Kynurenine 3-monooxygenase limits de novo NAD+ synthesis through dietary tryptophan in renal proximal tubule epithelial cell models.

Nicotinamide adenine dinucleotide (NAD+) is a pivotal coenzyme, essential for cellular reactions, metabolism, and mitochondrial function. Depletion of kidney NAD+ levels and reduced de novo NAD+ synthesis through the tryptophan-kynurenine pathway are linked to acute kidney injury (AKI), whereas augmenting NAD+ shows promise in reducing AKI. We investigated de novo NAD+ biosynthesis using in vitro, ex vivo, and in vivo models to understand its role in AKI. Two-dimensional (2-D) cultures of human primary renal proximal tubule epithelial cells (RPTECs) and HK-2 cells showed limited de novo NAD+ synthesis, likely due to low pathway enzyme gene expression. Using three-dimensional (3-D) spheroid culture model improved the expression of tubular-specific markers and enzymes involved in de novo NAD+ synthesis. However, de novo NAD+ synthesis remained elusive in the 3-D spheroid culture, regardless of injury conditions. Further investigation revealed that 3-D cultured cells could not metabolize tryptophan (Trp) beyond kynurenine (KYN). Intriguingly, supplementation of 3-hydroxyanthranilic acid into RPTEC spheroids was readily incorporated into NAD+. In a human precision-cut kidney slice (PCKS) ex vivo model, de novo NAD+ synthesis was limited due to substantially downregulated kynurenine 3-monooxygenase (KMO), which is responsible for KYN to 3-hydroxykynurenine conversion. KMO overexpression in RPTEC 3-D spheroids successfully reinstated de novo NAD+ synthesis from Trp. In addition, in vivo study demonstrated that de novo NAD+ synthesis is intact in the kidney of the healthy adult mice. Our findings highlight disrupted tryptophan-kynurenine NAD+ synthesis in in vitro cellular models and an ex vivo kidney model, primarily attributed to KMO downregulation.NEW & NOTEWORTHY Nicotinamide adenine dinucleotide (NAD+) is essential in regulating mitochondrial function. Reduced NAD+ synthesis through the de novo pathway is associated with acute kidney injury (AKI). Our study reveals a disruption in de novo NAD+ synthesis in proximal tubular models, but not in vivo, attributed to downregulation of enzyme kynurenine 3-monooxygenase (KMO). These findings highlight a crucial role of KMO in governing de novo NAD+ biosynthesis within the kidney, shedding light on potential AKI interventions.

Pan AMPK Activation Protects Tubules in Rat Ischemic Acute Kidney Injury.

Acute Kidney Injury (AKI) is characterized by an abrupt decline in kidney function and has been associated with excess risks of death, kidney disease progression, and cardiovascular events. The kidney has a high energetic demand with mitochondrial health being essential to renal function and damaged mitochondria has been reported across AKI subtypes. 5' adenosine monophosphate-activated protein kinase (AMPK) activation preserves cellular energetics through improvement of mitochondrial function and biogenesis when ATP levels are low such as under ischemia-induced AKI. We developed a selective potent small molecule pan AMPK activator, compound 1, and tested its ability to increase AMPK activity and preserve kidney function during ischemia/reperfusion injury in rats. A single administration of 1 caused sustained activation of AMPK for at least 24 hours, protected against acute tubular necrosis, and reduced clinical markers of tubular injury such as NephroCheck and Fractional Excretion of Sodium (FENa). Reduction in plasma creatinine and increased Glomerular Filtration Rate (GFR) indicated preservation of kidney function. Surprisingly, we observed a strong diuretic effect of AMPK activation associated with natriuresis both with and without AKI. Our findings demonstrate that activation of AMPK leads to protection of tubular function under hypoxic/ischemic conditions which holds promise as a potential novel therapeutic approach for AKI. Significance Statement No approved pharmacological therapies currently exist for acute kidney injury. We developed Compound 1 which dose-dependently activated AMPK in the kidney and protected kidney function and tubules after ischemic renal injury in the rat. This was accompanied by natriuresis in injured as well as uninjured rats.

