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.

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.

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.

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.

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.

PNPLA3 downregulation exacerbates the fibrotic response in human hepatic stellate cells.

Non-alcoholic steatohepatitis (NASH) results, in part, from the interaction of metabolic derangements with predisposing genetic variants, leading to liver-related complications and mortality. The strongest genetic determinant is a highly prevalent missense variant in patatin-like phospholipase domain-containing protein 3 (PNPLA3 p.I148M). In human liver hepatocytes PNPLA3 localizes to the surface of lipid droplets where the mutant form is believed to enhance lipid accumulation and release of pro-inflammatory cytokines. Less is known about the role of PNPLA3 in hepatic stellate cells (HSCs). Here we characterized HSC obtained from patients carrying the wild type (n = 8 C/C) and the heterozygous (n = 6, C/G) or homozygous (n = 6, G/G) PNPLA3 I148M and investigated the effect of genotype and PNPLA3 downregulation on baseline and TGF-ß-stimulated fibrotic gene expression. HSCs from all genotypes showed comparable baseline levels of PNPLA3 and expression of the fibrotic genes α-SMA, COL1A1, TIMP1 and SMAD7. Treatment with TGF-ß increased PNPLA3 expression in all 3 genotypes (~2-fold) and resulted in similar stimulation of the expression of several fibrogenic genes. In primary human HSCs carrying wild-type (WT) PNPLA3, siRNA treatment reduced PNPLA3 mRNA by 79% resulting in increased expression of α-SMA, Col1a1, TIMP1, and SMAD7 in cells stimulated with TGF-ß. Similarly, knock-down of PNPLA3 in HSCs carrying either C/G or G/G genotypes resulted in potentiation of TGF-ß induced expression of fibrotic genes. Knockdown of PNPLA3 did not impact fibrotic gene expression in the absence of TGF-ß treatment. Together, these data indicate that the presence of the I148M PNPLA3 mutation in HSC has no effect on baseline activation and that downregulation of PNPLA3 exacerbates the fibrotic response irrespective of the genotype.

GRK Inhibition Potentiates Glucagon-Like Peptide-1 Action.

The glucagon-like peptide-1 receptor (GLP-1R) is a G-protein-coupled receptor (GPCR) whose activation results in suppression of food intake and improvement of glucose metabolism. Several receptor interacting proteins regulate the signaling of GLP-1R such as G protein-coupled receptor kinases (GRK) and ß-arrestins. Here we evaluated the physiological and pharmacological impact of GRK inhibition on GLP-1R activity leveraging small molecule inhibitors of GRK2 and GRK3. We demonstrated that inhibition of GRK: i) inhibited GLP-1-mediated ß-arrestin recruitment, ii) enhanced GLP-1-induced insulin secretion in isolated islets and iii) has additive effect with dipeptidyl peptidase 4 in mediating suppression of glucose excursion in mice. These findings highlight the importance of GRK to modulate GLP-1R function in vitro and in vivo. GRK inhibition is a potential therapeutic approach to enhance endogenous and pharmacologically stimulated GLP-1R signaling.

Persistent or Transient Human ß Cell Dysfunction Induced by Metabolic Stress: Specific Signatures and Shared Gene Expression with Type 2 Diabetes.

Pancreatic ß cell failure is key to type 2 diabetes (T2D) onset and progression. Here, we assess whether human ß cell dysfunction induced by metabolic stress is reversible, evaluate the molecular pathways underlying persistent or transient damage, and explore the relationships with T2D islet traits. Twenty-six islet preparations are exposed to several lipotoxic/glucotoxic conditions, some of which impair insulin release, depending on stressor type, concentration, and combination. The reversal of dysfunction occurs after washout for some, although not all, of the lipoglucotoxic insults. Islet transcriptomes assessed by RNA sequencing and expression quantitative trait loci (eQTL) analysis identify specific pathways underlying ß cell failure and recovery. Comparison of a large number of human T2D islet transcriptomes with those of persistent or reversible ß cell lipoglucotoxicity show shared gene expression signatures. The identification of mechanisms associated with human ß cell dysfunction and recovery and their overlap with T2D islet traits provide insights into T2D pathogenesis, fostering the development of improved ß cell-targeted therapeutic strategies.

