Mitochondria play a crucial role in cellular energy metabolism and homeostasis and are strongly implicated in aging and age-related diseases. The outer mitochondrial membrane protein voltage-dependent anion channel (VDAC) plays multiple roles in mitochondrial homeostasis, including transport of metabolites, ATP, and Ca2+ Dysregulation of VDAC levels has been associated with cancer, neurodegeneration, metabolic disorders, and aging. Previously, we demonstrated that elevated VDAC-1 levels in Caenorhabditis elegans lead to increased mitochondrial permeability and reduced life span. Here we demonstrate that reduced VDAC-1 function extends life span through the activation of the mitochondrial unfolded protein response (UPRmt), a conserved stress response that maintains mitochondrial proteostasis and is linked to life span extension in multiple species. Leveraging unbiased genomic discovery, we identified genes encoding several proteins in the PeBoW complex as a critical mediator of UPRmt activation following VDAC-1 loss. More broadly, we demonstrated a universal requirement for several PeBoW component genes across diverse mitochondrial stressors in order to fully animate the UPRmt Our findings reveal a heretofore unappreciated role for PeBoW components in UPRmt induction and life span extension in response to mitochondrial stress, highlighting its essential function in mitochondrial quality control and longevity pathways.

Exposure to low levels of environmental challenges, known as hormetic stress, fosters subsequent stress resistance and promotes healthy aging in later life. However, specific mechanisms governing transcriptional reprogramming upon hormetic nutrient stress remain elusive. Here, we identify histone H3 lysine 27 acetylation (H3K27ac) as a crucial driver of transcriptomic adaptation to hormetic fasting. Beyond its immediate function of enhancing lipid catabolism for alternative energy sources, stress-induced H3K27ac activates lifelong antioxidant defenses, thereby reducing reactive oxygen species (ROS) produced by stress-induced fatty acid oxidation and their accumulation during aging. Induced H3K27ac at metabolic genes, mediated by the pioneer factor PHA-4/FOXA, the cooperating transcription factor NHR-49/HNF4, and the nucleoporin 50 (NPP-16/NUP50), is crucial for lifespan extension under hormetic nutrient stress in Caenorhabditis elegans. Our findings establish H3K27ac as a key transcriptional switch bridging nutrient status with transcriptomic reprogramming, underpinning the longevity of hormetic fasting through orchestrating lipid catabolism and antioxidant defenses.

The nuclear pore complex (NPC) is the single gateway between the nucleus and the cytoplasm, and in healthy cells there is a size threshold for passive diffusion across the NPC. In aging and disease, the NPC deteriorates, leading to promiscuous passive transport. We have previously showed that NPC protein expression is required for biguanide-induced lifespan extension, mTOR inhibition, and further that biguanide treatment leads to restriction of passive nuclear transport, but the underlying changes leading to this restriction were not identified. Here, we use fluorescent dextran transport and biochemical assays in HeLa cells to clarify the mechanism by which biguanide phenformin alters NPC permeability. We find phenformin treatment in HeLa cells leads to restricted passive nuclear transport in a dose and time-dependent manner. Multiple inhibitors of the mitochondrial electron transport chain also restrict passive nucleocytoplasmic transport. Critically, phenformin reduced expression of O-linked N-acetylglucosamine (O-GlcNAc) transferase, lowering global O-GlcNAcylation and locally decreasing O-GlcNAcylation of Nup98. O-GlcNAc transferase inhibition alone restricts passive transport, while increasing O-GlcNAcylation reverses phenformin's effects. These results identify O-GlcNAc as a mitochondrial-nuclear signal and show that electron transport chain inhibition rapidly modulates nucleocytoplasmic transport via NPC post-translational modification in human cancer cells.