GPR40-Mediated Gα12 Activation by Allosteric Full Agonists Highly Efficacious at Potentiating Glucose-Stimulated Insulin Secretion in Human Islets.

GPR40 is a clinically validated molecular target for the treatment of diabetes. Many GPR40 agonists have been identified to date, with the partial agonist fasiglifam (TAK-875) reaching phase III clinical trials before its development was terminated due to off-target liver toxicity. Since then, attention has shifted toward the development of full agonists that exhibit superior efficacy in preclinical models. Full agonists bind to a distinct binding site, suggesting conformational plasticity and a potential for biased agonism. Indeed, it has been suggested that alternative pharmacology may be required for meaningful efficacy. In this study, we described the discovery and characterization of Compound A, a newly identified GPR40 allosteric full agonist highly efficacious in human islets at potentiating glucose-stimulated insulin secretion. We compared Compound A-induced GPR40 activity to that induced by both fasiglifam and AM-1638, another allosteric full agonist previously reported to be highly efficacious in preclinical models, at a panel of G proteins. Compound A was a full agonist at both the Gαq and Gαi2 pathways, and in contrast to fasiglifam Compound A also induced Gα12 coupling. Compound A and AM-1638 displayed similar activity at all pathways tested. The Gα12/Gα13-mediated signaling pathway has been linked to protein kinase D activation as well as actin remodeling, well known to contribute to the release of insulin vesicles. Our data suggest that the pharmacology of GPR40 is complex and that Gα12/Gα13-mediated signaling, which may contribute to GPR40 agonists therapeutic efficacy, is a specific property of GPR40 allosteric full agonists.

Discovery of novel benzo[b]thiophene tetrazoles as non-carboxylate GPR40 agonists.

GPR40 partial agonism is a promising new mechanism for the treatment of type 2 diabetes mellitus with clinical proof of concept. Most of the GPR40 agonists in the literature have a carboxylic acid functional group, which may pose a risk for idiosyncratic drug toxicity. A novel series of GPR40 agonists containing a tetrazole as a carboxylic acid bioisostere was identified. This series of compounds features a benzo[b]thiophene as the center ring, which is prone to oxidation during phase 1 metabolism. Following SAR optimization targeting GPR40 agonist activity and intrinsic clearance in microsomes (human and rat), potent and metabolically stable compounds were selected for in vivo evaluation. The compounds are efficacious at lowering blood glucose in a SD rat oGTT model.

Discovery of a novel potent GPR40 full agonist.

Compound 12 is a GPR40 agonist that realizes the full magnitude of efficacy possible via GPR40 receptor agonism. In vitro and in vivo studies demonstrated superior glucose lowering by 12 compared to fasiglifam (TAK-875), in a glucose dependent manner. The enhanced efficacy observed with the full agonist 12 was associated with both direct and indirect stimulation of insulin secretion.

Metabolic tracing of monoacylglycerol acyltransferase-2 activity in vitro and in vivo.

