Intracellular Triglyceride Accumulation Research Articles

Statin-associated regulation of hepatic PNPLA3 in patients without known liver disease.

Statins are used for metabolic dysfunction-associated steatotic liver disease (MASLD) (NAFLD) treatment, but their role in this context is unclear. Genetic variants of patatin-like phospholipase domain containing 3 (PNPLA3) are associated with MASLD susceptibility and statin treatment efficacy. Access to liver biopsies before established MASLD is limited, and statins and PNPLA3 in early liver steatosis are thus difficult to study. Liver biopsies were collected from 261 patients without known liver disease at surgery and stratified based on statin use and criteria for the metabolic syndrome (MS). Genotypes and transcript levels were measured using Illumina and Affymetrix arrays, and metabolic and lipoprotein profiles by clinical assays. Statin effects on PNPLA3, de novo lipogenesis (DNL), and lipid accumulation were further studied in vitro. The PNPLA3I148M genetic variant was associated with significantly lower hepatic levels of cholesterol synthesis-associated transcripts. Patients with MS had significantly higher hepatic levels of MASLD and lipogenesis-associated transcripts than non-MS patients. Patients with MS on statin therapy had significantly higher hepatic levels of PNPLA3, acetyl-CoA carboxylase alpha, and ATP citrate lyase, and statin use was associated with higher plasma fasting glucose, insulin, and HbA1c. Exposure of hepatocyte-like HepG2 cells to atorvastatin promoted intracellular accumulation of triglycerides and lipogenesis-associated transcripts. Atorvastatin-exposure of HepG2, sterol O-acyltransferase (SOAT) 2-only-HepG2, primary human hepatic stellate, and hepatic stellate cell-like LX2 cells significantly increased levels of PNPLA3 and SREBF2-target genes, whereas knockdown of SREBF2 attenuated this effect. Collectively, these observations suggest statin-associated regulation of PNPLA3 and DNL in liver. The potential interaction between PNPLA3 genotype and metabolic status should be considered in future studies in the context of statin therapy for MASLD.

Purple Butter Clam (Saxidomus Purpurata) as a Potential Functional Food Source for Obesity Treatment.

Saxidomus purpurata extract (SPE) is a highly consumable seafood worldwide with known health-related benefits. However, there are no reports of its' anti-obesity effect. This study explores the potential of SPE for anti-obesity effects by modulating adipogenesis and lipolysis. SPE reduced intracellular lipid and triglyceride accumulation while increasing free glycerol release in adipocytes. SPE inhibited lipogenesis protein expressions and increased the phosphorylation of hormone-sensitive lipase and Adenosine monophosphate-activated protein kinase (AMPK) to promote lipolysis. In addition, SPE suppressed adipogenesis by downregulating protein expression of key adipogenic markers, peroxisome proliferator-activated receptor gamma (PPARγ), CCAAT/enhancer-binding protein α (C/EBPα), and sterol regulatory element-binding protein 1 (SREBP1) via Wnt/β-catenin signaling. SPE augmented the heme oxygenase-1 (HO-1) expression. Thus, pharmacological intervention with Zinc protoporphyrin (ZnPP-HO-1 antagonist) was employed to validate the HO-1 role. The presence of ZnPP increased the lipid accumulation and reduced the free glycerol release. At the molecular level, adipogenic transcription factors (PPARγ, C/EBPα, and SREBP1) expressions were restored in the presence of ZnPP. GC-MS analysis revealed that SPE was comprised of several fatty acids, contributing to its anti-obesity activity. SPE is an effective nutraceutical that can be used to reduce the progression of obesity. HO-1 expression during adipogenesis might be the mechanism of action for the anti-obesity effect of SPE.

Silibinin inhibits PM2.5-induced liver triglyceride accumulation through enhancing the function of mitochondrial Complexes I and II.

