Heat Shock Factor Research Articles

Clerodendranthus spicatus (Thunb.) Water Extracts Reduce Lipid Accumulation and Oxidative Stress in the Caenorhabditis elegans.

Clerodendranthus spicatus (Thunb.) (Kidney tea) is a very distinctive ethnic herbal medicine in China. Its leaves are widely used as a healthy tea. Many previous studies have demonstrated its various longevity-promoting effects; however, the safety and specific health-promoting effects of Clerodendranthus spicatus (C. spicatus) as a dietary supplement remain unclear. In order to understand the effect of C. spicatus on the longevity of Caenorhabditis elegans (C. elegans), we evaluated its role in C. elegans; C. spicatus water extracts (CSw) were analyzed for the major components and the effects on C. elegans were investigated from physiological and biochemical to molecular levels; CSw contain significant phenolic components (primarily rosmarinic acid and eugenolinic acid) and flavonoids (primarily quercetin and isorhamnetin) and can increase the lifespan of C. elegans. Further investigations showed that CSw modulate stress resistance and lipid metabolism through influencing DAF-16/FoxO (DAF-16), Heat shock factor 1 (HSF-1), and Nuclear Hormone Receptor-49 (NHR-49) signalling pathways; CSw can improve the antioxidant and hypolipidemic activity of C. elegans and prolong the lifespan of C. elegans (with the best effect at low concentrations). Therefore, the recommended daily use of C. spicatus should be considered when consuming it as a healthy tea on a daily basis.

The stress response regulator HSF1 modulates natural killer cell anti-tumour immunity.

Diverse cellular insults converge on activation of the heat shock factor 1 (HSF1), which regulates the proteotoxic stress response to maintain protein homoeostasis. HSF1 regulates numerous gene programmes beyond the proteotoxic stress response in a cell-type- and context-specific manner to promote malignancy. However, the role(s) of HSF1 in immune populations of the tumour microenvironment remain elusive. Here, we leverage an in vivo model of HSF1 activation and single-cell transcriptomic tumour profiling to show that augmented HSF1 activity in natural killer (NK) cells impairs cytotoxicity, cytokine production and subsequent anti-tumour immunity. Mechanistically, HSF1 directly binds and regulates the expression of key mediators of NK cell effector function. This work demonstrates that HSF1 regulates the immune response under the stress conditions of the tumour microenvironment. These findings have important implications for enhancing the efficacy of adoptive NK cell therapies and for designing combinatorial strategies including modulators of NK cell-mediated tumour killing.

Integrated transcriptomic and proteomic analysis unravels the response mechanisms of Saccharina japonica to heat stress

High temperature is a major abiotic stress that limits the growth and productivity of seaweed. Over the past decades, the cold-temperate seaweed Saccharina japonica has been domesticated and gradually adapted to subtropical regions with higher temperatures. However, there is limited knowledge regarding the molecular responses of S. japonica to heat stress. In this study, an integrated transcriptomic and proteomic analysis was performed to explore the molecular mechanism underlying the response to heat stress in S. japonica. A total of 6777 genes and 226 proteins were identified that exhibited differential expression in heat stress samples comparison to non-stress sample. GO analysis revealed that the GO molecular function (MF) terms related to unfolded protein binding/protein binding, and heat shock protein (HSP) binding/HSP90 binding were significantly enriched in the up-regulated DEGs/DEPs in S. japonica sporophytes under heat stress. Further analysis of the global gene and protein expression profiles unveiled the potential mechanisms in S. japonica to alleviate heat stress through up-regulation of the heat stress transcription factor-heat shock proteins-HSP70/HSP90 organising proteins (HSF-HSPs-HOPs) module, down-regulation of reactive oxygen species (ROS) generation and up-regulation of ROS scavenging. Our results provide novel insights into the heat stress acclimation mechanism in S. japonica, and would benefit future selection of heat-tolerant varieties.

Transglutaminase 2 Regulates HSF1 Gene Expression in the Acute Phase of Fish Optic Nerve Regeneration.

