoning and calciphylaxis, protects against IH in a mouse model of arterial restenosis and in human vein segments. STS acts as an H2S donor to limit VSMC migration and proliferation via microtubule depolymerisation. FUNDING: This work was supported by the Swiss National Science Foundation (grant FN-310030_176158 to FA and SD and PZ00P3-185927 to AL); the Novartis Foundation to FA; and the Union des Sociétés Suisses des Maladies Vasculaires to SD, and the Fondation pour la recherche en chirurgie vasculaire et thoracique.

The non-essential TSC complex component TBC1D7 restricts tissue mTORC1 signaling and brain and neuron growth.

The tuberous sclerosis complex (TSC) 1 and 2 proteins associate with TBC1D7 to form the TSC complex, which is an essential suppressor of mTOR complex 1 (mTORC1), a ubiquitous driver of cell and tissue growth. Loss-of-function mutations in TSC1 or TSC2, but not TBC1D7, give rise to TSC, a pleiotropic disorder with aberrant activation of mTORC1 in various tissues. Here, we characterize mice with genetic deletion of Tbc1d7, which are viable with normal growth and development. Consistent with partial loss of function of the TSC complex, Tbc1d7 knockout (KO) mice display variable increases in tissue mTORC1 signaling with increased muscle fiber size but with strength and motor defects. Their most pronounced phenotype is brain overgrowth due to thickening of the cerebral cortex, with enhanced neuron-intrinsic mTORC1 signaling and growth. Thus, TBC1D7 is required for full TSC complex function in tissues, and the brain is particularly sensitive to its growth-suppressing activities.

Multiomics assessment of dietary protein titration reveals altered hepatic glucose utilization.

Dietary protein restriction (PR) has rapid effects on metabolism including improved glucose and lipid homeostasis, via multiple mechanisms. Here, we investigate responses of fecal microbiome, hepatic transcriptome, and hepatic metabolome to six diets with protein from 18% to 0% of energy in mice. PR alters fecal microbial composition, but metabolic effects are not transferable via fecal transplantation. Hepatic transcriptome and metabolome are significantly altered in diets with lower than 10% energy from protein. Changes upon PR correlate with calorie restriction but with a larger magnitude and specific changes in amino acid (AA) metabolism. PR increases steady-state aspartate, serine, and glutamate and decreases glucose and gluconeogenic intermediates. 13C6 glucose and glycerol tracing reveal increased fractional enrichment in aspartate, serine, and glutamate. Changes remain intact in hepatic ATF4 knockout mice. Together, this demonstrates an ATF4-independent shift in gluconeogenic substrate utilization toward specific AAs, with compensation from glycerol to promote a protein-sparing response.

Intracellular H2S production is an autophagy-dependent adaptive response to DNA damage.

Hydrogen sulfide (H2S) is a gasotransmitter with broad physiological activities, including protecting cells against stress, but little is known about the regulation of cellular H2S homeostasis. We have performed a high-content small-molecule screen and identified genotoxic agents, including cancer chemotherapy drugs, as activators of intracellular H2S levels. DNA damage-induced H2S in vitro and in vivo. Mechanistically, DNA damage elevated autophagy and upregulated H2S-generating enzyme CGL; chemical or genetic disruption of autophagy or CGL impaired H2S induction. Importantly, exogenous H2S partially rescued autophagy-deficient cells from genotoxic stress. Furthermore, stressors that are not primarily genotoxic (growth factor depletion and mitochondrial uncoupler FCCP) increased intracellular H2S in an autophagy-dependent manner. Our findings highlight the role of autophagy in H2S production and suggest that H2S generation may be a common adaptive response to DNA damage and other stressors.