The standardized extract of milk thistle seeds, known as silibinin, has been utilized in herbal medicine for over two centuries, with the aim of safeguarding the liver against the deleterious effects of various toxic substances. However, the role of silibinin in Particulate Matter (PM2.5)-induced intrahepatic triglyceride accumulation remains unclear. This study seeks to investigate the impact of silibinin on PM2.5-induced intrahepatic triglyceride accumulation and elucidate potential underlying mechanisms. A model of intrahepatic triglyceride accumulation was established in male C57BL/6J mice through intratracheal instillation of PM2.5, followed by assessment of liver weight, body weight, liver index, and measurements of intrahepatic triglycerides and cholesterol after treatment with silibinin capsules. Hep G2 cells were exposed to PM2.5 suspension to create an intracellular triglyceride accumulation model, and after treatment with silibinin, cell viability, intracellular triglycerides and cholesterol, fluorescence staining for Nile Red (lipid droplets), and DCFH-DA (Reactive Oxygen Species, ROS), as well as proteomics, real-time PCR, and mitochondrial function assays, were performed to investigate the mechanisms involved in reducing triglycerides. PM2.5 exposure leads to triglyceride accumulation, increased ROS production, elevated expression of inflammatory factors, decreased expression of antioxidant factors, and increased expression of downstream genes of aryl hydrocarbon receptor. Silibinin can partially or fully reverse these factors, thereby protecting cells and animal livers from PM2.5-induced damage. In vitro studies show that silibinin exerts its protective effects by preserving oxidative phosphorylation of mitochondrial complexes I and II, particularly significantly enhancing the function of mitochondrial complex II. Succinate dehydrogenase (mitochondrial complex II) is a direct target of silibinin, but silibinin A and B exhibit different affinities for different subunits of complex II. Silibinin improved the accumulation of intrahepatic triglycerides induced by PM2.5, and this was, at least in part, explained by an enhancement of oxidative phosphorylation in mitochondrial Complexes I and II.

Piperlongumine inhibits the early stage of adipogenesis in 3T3-L1 cells

Piperlongumine (PLM), a natural compound isolated from long peppers, has been reported to possess multiple pharmacological roles, including anti-tumor and anti-diabetic. However, the pharmacological role of PLM on adipogenesis is still unknown. In this study, we found that PLM strongly inhibited 3T3-L1 adipocyte differentiation. This inhibition was determined by the accumulation of lipid droplets and intracellular triglycerides. In addition, PLM downregulated both the mRNA and protein expression of adipogenic transcription factors, including CCAAT-enhancer binding proteins β (C/EBPβ), C/EBPα, and peroxisome proliferator-activated receptor γ (PPARγ). Based on the time-course experiment, we found that the inhibitory effect of PLM on adipogenesis was mainly involved in the early stage of adipogenesis. Studying these differential effects could uncover new mechanisms for regulating adipogenesis and new chemicals for treating obesity.

Citrullus mucosospermus Extract Reduces Weight Gain in Mice Fed a High-Fat Diet.

This study aimed to investigate the therapeutic potential of Citrullus mucosospermus extract (CME) in counteracting adipogenesis and its associated metabolic disturbances in murine models. In vitro experiments utilizing 3T3-L1 preadipocytes revealed that CME potently inhibited adipocyte differentiation, as evidenced by a dose-dependent reduction in lipid droplet formation. Remarkably, CME also attenuated glucose uptake and intracellular triglyceride accumulation in fully differentiated adipocytes, suggesting its ability to modulate metabolic pathways in mature adipose cells. Translating these findings to an in vivo setting, we evaluated the effects of CME in C57BL/6N mice fed a high-fat diet (HFD) for 10 weeks. CME administration, concomitantly with the HFD, resulted in a significant attenuation of body weight gain compared to the HFD control group. Furthermore, CME treatment led to substantial reductions in liver weight, total fat mass, and deposits of visceral and retroperitoneal adipose tissue, underscoring its targeted impact on adipose expansion. Histological analyses revealed the remarkable effects of CME on hepatic steatosis. While the HFD group exhibited severe lipid accumulation within liver lobules, CME dose-dependently mitigated this pathology, with the highest dose virtually abolishing hepatic fat deposition. An examination of adipose tissue revealed a progressive reduction in adipocyte hypertrophy upon CME treatment, culminating in a near-normalization of adipocyte morphology at the highest dose. Notably, CME exhibited potent anti-inflammatory properties, significantly attenuating the upregulation of pro-inflammatory cytokines' mRNA levels (TNF-α, IL-1β and IL-6) in the livers of HFD-fed mice. This suggests a potential mechanism through which CME may exert protective effects against inflammation associated with obesity and fatty liver disease.

