Induction Of Activating Transcription Factor Research Articles

Thbs1 regulates skeletal muscle mass in a TGFβ-Smad2/3-ATF4-dependent manner

Loss of muscle mass is a feature of chronic illness and aging. Here, we report that skeletal muscle-specific thrombospondin-1 transgenic mice (Thbs1 Tg) have profound muscle atrophy with age-dependent decreases in exercise capacity and premature lethality. Mechanistically, Thbs1 activates transforming growth factor β (TGFβ)-Smad2/3 signaling, which also induces activating transcription factor 4 (ATF4) expression that together modulates the autophagy-lysosomal pathway (ALP) and ubiquitin-proteasome system (UPS) to facilitate muscle atrophy. Indeed, myofiber-specific inhibition of TGFβ-receptor signaling represses the induction of ATF4, normalizes ALP and UPS, and partially restores muscle mass in Thbs1 Tg mice. Similarly, myofiber-specific deletion of Smad2 and Smad3 or the Atf4 gene antagonizes Thbs1-induced muscle atrophy. More importantly, Thbs1-/- mice show significantly reduced levels of denervation- and caloric restriction-mediated muscle atrophy, along with blunted TGFβ-Smad3-ATF4 signaling. Thus, Thbs1-mediated TGFβ-Smad3-ATF4 signaling in skeletal muscle regulates tissue rarefaction, suggesting a target for atrophy-based muscle diseases and sarcopenia with aging.

Stem-loop-induced ribosome queuing in the uORF2/ATF4 overlap fine-tunes stress-induced human ATF4 translational control

Activating transcription factor 4 (ATF4) is a master transcriptional regulator of the integrated stress response, leading cells toward adaptation or death. ATF4's induction under stress was thought to be due to delayed translation reinitiation, where the reinitiation-permissive upstream open reading frame 1 (uORF1) plays a key role. Accumulating evidence challenging this mechanism as the sole source of ATF4 translation control prompted us to investigate additional regulatory routes. We identified a highly conserved stem-loop in the uORF2/ATF4 overlap, immediately preceded by a near-cognate CUG, which introduces another layer of regulation in the form of ribosome queuing. These elements explain how the inhibitory uORF2 can be translated under stress, confirming prior observations but contradicting the original regulatory model. We also identified two highly conserved, potentially modified adenines performing antagonistic roles. Finally, we demonstrated that the canonical ATF4 translation start site is substantially leaky scanned. Thus, ATF4's translational control is more complex than originally described, underpinning its key role in diverse biological processes.

ATF4 expression in thermogenic adipocytes is required for cold-induced thermogenesis in mice via FGF21-independent mechanisms

In brown adipose tissue (BAT), short-term cold exposure induces the activating transcription factor 4 (ATF4), and its downstream target fibroblast growth factor 21 (FGF21). Induction of ATF4 in BAT in response to mitochondrial stress is required for thermoregulation, partially by increasing FGF21 expression. In the present study, we tested the hypothesis that Atf4 and Fgf21 induction in BAT are both required for BAT thermogenesis under physiological stress by generating mice selectively lacking either Atf4 (ATF4 BKO) or Fgf21 (FGF21 BKO) in UCP1-expressing adipocytes. After 3 days of cold exposure, core body temperature was significantly reduced in ad-libitum-fed ATF4 BKO mice, which correlated with Fgf21 downregulation in brown and beige adipocytes, and impaired browning of white adipose tissue. Conversely, despite having reduced browning, FGF21 BKO mice had preserved core body temperature after cold exposure. Mechanistically, ATF4, but not FGF21, regulates amino acid import and metabolism in response to cold, likely contributing to BAT thermogenic capacity under ad libitum-fed conditions. Importantly, under fasting conditions, both ATF4 and FGF21 were required for thermogenesis in cold-exposed mice. Thus, ATF4 regulates BAT thermogenesis under fed conditions likely in a FGF21-independent manner, in part via increased amino acid uptake and metabolism.

