Inhibition Of DGAT1 Research Articles

Lipid droplet formation induced by icaritin derivative IC2 promotes a combination strategy for cancer therapy

BackgroundLipid metabolism is crucial in cancer progression. Lipid droplets (LDs) generated in cancer cells can act as protective mechanisms through alleviating lipotoxicity under stress conditions. We previously developed IC2 from the Chinese medicine icaritin as an inhibitor of stearoyl-CoA desaturase 1 (SCD1). IC2 has been shown to disrupt lipid metabolism and inhibits cancer cell proliferation. However, the impact of IC2 on intracellular LDs and the potential of targeting LD formation for combination cancer therapy remain unexplored.MethodsLD formation in cancer cells was analyzed with oil red O or BODIPY staining by microscopy. LD quantification was normalized to the cell number. IC2-induced cellular responses were revealed by transcriptional analysis, real-time PCR, and immunoblotting. Mitochondrial functions were assessed by measuring ATP production and oxygen consumption. The lipid source for LD formation was studied using lipid transporter inhibitors or lipid deprivation. The effect of inhibiting LD formation on IC2's anti-tumor effects was evaluated using MTT assays and apoptosis assays, which was subsequently validated in an in vivo xenografted tumor model.ResultsIC2 exerted anti-tumor effects, resulting in LD formation in various cancer cells. LD formation stimulated by IC2 was independent of extracellular lipid sources and did not result from increased de novo fatty acid (FA) synthesis within the cancer cells. Transcriptional analysis indicated that IC2 disturbed mitochondrial functions, which was confirmed by impaired mitochondrial membrane potential (MMP) and reduced capacity for ATP production and oxygen consumption. Moreover, IC2 treatment led to a greater accumulation of lipids in LDs outside the mitochondria compared with the control group. IC2 inhibited the proliferation of PC3 cells and promoted the apoptosis of the cancer cells. These effects were further enhanced after inhibiting the diacylglycerol acyltransferase 1 (DGAT1), a key intracellular enzyme involved in LD formation. In PC3-xenografted mice, the DGAT1 inhibitor augmented the IC2-induced reduction in tumor growth by modulating LD formation.ConclusionLD formation is a feedback response to IC2’s anti-tumor effects, which compromises the anti-tumor actions. IC2’s anti-tumor efficacy can be enhanced by combining it with inhibitors targeting LD formation. This strategy may be extended to other anti-tumor agents that regulate lipid metabolism.

Lipotoxicity of palmitic acid is associated with DGAT1 downregulation and abolished by PPARα activation in liver cells

Lipotoxicity refers to the harmful effects of excess fatty acids on metabolic health, and it can vary depending on the type of fatty acids involved. Saturated and unsaturated fatty acids exhibit distinct effects, though the precise mechanisms behind these differences remain unclear. Here, we investigated the lipotoxicity of palmitic acid (PA), a saturated fatty acid, compared with oleic acid (OA), a monounsaturated fatty acid, in the hepatic cell line HuH7. Our results demonstrated that PA, unlike OA, induces lipotoxicity, endoplasmic reticulum (ER) stress, and autophagy inhibition. Compared with OA, PA treatment leads to less lipid droplet (LD) accumulation and a significant reduction in the mRNA and protein level of diacylglycerol acyltransferase 1 (DGAT1), a key enzyme of triacylglycerol synthesis. Using modulators of ER stress and autophagy, we established that DGAT1 downregulation by PA is closely linked to these cellular pathways. Notably, the ER stress inhibitor 4-phenylbutyrate can suppress PA-induced DGAT1 downregulation. Furthermore, knockdown of DGAT1 by siRNA or with A922500, a specific DGAT1 inhibitor, resulted in cell death, even with OA. Both PA and OA increased the oxygen consumption rate; however, the increase associated with PA was only partially coupled to ATP synthesis. Importantly, treatment with GW7647 a specific PPARα agonist mitigated the lipotoxic effects of PA, restoring PA-induced ER stress, autophagy block, and DGAT1 suppression. In conclusion, our study highlights the crucial role of DGAT1 in PA-induced lipotoxicity, broadening the knowledge of the mechanisms underlying hepatic lipotoxicity and providing the basis for potential therapeutic interventions.