Single-cell atlas of ABCA7 loss-of-function reveals impaired neuronal respiration via choline-dependent lipid imbalances.

Loss-of-function (LoF) variants in the lipid transporter ABCA7 significantly increase the risk of Alzheimer's disease (odds ratio ∼2), yet the pathogenic mechanisms and the neural cell types affected by these variants remain largely unknown. Here, we performed single-nuclear RNA sequencing of 36 human post-mortem samples from the prefrontal cortex of 12 ABCA7 LoF carriers and 24 matched non-carrier control individuals. ABCA7 LoF was associated with gene expression changes in all major cell types. Excitatory neurons, which expressed the highest levels of ABCA7, showed transcriptional changes related to lipid metabolism, mitochondrial function, cell cycle-related pathways, and synaptic signaling. ABCA7 LoF-associated transcriptional changes in neurons were similarly perturbed in carriers of the common AD missense variant ABCA7 p.Ala1527Gly (n = 240 controls, 135 carriers), indicating that findings from our study may extend to large portions of the at-risk population. Consistent with ABCA7's function as a lipid exporter, lipidomic analysis of isogenic iPSC-derived neurons (iNs) revealed profound intracellular triglyceride accumulation in ABCA7 LoF, which was accompanied by a relative decrease in phosphatidylcholine abundance. Metabolomic and biochemical analyses of iNs further indicated that ABCA7 LoF was associated with disrupted mitochondrial bioenergetics that suggested impaired lipid breakdown by uncoupled respiration. Treatment of ABCA7 LoF iNs with CDP-choline (a rate-limiting precursor of phosphatidylcholine synthesis) reduced triglyceride accumulation and restored mitochondrial function, indicating that ABCA7 LoF-induced phosphatidylcholine dyshomeostasis may directly disrupt mitochondrial metabolism of lipids. Treatment with CDP-choline also rescued intracellular amyloid β -42 levels in ABCA7 LoF iNs, further suggesting a link between ABCA7 LoF metabolic disruptions in neurons and AD pathology. This study provides a detailed transcriptomic atlas of ABCA7 LoF in the human brain and mechanistically links ABCA7 LoF-induced lipid perturbations to neuronal energy dyshomeostasis. In line with a growing body of evidence, our study highlights the central role of lipid metabolism in the etiology of Alzheimer's disease.

Sexual Dimorphism's impact on adipogenesis: A three-dimensional in vitro model treated with 17β-estradiol and testosterone

Using a three-dimensional (3-D) in vitro culture model, we report the dose dependent effect of 17β-estradiol and testosterone on the adipogenic differentiation and maturation of human adipose derived stem cells (hASCs) obtained from female and male patients. Considering sexual dimorphism, we expected male and female adipocytes to respond differently to the sex steroids. Both male and female hASC spheroids were exposed to 100 nM and 500 nM of 17β-estradiol and testosterone either at the beginning of the adipogenic maturation (Phase I) to discourage intracellular triglyceride accumulation or exposed after adipogenic maturation (Phase II) to reduce the intracellular triglyceride accumulation. The results show that 17β-estradiol leads to a dose dependent reduction in intracellular triglyceride accumulation in female hASC spheroids compared to the both untreated and testosterone-treated cells. Affirming our hypothesis, 17β-estradiol prevented intracellular triglyceride accumulation during Phase I, while it stimulated lipolysis during Phase II. PPAR-γ and adiponectin gene expression also reduced upon 17β-estradiol treatment in female cells. Interestingly, 17β-estradiol and testosterone had only a modest effect on the male hASC spheroids. Collectively, our findings suggest that 17β-estradiol can prevent fat accumulation in adipocytes during early and late stages of maturation in females.

