Liver Lipid Metabolism Research Articles

Combination of Pioglitazone and Metformin Actions on Liver Lipid Metabolism in Obese Mice.

Despite the increasing prevalence rate of nonalcoholic fatty liver disease (NAFLD) worldwide, efficient pharmacotherapeutic regimens against NAFLD still need to be explored. Previous studies found that pioglitazone and metformin therapy could partly ameliorate NAFLD, but their combination therapy effects have not been researched. In the present study, we assessed the protective effects of metformin and pioglitazone combination therapy on liver lipid metabolism in high-fat diet (HFD)-fed mice and investigated the molecular mechanism. Male C57BL/6 mice were divided into five groups: normal control; HFD control; metformin monotherapy; pioglitazone monotherapy and combined therapy. After 8 weeks of pharmacological intervention, glucose and lipid metabolism characteristics, hepatic histology, lipidomics profiling and RNA-seq analysis were performed. The combination of pioglitazone and metformin significantly ameliorated HFD-induced metabolic disturbance and the hepatic oil red O area. A lipidomics analysis showed that combined therapy could significantly reduce the high levels of free fatty acids (FFA), diacylglycerol and triglycerides, while a set of glycerophospholipids and sphingolipids were increased in the combined therapy group. Consistently, an RNA-seq analysis also showed a remarkable reduction in genes associated with FFA uptake and de novo lipogenesis, including Cd36, Fads1, Fads2, Fasn, Scd1, Elovl5 and Pklr in the combined therapy group. Pioglitazone and metformin might have a synergistic protective effect on NAFLD by improving hepatic lipid profiles in HFD-induced mice. Further studies are needed to verify the clinical effects.

Chaperone-mediated autophagy dysregulation during aging impairs hepatic fatty acid oxidation via accumulation of NCoR1

ObjectiveAlterations in lipid metabolism are associated with aging and age-related diseases. Chaperone-mediated autophagy (CMA) is a lysosome-dependent process involved in specific protein degradation. Heat shock cognate 71 kDa protein (Hsc70) recognizes cytosolic proteins with KFERQ motif and allows them to enter the lysosome via lysosome-associated membrane glycoprotein 2 isoform A (LAMP2A). CMA deficiency is associated with dysregulated lipid metabolism in the liver. In this study, we examined the effect of CMA on lipid metabolism in the aged liver. Methods12-week-old and 88-week-old mice were employed to assess the effect of aging on hepatic CMA activity. We generated CMA-deficient mouse primary hepatocytes using siRNA for Lamp2a and liver-specific LAMP2A knockdown mice via adeno-associated viruses expressing short hairpin RNAs to investigate the influence of CMA on lipid metabolism. ResultsWe noted aging-induced progression toward fatty liver and a decrease in LAMP2A levels in total protein and lysosomes. The expression of genes associated with fatty acid oxidation was markedly downregulated in the aged liver, as verified in CMA-deficient mouse primary hepatocytes. In addition, the aged liver accumulated nuclear receptor corepressor 1 (NCoR1), a negative regulator of peroxisome proliferator-activated receptor α (PPARα). We found that Hsc70 binds to NCoR1 via the KFERQ motif. Lamp2a siRNA treatment accumulated NCoR1 and decreased the fatty acid oxidation rate. Pharmacological activation of CMA by AR7 treatment increased LAMP2A expression, leading to NCoR1 degradation. A liver-specific LAMP2A knockdown via adeno-associated viruses expressing short hairpin RNAs caused NCoR1 accumulation, inactivated PPARα, downregulated the expression of fatty acid oxidation-related genes and significantly increased liver triglyceride levels. ConclusionsOur results elucidated a novel PPARα regulatory mechanism involving CMA-mediated NCoR1 degradation during aging. These findings demonstrate that CMA dysregulation is crucial for the progression of aging-related fatty liver diseases.