GDF15 is required for cold-induced thermogenesis and contributes to improved systemic metabolic health following loss of OPA1 in brown adipocytes.

We previously reported that mice lacking the protein optic atrophy 1 (OPA1 BKO) in brown adipose tissue (BAT) display induction of the activating transcription factor 4 (ATF4), which promotes fibroblast growth factor 21 (FGF21) secretion as a batokine. FGF21 increases metabolic rates under baseline conditions but is dispensable for the resistance to diet-induced obesity (DIO) reported in OPA1 BKO mice (Pereira et al., 2021). To determine alternative mediators of this phenotype, we performed transcriptome analysis, which revealed increased levels of growth differentiation factor 15 (GDF15), along with increased protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) levels in BAT. To investigate whether ATF4 induction was mediated by PERK and evaluate the contribution of GDF15 to the resistance to DIO, we selectively deleted PERK or GDF15 in OPA1 BKO mice. Mice with reduced OPA1 and PERK levels in BAT had preserved ISR activation. Importantly, simultaneous deletion of OPA1 and GDF15 partially reversed the resistance to DIO and abrogated the improvements in glucose tolerance. Furthermore, GDF15 was required to improve cold-induced thermogenesis in OPA1 BKO mice. Taken together, our data indicate that PERK is dispensable to induce the ISR, but GDF15 contributes to the resistance to DIO, and is required for glucose homeostasis and thermoregulation in OPA1 BKO mice by increasing energy expenditure.

ATF3 induction prevents precocious activation of skeletal muscle stem cell by regulating H2B expression

Skeletal muscle stem cells (also called satellite cells, SCs) are important for maintaining muscle tissue homeostasis and damage-induced regeneration. However, it remains poorly understood how SCs enter cell cycle to become activated upon injury. Here we report that AP-1 family member ATF3 (Activating Transcription Factor 3) prevents SC premature activation. Atf3 is rapidly and transiently induced in SCs upon activation. Short-term deletion of Atf3 in SCs accelerates acute injury-induced regeneration, however, its long-term deletion exhausts the SC pool and thus impairs muscle regeneration. The Atf3 loss also provokes SC activation during voluntary exercise and enhances the activation during endurance exercise. Mechanistically, ATF3 directly activates the transcription of Histone 2B genes, whose reduction accelerates nucleosome displacement and gene transcription required for SC activation. Finally, the ATF3-dependent H2B expression also prevents genome instability and replicative senescence in SCs. Therefore, this study has revealed a previously unknown mechanism for preserving the SC population by actively suppressing precocious activation, in which ATF3 is a key regulator.

Activating mutations in EGFR and PI3K promote ATF4 induction for NSCLC cell survival during amino acid deprivation

Some oncoproteins along with stress kinase general control non-derepressible 2 (GCN2) can ensure the induction of activating transcription factor 4 (ATF4) to counteract amino acid deprivation; however, little is known regarding the role of the oncogenic EGFR-PI3K pathway. In this study, we demonstrate that both mutated EGFR and PIK3CA contribute to ATF4 induction following GCN2 activation in NSCLC cells. The inhibition of EGFR or PI3K mutant proteins, pharmacologically or through genetic knockdown, inhibited ATF4 induction without affecting GCN2 activation. A downstream analysis revealed that the oncogenic EGFR-PI3K pathway may utilize mTOR-mediated translation control mechanisms for ATF4 induction. Furthermore, in NSCLC cells harboring co-mutations in EGFR and PIK3CA, the combined inhibition of these oncoproteins markedly suppressed ATF4 induction and the subsequent gene expression program as well as cell viability during amino acid deprivation. Our findings establish a role for the oncogenic EGFR-PI3K pathway in the adaptive stress response and provide a strategy to improve EGFR-targeted NSCLC therapy.

Microglial activating transcription factor 3 upregulation: An indirect target to attenuate inflammation in the nervous system.