Oxysterol binding protein regulates the resolution of TLR-induced cytokine production in macrophages

Toll-like receptors (TLRs) on macrophages sense microbial components and trigger the production of numerous cytokines and chemokines that mediate the inflammatory response to infection. Although many of the components required for the activation of the TLR pathway have been identified, the mechanisms that appropriately regulate the magnitude and duration of the response and ultimately restore homeostasis are less well understood. Furthermore, a growing body of work indicates that TLR signaling reciprocally interacts with other fundamental cellular processes, including lipid metabolism but only a few specific molecular links between immune signaling and the macrophage lipidome have been studied in detail. Oxysterol-binding protein (Osbp) is the founding member of a family of lipid-binding proteins with diverse functions in lipid sensing, lipid transport, and cell signaling but its role in TLR responses is not well defined. Here, we demonstrate that altering the state of Osbp with its natural ligand, 25-hydroxycholesterol (25HC), or pharmacologically, sustains and thereby amplifies Tlr4-induced cytokine production in vitro and in vivo. CRISPR-induced knockdown of Osbp abrogates the ability of these ligands to sustain TLR responses. Lipidomic analysis suggested that the effect of Osbp on TLR signaling may be mediated by alterations in triglyceride production and treating cells with a Dgat1 inhibitor, which blocks triglyceride production and completely abrogates the effect of Osbp on TLR signaling. Thus, Osbp is a sterol sensor that transduces perturbations of the lipidome to modulate the resolution of macrophage inflammatory responses.

Diacylglycerol O-acyltransferase isoforms play a role in peridroplet mitochondrial fatty acid metabolism in bovine liver

Hepatocellular lipid accumulation characterizes fatty liver in dairy cows. Lipid droplets (LD), specialized organelles that store lipids and maintain cellular lipid homeostasis, are responsible for the ectopic storage of lipids associated with several metabolic disorders. In recent years, nonruminant studies have reported that LD-mitochondria interactions play an important role in lipid metabolism. Due to the role of diacylglycerol acyltransferase isoforms (DGAT1 and DGAT2) in LD synthesis, we explored mechanisms of mitochondrial fatty acid transport in ketotic cows using liver biopsies and isolated primary hepatocytes. Compared with healthy cows, cows with fatty liver had massive accumulation of LD and high protein expression of the triglyceride (TAG) synthesis-related enzymes DGAT1 and DGAT2, LD synthesis-related proteins perilipin 2 (PLIN2) and perilipin 5 (PLIN5), and the mitochondrial fragmentation-related proteins dynamin-related protein 1 (DRP1) and fission 1 (FIS1). In contrast, factors associated with fatty acid oxidation, mitochondrial fusion, and mitochondrial electron transport chain complex were lower compared with those in the healthy cows. In addition, transmission electron microscopy revealed significant contacts between LD-mitochondria in liver tissue from cows with fatty liver. Compared with isolated cytoplasmic mitochondria, expression of carnitine palmitoyl transferase 1A (CPT1A) and DRP1 was lower, but mitofusin 2 (MFN2) and mitochondrial electron transport chain complex was greater in isolated peridroplet mitochondria from hepatic tissue of cows with fatty liver. In vitro data indicated that exogenous free fatty acids (FFA) induced hepatocyte LD synthesis and mitochondrial dynamics consistent with in vivo results. Furthermore, DGAT2 inhibitor treatment attenuated the FFA-induced upregulation of PLIN2 and PLIN5 and rescued the impairment of mitochondrial dynamics. Inhibition of DGAT2 also restored mitochondrial membrane potential and reduced hepatocyte reactive oxygen species production. The present in vivo and in vitro results indicated functional differences are present among different types of mitochondria in the liver tissue of dairy cows with ketosis. Activity of DGAT2 may play a key role in maintaining liver mitochondrial function and lipid homeostasis in dairy cows during the transition period.

Biallelic variants in LARS1 induce steatosis in developing zebrafish liver via enhanced autophagy

BackgroundBiallelic pathogenic variants of LARS1 cause infantile liver failure syndrome type 1 (ILFS1), which is characterized by acute hepatic failure with steatosis in infants. LARS functions as a protein associated with mTORC1 and plays a crucial role in amino acid-triggered mTORC1 activation and regulation of autophagy. A previous study demonstrated that larsb-knockout zebrafish exhibit conditions resembling ILFS. However, a comprehensive analysis of larsb-knockout zebrafish has not yet been performed because of early mortality.MethodsWe generated a long-term viable zebrafish model carrying a LARS1 variant identified in an ILFS1 patient (larsb-I451F zebrafish) and analyzed the pathogenesis of the affected liver of ILFS1.ResultsHepatic dysfunction is most prominent in ILFS1 patients during infancy; correspondingly, the larsb-I451F zebrafish manifested hepatic anomalies during developmental stages. The larsb-I451F zebrafish demonstrates augmented lipid accumulation within the liver during autophagy activation. Inhibition of DGAT1, which converts fatty acids to triacylglycerols, improved lipid droplets in the liver of larsb-I451F zebrafish. Notably, treatment with an autophagy inhibitor ameliorated hepatic lipid accumulation in this model.ConclusionsOur findings suggested that enhanced autophagy caused by biallelic LARS1 variants contributes to ILFS1-associated hepatic dysfunction. Furthermore, the larsb-I451F zebrafish model, which has a prolonged survival rate compared with the larsb-knockout model, highlights its potential utility as a tool for investigating the pathophysiology of ILFS1-associated liver dysfunction.