Effects of copper exposure and recovery in juvenile yellowtail kingfish (Seriola lalandi): Histological, physiological and molecular responses

This study investigated the physiological responses to copper toxicity in liver and gill of juvenile yellowtail kingfish by exposing to 0 (control), 0.0672, 0.12, 0.214, 0.384, 0.672, 1.20, or 2.14 mg/L copper sulfate for 7 days and then recovered for 14 days. The histopathology, enzyme activities and differential gene expression were detected. The results indicate the safe concentration value of copper for juvenile yellowtail kingfish is 0.083 mg/L. Copper exposure caused structural damage to gill and liver, and could be disappeared or restored after the recovery period. Copper exposure enhanced the Cu,Zn superoxide dismutase (Cu/Zn-SOD) and glutathione peroxidase (GSH-Px) activities and the malondialdehyde (MDA) level in liver. However, with increasing copper concentrations, liver aspartate transaminase (AST) and gill Na+/K+-ATPase activities were at first enhanced and then inhibited. Serum lysozyme (LZM) and Cu/Zn-SOD activities were enhanced at low copper concentrations but inhibited at high concentrations, and the activities of alanine transaminase (ALT) and AST significantly increased at greater copper concentrations. After the recovery period, only liver Cu/Zn-SOD activity returned to control level. In contrast, liver GSH-Px activity and MDA levels and gill Na+/K+-ATPase activity, serum LZM and AST activities remained significantly higher than control, but liver AST activity, serum ALT, and Cu/Zn-SOD activities were significantly lower than control. We constructed the gene regulatory networks of molecular pathways in liver and gill in response to copper exposure. The DEGs were enriched mainly in the pathway of lipid metabolism in liver and pathway of oxidative phosphorylation in gill, indicating these respective pathways are involved in the response to copper stress in this species. The present results elucidate some histological, physiological, and molecular mechanisms underlying copper-exposure stresses in yellowtail kingfish, which could help guide the safe utilization of copper sulfate in disease control of farmed individuals.

L-carnitine promotes liver regeneration after hepatectomy by enhancing lipid metabolism

BackgroundLipid metabolism plays an important role in liver regeneration, but its regulation still requires further research. In this study, lipid metabolites involved in mouse liver regeneration at different time points were sequenced and analyzed to study their influence on liver regeneration and its mechanism.MethodsOur experiment was divided into two parts. The first part examined lipid metabolites during liver regeneration in mice. In this part, lipid metabolites were sequentially analyzed in the livers of 70% mouse hepatectomy models at 0, 1, 3and 7 days after operation to find the changes of lipid metabolites in the process of liver regeneration. We screened L-carnitine as our research object through metabolite detection. Therefore, in the second part, we analyzed the effects of carnitine on mouse liver regeneration and lipid metabolism during liver regeneration. We divided the mouse into four groups: control group (70% hepatectomy group); L-carnitine group (before operation) (L-carnitine were given before operation); L-carnitine group (after operation)(L-carnitine were given after operation) and L-carnitine + perhexiline maleate (before operation) group. Weighing was performed at 24 h, 36 and 48 h in each group, and oil red staining, HE staining and MPO staining were performed. Tunnel fluorescence staining, Ki67 staining and serological examination.ResultsSequencing analysis of lipid metabolites in 70% of mouse livers at different time points after hepatectomy showed significant changes in carnitine metabolites. The results showed that, compared with the control group the mouse in L-carnitine group (before operation) at 3 time points, the number of fat drops in oil red staining was decreased, the number of Ki67 positive cells was increased, the number of MPO positive cells was decreased, the number of Tunnel fluorescence positive cells was decreased, and the liver weight was increased. Serum enzymes were decreased. Compared with control group, L-carnitine group (after operation) showed similar trends in all indexes at 36 and 48 h as L-carnitine group (before operation). L-carnitine + perhexiline maleate (before operation) group compared with control group, the number of fat drops increased, the number of Ki67 positive cells decreased, and the number of MPO positive cells increased at 3 time points. The number of Tunnel fluorescent positive cells increased and serum enzyme increased. However, both liver weights increased.ConclusionL-carnitine can promote liver cell regeneration by promoting lipid metabolism and reduce aseptic inflammation caused by excessive lipid accumulation.

Effect of Omega-3 Rich High-Fat Diet on Markers of Tissue Lipid Metabolism in Glucocorticoid-Treated Mice.

Glucocorticoids (GCs) are some of the most widely prescribed therapies for treating numerous inflammatory diseases and multiple cancer types. With chronic use, GCs' therapeutic benefits are concurrent with deleterious metabolic side effects, which worsen when combined with a high-fat diet (HFD). One characteristic of the common Western HFD is the presence of high omega-6 polyunsaturated fatty acids (PUFAs) and a deficiency in omega-3 PUFAs. The aim of this experiment was to determine whether fat composition resulting from HFD affects glucocorticoid-induced alterations in lipid-handling by the liver and skeletal muscle. Male wild-type C57BL/6 mice were randomized into two groups: n-6 (45% fat 177.5 g lard) and n-3 (45% fat 177.5 g Menhaden oil). After 4 weeks on their diets, groups were divided to receive either daily injections of dexamethasone (3 mg/kg/day) or sterile PBS for 1 week while continuing diets. The n-3 HFD diet attenuated adipose and hepatic fatty accumulation and prevented GC-induced increases in liver lipid metabolism markers Cd36 and Fabp. N-3 HFD had little effect on markers of lipid metabolism in oxidative and glycolytic skeletal muscle and was unable to attenuate GC-induced gene expression in the muscle. The present study's result demonstrated that the change of fat composition in HFD could beneficially alter the fatty acid accumulation and associated lipid metabolism markers in mice treated with dexamethasone.