Fish retinal ganglion cells (RGCs) can regenerate after optic nerve lesions (ONLs). We previously reported that heat shock factor 1 (HSF1) and Yamanaka factors increased in the zebrafish retina 0.5-24 h after ONLs, and they led to cell survival and the transformation of neuro-stem cells. We also showed that retinoic acid (RA) signaling and transglutaminase 2 (TG2) were activated in the fish retina, performing neurite outgrowth 5-30 days after ONLs. In this study, we found that RA signaling and TG2 increased within 0.5 h in the zebrafish retina after ONLs. We examined their interaction with the TG2-specific morpholino and inhibitor due to the significantly close initiation time of TG2 and HSF1. The inhibition of TG2 led to the complete suppression of HSF1 expression. Furthermore, the results of a ChIP assay with an anti-TG2 antibody evidenced significant anti-TG2 immunoprecipitation of HSF1 genome DNA after ONLs. The inhibition of TG2 also suppressed Yamanaka factors' gene expression. This rapid increase in TG2 expression occurred 30 min after the ONLs, and RA signaling occurred 15 min before this change. The present study demonstrates that TG2 regulates Yamanaka factors via HSF1 signals in the acute phase of fish optic nerve regeneration.

Heat Shock Factor 1 forms nuclear condensates and restructures the yeast genome before activating target genes.

In insects and mammals, 3D genome topology has been linked to transcriptional states yet whether this link holds for other eukaryotes is unclear. Using both ligation proximity and fluorescence microscopy assays, we show that in Saccharomyces cerevisiae, Heat Shock Response (HSR) genes dispersed across multiple chromosomes and under the control of Heat Shock Factor (Hsf1) rapidly reposition in cells exposed to acute ethanol stress and engage in concerted, Hsf1-dependent intergenic interactions. Accompanying 3D genome reconfiguration is equally rapid formation of Hsf1-containing condensates. However, in contrast to the transience of Hsf1-driven intergenic interactions that peak within 10-20 min and dissipate within 1 h in the presence of 8.5% (v/v) ethanol, transcriptional condensates are stably maintained for hours. Moreover, under the same conditions, Pol II occupancy of HSR genes, chromatin remodeling, and RNA expression are detectable only later in the response and peak much later (>1 h). This contrasts with the coordinate response of HSR genes to thermal stress (39°C) where Pol II occupancy, transcription, histone eviction, intergenic interactions, and formation of Hsf1 condensates are all rapid yet transient (peak within 2.5-10 min and dissipate within 1 h). Therefore, Hsf1 forms condensates, restructures the genome and transcriptionally activates HSR genes in response to both forms of proteotoxic stress but does so with strikingly different kinetics. In cells subjected to ethanol stress, Hsf1 forms condensates and repositions target genes before transcriptionally activating them.

Protein Misfolding Releases Human HSF1 from HSP70 Latency Control

Heat shock factor 1 (HSF1) responds to stress to mount the heat shock response (HSR), a conserved transcriptional program that allows cells to maintain proteostasis by upregulating heat shock proteins (HSPs). The homeostatic stress regulation of HSF1 plays a key role in human physiology and health but its mechanism has remained difficult to pinpoint. Recent work in the budding yeast model has implicated stress-inducible chaperones of the HSP70 family as direct negative regulators of HSF1 activity. Here, we have investigated the latency control and activation of human HSF1 by HSP70 and misfolded proteins. Purified oligomeric HSF1-HSP70 (HSPA1A) complexes exhibited basal DNA binding activity that was inhibited by increasing the levels of HSP70 and, importantly, misfolded proteins reverted the inhibitory effect. Using site-specific UV photo-crosslinking, we monitored HSP70-HSF1 complexes in HEK293T cells. While HSF1 was bound by the substrate binding domain of HSP70 in unstressed cells, activation of HSF1 by heat shock as well as by inducing the misfolding of newly synthesized proteins resulted in release of HSF1 from the chaperone. Taken our results together, we conclude that latent HSF1 populate dynamic complexes with HSP70, which are sensitive to increased levels of misfolded proteins that compete for binding to the HSP70 substrate binding domain. Thus, human HSF1 is activated by various stress conditions that all titrate available HSP70.