Methyl Cinnamate (MC) Alleviates Free Fatty Acids (FFAs) Induced Lipid Accumulation Through the AMPK Pathway in HepG2 Cells.

AMP-activated protein kinase (AMPK) plays a critical role in energy metabolism. Its activation leads to the phosphorylation of downstream proteins such as acetyl-CoA carboxylase (ACC) and sterol regulatory element-binding protein-1 (SREBP1), subsequently inhibiting de novo fatty acid synthesis, thereby reducing intracellular triglyceride accumulation. MC is a compound found in extracts from Zanthoxylum armatum DC plants. Research has shown that MC can inhibit the differentiation of 3T3-L1 adipocytes through the CAMKK2-AMPK pathway. However, the biological effect of MC in HepG2 cells remains unknown. In this study, we utilized HepG2 cells to establish a model of MAFLD through FFAs stimulation. We investigated the biological effects of MC on HepG2 cells and studied its impact on lipid metabolism. Small interfering RNA was employed to explore the mechanism by which MC activates AMPK. Finally, molecular docking was conducted, establishing a model of the interaction between AMPK and MC. We observed that MC can alleviate triglyceride accumulation in HepG2 cells. We observed the elevated p-AMPK/AMPK, P-ACC/ ACC, and elevated CPT1a after treatment of MC in HepG2 cells. The interference of CAMKK2 mRNA did not impact the ability of MC to phosphorylate AMPK. Compound C attenuates the ability of MC to increase p-AMPK. Molecular docking results led us to hypothesize that MC directly interacts with AMPK, resulting in AMPK phosphorylation and improved lipid accumulation in HepG2 cells.

The impact of bed rest on human skeletal muscle metabolism

Insulin sensitivity and metabolic flexibility decrease in response to bed rest, but the temporal and causal adaptations in human skeletal muscle metabolism are not fully defined. Here, we use an integrative approach to assess human skeletal muscle metabolism during bed rest and provide a multi-system analysis of how skeletal muscle and the circulatory system adapt to short- and long-term bed rest (German Clinical Trials: DRKS00015677). We uncover that intracellular glycogen accumulation after short-term bed rest accompanies a rapid reduction in systemic insulin sensitivity and less GLUT4 localization at the muscle cell membrane, preventing further intracellular glycogen deposition after long-term bed rest. We provide evidence of a temporal link between the accumulation of intracellular triglycerides, lipotoxic ceramides, and sphingomyelins and an altered skeletal muscle mitochondrial structure and function after long-term bed rest. An intracellular nutrient overload therefore represents a crucial determinant for rapid skeletal muscle insulin insensitivity and mitochondrial alterations after prolonged bed rest.

Coffee pulp simulated digestion enhances its in vitro ability to decrease emulsification and digestion of fats, and attenuates lipid accumulation in HepG2 cell model

The coffee industry produces a considerable quantity of coffee pulp (CP), a by-product with high levels of caffeine, phenolic compounds, and dietary fiber, which are reportedly involved in the lipid homeostasis regulation required to maintain human health. This work's objective was to evaluate the hypolipidemic activity of coffee pulp flour (CPF) and aqueous extract (CPE) after static simulated digestion by the assessment of their in vitro capacity to decrease emulsification and digestion of fats, and lipid-lowering capacity in HepG2 cells after the induction of intracellular fat accumulation. The CPF and CPE digested fractions displayed in vitro hypolipidemic properties by preserving the reduction of micellar cholesterol solubility (27–34%) and the secondary bile acid-binding capacity (22–30%), increasing their primary bile acid-binding ability (2.7-fold and 2.4-fold, respectively), and inhibiting the lipase and the HMGCR (77–79% and 36–85%, respectively) activities. Moreover, the hypolipidemic properties of non-digested fractions enhanced the CPF potential to decrease lipid absorption. Both ingredients (CPF and CPE) demonstrated lipid-lowering effects since they effectively counteract the accumulation of intracellular triglycerides and cholesterol triggered by palmitic acid in hepatic cells after the simulated digestion. This study suggests that phenolic compounds, caffeine, and dietary fiber may be responsible for the lipid-lowering properties exhibited by the CP ingredients and their composition differences affect the above-mentioned properties exhibited in the simulated digestion. These results contribute to demonstrating that the CPF and the CPE may act as modulators of pathways involved in hepatic lipid accumulation and could be a key element in its prevention.