Monoacylglycerol acyltransferase 2 (MGAT2) catalyzes the synthesis of diacylglycerol (DAG) from free fatty acids (FFA) and sn-monoacylglycerol (MG), the two major hydrolysis products of dietary fat. To demonstrate MGAT2-mediated cellular activity of triglyceride (TG) synthesis, we utilized 1-oleoyl-glycerol-d5 as a substrate to trace MGAT2-driven 1-oleoyl-glycerol-d5 incorporation into TG in HEK293 cells stably expressing human MGAT2. The oleoyl-glycerol-d5 incorporated major TG species were then quantified by liquid chromatography electrospray ionization tandem mass spectrometry (LC/ESI/MS/MS) in a 96-well format. Conventional MGAT2 target-engagement in vivo assays measure the elevation of total plasma TG by orally dosing a bolus of TG oil. We developed a novel LC/ESI/MS/MS-based fat absorption assay to assess the ability of MGAT2 inhibitors to inhibit fat absorption in CD1 mice by a meal tolerance test consisting of a mixture of liquid Boost plus® and 0.59 g/kg U13C-TG oil. The newly resynthesized plasma heavy TGs containing three 13C in the glycerol backbone and two U13C-acyl-chains, which represented the digested, absorbed and resynthesized TGs, were then quantitated by LC/ESI/MS/MS. With this assay, we identified a potent MGAT2 inhibitor that blocked MGAT2-mediated activity in vitro and in vivo. The use of 1-oleoyl-glycerol-d5 and U13C-TG oil followed by LC/ESI/MS/MS detection of stable-isotopic labeled DAG, TG, or glycerol provides a wide range of applications to study pathophysiological regulation of the monoacylglycerol pathway and MGAT2 activity.

Acute hemodynamic and renal effects of glucagon-like peptide 1 analog and dipeptidyl peptidase-4 inhibitor in rats.

BACKGROUND: Glucagon-like peptide 1 (GLP-1) analogs and dipeptidyl peptidase-4 (DPP4) inhibitors are a newer class of antidiabetics named as incretin-based therapy. In addition to the homeostatic control of glucose, the incretin-based therapy has shown beneficial effects on the cardiovascular system in preclinical and clinical studies. However, there is limited information on their renal effects. To this end, we assessed the acute hemodynamic and renal effects of a GLP-1 analog, Liraglutide, and a DPP4 inhibitor, MK-0626. METHODS: Experiments were performed in anesthetized male Sprague-Dawley rats. Three ascending doses of Liraglutide (3, 9, and 27 nmol/kg/h) or MK-0626 (1 mg/kg) with or without GLP-1 peptide (2.4, 4.8, or 9.6 pmol/kg/min) were administered. Blood pressure (BP) and heart rate (HR) were recorded from an indwelling catheter. Glomerular filtration rate (GFR) and renal blood flow (RBF) were assessed by inulin and para-aminohippurate clearance, respectively. Renal excretory function was assessed in metabolic studies. RESULTS: Both Liraglutide and MK-0626 plus GLP-1 evoked significant diuretic and natriuretic responses and increased GFR. MK-0626 alone increased RBF. Liraglutide at 27 nmol//kg/h and MK-0626 plus GLP-1 at 9.6 pmol/kg/min also increased HR, whereas BP was not affected. CONCLUSION: The results of the present study demonstrated that a GLP-1 analog and a DPP4 inhibitor may have beneficial effects on renal sodium and water handling. Additionally, the DPP4 inhibitor, MK-0626, favorably affects renal hemodynamics by increasing RBF. However, exceedingly high levels of GLP-1 receptor agonists may adversely affect the cardiovascular system in acute setting, as demonstrated by an acute increase in HR.

Development of a neuromedin U-human serum albumin conjugate as a long-acting candidate for the treatment of obesity and diabetes. Comparison with the PEGylated peptide.

Neuromedin U (NMU) is an endogenous peptide implicated in the regulation of feeding, energy homeostasis, and glycemic control, which is being considered for the therapy of obesity and diabetes. A key liability of NMU as a therapeutic is its very short half-life in vivo. We show here that conjugation of NMU to human serum albumin (HSA) yields a compound with long circulatory half-life, which maintains full potency at both the peripheral and central NMU receptors. Initial attempts to conjugate NMU via the prevalent strategy of reacting a maleimide derivative of the peptide with the free thiol of Cys34 of HSA met with limited success, because the resulting conjugate was unstable in vivo. Use of a haloacetyl derivative of the peptide led instead to the formation of a metabolically stable conjugate. HSA-NMU displayed long-lasting, potent anorectic, and glucose-normalizing activity. When compared side by side with a previously described PEG conjugate, HSA-NMU proved superior on a molar basis. Collectively, our results reinforce the notion that NMU-based therapeutics are promising candidates for the treatment of obesity and diabetes.