Cadmium causes cerebral mitochondrial dysfunction through regulating mitochondrial HSF1

Mitochondria, as the powerhouse of the cell, play a vital role in maintaining cellular energy homeostasis and are known to be a primary target of cadmium (Cd) toxicity. The improper targeting of proteins to mitochondria can compromise the normal functions of the mitochondria. However, the precise mechanism by which protein localization contributes to the development of mitochondrial dysfunction induced by Cd is still not fully understood. For this research, Hy-Line white variety chicks (1-day-old) were used and equally distributed into 4 groups: the Control group (fed with a basic diet), the Cd35 group (basic diet with 35 mg/kg CdCl2), the Cd70 group (basic diet with 70 mg/kg CdCl2) and the Cd140 group (basic diet with 140 mg/kg CdCl2), respectively for 90 days. It was found that Cd caused the accumulation of heat shock factor 1 (HSF1) in the mitochondria, and the overexpression of HSF1 in the mitochondria led to mitochondrial dysfunction and neuronal damage. This process is due to the mitochondrial HSF1 (mtHSF1), causing mitochondrial fission through the upregulation of dynamin-related protein 1 (Drp1) content, while inhibiting oligomer formation of single-stranded DNA-binding protein 1 (SSBP1), resulting in the mitochondrial DNA (mtDNA) deletion. The findings unveil an unforeseen role of HSF1 in triggering mitochondrial dysfunction.

A human-specific cytotoxic neopeptide generated by the deafness gene Cingulin

Accumulation of mutant proteins in cells can induce proteinopathies and cause functional damage to organs. Recently, the Cingulin (CGN) protein has been shown to maintain the morphology of cuticular plates of inner ear hair cells and a frameshift mutation in CGN causes autosomal dominant non-syndromic hearing loss. Here, we find that the mutant CGN proteins form insoluble aggregates which accumulate intracellularly and lead to cell death. Expression of the mutant CGN in the inner ear results in severe hair cell death and hearing loss in mice, resembling the auditory phenotype in human patients. Interestingly, a human-specific residue (V1112) in the neopeptide generated by the frameshift mutation is critical for the aggregation and cytotoxicity of the mutant human CGN. Moreover, the expression of heat shock factor 1 (HSF1) decreases the accumulation of insoluble mutant CGN aggregates and rescues cell death. In summary, these findings identify mutant-specific toxic polypeptides as a disease-causing mechanism of the deafness mutation in CGN, which can be targeted by the expression of the cell chaperone response regulator HSF1.

Perinatal asphyxia leads to acute kidney damage and increased renal susceptibility in adulthood.

Perinatal asphyxia (PA) poses a significant threat to multiple organs, particularly the kidneys. Diagnosing PA-associated kidney injury remains challenging, and treatment options are inadequate. Furthermore, there is a lack of long-term follow-up data regarding the renal implications of PA. In this study, 7-day-old male Wistar rats were exposed to PA using a gas mixture (4% O2; 20% CO2 in N2 for 15 min) to investigate molecular pathways linked to renal tubular damage, hypoxia, angiogenesis, heat shock response, inflammation, and fibrosis in the kidney. In a second experiment, adult rats with a history of PA were subjected to moderate renal ischemia-reperfusion (IR) injury to test the hypothesis that PA exacerbates renal susceptibility. Our results revealed an increased gene expression of renal injury markers (kidney injury molecule-1 and neutrophil gelatinase-associated lipocalin), hypoxic and heat shock factors (hypoxia-inducible factor-1α, heat shock factor-1, and heat shock protein-27), proinflammatory cytokines (interleukin-1β, interleukin-6, tumor necrosis factor-α, and monocyte chemoattractant protein-1), and fibrotic markers (transforming growth factor-β, connective tissue growth factor, and fibronectin) promptly after PA. Moreover, a machine learning model was identified through random forest analysis, demonstrating an impressive classification accuracy (95.5%) for PA. Post-PA rats showed exacerbated functional decline and tubular injury and more intense hypoxic, heat shock, proinflammatory, and profibrotic response after renal IR injury compared with controls. In conclusion, PA leads to subclinical kidney injury, which may increase the susceptibility to subsequent renal damage later in life. In addition, the parameters identified through random forest analysis provide a robust foundation for future biomarker research in the context of PA.NEW & NOTEWORTHY This article demonstrates that perinatal asphyxia leads to subclinical kidney injury that permanently increases renal susceptibility to subsequent ischemic injury. We identified major molecular pathways involved in perinatal asphyxia-induced renal complications, highlighting potential targets of therapeutic approaches. In addition, random forest analysis revealed a model that classifies perinatal asphyxia with 95.5% accuracy that may provide a strong foundation for further biomarker research. These findings underscore the importance of multiorgan follow-up for perinatal asphyxia-affected patients.