Activating Transcription Factor 3 (ATF3) is upregulated in reaction to several cellular stressors found in a wide range of pathological conditions to coordinate a transcriptional response. ATF3 was first implicated in the transcriptional reaction to axotomy when its massive upregulation was measured in sensory and motor neuron cell bodies following peripheral nerve injury. It has since been shown to be critical for successful axon regeneration in the peripheral nervous system and a promising target to mitigate regenerative failure in the central nervous system. However, much of the research to date has focused on ATF3's function in neurons, leaving the expression, function, and therapeutic potential of ATF3 in glia largely unexplored. In the immunology literature ATF3 is seen as a master regulator of the innate immune system. Specifically, in macrophages following pathogen or damage associated molecular pattern receptor activation and subsequent cytokine release, ATF3 upregulation abrogates the inflammatory response. Importantly, ATF3 upregulation is not exclusively due to cellular stress exposure but has been achieved by the administration of several small molecules. In the central nervous system, microglia represent the resident macrophage population and are therefore of immediate interest with respect to ATF3 induction. It is our perspective that the potential of inducing ATF3 expression to dampen inflammatory microglial phenotype represents an unexplored therapeutic target and may have synergistic benefits when paired with concomitant neuronal ATF3 upregulation. This would be of particular benefit in pathologies that involve both detrimental inflammation and neuronal damage including spinal cord injury, multiple sclerosis, and stroke.

Activating transcription factor 3 inhibits NF‑κB p65 signaling pathway and mediates apoptosis and cell cycle arrest in cervical cancer cells

BackgroundAs a novel tumor suppressor mediator, activating transcription factor 3 (ATF3) has recently aroused an interest in its possible therapeutic applications in various cancers. In this study, we evaluated the effect of ATF3 overexpression on the cellular level of nuclear factor kappa B (NF-κB) in human papillomavirus (HPV)-infected Ca Ski cells. Further, we examined whether ATF3 could mediate cell cycle arrest and alter the apoptosis level of Ca Ski cells.MethodsThe biological behavior of Ca Ski cells was evaluated prior and subsequent to the overexpression of ATF3 by MTT assay, fluorescence microscopy, cell cycle and annexin V/PI flow cytometric analysis. The effect of ectopic ATF3 expression on the cellular level of NF-κB in HPV-positive cells was evaluated by western blotting assay.ResultsThe overexpression of ATF3 in Ca Ski cells led to significant apoptosis and cell cycle arrest in the G1 phase. Western blotting assay revealed a discernible reduction of NF-κB p65 level in cervical cancer cells.ConclusionATF3 acts as a tumor suppressor factor in HPV16-infected Ca Ski cells and exerts anti-cancer effects on HPV16-related cervical cancer cells potentially by hindering cell growth and inducing cell cycle arrest through the down-regulation of NF-κB. Our results suggest that ATF3 induction or NF-κB suppression may be useful targets for HPV16-related cervical cancer prevention and treatment.

Induction of the activating transcription factor-4 in the intratumoral CD8+ T cells sustains their viability and anti-tumor activities.

Immune suppressive factors of the tumor microenvironment (TME) undermine viability and exhaust the activities of the intratumoral cytotoxic CD8 + T lymphocytes (CTL) thereby evading anti-tumor immunity and decreasing the benefits of immune therapies. To counteract this suppression and improve the efficacy of therapeutic regimens, it is important to identify and understand the critical regulators within CD8 + T cells that respond to TME stress and tumor-derived factors. Here we investigated the regulation and importance of activating transcription factor-4 (ATF4) in CTL using a novel Atf4ΔCD8 mouse model lacking ATF4 specifically in CD8 + cells. Induction of ATF4 in CD8 + T cells occurred in response to antigenic stimulation and was further increased by exposure to tumor-derived factors and TME conditions. Under these conditions, ATF4 played a critical role in the maintenance of survival and activities of CD8 + T cells. Conversely, selective ablation of ATF4 in CD8 + T cells in mice rendered these Atf4ΔCD8 hosts prone to accelerated growth of implanted tumors. Intratumoral ATF4-deficient CD8 + T cells were under-represented compared to wild-type counterparts and exhibited impaired activation and increased apoptosis. These findings identify ATF4 as an important regulator of viability and activity of CD8 + T cells in the TME and argue for caution in using agents that could undermine these functions of ATF4 for anti-cancer therapies.