A novel approach to pH-Responsive targeted cancer Therapy: Inhibition of FaDu cancer cell proliferation with a pH low insertion Peptide-Conjugated DGAT1 inhibitor

Targeting enzymes involved in lipid metabolism is increasingly recognized as a promising anticancer strategy. Efficient inhibition of diacylglycerol O-transferase 1 (DGAT1) can block fatty acid (FA) storage. This, in turn, triggers an increase in free polyunsaturated FA concentration, leading to peroxidation and ferroptosis. In this study, we report the development of a pH-sensitive peptide (pHLIP)-drug conjugate designed to selectively deliver DGAT1 inhibitors to cancer cells nested within the acidic microenvironment of tumors. We utilized two previously established pHLIP sequences for coupling with drugs. The study of DGAT1 conjugates in large unilamellar vesicles (LUVs) of different compositions did not reveal enhanced pH-dependent insertion compared to POPC LUVs. However, using in vitro 3D tumor spheroids, significant antiproliferative effects were observed upon exposure to pHLIP-T863 (DGAT1 inhibitor) conjugates, surpassing the inhibitory activity of T863 alone. In conclusion, our study provides the first evidence that pHLIP-based conjugates with DGAT1 inhibitors have the potential to specifically target the acidic compartment of tumors. Moreover, it sheds light on the limitations of LUV models in capturing the pH-dependency of such conjugates.

DGAT2 inhibition blocks SREBP-1 cleavage and improves hepatic steatosis by increasing phosphatidylethanolamine in the ER

Diacylglycerol acyltransferase 2 (DGAT2) catalyzes the final step of triglyceride (TG) synthesis. DGAT2 deletion in mice lowers liver TGs, and DGAT2 inhibitors are under investigation for the treatment of fatty liver disease. Here, we show that DGAT2 inhibition also suppressed SREBP-1 cleavage, reduced fatty acid synthesis, and lowered TG accumulation and secretion from liver. DGAT2 inhibition increased phosphatidylethanolamine (PE) levels in the endoplasmic reticulum (ER) and inhibited SREBP-1 cleavage, while DGAT2 overexpression lowered ER PE concentrations and increased SREBP-1 cleavage invivo. ER enrichment with PE blocked SREBP-1 cleavage independent of Insigs, which are ER proteins that normally retain SREBPs in the ER. Thus, inhibition of DGAT2 shunted diacylglycerol into phospholipid synthesis, increasing the PE content of the ER, resulting in reduced SREBP-1 cleavage and less hepatic steatosis. This study reveals a new mechanism that regulates SREBP-1 activation and lipogenesis that is independent of sterols and SREBP-2 in liver.

Direct effects of adipocyte lipolysis on AMPK through intracellular long-chain acyl-CoA signaling

Long-chain acyl-CoAs (LC-acyl-CoAs) are important intermediary metabolites and are also thought to function as intracellular signaling molecules; however, the direct effects of LC-acyl-CoAs have been difficult to determine in real-time and dissociate from Protein Kinase A (PKA) signaling. Here, we examined the direct role of lipolysis in generating intracellular LC-acyl-CoAs and activating AMPK in white adipocytes by pharmacological activation of ABHD5 (also known as CGI-58), a lipase co-activator. Activation of lipolysis in 3T3-L1 adipocytes independent of PKA with synthetic ABHD5 ligands, resulted in greater activation of AMPK compared to receptor-mediated activation with isoproterenol, a β-adrenergic receptor agonist. Importantly, the effect of pharmacological activation of ABHD5 on AMPK activation was blocked by inhibiting ATGL, the rate-limiting enzyme for triacylglycerol hydrolysis. Utilizing a novel FRET sensor to detect intracellular LC-acyl-CoAs, we demonstrate that stimulation of lipolysis in 3T3-L1 adipocytes increased the production of LC-acyl-CoAs, an effect which was blocked by inhibition of ATGL. Moreover, ATGL inhibition blocked AMPKβ1 S108 phosphorylation, a site required for allosteric regulation. Increasing intracellular LC-acyl-CoAs by removal of BSA in the media and pharmacological inhibition of DGAT1 and 2 resulted in greater activation of AMPK. Finally, inhibiting LC-acyl-CoA generation reduced activation of AMPK; however, did not lower energy charge. Overall, results demonstrate that lipolysis in white adipocytes directly results in allosteric activation of AMPK through the generation of LC-acyl-CoAs.