Potential use of HSP70 as an indicator of heat stress in dairy cows – a review

Heat stress (HS) poses significant challenges to the dairy industry, resulting in reduced milk production, impaired reproductive performance, and compromised animal welfare. Therefore, understanding the molecular mechanisms underlying cellular responses to HS is crucial for developing effective strategies to mitigate its adverse effects. Heat shock protein 70 (HSP70) has emerged as a potential player involved in cellular thermotolerance in dairy cows. This review provides a comprehensive overview of the role of HSP70 as a molecular chaperone in cellular thermotolerance in dairy cows under HS. HSP70 facilitates proper protein folding and prevents the aggregation of denatured proteins. By binding to misfolded proteins, it helps maintain protein homeostasis and prevents the accumulation of damaged proteins during HS. Additionally, HSP70 interacts with various regulatory proteins and signaling pathways, contributing to the cellular adaptive response to HS. The upregulation of HSP70 expression in response to HS is regulated by a complex network involving heat-shock factors (HSFs), heat-shock element-binding proteins, and HSF co-chaperones. Therefore, HSP70 holds the potential to be a useful indicator of tissue stress due to its role in maintaining cellular balance, and as it is released both inside and outside cells in response to stress. Traditional methods of measuring HSP70 in blood samples are labor-intensive, and with the process being potentially stressful for the animals and may subsequently affect the results. Therefore, measuring HSP expression in cow's milk has shown promise as an easy, non-invasive, and accurate way to detect HS in dairy cows. Monitoring HSP70 levels in milk can be applied as a supplementary approach to identify HS or HS resistance of individual cows, selection of suitable animals and to guide targeted management strategies. However, despite the potential advantages of using HSP70 as a biomarker for monitoring HS on dairy cows, challenges remain in standardizing measurement protocols, establishing species-specific reference ranges, addressing inter-individual variations, and determining the specificity of changes in HSP70 due to HS. Future research should focus on developing non-invasive techniques for HSP70 detection, with consideration of climatic conditions, and unravelling the molecular interactions and regulatory networks involving HSP70.

The Effect of HSF1 Activity Inhibitor of the Cardenolide Group (CL-43) on Tumor and Nontransformed Cells

The Effect of HSF1 Activity Inhibitor of the Cardenolide Group (CL-43) on Tumor and Nontransformed Cells

Heat Shock Related Protein Expression in Abdominal Testes of Asian Elephant (Elephas maximus).