MiR-889-3p Facilitates the Browning Process of White Adipocyte Precursors by Targeting the SON Gene.

It is well-established that beige/brown adipose tissue can dissipate stored energy through thermogenesis; hence, the browning of white adipocytes (WAT) has garnered significant interest in contemporary research. Our preceding investigations have identified a marked downregulation of miR-889-3p concurrent with the natural maturation of brown adipose tissue. However, the specific role and underlying molecular mechanisms of miR-889-3p in the browning process of white adipose tissue warrant further elucidation. In this research, we initially delved into the potential role of miR-889-3p in preadipocyte growth via flow cytometry and CCK-8 assay, revealing that miR-889-3p can stimulate preadipocyte growth. To validate the potential contribution of miR-889-3p in the browning process of white adipose tissue, we established an in vitro rabbit white adipocyte browning induction, which exhibited a significant upregulation of miR-889-3p during the browning process. RT-qPCR and Western blot analysis indicated that miR-889-3p overexpression significantly amplified the mRNA levels of UCP1, PRDM16, and CIDEA, as well as UCP1 protein levels. Furthermore, miR-889-3p overexpression fostered intracellular triglyceride accumulation. Conversely, the downregulation of miR-889-3p hindered the browning of rabbit preadipocytes. Subsequently, based on target gene prediction and luciferase reporter gene determination, we demonstrated that miR-889-3p directly targets the 3'-UTR region of SON. Lastly, we observed that inhibiting SON could facilitate the browning of rabbit preadipocytes. In conclusion, our findings suggest that miR-889-3p facilitates the browning process of white adipocyte precursors by specifically targeting the SON gene.

Caveolin-1 deficiency alleviates palmitate-induced intracellular lipid accumulation and inflammation in pancreatic β cells.

Lipotoxicity-induced pancreatic β cell damage is a strong predictor of type 2 diabetes mellitus (T2DM). Our previous work showed that Caveolin-1 (Cav-1) depletion decreased β-cell apoptosis and improved β-cell viability. Further microarray analysis indicated significant changes in the expression of genes related to fatty acid metabolism and inflammation. The objective of this study was to explore the role of Cav-1 in intracellular lipid accumulation and inflammation in β cells under lipotoxic conditions. Here, we established a β-cell-specific Cav-1 knockout (β-Cav-1 KO) mouse model and a CAV-1 depleted β cell line (NIT-1). We found that Cav-1 silencing significantly reduced palmitate (PA)-induced intracellular triglyceride (TG) accumulation and decreased proinflammatory factor expression in both the mouse and cell models. Further mechanistic investigation revealed that amelioration of lipid metabolism was achieved through the downregulation of lipogenic markers (SREBP-1c, FAS and ACC) and upregulation of a fatty acid oxidation marker (CPT-1). Meanwhile, decrease of inflammatory cytokines (IL-6, TNF-α, and IL-1β) secretion was found with the involvement of the IKKβ/NF-κB signaling pathways. Our findings suggest that Cav-1 is of considerable importance in regulating lipotoxicity-induced β-cell intracellular lipid accumulation and inflammation.