Discovery of biomarkers related to abnormal lipid metabolism in liver and serum and intervention mechanism of ginsenoside Rb_1 in hyperlipidemia rats based on non-targeted metabolomics

Through the non-targeted metabolomics study of endogenous substances in the liver and serum of hyperlipidemia rats, the biomarkers related to abnormal lipid metabolism in hyperlipidemia rats were found, and the target of ginsenoside Rb_1 in improving hyperlipidemia was explored and its mechanism was elucidated. The content of serum biochemical indexes of rats in each group was detected by the automatic biochemical analyzer. The metabolite profiles of liver tissues and serum of rats were analyzed by HPLC-MS. Principal component analysis(PCA) and orthogonal partial least squares-discriminant analysis(OPLS-DA) were used to compare and analyze the metabolic data in the normal group, the hyperlipidemia group, and the ginsenoside Rb_1 group, and screen potential biomar-kers. The related metabolic pathways were further constructed by KEGG database analysis. The results showed that hyperlipemia induced dyslipidemia in rats, which was alleviated by ginsenoside Rb_1. The non-targeted metabolomics results showed that there were 297 differential metabolites in the liver tissues of hyperlipidemia rats, 294 differential metabolites in the serum samples, and 560 diffe-rential metabolites in the hyperlipidemia rats treated by ginsenoside Rb_1. Perillic acid and N-ornithyl-L-taurine were common metabolites in the liver and serum samples, which could be used as potential biomarkers for ginsenoside Rb_1 in the improvement of hyperlipidemia. As revealed by pathway enrichment in the liver and serum, ginsenoside Rb_1 could participate in the metabolic pathway of choline in both the liver and serum. In addition, ginsenoside Rb_1 also participated in the ABC transporter, alanine, aspartic acid, and glutamate metabolism, protein digestion and absorption, β-alanine metabolism, taurine and hypotaurine metabolism, caffeine metabolism, valine, leucine, and isoleucine biosynthesis, arachidonic acid metabolism, and methionine and cysteine metabolism to improve dyslipidemia in rats.

Effects of Different Dietary Starch Sources and Digestible Lysine Levels on Carcass Traits, Serum Metabolites, Liver Lipid and Breast Muscle Protein Metabolism in Broiler Chickens.

This study investigated the effects of digestible lysine (dLys) in different dietary starch sources on liver lipid metabolism and breast muscle protein metabolism in broiler chickens. The experimental design was a 3 × 3 two-factor completely randomized design. A total of 702 one-day-old male Arbor Acres Plus broilers were randomly divided into nine treatments of six replicate cages with thirteen birds each. The treatments consisted of three different starch sources (corn, cassava and waxy corn) with three different dLys levels (1.08%, 1.20% and 1.32%). The trial lasted from 1 to 21 days. Carcass traits, serum metabolites, breast muscle protein and liver lipid metabolism were evaluated. A significant interaction effect (p < 0.05) for dietary starch sources and dLys levels was noted in the percentage of abdominal fat and gene expression related to breast muscle protein metabolism throughout the experimental period. The waxy corn starch diet and a 1.08% dLys level in the diet increased both the percentage of abdominal fat (p < 0.01) and blood total cholesterol (p < 0.05) in the broilers. The waxy corn starch diet significantly upregulated the mRNA expressions of Eif4E, AMPK, FABP1, ACC and CPT1 (p < 0.05). The 1.32% dLys level significantly upregulated the mRNA expressions of mTOR, S6K1, Eif4E, AMPK and PPARα (p < 0.05) and significantly downregulated the mRNA expressions of MuRF and Atrogin-1 (p < 0.05). In summary, the waxy corn starch diet resulted in significantly higher expression levels of fat-synthesis-related genes than lipolysis-related genes, leading to abdominal fat deposition in broilers. Increasing the level of dLys in the diet increased the protein content in muscle by promoting protein synthesis and inhibiting protein degradation and also promoted the expression of lipolysis-related genes, thereby degrading the generation of abdominal fat in broilers. Our findings signify that increasing the dLys level to 1.32% when using the waxy corn starch diet could improve carcass traits.