The abdominal testes of Asian elephants show normal spermatogenesis. Heat shock in cryptorchid testes elevates heat shock factor (HSF) expression, leading to germ cell apoptosis, while increased heat shock proteins (HSPs) levels provide protection. To investigate how heat shock affects elephant spermatogenic cells, focusing on heat shock-related molecules and the cell death mechanism, immunohistochemistry and TUNEL staining were employed to assess the immunoexpression of several heat shock-related molecules and the status of apoptosis in elephant fibroblasts (EF) induced by heat shock stimulus. Additionally, the immunoexpression of heat shock-related molecules and cell proliferation status in the elephant spermatogenic cells. Our finding indicated that heat shock-induced HSF1 immunoexpression in EF leads to apoptosis mediated by T-cell death-associated gene 51 (TDAG51) while also upregulating HSP70 to protect damaged cells. In elephant spermatogenic cells, immunostaining revealed a predominance of proliferating cell nuclear antigen (PCNA)-positive cells with minimal TDAG51- and TUNEL-positive cells, suggesting active proliferation and apoptosis suppression during normal spermatogenesis in the abdominal testis. Interestingly, spermatogonia co-immunoexpressed HSF1 and HSP90, potentially reducing apoptosis through protective mechanisms different from those observed in other mammals. Spermatogenic cells did not show immunolocalisation of HSP70, and hence, it may not contribute to protecting the spermatogonia from heat shock because the transcriptional activity of HSF1 is suppressed by HSP90A binding. This study provides insight into the specific heat shock response and defence mechanisms in elephant spermatogenic cells and may contribute to our understanding of species-specific adaptation to environmental stresses of the testis.

Bag-1-mediated HSF1 phosphorylation regulates expression of heat shock proteins in breast cancer cells.

According to the World Health Organization in 2022, 2.3 million women were diagnosed with breast cancer. Investigating the interaction networks between Bcl-2-associated athanogene (Bag)-1 and other chaperone proteins may further the current understanding of the regulation of protein homeostasis in breast cancer cells and contribute to the development of treatment options. The present study aimed to determine the interactions between Bag-1 and heat shock proteins (HSPs); namely, HSP90, HSP70 and HSP27, to elucidate their role in promoting heat shock factor-1 (HSF1)-dependent survival of breast cancer cells. HER2-negative (MCF-7) and HER2-positive (BT-474) cell lines were used to examine the impact of Bag-1 expression on HSF1 and HSPs. We demonstrated that Bag-1 overexpression promoted HER2 expression in breast cancer cells, thereby resulting in the concurrent constitutive activation of the HSF1-HSP axis. The activation of HSP results in the stabilization of several tumor-promoting HSP clients such as AKT, mTOR and HSF1 itself, which substantially accelerates tumor development. Our results suggest that Bag-1 can modulate the chaperone activity of HSPs, such as HSP27, by directly or indirectly regulating the phosphorylation of HSF1. This modulation of chaperone activity can influence the activation of genes involved in cellular homeostasis, thereby protecting cells against stress.

HOP stabilizes the HSFA1a and plays a main role in the onset of thermomorphogenesis.

Living organisms have the capacity to respond to environmental stimuli, including warm conditions. Upon sensing mild temperature, plants launch a transcriptional response that promotes morphological changes, globally known as thermomorphogenesis. This response is orchestrated by different hormonal networks and by the activity of different transcription factors, including the heat shock factor A1(HSFA1) family. Members of this family interact with heat shock protein70(HSP70) and heat shock protein 90(HSP90); however, the effect of this binding on the regulation of HSFA1 activity or of the role ofcochaperones, such as the HSP70-HSP90 organizing protein (HOP) on HSFA1 regulation, remains unknown.Here, we show that AtHOPs are involved in the folding and stabilization of the HSFA1a and are required for the onset of the transcriptional response associated to thermomorphogenesis. Our results demonstrate that the three members of the AtHOP family bind in vivo to the HSFA1a and that the expression of multiple HSFA1a-responsive-responsive genes is altered in the hop1 hop2 hop3 mutant under warm temperature. Interestingly, HSFA1a is accumulated at lower levels in the hop1 hop2 hop3 mutant, while control levels are recovered in the presence of the proteasome inhibitor MG132 or the synthetic chaperone tauroursodeoxycholic acid (TUDCA). This uncovers the HSFA1a as a client of HOP complexes in plants and reveals the participation of HOPs in HSFA1a stability.