Discovery of a GPR40 Superagonist: The Impact of Aryl Propionic Acid α-Fluorination.

GPR40 is a G-protein-coupled receptor which mediates fatty acid-induced glucose-stimulated insulin secretion from pancreatic beta cells and incretion release from enteroendocrine cells of the small intestine. GPR40 full agonists exhibit superior glucose lowering compared to partial agonists in preclinical species due to increased insulin and GLP-1 secretion, with the added benefit of promoting weight loss. In our search for potent GPR40 full agonists, we discovered a superagonist which displayed excellent in vitro potency and superior efficacy in the Gαs-mediated signaling pathway. Most synthetic GPR40 agonists have a carboxylic acid headgroup, which may cause idiosyncratic toxicities, including drug-induced-liver-injury (DILI). With a methyl group and a fluorine atom substituted at the α-C of the carboxylic acid group, 19 is not only highly efficacious in lowering glucose and body weight in rodent models but also has a low DILI risk due to its stable acylglucuronide metabolite.

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 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.

A comparative study of the binding properties, dipeptidyl peptidase-4 (DPP-4) inhibitory activity and glucose-lowering efficacy of the DPP-4 inhibitors alogliptin, linagliptin, saxagliptin, sitagliptin and vildagliptin in mice.

AIMS: Since 2006, DPP-4 inhibitors have become established therapy for the treatment of type 2 diabetes. Despite sharing a common mechanism of action, considerable chemical diversity exists amongst members of the DPP-4 inhibitor class, raising the question as to whether structural differences may result in differentiated enzyme inhibition and antihyperglycaemic activity. METHODS: We have compared the binding properties of the most commonly used inhibitors and have investigated the relationship between their inhibitory potency at the level of the enzyme and their acute glucose-lowering efficacy. RESULTS: Firstly, using a combination of published crystal structures and in-house data, we demonstrated that the binding site utilized by all of the DPP-4 inhibitors assessed was the same as that used by neuropeptide Y, supporting the hypothesis that DPP-4 inhibitors are able to competitively inhibit endogenous substrates for the enzyme. Secondly, we ascertained that the enzymatic cleft of DPP-4 is a relatively large cavity which displays conformational flexibility to accommodate structurally diverse inhibitor molecules. Finally, we found that for all inhibitors, irrespective of their chemical structure, the inhibition of plasma DPP-4 enzyme activity correlates directly with acute plasma glucose lowering in mice. CONCLUSION: The common binding site utilized by different DPP-4 inhibitors enables similar competitive inhibition of the cleavage of the endogenous DPP-4 substrates. Furthermore, despite chemical diversity and a range of binding potencies observed amongst the DPP-4 inhibitors, a direct relationship between enzyme inhibition in the plasma and glucose lowering is evident in mice for each member of the classes studied.

Fasiglifam (TAK-875): Mechanistic Investigation and Retrospective Identification of Hazards for Drug Induced Liver Injury.

TAK-875, a GPR40 agonist, was withdrawn from Phase III clinical trials due to drug-induced liver injury (DILI). Mechanistic studies were conducted to identify potential DILI hazards (covalent binding burden (CVB), hepatic transporter inhibition, mitochondrial toxicity, and liver toxicity in rats) associated with TAK-875. Treatment of hepatocytes with radiolabeled TAK-875 resulted in a CVB of 2.0 mg/day, which is above the threshold of 1 mg/day considered to be a risk for DILI. Covalent binding to hepatocytes was due to formation of a reactive acyl glucuronide (AG) and, possibly, an acyl-CoA thioester intermediate. Formation of TAK-875AG in hepatocytes and/or in vivo was in the order of non-rodents > human (in vitro only) > rat. These data suggest that non-rodents, and presumably humans, form TAK-875AG more efficiently than rats, and that AG-mediated toxicities in rats may only occur at high doses. TAK-875 (1000 mg/kg/day) formed significant amounts of AG metabolite (≤32.7 µM) in rat liver that was associated with increases in ALT (×4), bilirubin (×9), and bile acids (×3.4), and microscopic findings of hepatocellular hypertrophy and single cell necrosis. TAK-875 and TAK-875AG had similar potencies (within 3-fold) for human multi-drug resistant associated protein 2/4 (MRP2/4) and bile salt export pump, but TAK-875AG was exceptionally potent against MRP3 (0.21 µM). Inhibition of MRPs may contribute to liver accumulation of TAK-875AG. TAK-875 also inhibited mitochondrial respiration in HepG2 cells, and mitochondrial Complex 1 and 2 activities in isolated rat mitochondria. In summary, formation of TAK-875AG, and possibly TAK-875CoA in hepatocytes, coupled with inhibition of hepatic transporters and mitochondrial respiration may be key contributors to TAK-875-mediated DILI.