Impacts of polypropylene microplastics on lipid profiles of mouse liver uncovered by lipidomics analysis and Raman spectroscopy

Microplastics (MPs) are emerging contaminants, and there are only limited studies reporting the impacts of some MPs on liver lipid metabolism in animals. In this study, we investigated the accumulation of polypropylene-MPs in mouse liver and unraveled the change in lipid metabolic profiles by both lipidomics and Raman spectroscopy. Polypropylene-MP exposure did not cause obvious health symptoms, but hematoxylin-eosin staining showed pathological changes that polypropylene-MPs induced lipid droplet accumulation in liver. Lipidomics results showed a significant change in lipid metabolic profiles and the most influenced categories were triglycerides, fatty acids, free fatty acids and lysophosphatidylcholine, implying the effects of polypropylene-MPs on the hemostasis of lipid droplet biogenesis and catabolism. Most altered lipids contained unsaturated bonds and polyunsaturated phospholipids, possibly affecting the fluidity and curvature of membrane surfaces. Raman spectroscopy confirmed that the major spectral alterations of liver tissues were related to lipids, evidencing the altered lipid metabolism and cell membrane components in the presence of polypropylene-MPs. Our findings firstly disclosed the impacts of polypropylene-MPs on lipid metabolisms in mouse liver and hinted at their detrimental disturbance on membrane properties, cellular lipid storage and oxidation regulation, helping our deeper understanding on the toxicities and corresponding risks of polypropylene-MPs to mammals.

Pyridylnidulin exerts anti-diabetic properties and improves non-alcoholic fatty liver disease in diet-induced obesity mice.

Introduction: Non-alcoholic fatty liver disease (NAFLD) is one of the metabolic disorders related to the pathophysiology of type 2 diabetes mellitus (T2DM). Therapeutic strategies are focused on the improvement of energy balance and lifestyle modification. Additionally, the derivative of the bioactive fungal metabolite is of interest to provide health benefits, especially in obese and pre-diabetic conditions. In our screening of anti-diabetic compounds from fungal metabolites and semisynthetic derivatives, a depsidone derivative, namely pyridylnidulin (PN), showed potent glucose uptake-inducing activity. The present study aimed to investigate the liver lipid metabolism and anti-diabetic properties of PN in diet-induced obesity mice. Methods: Male C57BL/6 mice were induced obesity and pre-diabetic conditions by dietary intervention with a high-fat diet (HFD) for 6 weeks. These obese mice were orally administered with PN (40 or 120 mg/kg), metformin (150 mg/kg), or vehicle for 4 weeks. Glucose tolerance, plasma adipocytokines, hepatic gene and protein expressions were assessed after treatment. Results: Improved glucose tolerance and reduced fasting blood glucose levels were found in the PN and metformin-treated mice. Additionally, hepatic triglyceride levels were consistent with the histopathological steatosis score regarding hepatocellular hypertrophy in the PN and metformin groups. The levels of plasma adipocytokines such as tumor necrosis factor-α (TNF-α) and monocyte chemoattractant protein-1 (MCP-1) were reduced in the PN (120 mg/kg) and metformin-treated mice. In addition, hepatic gene expression involved in lipid metabolism, including lipogenic enzymes was significantly reduced in the PN (120 mg/kg) and metformin-treated mice. The increased protein expression levels of phosphorylated AMP-activated protein kinase (p-AMPK) was also found in PN and metformin-treated mice. Discussion: Considering the increased p-AMPK protein expression levels in PN and metformin-treated mice were revealed as the underlying mechanisms to improve metabolic parameters. These results suggested that PN provided the health benefit to slow the progression of NAFLD and T2DM in obese and pre-diabetic conditions.