Organisms utilize sophisticated neurocircuitry to select optimal food sources. Methylobacterium is a lifespan-promoting diet for C. elegans that drives faster development and longevity; however, after ingestion, C. elegans consistently choose any other food option available. A screen for genetic regulators of this avoidance behavior toward Methylobacterium identified the AWB/AWC sensory neurons and the odr-1 guanylate cyclase expressed in those four ciliated neurons as mediators of the aversive response. Metabolic profiling of the Methylobacterium diet reveals enrichment for saturated fats, and here, we show that C. elegans sense and integrate signals related to these ingested lipids that subsequently cue food-related behaviors. Moreover, disruption of endogenous lipid metabolism modifies the intensity of the avoidance from Methylobacterium, suggesting the current state of lipid homeostasis influences food preference. Taken together, our work reveals that C. elegans modify food choice contemporaneously based in part on the chemosensory capacity to detect and utilize dietary lipids, which has longevity-promoting consequences.

Biguanides, including metformin, the world's most prescribed oral hypoglycemic, extend health span and lifespan in vertebrates and invertebrates. Given the widespread use and apparent safety of metformin, it is assumed that its effects are not associated with toxicity, except when in marked excess. Here, we determine that accumulation of damaging reducing equivalents is an unanticipated toxicity associated with biguanides, defense against which requires post-transcriptional protection of de novo lipogenesis. We demonstrate that biguanide treatment during impaired lipogenesis drives NADPH toxicity, leading to catastrophic elevation of NADH/GSH reducing equivalents and accelerated death across metazoans. Multiple NADPH-generating interventions require de novo lipogenesis to prevent markedly shortened survival, indicating that this defense mechanism is broadly leveraged. We propose that fatty acid biosynthesis is a tunable rheostat that can minimize biguanide-induced reductive stress while maximizing its pro-longevity outcomes and can serve as an exploitable vulnerability in reductive stress-sensitive cancers.

Exposure to low levels of environmental challenges, known as hormetic stress, such as nutrient deprivation and heat shock, fosters subsequent stress resistance and promotes healthy aging in later life. However, specific mechanisms governing transcriptional reprogramming upon hormetic nutrient stress remain elusive. In this study, we identified histone H3 lysine 27 acetylation (H3K27ac) as a crucial driver of transcriptomic adaptation to hormetic fasting. Beyond its immediate function of enhancing lipid catabolism for alternative energy sources, stress-induced H3K27ac activates lifelong antioxidant defenses, thereby reducing reactive oxygen species (ROS) produced by stress-induced fatty acid oxidation and their accumulation during aging. The increase in H3K27ac, mediated by pioneer factor PHA-4/FOXA and cooperating transcription factor NHR-49/HNF4, is crucial for lifespan extension under hermetic nutrient stress in Caenorhabditis elegans. Our findings establish H3K27ac as a key transcriptional switch that bridges nutrient status with transcriptomic reprogramming, underpinning the pro-longevity effects of hormetic fasting through orchestrating lipid catabolism and antioxidative defenses.

As the only gateway governing nucleocytoplasmic transport, the nuclear pore complex (NPC) maintains fundamental cellular processes and deteriorates with age. However, the study of age-related roles of single NPC components remains challenging owing to the complexity of NPC composition. Here, we demonstrate that the central energy sensor, AMP-activated protein kinase (AMPK), post-translationally regulates the abundance of the nucleoporin NPP-16/NUP50 in response to nutrient availability and energetic stress. In turn, NPP-16/NUP50 promotes transcriptional activation of lipid catabolism to extend the lifespan of Caenorhabditis elegans independently of its role in nuclear transport. Rather, the intrinsically disordered region (IDR) of NPP-16/NUP50, through direct interaction with the transcriptional machinery, transactivates the promoters of catabolic genes. Remarkably, elevated NPP-16/NUP50 levels are sufficient to promote longevity and metabolic stress defenses. AMPK-NUP50 signaling is conserved in humans, indicating that bridging energy sensing to metabolic adaptation is an ancient role of this signaling axis.