Hypoxia-Mediated ATF4 Induction Promotes Survival in Detached Conditions in Metastatic Murine Mammary Cancer Cells.

Regions of hypoxia are common in solid tumors and drive changes in gene expression that increase risk of cancer metastasis. Tumor cells must respond to the stress of hypoxia by activating genes to modify cell metabolism and antioxidant response to improve survival. The goal of the current study was to determine the effect of hypoxia on cell metabolism and markers of oxidative stress in metastatic (metM-Wntlung) compared with nonmetastatic (M-Wnt) murine mammary cancer cell lines. We show that hypoxia induced a greater suppression of glutamine to glutamate conversion in metastatic cells (13% in metastatic cells compared to 7% in nonmetastatic cells). We also show that hypoxia increased expression of genes involved in antioxidant response in metastatic compared to nonmetastatic cells, including glutamate cysteine ligase catalytic and modifier subunits and malic enzyme 1. Interestingly, hypoxia increased the mRNA level of the transaminase glutamic pyruvic transaminase 2 (Gpt2, 7.7-fold) only in metM-Wntlung cells. The change in Gpt2 expression was accompanied by transcriptional (4.2-fold) and translational (6.5-fold) induction of the integrated stress response effector protein activating transcription factor 4 (ATF4). Genetic depletion ATF4 demonstrated importance of this molecule for survival of hypoxic metastatic cells in detached conditions. These findings indicate that more aggressive, metastatic cancer cells utilize hypoxia for metabolic reprogramming and induction of antioxidant defense, including activation of ATF4, for survival in detached conditions.

Translational control by heme-regulated elF2α kinase during erythropoiesis.

HRI is the heme-regulated elF2α kinase that phosphorylates the α-subunit of elF2. Although the role of HRI in inhibiting globin synthesis in erythroid cells is well established, broader roles of HRI in translation have been uncovered recently. This review is to summarize the new discoveries of HRI in stress erythropoiesis and in fetal γ-globin expression. HRI and activating transcription factor 4 (ATF4) mRNAs are highly expressed in early erythroblasts. Inhibition of protein synthesis by HRI-phosphorylated elF2α (elF2αP) is necessary to maintain protein homeostasis in both the cytoplasm and mitochondria. In addition, HRI-elF2αP specifically enhances translation of ATF4 mRNA leading to the repression of mechanistic target of rapamycin complex 1 (mTORC1) signaling. ATF4-target genes are most highly activated during iron deficiency to maintain mitochondrial function, redox homeostasis, and to enable erythroid differentiation. HRI is therefore a master translation regulator of erythropoiesis sensing intracellular heme concentrations and oxidative stress for effective erythropoiesis. Intriguingly, HRI-elF2αP-ATF4 signaling also inhibits fetal hemoglobin production in human erythroid cells. The primary function of HRI is to maintain protein homeostasis accompanied by the induction of ATF4 to mitigate stress. Role of HRI-ATF4 in γ-globin expression raises the potential of HRI as a therapeutic target for hemoglobinopathy.