An HSF1–JMJD6–HSP feedback circuit promotes cell adaptation to proteotoxic stress

Heat Shock Factor 1 (HSF1) is best known as the master transcriptional regulator of the heat-shock response (HSR), a conserved adaptive mechanism critical for protein homeostasis (proteostasis). Combining a genome-wide RNAi library with an HSR reporter, we identified Jumonji domain-containing protein 6 (JMJD6) as an essential mediator of HSF1 activity. In follow-up studies, we found that JMJD6 is itself a noncanonical transcriptional target of HSF1 which acts as a critical regulator of proteostasis. In a positive feedback circuit, HSF1 binds and promotes JMJD6 expression, which in turn reduces heat shock protein 70 (HSP70) R469 monomethylation to disrupt HSP70-HSF1 repressive complexes resulting in enhanced HSF1 activation. Thus, JMJD6 is intricately wired into the proteostasis network where it plays a critical role in cellular adaptation to proteotoxic stress.

Regulatory mechanism of cold-inducible diapause in Caenorhabditis elegans

Temperature is a critical environmental cue that controls the development and lifespan of many animal species; however, mechanisms underlying low-temperature adaptation are poorly understood. Here, we describe cold-inducible diapause (CID), another type of diapause induced by low temperatures in Caenorhabditis elegans. A premature stop codon in heat shock factor 1 (hsf-1) triggers entry into CID at 9 °C, whereas wild-type animals enter CID at 4 °C. Furthermore, both wild-type and hsf-1(sy441) mutant animals undergoing CID can survive for weeks, and resume growth at 20 °C. Using epistasis analysis, we demonstrate that neural signalling pathways, namely tyraminergic and neuromedin U signalling, regulate entry into CID of the hsf-1 mutant. Overexpression of anti-ageing genes, such as hsf-1, XBP1/xbp-1, FOXO/daf-16, Nrf2/skn-1, and TFEB/hlh-30, also inhibits CID entry of the hsf-1 mutant. Based on these findings, we hypothesise that regulators of the hsf-1 mutant CID may impact longevity, and successfully isolate 16 long-lived mutants among 49 non-CID mutants via genetic screening. Furthermore, we demonstrate that the nonsense mutation of MED23/sur-2 prevents CID entry of the hsf-1(sy441) mutant and extends lifespan. Thus, CID is a powerful model to investigate neural networks involving cold acclimation and to explore new ageing mechanisms.

Multifaceted roles of Arabidopsis heat shock factor binding protein in plant growth, development, and heat shock response

Heat shock factor-binding protein (HSBP) is a 10 kDa protein in plants and animals and consists exclusively of a coiled-coil domain. During the recovery phase following a heat shock response, HSBP relocates from the cytoplasm to the nucleus in Arabidopsis (Arabidopsis thaliana), where it interacts with heat shock factors (HSFs). Here, we found that 16 out of the 19 functional known HSFs can interact with HSBP. Besides, our results indicate that HSBP negatively regulates HSF gene expression during normal growth conditions and recovery from heat shock. Expanding our understanding of HSBP's physiological functions during regular growth, co-immunoprecipitation and mass spectrometry analysis identified 16 coiled-coil domain-containing proteins co-immunoprecipitated with HSBP. These proteins encompass HSP70s, all components of the MAIGO2 complex, COP1-interactive protein1 (CIP1), CIP1-like protein, kinesin-like protein for actin-based chloroplast movement1 and 2 (KAC1/2), and HSBP itself. By examining mutant plants lacking HSBP and its interacting proteins, we elucidated their functional relationships. Our findings underscore the indispensability of the HSBP coiled-coil heptad repeat for interacting with its partners and its crucial role in growth, development, and the heat shock response. In addition to its involvement in heat shock response, Arabidopsis HSBP, a discreet small regulatory protein, exerts multiple regulatory functions in hypocotyl elongation, flowering time, chloroplast photo-relocation, and seed development.

JRK binds satellite III DNA and is necessary for the heat shock response.