Brain glucose metabolism controls the hepatic secretion of triglyceride-rich lipoproteins.

Increased production of very low-density lipoprotein (VLDL) is a critical feature of the metabolic syndrome. Here we report that a selective increase in brain glucose lowered circulating triglycerides (TG) through the inhibition of TG-VLDL secretion by the liver. We found that the effect of glucose required its conversion to lactate, leading to activation of ATP-sensitive potassium channels and to decreased hepatic activity of stearoyl-CoA desaturase-1 (SCD1). SCD1 catalyzed the synthesis of oleyl-CoA from stearoyl-CoA. Curtailing the liver activity of SCD1 was sufficient to lower the hepatic levels of oleyl-CoA and to recapitulate the effects of central glucose administration on VLDL secretion. Notably, portal infusion of oleic acid restored hepatic oleyl-CoA to control levels and negated the effects of both central glucose and SCD1 deficiency on TG-VLDL secretion. These central effects of glucose (but not those of lactate) were rapidly lost in diet-induced obesity. These findings indicate that a defect in brain glucose sensing could play a critical role in the etiology of the metabolic syndrome.

VEPTP inhibition with an extracellular domain targeting antibody did not restore albuminuria in a mouse model of diabetic kidney disease.

Diabetic kidney disease (DKD) is the leading cause of end-stage kidney disease. DKD is a heterogeneous disease with complex pathophysiology where early endothelial dysfunction is associated with disease progression. The Tie2 receptor and Angiopoietin 1 and 2 ligands are critical for maintaining endothelial cell permeability and integrity. Tie2 signaling is negatively regulated by the endothelial specific transmembrane receptor Vascular Endothelial Protein Tyrosine Phosphatase (VEPTP). Genetic deletion of VEPTP protects from hypertension and diabetes induced renal injury in a mouse model of DKD. Here, we show that VEPTP inhibition with an extracellular domain targeting VEPTP antibody induced Tie2 phosphorylation and improved VEGF-A induced vascular permeability both in vitro and in vivo. Treatment with the VEPTP blocking antibody decreased the renal expression of endothelial activation markers (Angpt2, Edn1, and Icam1) but failed to improve kidney function in db/db uninephrectomized ReninAAV DKD mice.

A PEGylated analog of the gut hormone oxyntomodulin with long-lasting antihyperglycemic, insulinotropic and anorexigenic activity.

Peptide agonists of the glucagon-like peptide 1 (GLP-1) receptor (GLP1R) are rapidly gaining favor as antidiabetic agents, since in addition to increasing glucose-dependent insulin secretion, they also cause weight loss. Oxyntomodulin (OXM), a natural peptide with sequence homology to both glucagon and GLP-1, has glucose-lowering activity in rodents and anorectic activity in rodents and humans, but its clinical utility is limited by a short circulatory half-life due to rapid renal clearance and degradation by dipeptidyl peptidase IV (DPP-IV). Here, we describe the development of a novel DPP-IV-resistant, long-acting GLP1R agonist, based on derivatization of a suitably chosen OXM analog with high molecular weight polyethylene glycol (PEG) ('PEGylation'). PEG-OXM exerts an anti-hyperglycemic effect in diet-induced obese (DIO) mice in a glucose-dependent manner, with a maximally efficacious dose of 0.1mg/kg, and reduces food intake and body weight with a minimally efficacious dose of 1mg/kg. If this pharmacology is recapitulated in patients with type 2 diabetes, these results indicate PEG-OXM as a potential novel once-weekly GLP-1 mimetic with both glucose-lowering activity and weight loss efficacy.

G protein-coupled receptors and obesity.