69-OR: Multiancestry Genome-Wide Association Study Reveals Distinct Biological Pathways and Cell Types Driving Type 2 Diabetes Risk with Heterogeneous Effects across Diverse Populations

The Type 2 Diabetes Global Genomics Initiative aggregated genome-wide association studies (GWAS) of type 2 diabetes (T2D) in 428,452 affected individuals and 2,107,149 controls from five major ancestries (39.7% non-European ancestry) to enhance power for locus discovery and improve understanding of the pathophysiological basis of the disease. We identified 1,289 independent association signals (P &amp;lt; 5 x 10-8) at 611 loci, of which 145 were previously unreported. We applied K-means clustering to the signals based on their associations with 37 cardiometabolic traits and identified eight clusters with distinct profiles. Three clusters were related to insulin secretion and had a positive, negative, or no effect on proinsulin levels. These clusters were significantly enriched (P &amp;lt; 0.05/222, Bonferroni correction for the number of cell types) for epigenomic annotations from single-cell ATAC-seq data in pancreatic islets but had different enrichment in pancreatic ductal cells and intestinal endocrine cells. The remaining five clusters related to insulin resistance (IR) physiology: increased body fat; unfavourable fat distribution (decreased gluteofemoral fat and increased visceral fat); obesity (increased body mass index); lipodystrophy (increased fasting insulin and decreased body fat); and liver lipid metabolism (increased liver fat and liver enzymes). IR clusters showed more diverse patterns of enrichment: obesity in adrenal cells; fat distribution in endothelial cells and pericytes in blood vessels; and lipodystrophy in adipocytes. T2D signals were more strongly enriched for ancestry-correlated heterogeneity in effect sizes in insulin secretion clusters than IR clusters. These data demonstrate distinct clusters of T2D association signals representing different trajectories to disease with observable differences in effect sizes across ancestry groups. Disclosure K.Suzuki: None. Diamante consortium: n/a. Va million veteran program: n/a. J.B.Meigs: Consultant; Quest Diagnostics. M.I.Mccarthy: Employee; Genentech, Inc., Stock/Shareholder; Roche Pharmaceuticals. A.Mahajan: Employee; Genentech, Inc. J.M.Mercader: None. C.Spracklen: None. M.Boehnke: None. M.Vujkovic: None. J.I.Rotter: None. K.Hatzikotoulas: None. B.F.Voight: None. E.Zeggini: None. A.Morris: None. Type 2 diabetes global genomics initiative: n/a. L.Southam: None. X.Yin: None. K.Lorenz: None. R.Mandla: None. H.J.Taylor: None. A.Huerta: None. N.W.Rayner: None.

1573-P: Association of Thyroid Hormone Levels with NAFLD in Recent-Onset Diabetes

Nonalcoholic fatty liver disease (NAFLD) is a growing health concern associated with obesity and type 2 diabetes (T2D). Thyroid hormones are important regulators of lipid homeostasis. Indeed, people with hypothyroidism are at greater risk for NAFLD and it has been suggested that humans with T2D are more likely to develop hypothyroidism. However, little is known about the relationship of thyroid function with NAFLD in type 1 diabetes (T1D) and T2D. We hypothesized that (i) low free thyroxin (fT4) and high thyroid-stimulating hormone (TSH) levels are associated with increased hepatocellular lipid content (HCL) and (ii) that fT4 and TSH can identify persons at high-risk for hepatic steatosis in people with recent-onset diabetes. We examined people with T1D (n=358) or T2D (n=596) and 175 healthy participants of the German Diabetes Study (GDS). First, validation of the fatty liver index (FLI) against quantification of HCL by 1H-magnetic resonance spectroscopy revealed a close correlation (ß=0.715, p&amp;lt;0.001; n=446). Second, FLI negatively correlated with fT4 in males (ß=−0.139, p&amp;lt;0.01), but not in females with T2D (ß=−0.086, p=0.26). Likewise, TSH associated positively with FLI in males (ß=0.116, p&amp;lt;0.05), but not in females with T2D (ß=−0.057, p=0.45). TSH and FLI were differently associated between males and females before (p&amp;lt;0.05), but not after (p=0.82) adjustment for BMI. Finally, fT4 had a low diagnostic precision for steatosis (by FLI) as derived from the respective area under the receiver operating curve (0.57, 95% CI [0.53;0.62]; p&amp;lt;0.001) in T2D and was not significant in other groups. TSH had no diagnostic precision for steatosis in any groups. In conclusion, FLI can be used as a surrogate measure of HCL in recent-onset diabetes. The correlation of FLI with lower thyroid function in male T2D suggests a sex-specific interaction between thyroid and liver lipid metabolism, which is mainly driven by body mass. Both, fT4 and TSH offer no relevant efficacy to detect steatosis at least in recent-onset diabetes. Disclosure N. Saatmann: None. M. Schön: None. O.P. Zaharia: None. M. Huttasch: None. K. Strassburger: None. S. Trenkamp: None. Y. Kupriyanova: None. V. Schrauwen-Hinderling: None. S. Kahl: None. V. Burkart: None. R. Wagner: Speaker's Bureau; Novo Nordisk, Sanofi. Advisory Panel; Daiichi Sankyo. M. Roden: Advisory Panel; Eli Lilly and Company. Research Support; Boehringer-Ingelheim, Novo Nordisk, Novartis. Consultant; TARGET PharmaSolutions, Inc. Research Support; Sanofi.