Porcine enteric alphacoronavirus infection increases lipid droplet accumulation to facilitate the virus replication

Coronaviruses are widely transmissible between humans and animals, causing diseases of varying severity. Porcine enteric alphacoronavirus (PEAV) is a newly-discovered pathogenic porcine enteric coronavirus in recent years, which causes watery diarrhea in newborn piglets. The host inflammatory responses to PEAV and its metabolic regulation mechanisms remain unclear, and no antiviral studies have been reported. Therefore, we investigated the pathogenic mechanism and antiviral drugs of PEAV. The transcriptomic analysis of PEAV-infected host cells revealed that PEAV could upregulate lipid metabolism pathways. In lipid metabolism, steady-state energy processes, which can be mediated by lipid droplets (LDs), are the main functions of organelles. LDs are also important in viral infection and inflammation. In infected cells, PEAV increased LD accumulation, upregulated NF-κB signaling, promoted the production of the inflammatory cytokines IL-1β and IL-8, and induced cell death. Inhibiting LD accumulation with a DGAT-1 inhibitor significantly inhibited PEAV replication, downregulated the NF-κB signaling pathway, reduced the production of IL-1β and IL-8, and inhibited cell death. The NF-κB signaling pathway inhibitor BAY11-7082 significantly inhibited LD accumulation and PEAV replication. Metformin hydrochloride also exerted anti-PEAV effects and significantly inhibited LD accumulation, downregulated the NF-κB signaling pathway, reduced the production of IL-1β and IL-8, and inhibited cell death. LD accumulation in the lipid metabolism pathway therefore plays an important role in the replication and pathogenesis of PEAV, and metformin hydrochloride inhibits LD accumulation and the inflammatory response to exert anti-PEAV activity and reducing pathological injury. These findings contribute new targets for developing treatments for PEAV infections.

The roles of DGAT1 and DGAT2 in human myotubes are dependent on donor patho-physiological background.

The roles of DGAT1 and DGAT2 in lipid metabolism and insulin responsiveness of human skeletal muscle were studied using cryosections and myotubes prepared from muscle biopsies from control, athlete, and impaired glucose regulation (IGR) cohorts of men. The previously observed increases in intramuscular triacylglycerol (IMTG) in athletes and IGR were shown to be related to an increase in lipid droplet (LD) area in type I fibers in athletes but, conversely, in type II fibers in IGR subjects. Specific inhibition of both diacylglycerol acyltransferase (DGAT) 1 and 2 decreased fatty acid (FA) uptake by myotubes, whereas only DGAT2 inhibition also decreased fatty acid oxidation. Fatty acid uptake in myotubes was negatively correlated with the lactate thresholds of the respective donors. DGAT2 inhibition lowered acetate uptake and oxidation in myotubes from all cohorts whereas DGAT1 inhibition had no effect. A positive correlation between acetate oxidation in myotubes and resting metabolic rate (RMR) from fatty acid oxidation in vivo was observed. Myotubes from athletes and IGR had higher rates of de novo lipogenesis from acetate that were normalized by DGAT2 inhibition. Moreover, DGAT2 inhibition in myotubes also resulted in increased insulin-induced Akt phosphorylation. The differential effects of DGAT1 and DGAT2 inhibition suggest that the specialized role of DGAT2 in esterifying nascent diacylglycerols and de novo synthesized FA is associated with synthesis of a pool of triacylglycerol, which upon hydrolysis results in effectors that promote mitochondrial fatty acid oxidation but decrease insulin signaling in skeletal muscle cells.