Understanding the complex factors influencing mammalian metabolism and body weight homeostasis is a long-standing challenge requiring knowledge of energy intake, absorption and expenditure. Using measurements of respiratory gas exchange, indirect calorimetry can provide non-invasive estimates of whole-body energy expenditure. However, inconsistent measurement units and flawed data normalization methods have slowed progress in this field. This guide aims to establish consensus standards to unify indirect calorimetry experiments and their analysis for more consistent, meaningful and reproducible results. By establishing community-driven standards, we hope to facilitate data comparison across research datasets. This advance will allow the creation of an in-depth, machine-readable data repository built on shared standards. This overdue initiative stands to markedly improve the accuracy and depth of efforts to interrogate mammalian metabolism. Data sharing according to established best practices will also accelerate the translation of basic findings into clinical applications for metabolic diseases afflicting global populations. The authors highlight inconsistencies and divergencies in the literature reporting data on indirect calorimetry for studies on whole-body energy homeostasis, and propose harmonization of standards to facilitate data comparison and interpretation across different datasets.

Despite being principally prescribed to treat type 2 diabetes, biguanides, especially metformin and phenformin, have been shown to extend lifespan and healthspan in preclinical models. Although there have been conflicting results in studies involving rodents and humans, consistent evidence indicates metformin and phenformin's ability to significantly extend lifespan in Caenorhabditis elegans. We find that variation in agar from lot-to-lot or from different manufacturers influences metformin's ability to extend lifespan in diverse Caenorhabditis species. Using unbiased metabolomics and genetics, we traced the ability of certain agars to interfere with metformin-prompted lifespan extension to differences in glucose, dipeptide, and trace element levels. These compounds act directly in the worm, independently of the bacterial food source, preventing longevity through action downstream of longevity effectors skn-1 and AMPK. In contrast, phenformin prompts robust lifespan extension in the face of environmental changes and exhibits broad positive effects in aging across genetically diverse Caenorhabditis species.

Biguanides, including metformin, the world's most prescribed oral hypoglycemic, extend health-span and lifespan in vertebrates and invertebrates. Given the widespread use and apparent safety of metformin, it is assumed that its effects are not associated with toxicity, except when in marked excess. Here we determine that accumulation of damaging reducing equivalents is an unanticipated toxicity associated with biguanides, the defense against which requires post-transcriptional protection of de novo fatty acid biosynthesis. We demonstrate that biguanide treatment during impaired fatty acid biosynthesis drives NADPH toxicity, leading to catastrophic elevation of NADH/GSH reducing equivalents and accelerated death across metazoans. Multiple NADPH-generating interventions require fatty acid biosynthesis to prevent markedly shortened survival, indicating that this defense mechanism is broadly leveraged. We propose that fatty acid biosynthesis is a tunable rheostat which can minimize biguanide-induced reductive stress whilst maximizing its pro-longevity outcomes and serve as an exploitable vulnerability in reductive stress sensitive cancers. HIGHLIGHTS:Biguanides inhibit cytosolic mRNA translation to extend lifespan in C. elegans . Fatty acid synthesis is translationally protected by eIF3 complex subunits. pod-2 / fasn-1 inactivation amplifies biguanide-induced reductive stress and death. NADPH-generating insults require fatty acid synthesis to buffer reductive stress.

Elevated glucocorticoid levels are common in conditions such as aging, chronic stress, Cushing syndrome, and glucocorticoid therapy. While glucocorticoids suppress inflammation through the glucocorticoid receptor (GR), they also cause metabolic side effects. Investigating alternative pathways beyond GR activation is crucial for reducing these side effects. Our phosphoproteomics analysis revealed that glucocorticoid exposure promotes phosphorylation at the RxxS motifs of multiple proteins in preadipocytes, including those mediated by serum- and glucocorticoid-induced kinase 3 (SGK3). SGK3 is a key mediator of glucocorticoid-induced adipogenesis, as shown by impaired adipogenesis after SGK3 inhibition or genetic ablation. Sgk3-KO mice were resistant to obesity induced by glucocorticoid or a high-fat diet, and proteolysis targeting chimeras (PROTAC) targeting SGK3 reduced adipogenesis in both obese mice and in a thyroid eye disease cell line. Mechanistically, SGK3 translocated to the nucleus upon glucocorticoid stimulation, interacted with and phosphorylated the BRG1 subunit of the BAF complex, and prevented BRG1 degradation, promoting chromatin remodeling necessary for adipogenesis. These findings highlight SGK3 as a potential therapeutic target to mitigate metabolic side effects of elevated glucocorticoid levels.