Taurine Protects against the Fatty Liver Hemorrhagic Syndrome in Laying Hens through the Regulation of Mitochondrial Homeostasis.

Metabolic-associated fatty liver disease (MAFLD) is a chronic liver disease caused by fat deposition in the liver of humans and mammals, while fatty liver hemorrhagic syndrome (FLHS) is a fatty liver disease in laying hens which can increase the mortality and cause severe economic losses to the laying industry. Increasing evidence has shown a close relationship between the occurrence of fatty liver disease and the disruption of mitochondrial homeostasis. Studies have proven that taurine can regulate hepatic fat metabolism, reduce hepatic fatty deposition, inhibit oxidative stress, and alleviate mitochondrial dysfunction. However, the mechanisms by which taurine regulates mitochondrial homeostasis in hepatocytes need to be further studied. In this study, we determined the effects and mechanisms of taurine on high-energy low-protein diet-induced FLHS in laying hens and in cultured hepatocytes in free fatty acid (FFA)-induced steatosis. The liver function, lipid metabolism, antioxidant capacity, mitochondrial function, mitochondrial dynamics, autophagy, and biosynthesis were detected. The results showed impaired liver structure and function, mitochondrial damage and dysfunction, lipid accumulation, and imbalance between mitochondrial fusion and fission, mitochondrial autophagy, and biosynthesis in both FLHS hens and steatosis hepatocytes. Taurine administration can significantly inhibit the occurrence of FLHS, protect mitochondria in hepatocytes from disease induced by lipid accumulation and FFA, up-regulate the expression levels of Mfn1, Mfn2, Opa1, LC3I, LC3II, PINK1, PGC-1α, Nrf1, Nrf2, and Tfam, and down-regulate the expression levels of Fis1, Drp1, and p62. In conclusion, taurine can protect laying hens from FLHS through the regulation of mitochondrial homeostasis, including the regulation of mitochondrial dynamics, autophagy, and biosynthesis.

1572-P: USP30 Regulates Hepatic Mitophagy and Mitochondrial Function

Mitochondria regulate several cellular and metabolic processes, necessitating cells to employ highly evolved quality control mechanisms to remove damaged mitochondria through a selective form of autophagy called mitophagy. Mitophagy is a mitochondrial quality control pathway, mediated in part by the ubiquitination of outer mitochondrial membrane (OMM) proteins. USP30 is an OMM deubiquitinase that regulates mitophagy by degrading OMM ubiquitin, thus inhibiting mitophagy. Hepatic mitophagy is reduced in obese fatty liver and is associated with mitochondrial dysfunction. Whether USP30 regulates mitophagy in hepatocytes in vivo and affects mitochondrial function is currently unknown. Therefore, we utilized the Cre-lox system to modulate USP30 expression in vivo to determine the effects on hepatic mitochondrial and metabolic function. Tail-vain injection of AAV8-TBG-Cre into USP30fl/fl and wild-type C57BL6 male mice was used to generate liver-specific USP30 knockout (LKO) and control mice (WT). Mice were fed regular chow and sacrificed at 16 weeks of age, 6 weeks after AAV dosing. Mitochondrial respiratory capacity was assessed using purified liver mitochondria and lipid metabolism was assessed biochemically in mice liver and plasma. USP30 liver-specific deletion in mice resulted in an impaired mitochondrial respiratory capacity as evidenced by increased mitochondrial leak respiration and reduced ADP-stimulated respiration in LKO mice compared to WT, such that the respiratory control ratio was significantly reduced in LKO mice. There were no differences in liver triglyceride levels, nor plasma triglycerides or fatty acids, while plasma cholesterol levels were 25% reduced in LKO mice (p=0.051). These data demonstrate that USP30 is an important regulator of mitochondrial respiratory capacity in hepatocytes in vivo, and that USP30 may normally inhibit excessive non-selective mitophagy in otherwise healthy mice. Disclosure O.G.Fagunloye: None. F.Bello: None. A.Vandevender: None. I.J.Sipula: None. M.J.Jurczak: None. Funding National Institute of Diabetes and Digestive and Kidney Diseases (R01DK114012)