Preferential lipolysis of DGAT1 over DGAT2 generated triacylglycerol in Huh7 hepatocytes

Two distinct diacylglycerol acyltransferases (DGAT1 and DGAT2) catalyze the final committed step of triacylglycerol (TG) synthesis in hepatocytes. After its synthesis in the endoplasmic reticulum (ER) TG is either stored in cytosolic lipid droplets (LDs) or is assembled into very low-density lipoproteins in the ER lumen. TG stored in cytosolic LDs is hydrolyzed by adipose triglyceride lipase (ATGL) and the released fatty acids are converted to energy by oxidation in mitochondria. We hypothesized that targeting/association of ATGL to LDs would differ depending on whether the TG stores were generated through DGAT1 or DGAT2 activities. Individual inhibition of DGAT1 or DGAT2 in Huh7 hepatocytes incubated with oleic acid did not yield differences in TG accretion while combined inhibition of both DGATs completely prevented TG synthesis suggesting that either DGAT can efficiently esterify exogenously supplied fatty acid. DGAT2-made TG was stored in larger LDs, whereas TG formed by DGAT1 accumulated in smaller LDs. Inactivation of DGAT1 or DGAT2 did not alter expression (mRNA or protein) of ATGL, the ATGL activator ABHD5/CGI-58, or LD coat proteins PLIN2 or PLIN5, but inactivation of both DGATs increased PLIN2 abundance despite a dramatic reduction in the number of LDs. ATGL was found to preferentially target to LDs generated by DGAT1 and fatty acids released from TG in these LDs were also preferentially used for fatty acid oxidation. Combined inhibition of DGAT2 and ATGL resulted in larger LDs, suggesting that the smaller size of DGAT1-generated LDs is the result of increased lipolysis of TG in these LDs.

Porcine reproductive and respiratory syndrome virus regulates lipid droplet accumulation in lipid metabolic pathways to promote viral replication

Porcine reproductive and respiratory syndrome (PRRS) is a severe respiratory disease caused by porcine reproductive and respiratory syndrome virus (PRRSV) that can lead to the abortion of pregnant sows and decreased boar semen quality. However, the mechanisms of PRRSV replication in the host have not yet been fully elucidated. As lipid metabolism and lipid droplets (LDs) have been reported to play important roles in the replication of various viruses, we aimed to explore the mechanisms through which LDs affect PRRSV replication. Laser confocal and transmission electron microscopy revealed that PRRSV infection promoted intracellular LD accumulation, which was significantly reduced by treatment with the NF-κB signaling pathway inhibitors BAY11–7082 and metformin hydrochloride (MH). In addition, treatment with a DGAT1 inhibitor significantly reduced the protein expression of Phosphorylated NF-ΚB P65and PIκB and the transcription of IL-1β and IL-8 in the NF-κB signaling pathway. Furthermore, we showed that the reduction of the NF-κB signaling pathway and LDs significantly reduced PRRSV replication. Together, the findings of this study suggest a novel mechanism through which PRRSV regulates the NF-κB signaling pathway to increase LD accumulation and promote viral replication. Moreover, we demonstrated that both BAY11–7082 and MH can reduce PRRSV replication by reducing the NF-κB signaling pathway and LD accumulation. This study lays a theoretical foundation for research on the mechanism of PRRS prevention and control, as well as the research and development of antiviral drugs.

Metabolic reprogramming and lipid droplets are involved in Zika virus replication in neural cells

Zika virus (ZIKV) infection is a global public health concern linked to adult neurological disorders and congenital diseases in newborns. Host lipid metabolism, including lipid droplet (LD) biogenesis, has been associated with viral replication and pathogenesis of different viruses. However, the mechanisms of LD formation and their roles in ZIKV infection in neural cells are still unclear. Here, we demonstrate that ZIKV regulates the expression of pathways associated with lipid metabolism, including the upregulation and activation of lipogenesis-associated transcription factors and decreased expression of lipolysis-associated proteins, leading to significant LD accumulation in human neuroblastoma SH-SY5Y cells and in neural stem cells (NSCs). Pharmacological inhibition of DGAT-1 decreased LD accumulation and ZIKV replication in vitro in human cells and in an in vivo mouse model of infection. In accordance with the role of LDs in the regulation of inflammation and innate immunity, we show that blocking LD formation has major roles in inflammatory cytokine production in the brain. Moreover, we observed that inhibition of DGAT-1 inhibited the weight loss and mortality induced by ZIKV infection in vivo. Our results reveal that LD biogenesis triggered by ZIKV infection is a crucial step for ZIKV replication and pathogenesis in neural cells. Therefore, targeting lipid metabolism and LD biogenesis may represent potential strategies for anti-ZIKV treatment development.

Differential Kendrick's Plots as an Innovative Tool for Lipidomics in Complex Samples: Comparison of Liquid Chromatography and Infusion-Based Methods to Sample Differential Study.