G protein-coupled receptors (GPCRs) have emerged as important drug targets for various chronic diseases, including obesity and diabetes. Obesity is a complex chronic disease that requires long term management predisposing to type 2 diabetes, heart disease, and some cancers. The therapeutic landscape for GPCR as targets of anti-obesity medications has undergone significant changes with the approval of semaglutide, the first peptide glucagon like peptide 1 receptor agonist (GLP-1RA) achieving double digit weight loss (≥10%) and cardiovascular benefits. The enhanced weight loss, with the expected beneficial effect on obesity-related complications and reduction of major adverse cardiovascular events (MACE), has propelled the commercial opportunity for the obesity market leading to new players entering the space. Significant progress has been made on approaches targeting GPCRs such as single peptides that simultaneously activate GIP and/or GCGR in addition to GLP1, oral tablet formulation of GLP-1, small molecules nonpeptidic oral GLP1R and fixed-dose combination as well as add-on therapy for patients already treated with a GLP-1 agonist.

Metabolism-based approaches for autosomal dominant polycystic kidney disease.

Autosomal Dominant Polycystic Kidney Disease (ADPKD) leads to end stage kidney disease (ESKD) through the development and expansion of multiple cysts throughout the kidney parenchyma. An increase in cyclic adenosine monophosphate (cAMP) plays an important role in generating and maintaining fluid-filled cysts because cAMP activates protein kinase A (PKA) and stimulates epithelial chloride secretion through the cystic fibrosis transmembrane conductance regulator (CFTR). A vasopressin V2 receptor antagonist, Tolvaptan, was recently approved for the treatment of ADPKD patients at high risk of progression. However additional treatments are urgently needed due to the poor tolerability, the unfavorable safety profile, and the high cost of Tolvaptan. In ADPKD kidneys, alterations of multiple metabolic pathways termed metabolic reprogramming has been consistently reported to support the growth of rapidly proliferating cystic cells. Published data suggest that upregulated mTOR and c-Myc repress oxidative metabolism while enhancing glycolytic flux and lactic acid production. mTOR and c-Myc are activated by PKA/MEK/ERK signaling so it is possible that cAMPK/PKA signaling will be upstream regulators of metabolic reprogramming. Novel therapeutics opportunities targeting metabolic reprogramming may avoid or minimize the side effects that are dose limiting in the clinic and improve on the efficacy observed in human ADPKD with Tolvaptan.

Impaired regulation of hepatic glucose production in mice lacking the forkhead transcription factor Foxo1 in liver.

The hallmark of type 2 diabetes is excessive hepatic glucose production. Several transcription factors and coactivators regulate this process in cultured cells. But gene ablation experiments have yielded few clues as to the physiologic mediators of this process in vivo. We show that inactivation of the gene encoding forkhead protein Foxo1 in mouse liver results in 40% reduction of glucose levels at birth and 30% reduction in adult mice after a 48 hr fast. Gene expression and glucose clamp studies demonstrate that Foxo1 ablation impairs fasting- and cAMP-induced glycogenolysis and gluconeogenesis. Pgc1alpha is unable to induce gluconeogenesis in Foxo1-deficient hepatocytes, while the cAMP response is significantly blunted. Conversely, Foxo1 deletion in liver curtails excessive glucose production caused by generalized ablation of insulin receptors and prevents neonatal diabetes and hepatosteatosis in insulin receptor knockout mice. The data provide a unifying mechanism for regulation of hepatic glucose production by cAMP and insulin.

Differential effects of oxyntomodulin and GLP-1 on glucose metabolism.