Investigating the Levels of Liver Lipogenic and Lipolytic Enzymes in Rat with High-Fat Diet and Sucrose Solution Underwent Progressive Resistance Training

Objective: Consuming too much fat or carbohydrates stimulates lipogenesis and excess fat is stored in non-fat tissues, including the liver, and manifests as obesity and fatty liver disease. This study aimed to investigate the effect of eight weeks of progressive resistance training (PRT) on the liver levels of some enzymes affecting lipid metabolism in rats fed a high-fat diet and sucrose solution.&#x0D; Materials and Methods: Twenty-four male wistar rats with 5 weeks of age were randomly divided into two groups: standard diet (SD) (n=8) and high-fat diet and sucrose solution (HFDS) (n=16). Twelve weeks later, HFDS group was divided into two groups: sedentary (HS) and PRT (HPRT). The PRT program was implemented 3 days a week for 8 weeks. Gene expression of AMPKα1, SCD-1, ATGL and FASN enzymes affecting lipid metabolism in liver tissue and its fat content were investigated.&#x0D; Results: HFDS significantly increased the body weight (P: 0.001) and significantly decreased the liver expression of ATGL and FASN (P: 0.001, P: 0.011). Eight weeks of PRT did not show a significant difference in the expression of AMPKα1, SCD-1, ATGL and FASN genes. Rats fed HFDS had considerably higher levels of triglyceride (TG) and total cholesterol (TC) in their liver tissue (P: 0.004, P: 0.001) and PRT did not affect them (P: 0.959, P: 0.809 respectively).&#x0D; Conclusion: It seems that eight weeks of PRT will not change liver lipid metabolism enzymes. Therefore, modifying the diet and changing it, will probably show different results after PRT.

245-LB: Glucagon Infusion into the Mediobasal Hypothalamus Lowers Hepatic Triglyceride Secretion

Diabetes and obesity are associated with dysregulated liver lipid metabolism. Glucagon has an essential role in lipid metabolism, in addition to regulating glucose homeostasis. The brain is a key coordinator of whole-body metabolism. Glucagon signalling in the mediobasal hypothalamus (MBH), a forebrain region that senses hormones and nutrients to regulate metabolism, lowers hepatic glucose production and appetite. However, whether glucagon action in the MBH affects lipid homeostasis in healthy and high-fat diet (HFD)-fed animals remains unknown. We assessed glucagon signaling in the MBH in regulating hepatic TG secretion in chow-fed normolipidemic and HFD-induced hyperlipidemic rats. Stereotaxic MBH cannulation and vascular catheterizations in Sprague-Dawley rats allowed for direct MBH infusions, intravenous (IV) injections, and blood sampling. Plasma TGs were measured in 10h-fasted rats after IV poloxamer injection with concurrent MBH infusions. MBH glucagon decreased TG secretion compared to MBH vehicle control animals. This hypothalamic effect of glucagon was blocked by a simultaneous MBH co-infusion of an antagonist to the glucagon receptor (GCGR) and by protein kinase A (PKA) inhibition in the MBH in chow-fed rats. Direct MBH infusion of a PKA activator recapitulated the TG-lowering effects of MBH glucagon. Together, this suggests that MBH glucagon signals via a MBH GCGR-PKA pathway. Interestingly, both MBH glucagon and MBH PKA activation lowered TG secretion and plasma FFAs in HFD-induced hyperlipidemic animals. However, MBH glucagon did not affect hepatic TG content or liver lipogenic protein levels of MTP, FAS, or pACC:ACC of RC and HFD rats compared to their MBH vehicle controls. We demonstrate that hypothalamic glucagon modulates hepatic TG secretion via a MBH GCGR-PKA signalling pathway, and activating MBH glucagon signalling improves lipid metabolism in hyperlipidemic rats. These findings may provide therapeutic insight on lowering lipids in hyperlipidemia. Disclosure M. S. Grewal: None. B. Vasilev: None. C. M. Soltys: None. B. Lum: None. J. T. Yue: None. Funding Canadian Institutes of Health Research (PJT-378765); Canada Foundation for Innovation (37267); Canada Research Chairs Tier 2 in Brain Regulation of Metabolism

Mettl3-mediated m6A modification plays a role in lipid metabolism disorders and progressive liver damage in mice by regulating lipid metabolism-related gene expression.