Lipidomics has developed rapidly over the past decade. Nontargeted lipidomics from biological samples remains a challenge due to the high structural diversity, the concentration range of lipids, and the complexity of biological samples. We introduce here the use of differential Kendrick's plots as a rapid visualization tool for a qualitative nontargeted analysis of lipids categories and classes from data generated by either liquid chromatography-mass spectrometry (LC-MS) or direct infusion (nESI-MS). Each lipid class is easily identified by comparison with the theoretical Kendrick plot pattern constructed from exact mass measurements and by using MSKendrickFilter, an in-house Python software. The lipids are identified with the LIPID MAPS database. In addition, in LC-MS, the software based on the Kendrick plots returns the retention time from all the lipids belonging to the same series. Lipid extracts from a yeast (Saccharomyces cerevisiae) are used as a model. An on/off case comparing Kendrick plots from two cell lines (prostate cancer cell lines treated or not with a DGAT2 inhibition) clearly shows the effect of the inhibition. Our study demonstrates the good performance of direct infusion as a fast qualitative screening method as well as for the analysis of chromatograms. A fast screening semiquantitative approach is also possible, while the targeted mode remains the golden standard for precise quantitative analysis.

S523 A Phase 2, Randomized, Double-Blind, Placebo-Controlled Study to Assess the Efficacy, Safety and Tolerability of ANJ908, a Novel DGAT1 Inhibitor, in Patients With Chronic Idiopathic Constipation

Introduction: Current treatment for chronic idiopathic constipation (CIC) is limited by: 1. Low magnitude of drug effect (increase of only ∼1-1.5 SBM per week) 2. Low overall response rate (typically < 20%) 3. Loss of efficacy within 3-6 months. Real-world experience where CIC patients often cycle through 2-4 pharmacotherapies shows the need for new medications with better efficacy. ANJ908 is a safe and well-tolerated diacylglycerol acyltransferase (DGAT) 1 inhibitor which increases bowel movement number and softens the stool in humans. We designed and conducted a phase 2 study of ANJ908 in CIC patients and present the data here for the first time. Methods: ANJ908C2102 was a multicenter, randomized, placebo-controlled, double-blind study designed to assess the efficacy and safety of ANJ908 in CIC patients. Patients meeting ROME IV diagnostic criteria for functional constipation (a.k.a. CIC) and had < 3 SBMs/week for prior the two weeks were eligible. Enrolled patients underwent a 2 week run-in period where all previous constipation treatment was stopped. After run-in, patients were randomized (1:1:1) to 4 weeks of once daily treatment with placebo, ANJ908 at 20 or 40 mg. Bowel movement timing, stool details, and patient reported constipation symptoms were recorded in an eDiary. Patient safety labs and AEs were monitored throughout the study. Results: A total of 191 patients were randomized. Patients were 77.9% female, with a mean (SD) age and BMI of 42.2 (14.4) years and 23.2 (4.36) kg/m2, respectively. ANJ908 increased the mean (SE) placebo-subtracted number of SBM/week by +2.28 (0.75; p = 0.0027) and +3.10 (0.74; p < 0.0001) and CSBM/week by +1.53 (0.70; p = 0.03) and +1.81 (0.70; p = 0.01), respectively at week 4. ANJ908 improved stool consistency, provided constipation relief, decreased straining severity, and reduced rescue medication use. ANJ908 was safe and generally well tolerated. Mild diarrhea, nausea and vomiting were the most common AEs with a total of 7 (out of 64) patients in the ANJ908 20 mg group discontinuing treatment due to AEs. Conclusion: ANJ908 demonstrated increases in weekly SBMs and CSBMs to a magnitude greater than previous treatments. Furthermore, ANJ908 improved patient reported stool consistency and relief of constipation symptoms. The benefit-risk profile of ANJ908 justifies advancement into phase 3 clinical development to confirm its efficacy and safety. If this profile is confirmed, ANJ908 may become a treatment better able to address the unmet need in CIC patients.

Sulfur mustard analog 2-chloroethyl ethyl sulfide increases triglycerides by activating DGAT1-dependent biogenesis and inhibiting PGC1ɑ-dependent fat catabolism in immortalized human bronchial epithelial cells

Using sulfur mustard analog 2-chloroethyl ethyl sulfide (CEES), we established an in vitro model by poisoning cultured immortalized human bronchial epithelial cells. Nile Red staining revealed lipids accumulated 24 h after a toxic dose of CEES (0.9 mM). Lipidomics analysis showed most of the increased lipids were triglycerides (TGs), and the increase in TGs was further confirmed using a Triglyceride-Glo™ Assay kit. Protein and mRNA levels of DGAT1, an important TG biogenesis enzyme, were increased following 0.4 mM CEES exposure. Under higher dose CEES (0.9 mM) exposure, protein and mRNA levels of PPARγ coactivator-1ɑ (PGC-1ɑ), a well-known transcription factor that regulates fatty acid oxidation, were decreased. Finally, application with DGAT1 inhibitor A 922500 or PGC1ɑ agonist ZLN005 was able to block the CEES-induced TGs increase. Overall, our dissection of CEES-induced TGs accumulation provides new insight into energy metabolism dysfunction upon vesicant exposure. HIGHLIGHTS In CEES (0.9 mM)-injured cells: Triglycerides (TGs) were abundant in the accumulated lipids. Expression of DGAT1, not DGAT2, was increased. Expression of PGC1ɑ, not PGC1β, was reduced. DGAT1 inhibitor or PGC1ɑ agonist blocked the CEES-mediated increase in TGs.