Glucagon-like peptide-1 (GLP-1) and oxyntomodulin (OXM) are peptide hormones secreted postprandially from the gut that stimulate insulin secretion in a glucose-dependent manner. OXM activates both the GLP-1 receptor (GLP1R) and the glucagon receptor (GCGR). It has been suggested that OXM acutely modulates glucose metabolism solely through GLP1R agonism. Because OXM activates the GLP1R with lower affinity than GLP-1, we generated a peptide analog (Q→E, OXMQ3E) that does not exhibit glucagon receptor agonist activity but retains the same affinity as OXM for GLP1R. We compared the effects of OXM and OXMQ3E in a glucose tolerance test and, to better characterize the effect on glucose metabolism, we performed controlled infusions of OXM or OXMQ3E during a hyperglycemic clamp performed in wild-type, Glp1r(-/-), and Gcgr(-/-) mice. Our findings show that OXM, but not OXMQ3E, activates the GCGR in vivo. Second, OXM and OXMQ3E improve glucose tolerance following an acute glucose challenge and during a hyperglycemic clamp in mice. Finally, OXM infusion during a glucose clamp reduces the glucose infusion rate (GIR) despite a simultaneous increase in insulin levels in Glp1r(-/-) mice, whereas OXM and OXMQ3E increase GIR to a similar extent in Gcgr(-/-) mice. In conclusion, activation of the GCGR seems to partially attenuate the acute beneficial effects on glucose and contributes to the insulinotropic action of oxyntomodulin.

PEGylation of Neuromedin U yields a promising candidate for the treatment of obesity and diabetes.

Neuromedin U (NMU) is an endogenous peptide, whose role in the regulation of feeding and energy homeostasis is well documented. Two NMU receptors have been identified: NMUR1, expressed primarily in the periphery, and NMUR2, expressed predominantly in the brain. We recently demonstrated that acute peripheral administration of NMU exerts potent but acute anorectic activity and can improve glucose homeostasis, with both actions mediated by NMUR1. Here, we describe the development of a metabolically stable analog of NMU, based on derivatization of the native peptide with high molecular weight poly(ethylene) glycol (PEG) ('PEGylation'). PEG size, site of attachment, and conjugation chemistry were optimized, to yield an analog which displays robust and long-lasting anorectic activity and significant glucose-lowering activity in vivo. Studies in NMU receptor-deficient mice showed that PEG-NMU displays an expanded pharmacological profile, with the ability to engage NMUR2 in addition to NMUR1. In light of these data, PEGylated derivatives of NMU represent promising candidates for the treatment of obesity and diabetes.

Hypothalamic sensing of circulating fatty acids is required for glucose homeostasis.

Increased glucose production is a hallmark of type 2 diabetes and alterations in lipid metabolism have a causative role in its pathophysiology. Here we postulate that physiological increments in plasma fatty acids can be sensed within the hypothalamus and that this sensing is required to balance their direct stimulatory action on hepatic gluconeogenesis. In the presence of physiologically-relevant increases in the levels of plasma fatty acids, negating their central action on hepatic glucose fluxes through (i) inhibition of the hypothalamic esterification of fatty acids, (ii) genetic deletion (Sur1-deficient mice) of hypothalamic K(ATP) channels or pharmacological blockade (K(ATP) blocker) of their activation by fatty acids, or (iii) surgical resection of the hepatic branch of the vagus nerve led to a marked increase in liver glucose production. These findings indicate that a physiological elevation in circulating lipids can be sensed within the hypothalamus and that a defect in hypothalamic lipid sensing disrupts glucose homeostasis.

A FFAR1 full agonist restores islet function in models of impaired glucose-stimulated insulin secretion and diabetic non-human primates.