Theaflavin inhibits African swine fever virus replication by disrupting lipid metabolism through activation of the AMPK signaling pathway in virto

African swine fever virus (ASFV) is the etiological agent of African swine fever (ASF), which is one of the most harmful swine diseases in the pig industry because of its nearly 100% mortality rate in domestic pigs and results in incalculable economic loss. Ever since ASF was initially reported, scientists have worked to develop anti-ASF vaccines; however, currently no clinically effective vaccine for ASF is available. Therefore, the development of novel measures to prevent ASFV infection and transmission is essential. In this study, we aimed to investigate the anti-ASF activity of theaflavin (TF), a natural compound mainly isolated from black tea. We found that TF potently inhibited ASFV replication at non-cytotoxic concentrations ex vivo in primary porcine alveolar macrophages (PAMs). Mechanistically, we found that TF inhibited ASFV replication by acting on cells rather than interacting directly with ASFV to inhibit viral replication. Further, we found that TF upregulated the AMPK (5′-AMP-activated protein kinase) signaling pathway in ASFV-infected and uninfected cells, and treatment with the AMPK agonist MK8722 upregulated the AMPK signaling pathway and inhibited ASFV proliferation in a dose-dependent manner. Notably, the effects of TF on AMPK activation and ASFV inhibition were partially reversed by the AMPK inhibitor dorsomorphin. In addition, we found that TF down-regulated the expression of genes related to lipid synthesis and decreased the intracellular accumulation of total cholesterol and total triglycerides in ASFV-infected cells, suggesting that TF may inhibit ASFV replication by disrupting lipid metabolism. In summary, our results demonstrated that TF is an ASFV infection inhibitor and revealed the mechanism by which ASFV replication is inhibited, providing a novel mechanism and potential lead compound for the development of anti-ASFV drugs.

Suppression of Lipid Accumulation in the Differentiation of 3T3-L1 Preadipocytes and Human Adipose Stem Cells into Adipocytes by TAK-715, a Specific Inhibitor of p38 MAPK

Excessive preadipocyte differentiation is linked with obesity. Although previous studies have shown that p38 MAPK is associated with adipogenesis, the regulation of preadipocyte differentiation by TAK-715, an inhibitor of p38 mitogen-activated protein kinase (MAPK), remains unclear. Interestingly, TAK-715 at 10 μM vastly suppressed the accumulation of lipid and intracellular triglyceride (TG) content with no cytotoxicity during 3T3-L1 preadipocyte differentiation. On mechanistic levels, TAK-715 significantly decreased the expressions of the CCAAT/enhancer-binding protein-α (C/EBP-α), peroxisome proliferator-activated receptor gamma (PPAR-γ), fatty acid synthase (FAS), and perilipin A. Similarly, the phosphorylation of the signal transducer and activator of transcription-3 (STAT-3) in differentiating 3T3-L1 cells was also reduced with TAK-715 treatment. Moreover, TAK-715 significantly blocked the phosphorylation of activating transcription factor-2 (ATF-2), a p38 MAPK downstream molecule, during 3T3-L1 preadipocyte differentiation. Of importance, TAK-715 also markedly impeded the phosphorylation of p38 MAPK and suppressed lipid accumulation during the adipocyte differentiation of human adipose stem cells (hASCs). Concisely, this is the first report that TAK-715 (10 μM) has potent anti-adipogenic effects on the adipogenesis process of 3T3-L1 cells and hASCs through the regulation of the expression and phosphorylation of p38 MAPK, C/EBP-α, PPAR-γ, STAT-3, FAS, and perilipin A.

Cydonia oblonga Miller fruit extract exerts an anti-obesity effect in 3T3-L1 adipocytes by activating the AMPK signaling pathway.