DGAT2 Inhibition Potentiates Lipid Droplet Formation To Reduce Cytotoxicity in APOL1 Kidney Risk Variants.

Two variants in the gene encoding apolipoprotein L1 (APOL1) that are highly associated with African ancestry are major contributors to the large racial disparity in rates of human kidney disease. We previously demonstrated that recruitment of APOL1 risk variants G1 and G2 from the endoplasmic reticulum to lipid droplets leads to reduced APOL1-mediated cytotoxicity in human podocytes. We used CRISPR-Cas9 gene editing of induced pluripotent stem cells to develop human-derived APOL1G0/G0 and APOL1G2/G2 kidney organoids on an isogenic background, and performed bulk RNA sequencing of organoids before and after treatment with IFN-γ. We examined the number and distribution of lipid droplets in response to treatment with inhibitors of diacylglycerol O-acyltransferases 1 and 2 (DGAT1 and DGAT2) in kidney cells and organoids. APOL1 was highly upregulated in response to IFN-γ in human kidney organoids, with greater increases in organoids of high-risk G1 and G2 genotypes compared with wild-type (G0) organoids. RNA sequencing of organoids revealed that high-risk APOL1G2/G2 organoids exhibited downregulation of a number of genes involved in lipogenesis and lipid droplet biogenesis, as well as upregulation of genes involved in fatty acid oxidation. There were fewer lipid droplets in unstimulated high-risk APOL1G2/G2 kidney organoids than in wild-type APOL1G0/G0 organoids. Whereas DGAT1 inhibition reduced kidney organoid lipid droplet number, DGAT2 inhibition unexpectedly increased organoid lipid droplet number. DGAT2 inhibition promoted the recruitment of APOL1 to lipid droplets, with associated reduction in cytotoxicity. Lipogenesis and lipid droplet formation are important modulators of APOL1-associated cytotoxicity. Inhibition of DGAT2 may offer a potential therapeutic strategy to attenuate cytotoxic effects of APOL1 risk variants.

Role of diacylglycerol O-acyltransferase (DGAT) isoforms in bovine hepatic fatty acid metabolism

Fatty acid accumulation in hepatocytes induced by high concentrations of fatty acids due to lipolysis and the associated oxidative damage they cause occur most frequently after calving. Because of their role in esterification of fatty acids, diacylglycerol acyltransferase isoforms (DGAT1 and DGAT2) could play a role in the susceptibility of dairy cows to develop fatty liver. To gain mechanistic insights, we performed in vivo and in vitro analyses using liver biopsies or isolated primary hepatocytes. The in vivo study (n = 5 cows/group) involved healthy cows [average liver triacylglycerol (TAG) = 0.78%; 0.58 to 0.93%, ratio of triglyceride weight to wet liver weight] or cows diagnosed with fatty liver (average TAG = 7.60%; 5.31 to 10.54%). In vitro, hepatocytes isolated from 3 healthy female calves (1 d old, 44 to 53 kg) were challenged with (fatty acids) or without (control) a 1.2 mM mixture of fatty acids in an attempt to induce metabolic stress. Furthermore, hepatocytes were treated with DGAT1 inhibitor or DGAT2 inhibitor for 2 h followed by a challenge with (DGAT1 inhibitor + fatty acids or DGAT2 inhibitor + fatty acids) or without (DGAT1 inhibitor or DGAT2 inhibitor) the 1.2 mM mixture of fatty acids for 12 h. Data analysis of liver biopsies was compared using a 2-tailed unpaired Student's t-test. Data from calf hepatocyte treatment comparisons were assessed by one-way ANOVA, and multiplicity for each experiment was adjusted by the Holm's procedure. Data indicated that both fatty liver and in vitro challenge with fatty acids were associated with greater mRNA and protein abundance of SREBF1, FASN, DGAT1, and DGAT2. In contrast, mRNA and protein abundance of CPT1A and very low-density lipoprotein synthesis-related proteins MTTP and APOB were markedly lower. However, compared with fatty acid challenge alone, DGAT1 inhibitor + fatty acids led to greater mRNA and protein abundance of CPT1A and APOB, and greater mRNA abundance of SREBF1 and MTTP. Furthermore, this treatment led to lower mRNA abundance of FASN and DGAT2 and TAG concentrations. Compared with fatty acid challenge alone, DGAT2 inhibitor + fatty acids led to greater mRNA and protein abundance of CPT1A, MTTP, and APOB, and lower mRNA and protein abundance of SREBF1 and FASN. In addition, compared with control and fatty acids, there was greater protein abundance of GRP78 and PERK in both DGAT1 and DGAT2 inhibitor with or without fatty acids. Furthermore, compared with control and fatty acids, reactive oxygen species concentrations in the DGAT1 inhibitor with or without fatty acid group was greater. Overall, data suggested that DGAT1 is particularly relevant in the context of hepatocyte TAG synthesis from exogenous fatty acids. Disruption of both DGAT1 and DGAT2 altered lipid homeostasis, channeling fatty acids toward oxidation and generation of reactive oxygen species. Both DGAT isoforms play a role in promoting fatty acid storage into TAG and lipid droplets to protect hepatocytes from oxidative damage.