The free fatty acid receptor 1 (FFAR1/GPR40) mediates fatty acid-induced insulin secretion from pancreatic ß-cells. At least 3 distinct binding sites exist on the FFAR1 receptor and numerous synthetic ligands have been investigated for their anti-diabetic actions. Fasiglifam, binds to site-1 and stimulates intra-cellular calcium release and improves glycemic control in diabetic patients. Recently, small molecule FFAR1 agonists were discovered which bind to site-3, stimulating both intra-cellular calcium and cAMP, resulting in insulin and glucagon-like peptide-1 (GLP-1) secretion. The ability of our site-3 FFAR1 agonist (compound A) to control blood glucose was evaluated in spontaneously diabetic cynomolgus monkeys during an oral glucose tolerance test. In type-2 diabetic (T2D) animals, significant reductions in blood glucose and insulin were noted. To better understand the mechanism of these in vivo findings, we evaluated the effect of compound A in islets under several conditions of dysfunction. First, healthy human and non-human primate islets were treated with compound A and showed potentiation of insulin and glucagon secretion from both species. Next, we determined glucose-responsive insulin secretion under gluco-lipotoxic conditions and from islets isolated from type-2 diabetic humans. Despite a dysfunctional phenotype that failed to secrete insulin in response to glucose, site-3 FFAR1 agonism not only enhanced insulin secretion, but restored glucose responsiveness across a range of glucose concentrations. Lastly, we treated ex vivo human islets chronically with a sulfonylurea to induce secondary beta-cell failure. Again, this model showed reduced glucose-responsive insulin secretion that was restored and potentiated by site-3 FFAR1 agonism. Together these data suggest a mechanism for FFAR1 where agonists have direct effects on islet hormone secretion that can overcome a dysfunctional T2D phenotype. These unique characteristics of FFAR1 site-3 agonists make them an appealing potential therapy to treat type-2 diabetes.

The mitochondrial fission protein Drp1 in liver is required to mitigate NASH and prevents the activation of the mitochondrial ISR.

OBJECTIVE: The mitochondrial fission protein Drp1 was proposed to promote NAFLD, as inhibition of hepatocyte Drp1 early in life prevents liver steatosis induced by high-fat diet in mice. However, whether Drp1-knockdown in older mice can reverse established NASH is unknown. METHODS: N-acetylgalactosamine-siRNA conjugates, an FDA approved method to deliver siRNA selectively to hepatocytes, were used to knockdown hepatocyte-Drp1 in mice (NAG-Drp1si). NASH was induced in C57BL/6NTac mice by Gubra-Amylin-NASH diet (D09100310, 40% fat, 22% fructose and 2% cholesterol) and treatment with NAG-Drp1si was started at week 24 of diet. Circulating transaminases, liver histology, gene expression of fibrosis and inflammation markers, and hydroxyproline synthesis determined NASH severity. Liver NEFA and triglycerides were quantified by GC/MS. Mitochondrial function was determined by respirometry. Western blots of Oma1, Opa1, p-eIf2α, as well as transcriptional analyses of Atf4-regulated genes determined ISR engagement. RESULTS: NAG-Drp1si treatment decreased body weight and induced liver inflammation in adult healthy mice. Increased hepatic Gdf15 production was the major contributor to body-weight loss caused by NAG-Drp1si treatment, as Gdf15 receptor deletion (Gfral KO) prevented the decrease in food intake and mitigated weight loss. NAG-Drp1si activated the Atf4-controlled integrated stress response (ISR) to increase hepatic Gdf15 expression. NAG-Drp1si in healthy mice caused ER stress and activated the mitochondrial protease Oma1, which are the ER and mitochondrial triggers that activate the Atf4-controlled ISR. Remarkably, induction of NASH was not sufficient to activate Oma1 in liver. However, NAG-Drp1si treatment was sufficient to activate Oma1 in adult mice with NASH, as well as exacerbating NASH-induced ER stress. Consequently, NAG-Drp1si treatment in mice with NASH led to higher ISR activation, exacerbated inflammation, fibrosis and necrosis. CONCLUSION: Drp1 mitigates NASH by decreasing ER stress, preventing Oma1 activation and ISR exacerbation. The elevation in Gdf15 actions induced by NAG-Drp1si might represent an adaptive response decreasing the nutrient load to liver when mitochondria are misfunctional. Our study argues against blocking Drp1 in hepatocytes to combat NASH.