Induction of heparanase 2 (Hpa2) expression by stress is mediated by ATF3

Activity of heparanase, endoglycosidase that cleaves heparan sulfate side chains in heparan sulfate proteoglycans, is highly implicated in tumor progression and metastasis. Heparanase inhibitors are therefore being evaluated clinically as anti-cancer therapeutics. Heparanase 2 (Hpa2) is a close homolog of heparanase that lacks HS-degrading activity and functions as an endogenous inhibitor of heparanase. As a result, Hpa2 appears to attenuate tumor growth but mechanisms that regulate Hpa2 expression and determine the ratio between heparanase and Hpa2 are largely unknown. We have recently reported that the expression of Hpa2 is induced by endoplasmic reticulum (ER) and proteotoxic stresses, but the mechanism(s) underlying Hpa2 gene regulation was obscure. Here we expand the notion that Hpa2 is regulated by conditions of stress. We report that while ER and hypoxia, each alone, resulted in a 3-7 fold increase in Hpa2 expression, combining ER stress and hypoxia resulted in a noticeable, over 40-fold increase in Hpa2 expression. A prominent induction of Hpa2 expression was also quantified in cells exposed to heat shock, proteotoxic stress, lysosomal stress, and chemotherapy (cisplatin), strongly implying that Hpa2 is regulated by conditions of stress. Furthermore, analyses of the Hpa2 gene promoter led to the identification of activating-transcription-factor 3 (ATF3) as a transcription factor that mediates Hpa2 induction by stress, thus revealing, for the first time, a molecular mechanism that underlies Hpa2 gene regulation. Induction of Hpa2 and ATF3 by conditions of stress that often accompany the rapid expansion of tumors is likely translated to improved survival of cancer patients.

OS06.3A LSD1-directed therapy curtails glioblastoma tumorigenicity by limiting the adaptation of tumor initiating cells to stressful environments

Abstract BACKGROUND Glioblastoma (GBM) is a fatal tumor whose aggressiveness, heterogeneity, therapy resistance and poor blood-brain-barrier penetration hinder the amelioration of the standard-of-care. Included in the GBM mass are the tumor initiating cells (TICs), representing the driver of GBM growth and relapse in virtue of their stem-like traits and therapy-resistance. Being constantly exposed to environmental stress, including nutrients deficiency, hypoxia and therapeutic insults, all GBM cells -and TICs in particular- have to be highly adaptive in order to survive. Thus, their ability to cope with stress could be targeted to curtail TICs maintenance and the whole GBM aggressiveness. The key of TICs adaptation relies, among the others, on their epigenetic plasticity, hence encouraging epigenetic drugs testing. MATERIAL AND METHODS By exploiting patient-derived GBM TICs and orthotopic xenograft models, we tested the antitumorigenic features of a novel, selective, orally bioavailable and brain-penetrant Lysine-specific histone demethylase 1 inhibitor (LSD1i). We confirmed the specificity of its effects by LSD1 genetic targeting. A combination of RNA-seq, Chromatin Immunoprecipitation(ChIP)-seq, Mass Spectrometry and reverse genetic experiments unraveled LSD1 molecular players in GBM TICs. RESULTS We identified LSD1 as a druggable target in human GBM: LSD1i treatment, mirrored by LSD1 genetic targeting, impairs growth, viability, stem-like traits and in vivo tumorigenicity of GBM TICs. Mechanistically, LSD1 is crucial for the expression of the activating transcription factor 4 (ATF4), which coordinates the integrated stress response (ISR) to manage stressful stimuli as nutrient deprivation and endoplasmic reticulum stress. By mimicking these stress cues in vitro, we found that LSD1i triggers a delayed but unabated ATF4 translation which provokes an over-lasting ISR, eventually culminating in GBM TICs apoptosis. Lastly, LSD1 demethylase activity is dispensable for ATF4 induction. Rather, LSD1i exerts its anti-tumorigenic potential by interfering with LSD1 scaffolding function in GBM TICs. CONCLUSION LSD1-directed therapy is likely a promising strategy to hinder GBM. By sensitizing GBM TICs to stress, LSD1i endangers the GBM TICs pool. The effectiveness of LSD1i administration in different patient-derived GBM TICs and xenografts, regardless of their molecular profile, places a strong rationale toward the clinical translation of this approach for GBM management. FUNDING Italian association for Cancer Research (AIRC) and Italian Ministry of Health