Organisms utilize sophisticated neurocircuitry to select optimal food sources within their environment. Methylobacterium is a lifespan-promoting bacterial diet for C. elegans that drives faster development and longevity, however after ingestion, C. elegans consistently choose any other food option available. A screen for genetic regulators of the avoidance behavior toward Methylobacterium identified the AWB and AWC sensory neurons and the odr-1 guanylate cyclase expressed exclusively in those four ciliated neurons as mediators of the antipathy response. Metabolic profiling of the Methylobacterium diet reveals a macromolecular profile enriched in saturated fats and here we show that C. elegans sense and integrate signals related to the type of ingested lipids that subsequently cues food-related behaviors. Moreover, disruption of endogenous lipid metabolism modifies the intensity of antipathy toward Methylobacterium which suggests that the current state of lipid homeostasis influences food preference. Enhanced expression of the sphingolipid degradation enzyme Saposin/ spp-9 enhances antipathy behaviors and activation of the sphingosine rheostat and more specifically modulation of the bioactive lipid mediator sphingosine-1-phosphate (S1P) acts as a signal to promote avoidance of Methylobacterium . Taken together, our work reveals that C. elegans modify food choices contemporaneously based on the availability of dietary lipids and the ability to metabolize dietary lipids.HIGHLIGHTS ### Competing Interest StatementThe authors have declared no competing interest.

Metformin, an oral antihyperglycemic drug that has been in use for over 60 years, remains a first-line therapy for type 2 diabetes (T2D). Numerous studies have suggested that metformin promotes health benefits beyond T2D management, including weight loss, cancer prevention and treatment, and anti-aging, through several proposed mechanistic targets. Here we discuss the established effects of metformin and the progress made in identifying its direct targets. Additionally, we emphasize the importance of elucidating the structural bases of the drug and its direct targets. Ultimately, this review aims to highlight the current state of knowledge regarding metformin and its related emerging discoveries, while also outlining critical future research directions.

Metabolic dysfunction-associated steatotic liver disease (MASLD, formerly known as nonalcoholic fatty liver disease (NAFLD)) and metabolic dysfunction-associated steatohepatitis (MASH, formerly known as nonalcoholic steatohepatitis (NASH)) are leading chronic liver diseases, driving cirrhosis, hepatocellular carcinoma, and mortality. MASLD/MASH is associated with increased senescence proteins, including Activin A, and senolytics have been proposed as a therapeutic approach. To test the role of Activin A, we induced hepatic expression of Activin A in a murine MASLD/MASH model. Surprisingly, overexpression of hepatic Activin A dramatically mitigated MASLD, reducing liver steatosis and inflammation as well as systemic fat accumulation, while improving insulin sensitivity. Further studies identified a dramatic decrease in the lipid-associated macrophages (LAM) marker glycoprotein NMB (Gpnmb) by Activin A and Gpnmb knockdown in the same model produced similar benefits and transcriptional changes to Activin A expression. These studies reveal a surprising protective role for Activin A in MASLD and the potential for SASP proteins to have context-specific beneficial effects. Moreover, they implicate both Activin A and Gpnmb as potential therapeutic targets for this condition.

AbstractDespite being principally prescribed to treat type 2 diabetes, biguanides, especially metformin and phenformin, have been shown to extend lifespan and healthspan in preclinical models, and to reduce the impact of aging-associated diseases such as cancer. While there have been conflicting results in studies involving rodents and humans, consistent evidence from laboratories worldwide, including our own, indicates metformin and phenformin’s ability to significantly extend lifespan inC. elegans. However, the pro-longevity effect of metformin can vary depending on environmental conditions. Specifically, the choice of agar from different manufacturers or batches influences metformin’s ability to extend lifespan inC. elegans. We traced ability of certain agar batches to interfere with metformin-prompted lifespan extension to the presence of a factor that acts directly in the worm, independently of the bacterial food source, that prevents longevity promoting effects downstream of longevity effectorsskn-1and AMPK. In contrast, phenformin prompts robust lifespan extension in the face of environmental changes and exhibits broad positive effects in aging across genetically diverseCaenorhabditisspecies where the impact of metformin is highly variable. Thus metformin effects in aging are impacted by heretofore unappreciated environmental factors. Phenformin may represent a more robust agent with which to understand the longevity promoting mechanisms downstream of biguanides.