The fruit of Cydonia oblonga Miller (COM) is used traditionally in Mediterranean region medicine to prevent or treat obesity, but its mechanism of action is still unclear. Beyond a demonstrated anti-obesity effect, the fruit was tested for the mechanism of adipogenesis in 3T3-L1 preadipocytes. 3T3-L1 preadipocytes were cultured for 8 days with COM fruit extract (COME) at different concentrations (0-600 µg/mL) with adipocyte differentiation medium. The cell viability was measured using an MTT assay; triglyceride (TG) was stained with Oil Red O. The expression levels of the adipogenesis-related genes and protein expression were analyzed by reverse transcription polymerase chain reaction and Western blotting, respectively. COME inhibited intracellular TG accumulation during adipogenesis. A COME treatment in 3T3-L1 cells induced upregulation of the adenosine monophosphate-activated protein kinase (AMPK)α phosphorylation and downregulation of the adipogenic transcription factors, such as sterol regulatory element-binding protein 1c, peroxisome proliferator-activated receptor γ, and CCAAT/enhancer binding protein α. The COME treatment reduced the mRNA expression of fatty acyl synthetase, adenosine triphosphate-citrate lyase, adipocyte protein 2, and lipoprotein lipase. It increased the mRNA expression of hormone-sensitive lipase and carnitine palmitoyltransferase I in 3T3-L1 cells. COME inhibits adipogenesis via the AMPK signaling pathways. COME may be used to prevent and treat obesity.

MTORC1 regulates a lysosome-dependent adaptive shift in intracellular lipid species.

The mechanistic target of rapamycin complex 1 (mTORC1) senses and relays environmental signals from growth factors and nutrients to metabolic networks and adaptive cellular systems to control the synthesis and breakdown of macromolecules; however, beyond inducing de novo lipid synthesis, the role of mTORC1 in controlling cellular lipid content remains poorly understood. Here we show that inhibition of mTORC1 via small molecule inhibitors or nutrient deprivation leads to the accumulation of intracellular triglycerides in both cultured cells and a mouse tumor model. The elevated triglyceride pool following mTORC1 inhibition stems from the lysosome-dependent, but autophagy-independent, hydrolysis of phospholipid fatty acids. The liberated fatty acids are available for either triglyceride synthesis or β-oxidation. Distinct from the established role of mTORC1 activation in promoting de novo lipid synthesis, our data indicate that mTORC1 inhibition triggers membrane phospholipid trafficking to the lysosome for catabolism and an adaptive shift in the use of constituent fatty acids for storage or energy production.

Identification of motifs and mechanisms for lipid droplet targeting of the lipolytic inhibitors G0S2 and HIG2.

G0S2 and HIG2 are two selective inhibitors of ATGL (also known as PNPLA2), the key enzyme for intracellular lipolysis. Whereas G0S2 regulates triglyceride (TG) mobilization in adipocytes and hepatocytes, HIG2 functions to enhance intracellular TG accumulation under hypoxic conditions. A homologous hydrophobic domain (HD) is shared by G0S2 and HIG2 (also known as HILPDA) for binding to ATGL. However, the determinants of their lipid droplet (LD) localization are unknown. Here, we study how G0S2 and HIG2 are targeted to LDs, and identify both ATGL-independent and -dependent mechanisms. Structural prediction and studies in cells reveal that ATGL-independent localization of G0S2 to both the endoplasmic reticulum (ER) and LDs is mediated by a hairpin structure consisting of two hydrophobic sequences. Positively charged residues in the hinge region play a crucial role in sorting G0S2, which initially localizes to ER, to LDs. Interestingly, the role of these positive charges becomes dispensable when ATGL is co-expressed. In comparison, HIG2, which lacks a similar hairpin structure, is dependent on ATGL for its full LD targeting. Thus, our studies identify specific structural features and mechanisms for mediating accumulation of these two ATGL inhibitors on LDs.

Hesperidin methyl chalcone ameliorates lipid metabolic disorders by activating lipase activity and increasing energy metabolism

Obesity has become an increasingly serious health issue with the continuous improvement in living standards. Its prevalence has become an economic burden on health care systems worldwide. Flavonoids have been shown to be beneficial in the prevention and treatment of obesity. Here, we evaluated the therapeutic potential of the flavonoid hesperidin methyl chalcone (HMC) on mice with high-fat diet (HFD)-induced hepatic steatosis in vivo and in vitro. Treatment with HMC reduced oleic and palmitic acid-induced increases in intracellular triglyceride accumulation in HepG2, AML12 and LMH cells. HMC also enhanced energy metabolism and lowered oxidative stress. We used Discovery studio to dock key proteins associated with lipid metabolism disorders to HMC, and found that HMC interacted with lipase. Furthermore, we demonstrated that HMC improved lipase activity and lipolysis. In addition, we found that HMC promoted glucose absorption, alleviated lipid metabolic disorders, improved HFD-induced liver injury, and regulated HFD-induced changes in energy metabolism. In conclusion, our study demonstrated that HMC ameliorated HFD-induced obesity and its complications by promoting lipase activity, and provides a novel approach for the prevention and treatment of obesity and related diseases.