Insulin-induced genes INSIG1 and INSIG2 mediate oxysterol-dependent activation of the PERK–eIF2α–ATF4 axis

Insulin-induced genes (INSIGs) encode endoplasmic reticulum–resident proteins that regulate intracellular cholesterol metabolism. Oxysterols are oxygenated derivatives of cholesterol, some of which orchestrate lipid metabolism via interaction with INSIGs. Recently, it was reported that expression of activating transcription factor-4 (ATF4) was induced by certain oxysterols; the precise of mechanism is unclear. Herein, we show that INSIGs mediate ATF4 upregulation upon interaction with oxysterol. Oxysterols that possess a high affinity for INSIG, such as 27- and 25-hydroxycholesterol (25HC), markedly induced the increase of ATF4 protein when compared with other oxysterols. In addition, ATF4 upregulation by these oxysterols was attenuated in INSIG1/2-deficient Chinese hamster ovary cells and recovered by either INSIG1 or INSIG2 rescue. Mechanistic studies revealed that the binding of 25HC to INSIG is critical for increased ATF4 protein via activation of protein kinase RNA-activated–like ER kinase and eukaryotic translation initiation factor 2α. Knockout of INSIG1 or INSIG2 in human hepatoma Huh7 cells attenuated ATF4 protein upregulation, indicating that only one of the endogenous INSIGs, unlike overexpression of intrinsic INSIG1 or INSIG2, was insufficient for ATF4 induction. Furthermore, ATF4 proactively upregulated the cell death–inducible gene expression, such as Chop, Chac1, and Trb3, thereby markedly reducing cell viability with 25HC. These findings support a model whereby that INSIGs sense an increase in oxysterol in the endoplasmic reticulum and induce an increase of ATF4 protein via the protein kinase RNA-activated–like ER kinase–eukaryotic translation initiation factor 2α pathway, thereby promoting cell death.

Activating transcription factor 4 mediates adaptation of human glioblastoma cells to hypoxia and temozolomide

The integrated stress response (ISR) is a central cellular adaptive program that is activated by diverse stressors including ER stress, hypoxia and nutrient deprivation to orchestrate responses via activating transcription factor 4 (ATF4). We hypothesized that ATF4 is essential for the adaptation of human glioblastoma (GB) cells to the conditions of the tumor microenvironment and is contributing to therapy resistance against chemotherapy. ATF4 induction in GB cells was modulated pharmacologically and genetically and investigated in the context of temozolomide treatment as well as glucose and oxygen deprivation. The relevance of the ISR was analyzed by cell death and metabolic measurements under conditions to approximate aspects of the GB microenvironment. ATF4 protein levels were induced by temozolomide treatment. In line, ATF4 gene suppressed GB cells (ATF4sh) displayed increased cell death and decreased survival after temozolomide treatment. Similar results were observed after treatment with the ISR inhibitor ISRIB. ATF4sh and ISRIB treated GB cells were sensitized to hypoxia-induced cell death. Our experimental study provides evidence for an important role of ATF4 for the adaptation of human GB cells to conditions of the tumor microenvironment characterized by low oxygen and nutrient availability and for the development of temozolomide resistance. Inhibiting the ISR in GB cells could therefore be a promising therapeutic approach.

Abstract 2325: Hypoxia-mediated ATF4 induction promotes survival in detached conditions in metastatic murine mammary cancer cells