JRK is a DNA-binding protein of the pogo superfamily of transposons, which includes the well-known centromere binding protein B (CENP-B). Jrk null mice exhibit epilepsy, and growth and reproductive disorders, consistent with its relatively high expression in the brain and reproductive tissues. Human JRK DNA variants and gene expression levels are implicated in cancers and neuropsychiatric disorders. JRK protein modulates β-catenin-TCF activity but little is known of its cellular functions. Based on its homology to CENP-B, we determined whether JRK binds centromeric or other satellite DNAs. We show that human JRK binds satellite III DNA, which is abundant at the chromosome 9q12 juxtacentromeric region and on Yq12, both sites of nuclear stress body assembly. Human JRK-GFP overexpressed in HeLa cells strongly localises to 9q12. Using an anti-JRK antiserum we show that endogenous JRK co-localises with a subset of centromeres in non-stressed cells, and with heat shock factor 1 following heat shock. Knockdown of JRK in HeLa cells proportionately reduces heat shock protein gene expression in heat-shocked cells. A role for JRK in regulating the heat shock response is consistent with the mouse Jrk null phenotype and suggests that human JRK may act as a modifier of diseases with a cellular stress component.

HSFA1 heat shock factors integrate warm temperature and heat signals in plants

Warm temperatures and heat stress trigger distinct plant responses. Recently, Li et al. and Tan et al. identified HSFA1 heat shock transcription factors (HSFs) as central gatekeepers of high-temperature signaling, integrating warm temperature and heat shock responses (HSRs) in arabidopsis (Arabidopsis thaliana). HSFA1d stabilizes phytochrome-interacting factor 4 (PIF4) and activates HSFA2, establishing a crosstalk between thermomorphogenesis and thermotolerance.

PANX1-mediated ATP release confers FAM3A’s suppression effects on hepatic gluconeogenesis and lipogenesis

BackgroundExtracellular adenosine triphosphate (ATP) is an important signal molecule. In previous studies, intensive research had revealed the crucial roles of family with sequence similarity 3 member A (FAM3A) in controlling hepatic glucolipid metabolism, islet β cell function, adipocyte differentiation, blood pressure, and other biological and pathophysiological processes. Although mitochondrial protein FAM3A plays crucial roles in the regulation of glucolipid metabolism via stimulating ATP release to activate P2 receptor pathways, its mechanism in promoting ATP release in hepatocytes remains unrevealed.Methodsdb/db, high-fat diet (HFD)-fed, and global pannexin 1 (PANX1) knockout mice, as well as liver sections of individuals, were used in this study. Adenoviruses and adeno-associated viruses were utilized for in vivo gene overexpression or inhibition. To evaluate the metabolic status in mice, oral glucose tolerance test (OGTT), pyruvate tolerance test (PTT), insulin tolerance test (ITT), and magnetic resonance imaging (MRI) were conducted. Protein–protein interactions were determined by coimmunoprecipitation with mass spectrometry (MS) assays.ResultsIn livers of individuals and mice with steatosis, the expression of ATP-permeable channel PANX1 was increased (P < 0.01). Hepatic PANX1 overexpression ameliorated the dysregulated glucolipid metabolism in obese mice. Mice with hepatic PANX1 knockdown or global PANX1 knockout exhibited disturbed glucolipid metabolism. Restoration of hepatic PANX1 rescued the metabolic disorders of PANX1-deficient mice (P < 0.05). Mechanistically, ATP release is mediated by the PANX1-activated protein kinase B-forkhead box protein O1 (Akt-FOXO1) pathway to inhibit gluconeogenesis via P2Y receptors in hepatocytes. PANX1-mediated ATP release also activated calmodulin (CaM) (P < 0.01), which interacted with c-Jun N-terminal kinase (JNK) to inhibit its activity, thereby deactivating the transcription factor activator protein-1 (AP1) and repressing fatty acid synthase (FAS) expression and lipid synthesis (P < 0.05). FAM3A stimulated the expression of PANX1 via heat shock factor 1 (HSF1) in hepatocytes (P < 0.05). Notably, FAM3A overexpression failed to promote ATP release, inhibit the expression of gluconeogenic and lipogenic genes, and suppress gluconeogenesis and lipid deposition in PANX1-deficient hepatocytes and livers.ConclusionsPANX1-mediated release of ATP plays a crucial role in maintaining hepatic glucolipid homeostasis, and it confers FAM3A’s suppressive effects on hepatic gluconeogenesis and lipogenesis.