Biguanides, including the world's most prescribed drug for type 2 diabetes, metformin, not only lower blood sugar, but also promote longevity in preclinical models. Epidemiologic studies in humans parallel these findings, indicating favorable effects of metformin on longevity and on reducing the incidence and morbidity associated with aging-related diseases. Despite this promise, the full spectrum of molecular effectors responsible for these health benefits remains elusive. Through unbiased screening in Caenorhabditis elegans, we uncovered a role for genes necessary for ether lipid biosynthesis in the favorable effects of biguanides. We demonstrate that biguanides prompt lifespan extension by stimulating ether lipid biogenesis. Loss of the ether lipid biosynthetic machinery also mitigates lifespan extension attributable to dietary restriction, target of rapamycin (TOR) inhibition, and mitochondrial electron transport chain inhibition. A possible mechanistic explanation for this finding is that ether lipids are required for activation of longevity-promoting, metabolic stress defenses downstream of the conserved transcription factor skn-1/Nrf. In alignment with these findings, overexpression of a single, key, ether lipid biosynthetic enzyme, fard-1/FAR1, is sufficient to promote lifespan extension. These findings illuminate the ether lipid biosynthetic machinery as a novel therapeutic target to promote healthy aging.

ABSTRACT Both hedgehog (Hh) and target of rapamycin complex 2 (TORC2) are central, evolutionarily conserved signaling pathways that regulate development and metabolism. In C. elegans, loss of the essential TORC2 component RICTOR (rict-1) causes delayed development, shortened lifespan, reduced brood, small size and increased fat. Here, we report that knockdown of both the hedgehog-related morphogen grd-1 and its patched-related receptor ptr-11 rescues delayed development in TORC2 loss-of-function mutants, and grd-1 and ptr-11 overexpression delays wild-type development to a similar level to that in TORC2 loss-of-function animals. These findings potentially indicate an unexpected role for grd-1 and ptr-11 in slowing developmental rate downstream of a nutrient-sensing pathway. Furthermore, we implicate the chronic stress transcription factor pqm-1 as a key transcriptional effector in this slowing of whole-organism growth by grd-1 and ptr-11. We propose that TORC2, grd-1 and ptr-11 may act linearly or converge on pqm-1 to delay organismal development.

The mitochondrial permeability transition (mPT) describes a Ca 2+ -dependent and cyclophilin D (CypD)-facilitated increase of inner mitochondrial membrane permeability that allows diffusion of molecules up to 1.5 kDa in size. It is mediated by a non-selective channel, the mitochondrial permeability transition pore (mPTP). Sustained mPTP opening causes mitochondrial swelling, which ruptures the outer mitochondrial membrane leading to subsequent apoptotic and necrotic cell death, and is implicated in a range of pathologies. However, transient mPTP opening at various sub-conductance states may contribute several physiological roles such as alterations in mitochondrial bioenergetics and rapid Ca 2+ efflux. Since its discovery decades ago, intensive efforts have been made to identify the exact pore-forming structure of the mPT. Both the adenine nucleotide translocase (ANT) and, more recently, the mitochondrial F 1 F O (F)-ATP synthase dimers, monomers or c-subunit ring alone have been implicated. Here we share the insights of several key investigators with different perspectives who have pioneered mPT research. We critically assess proposed models for the molecular identity of the mPTP and the mechanisms underlying its opposing roles in the life and death of cells. We provide in-depth insights into current controversies, seeking to achieve a degree of consensus that will stimulate future innovative research into the nature and role of the mPTP.