Abstract Regions of hypoxia are common in solid tumors and drive changes in gene expression that increase risk of cancer metastasis. Tumor cells must respond to the stress of hypoxia by activating genes to modify cell metabolism and antioxidant response to improve survival. The goal of the current studies was to determine the effect of hypoxia on cell metabolism and markers of oxidative stress in non-metastatic M-Wnt compared to metastatic metM-Wntlung murine mammary cancer cell lines. We show that hypoxia increased expression of genes involved in antioxidant response in metastatic compared to non-metastatic cells, including glutamate cysteine ligase catalytic and modifier subunits and malic enzyme 1. Further, hypoxia induces a greater suppression of glutamine to glutamate conversion in metastatic cells (15% in metastatic cells compared to 9% in non-metastatic cells). Interestingly, hypoxia increased the mRNA level of the transaminase glutamic pyruvic transaminase 2 (Gpt2, 7.7-fold) only in metM-Wntlung cells. The change in Gpt2 expression was accompanied by transcriptional (4.2-fold) and translational (6.5-fold) induction of the integrated stress response effector protein activating transcription factor 4 (ATF4), which mediated survival of hypoxic metastatic cells in detached conditions, as shown by genetic ATF4 depletion. These data indicate that more aggressive, metastatic cancer cells rely on metabolic reprogramming and induction of antioxidant defense, including activation of ATF4, in hypoxia for survival in detached conditions. Citation Format: Violet A. Kiesel, Stephen D. Hursting, Dorothy Teegarden. Hypoxia-mediated ATF4 induction promotes survival in detached conditions in metastatic murine mammary cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2325.

199-LB: ATF4 Deletion in Brown Adipose Tissue Impairs Adaptive Thermogenesis but Attenuates Diet-Induced Insulin Resistance

Short-term cold exposure induces the integrated stress response (ISR) and its main effector, the activating transcription factor 4 (ATF4), in brown adipose tissue (BAT). Furthermore, induction of ATF4 in BAT in response to mitochondrial stress is required for systemic metabolic adaptations mediated by BATokine secretion. To test the hypothesis that ATF4 in BAT is required for cold-induced thermogenesis and to promote BATokine secretion, we selectively deleted ATF4 in BAT by crossing Atf4 floxed mice with mice harboring the Cre recombinase under the control of the Ucp1 promoter (ATF4 BAT KO). KO mice experienced a significant reduction in core body temperature after 3 days of cold exposure relative to wild type (WT) mice. Impaired thermogenesis correlated with reduced BAT mass, and decreased UCP1 and tyrosine hydroxylase protein levels. In addition, KO mice had impaired cold-induced browning of white adipose tissue (WAT) and reduced expression of fibroblast growth factor 21 (Fgf21) and growth and differentiation factor 15 (Gdf15), BATokines shown to be induced in response to BAT thermogenic activation. Surprisingly, although KO mice had equivalent weight gain and fat mass relative to WT mice after 12 weeks of high-fat feeding, glucose and insulin intolerance were ameliorated, and fasting insulin levels were significantly reduced, indicating increased insulin sensitivity. Improved glucose clearance correlated with increased levels of glucose transporters 1 and 4 (GLUT1 and GLUT4) in BAT, but not in WAT or skeletal muscle. Taken together, our data indicate that Atf4 expression is required for full thermogenic activation of BAT and to regulate FGF21 and GDF15 release as BATokines, likely coordinating BAT thermogenesis with systemic adaptations. Finally, our data suggest that in diet-induced obesity, ATF4 downregulation may increase glucose utilization in BAT, contributing to improved glucose homeostasis and insulin sensitivity. Disclosure S. Bjorkman: None. A. A. Marti: None. E. T. Weatherford: None. L. M. García-peña: None. M. J. Potthoff: None. E. D. Abel: Board Member; Self; MyoKardia, Other Relationship; Self; AstraZeneca. R. Pereira: None. Funding National Institutes of Health (R01DK125405)

OPA1 deletion in brown adipose tissue improves thermoregulation and systemic metabolism via FGF21.