Acyl-CoA:diacylglycerol acyltransferase 1 inhibition in the small intestine increases plasma transaminase activity via the activation of protein kinase C pathway.

Acyl-CoAdiacylglycerol acyltransferase 1 (DGAT1) is a key enzyme in the fat absorption step in enterocytes. We previously reported that the pharmacological inhibition of DGAT1 increased plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activity in corn oil-loaded rats without any sign of hepatotoxicity. In this study, we investigated this mechanism. We found that this elevation occurred only during the pharmacologically active period of a DGAT1 inhibitor and the magnitude did not depend on the volume of corn oil. In addition, this elevation was not accompanied by increases in ALT or AST mRNA levels in the small intestine and liver. To clarify a lipid component responsible for this elevation, rats were treated with free fatty acids instead of corn oil and no plasma ALT elevation was observed. Next, rats were pretreated with inhibitors of monoacylglycerol acyltransferase 2 and intestinal microsomal triglyceride transfer protein instead of the DGAT1 inhibitor, but no plasma ALT elevation was observed after corn oil loading. Since the results suggested a possible role of diacylglycerol (DAG), which activates protein kinase C (PKC), we measured PKC activity in the small intestine and found that the activity was increased by treatment with the DGAT1 inhibitor and corn oil. Moreover, rats pretreated with a PKC inhibitor in combination with the DGAT1 inhibitor showed suppression of plasma ALT elevation. Taken together, the present results suggest that DAG accumulation induced by pharmacological DGAT1 inhibition and resultant PKC activation in enterocytes are involved in the increase in plasma ALT and AST activity in rats.

ATF6-DGAT pathway is involved in TLR7-induced innate immune response in Ctenopharyngodon idellus kidney cells

DGAT1 and DGAT2 are two acyl-CoA:diacylglycerol O-acyltransferase (DGAT) enzymes that catalyze the final step in triglyceride (TG) synthesis. TGs are the primary constituents of lipid droplets (LDs). Although it has been demonstrated that LDs modulate immune and inflammatory responses in CIK cells, little is known about whether DGAT1 and DGAT2 involve in this process. Firstly, grass carp DGAT2 was isolated and characterized, encoding 361 amino acids, and all DGAT2 proteins in genomic structures are conserved in vertebrates. Then, using TLR7 agonist, we induced LDs accumulation in CIK cells. Only DGAT1b and DGAT2 were upregulated in forming TLR7 agonist induced-LDs. Next, we utilized small-molecule inhibitors of DGAT1 and DGAT2. The results indicated that DGAT1 inactivation attenuated TG content and the relative expressions of IFNα3, NF-κB, IL-1β, and TNFα genes, whereas DGAT2 inhibition decreased TG content and the relative expressions of MyD88, IRF7, IFNα3, NF-κB, IL-1β, and TNFα genes, implying that DGAT1-generated LDs and DGAT2-generated LDs contribute to TLR7-induced immune response via different signaling pathways. Finally, inhibiting ATF6 effectively decreased DGAT-generated LDs accumulation and the expression of TLR7 signaling-related genes induced by TLR7 agonist, implying that ATF6 UPR pathway may mediate the role of DGAT-generated LDs in TLR7 signaling. Overall, we demonstrate that DGAT1 and DGAT2-catalyzed TAG synthesis may generate different LDs to provide distinct signaling platforms for innate TLR7 signaling.