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.

Glucagon like receptor 1/ glucagon dual agonist acutely enhanced hepatic lipid clearance and suppressed de novo lipogenesis in mice.

Lipid lowering properties of glucagon have been reported. Blocking glucagon signaling leads to rise in plasma LDL levels. Here, we demonstrate the lipid lowering effects of acute dosing with Glp1r/Gcgr dual agonist (DualAG). All the experiments were performed in 25 week-old male diet-induced (60% kCal fat) obese mice. After 2 hrs of fasting, mice were injected subcutaneously with vehicle, liraglutide (25nmol/kg) and DualAG (25nmol/kg). De novo cholesterol and palmitate synthesis was measured by deuterium incorporation method using D2O. 13C18-oleate infusion was used for measuring fatty acid esterification. Simultaneous activation of Glp1r and Gcgr resulted in decrease in plasma triglyceride and cholesterol levels. DualAG enhanced hepatic LDLr protein levels, along with causing decrease in content of plasma ApoB48 and ApoB100. VLDL secretion, de novo palmitate synthesis and fatty acid esterification decreased with acute DualAG treatment. On the other hand, ketone levels were elevated with DualAG treatment, indicating increased fatty acid oxidation. Lipid relevant changes were absent in liraglutide treated group. In an acute treatment, DualAG demonstrated significant impact on lipid homeostasis, specifically on hepatic uptake, VLDL secretion and de novo synthesis. These effects collectively reveal that lipid lowering abilities of DualAG are primarily through glucagon signaling and are liver centric.

Novel LC/MS/MS and High-Throughput Mass Spectrometric Assays for Monoacylglycerol Acyltransferase Inhibitors.

Monoacylglycerol acyltransferase enzymes (MGAT1, MGAT2, and MGAT3) convert monoacylglycerol to diacylglycerol (DAG). MGAT1 and MGAT2 are both implicated in obesity-related metabolic diseases. Conventional MGAT enzyme assays use radioactive substrates, wherein the product of the MGAT-catalyzed reaction is usually resolved by time-consuming thin layer chromatography (TLC) analysis. Furthermore, microsomal membrane preparations typically contain endogenous diacylglycerol acyltransferase (DGAT) from the host cells, and these DGAT activities can further acylate DAG to form triglyceride (TG). Our mass spectrometry (liquid chromatography-tandem mass spectrometry, or LC/MS/MS) MGAT2 assay measures human recombinant MGAT2-catalyzed formation of didecanoyl-glycerol from 1-decanoyl-rac-glycerol and decanoyl-CoA, to produce predominantly 1,3-didecanoyl-glycerol. Unlike 1,2-DAG, 1,3-didecanoyl-glycerol is proved to be not susceptible to further acylation to TG. 1,3-Didecanoyl-glycerol product can be readily solubilized and directly subjected to high-throughput mass spectrometry (HTMS) without further extraction in a 384-well format. We also have established the LC/MS/MS MGAT activity assay in the intestinal microsomes from various species. Our assay is proved to be highly sensitive, and thus it allows measurement of endogenous MGAT activity in cell lysates and tissue preparations. The implementation of the HTMS MGAT activity assay has facilitated the robust screening and evaluation of MGAT inhibitors for the treatment of metabolic diseases.

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.