Apol9a regulates myogenic differentiation via the ERK1/2 pathway in C2C12 cells.

Background: The rising prevalence of obesity and its complications is a big challenge for the global public health. Obesity is accompanied by biological dysfunction of skeletal muscle and the development of muscle atrophy. The deep knowledge of key molecular mechanisms underlying myogenic differentiation is crucial for discovering novel targets for the treatment of obesity and obesity-related muscle atrophy. However, no effective target is currently known for obesity-induced skeletal muscle atrophy. Methods: Transcriptomic analyses were performed to identify genes associated with the regulation of myogenic differentiation and their potential mechanisms of action. C2C12 cells were used to assess the myogenic effect of Apol9a through immunocytochemistry, western blotting, quantitative polymerase chain reaction, RNA interference or overexpression, and lipidomics. Results: RNA-seq of differentiated and undifferentiated C2C12 cells revealed that Apol9a expression significantly increased following myogenic differentiation and decreased during obesity-induced muscle atrophy. Apol9a silencing in these C2C12 cells suppressed the expression of myogenesis-related genes and reduced the accumulation of intracellular triglycerides. Furthermore, RNA-seq and western blot results suggest that Apol9a regulates myogenic differentiation through the activation of extracellular signal-regulated kinase 1/2 (ERK1/2). This assumption was subsequently confirmed by intervention with PD98059. Conclusion: In this study, we found that Apol9a regulates myogenic differentiation via the ERK1/2 pathway. These results broaden the putative function of Apol9a during myogenic differentiation and provide a promising therapeutic target for intervention in obesity and obesity-induced muscle atrophy.

Lactic acid bacteria–derived γ-linolenic acid metabolites are PPARδ ligands that reduce lipid accumulation in human intestinal organoids

Gut microbiota regulate physiological functions in various hosts, such as energy metabolism and immunity. Lactic acid bacteria, including Lactobacillus plantarum, have a specific polyunsaturated fatty acid saturation metabolism that generates multiple fatty acid species, such as hydroxy fatty acids, oxo fatty acids, conjugated fatty acids, and trans-fatty acids. How these bacterial metabolites impact host physiology is not fully understood. Here, we investigated the ligand activity of lactic acid bacteria-produced fatty acids in relation to nuclear hormone receptors expressed in the small intestine. Our reporter assays revealed two bacterial metabolites of γ-linolenic acid (GLA), 13-hydroxy-cis-6,cis-9-octadecadienoic acid (γHYD), and 13-oxo-cis-6,cis-9-octadecadienoic acid (γKetoD) activated peroxisome proliferator-activated receptor delta (PPARδ) more potently than GLA. We demonstrate that both γHYD and γKetoD bound directly to the ligand-binding domain of human PPARδ. A docking simulation indicated that four polar residues (T289, H323, H449, and Y473) of PPARδ donate hydrogen bonds to these fatty acids. Interestingly, T289 does not donate a hydrogen bond to GLA, suggesting that bacterial modification of GLA introducing hydroxy and oxo group determines ligand selectivity. In human intestinal organoids, we determined γHYD and γKetoD increased the expression of PPARδ target genes, enhanced fatty acid β-oxidation, and reduced intracellular triglyceride accumulation. These findings suggest that γHYD and γKetoD, which gut lactic acid bacteria could generate, are naturally occurring PPARδ ligands in the intestinal tract and may improve lipid metabolism in the human intestine.