Adrenergic stimulation of brown adipocytes alters mitochondrial dynamics, including the mitochondrial fusion protein optic atrophy 1 (OPA1). However, direct mechanisms linking OPA1 to brown adipose tissue (BAT) physiology are incompletely understood. We utilized a mouse model of selective OPA1 deletion in BAT (OPA1 BAT KO) to investigate the role of OPA1 in thermogenesis. OPA1 is required for cold-induced activation of thermogenic genes in BAT. Unexpectedly, OPA1 deficiency induced fibroblast growth factor 21 (FGF21) as a BATokine in an activating transcription factor 4 (ATF4)-dependent manner. BAT-derived FGF21 mediates an adaptive response by inducing browning of white adipose tissue, increasing resting metabolic rates, and improving thermoregulation. However, mechanisms independent of FGF21, but dependent on ATF4 induction, promote resistance to diet-induced obesity in OPA1 BAT KO mice. These findings uncover a homeostatic mechanism of BAT-mediated metabolic protection governed in part by an ATF4-FGF21 axis, which is activated independently of BAT thermogenic function.

O‐GlcNAc controls mitochondrial quality control mechanisms and mitochondrial stress response proteins

The accumulation of dysfunctional mitochondria is closely associated with Alzheimer Disease (AD). Mitochondrial dysfunction is an early feature of the disease and impaired turnover of damaged mitochondria increases cellular toxicity leading to neuronal damage. Importantly, AD exhibited a marked reduction in global post-translational modification β-N-acetylglucosamine (O-GlcNAc) level in post-mortem human brain tissue with AD and triple transgenic mouse model of AD (3xTg-AD). O-GlcNAc is a ubiquitous single sugar modification found on nuclear, cytoplasmic, and mitochondrial proteins. Cells maintain a homeostatic level of O-GlcNAc by cycling the addition and removal of the sugar by O-GlcNAc transferase (OGT) or O-GlcNAcase (OGA) respectively. Previously, we reported that sustained alterations in O-GlcNAcylation by pharmacologically or genetically manipulation impaired mitochondrial function. Here, we have established an essential role for O-GlcNAc in inducing the expression of several mitochondrial quality control mechanisms using cell lines and in vitro models of AD (differentiated organoids isolated from AD and control patients). We established that sustained alterations in O-GlcNAcylation by pharmacological inhibiting OGA induces the expression of two mitochondrial stress response proteins Activating Transcription Factor 4 (ATF4) and heat shock protein family A (GRP75). ATF4 is a key transcription factor to initiate mitophagy (mitochondria-selective autophagy). Consistence with the ATF4 induction, several mitophagy proteins are increased in the sustained alterations in O-GlcNAcylation. This post-translational modification plays crucial role in AD and restoring mitochondrial quality control mechanisms is important to prevent the accumulation of dysfunctional mitochondria.

Mitochondrial translation inhibition triggers ATF4 activation, leading to integrated stress response but not to mitochondrial unfolded protein response.

Mitochondrial–nuclear communication, known as retrograde signaling, is important for regulating nuclear gene expression in response to mitochondrial dysfunction. Previously, we have found that p32/C1qbp-deficient mice, which have a mitochondrial translation defect, show endoplasmic reticulum (ER) stress response and integrated stress response (ISR) gene expression in the heart and brain. However, the mechanism by which mitochondrial translation inhibition elicits these responses is not clear. Among the transcription factors that respond to mitochondrial stress, activating transcription factor 4 (ATF4) is a key transcription factor in the ISR. Herein, chloramphenicol (CAP), which inhibits mitochondrial DNA (mtDNA)-encoded protein expression, induced eukaryotic initiation factor 2 α subunit (eIF2α) phosphorylation and ATF4 induction, leading to ISR gene expression. However, the expression of the mitochondrial unfolded protein response (mtUPR) genes, which has been shown in Caenorhabditis elegans, was not induced. Short hairpin RNA-based knockdown of ATF4 markedly inhibited the CAP-induced ISR gene expression. We also observed by ChIP analysis that induced ATF4 bound to the promoter region of several ISR genes, suggesting that mitochondrial translation inhibition induces ISR gene expression through ATF4 activation. In the present study, we showed that mitochondrial translation inhibition induced the ISR through ATF4 activation rather than the mtUPR.