N6-methyladenosine (m6A), the most abundant and conserved epigenetic modification of mRNA, participates in various physiological and pathological processes. However, the roles of m6A modification in liver lipid metabolism have yet to be understood entirely. We aimed to investigate the roles of the m6A "writer" protein methyltransferase-like 3 (Mettl3) in liver lipid metabolism and the underlying mechanisms. We assessed the expression of Mettl3 in liver tissues of diabetes (db/db) mice, obese (ob/ob) mice, high saturated fat-, cholesterol-, and fructose-induced non-alcoholic fatty liver disease (NAFLD) mice, and alcohol abuse and alcoholism (NIAAA) mice by quantitative reverse-transcriptase PCR (qRT-PCR). Hepatocyte-specific Mettl3 knockout mice were used to evaluate the effects of Mettl3 deficiency in mouse liver. The molecular mechanisms underlying the roles of Mettl3 deletion in liver lipid metabolism were explored by multi-omics joint analysis of public data from the Gene Expression Omnibus database and further validated by qRT-PCR and Western blot. Significantly decreased Mettl3 expression was associated with NAFLD progression. Hepatocyte-specific knockout of Mettl3 resulted in significant lipid accumulation in the liver, increased serum total cholesterol levels, and progressive liver damage in mice. Mechanistically, loss of Mettl3 significantly downregulated the expression levels of multiple m6A-modified mRNAs related to lipid metabolism, including Adh7, Cpt1a, and Cyp7a1, further promoting lipid metabolism disorders and liver injury in mice. In summary, our findings demonstrate that the expression alteration of genes related to lipid metabolism by Mettl3-mediated m6A modification contributes to the development of NAFLD.

Integrated serum pharmacochemistry, 16S rRNA sequencing and metabolomics to reveal the material basis and mechanism of Yinzhihuang granule against non-alcoholic fatty liver disease.

Yinzhihuang granule (YZHG) has liver protective effect and can be used for clinical treatment of non-alcoholic fatty liver disease (NAFLD), but its material basis and mechanism need to be further clarified. This study aims to reveal the material basis and mechanism of YZHG treating NAFLD. Serum pharmacochemistry were employed to identify the components from YZHG. The potential targets of YZHG against NAFLD were predicted by system biology and then preliminarily verified by molecular docking. Furthermore, the functional mechanism of YZHG in NAFLD mice was elucidated by 16S rRNA sequencing and untargeted metabolomics. From YZHG, 52 compounds were identified, of which 42 were absorbed into the blood. Network pharmacology and molecular docking showed that YZHG treats NAFLD with multi-components and multi-targets. YZHG can improve the levels of blood lipids, liver enzymes, lipopolysaccharide (LPS), and inflammatory factors in NAFLD mice. YZHG can also significantly improve the diversity and richness of intestinal flora and regulate glycerophospholipid and sphingolipid metabolism. Moreover, Western Blot experiment showed that YZHG can regulate liver lipid metabolism and enhance intestinal barrier function. YZHG may treat NAFLD by improving the disruption of intestinal flora and enhancing the intestinal barrier. This will reduce the invasion of LPS into the liver subsequently regulate liver lipid metabolism and reduce liver inflammation.

Differential expression of lipid metabolic genes in hypercholesterolemic rabbit placenta predisposes the offspring to develop atherosclerosis in early adulthood

AimsMaternal hypercholesterolemia (MHC) is a pathological condition that may cause atherosclerosis in the adulthood of the offspring. The study aims to identify the role of in-utero programming by the placenta in atherogenesis and associated liver pathology in offspring. Main methodsFemale New Zealand white rabbits with normal lipid profiles were fed a 0.3 % HFD after mating. Lipid levels were monitored, and pregnant rabbits were sacrificed at the end of trimester 1, trimester 2, and trimester 3. Placental histology and expression of lipid metabolism genes were studied. Lipid levels, aortic lesions, and mRNA expression of cholesterol synthesis genes were investigated in fetuses at the end of gestation. A group of fetuses was allowed to attain early adulthood to investigate the liver lipid metabolism and atherogenesis with and without an HFD. Key findingsElevated maternal lipid levels and placental gene expression were differentially modulated in HFD-fed mothers. HFD-fed rabbits demonstrated differential expression of the placental genes involved in receptor-mediated endocytosis of cholesterol, lipogenesis, and lipolysis in all three trimesters. It resulted in significant lipid depositions in the placenta, hyperlipidemia, and a decrease in hepatic cholesterol synthesis in fetuses at the end of gestation. There was no atherogenesis in the aorta of offspring at trimester 3, but such offspring of HFD-fed mothers developed atherosclerosis and non-alcoholic fatty liver (NAFL) with profound steatosis in their early adulthood with and without HFD. SignificanceDiet-induced MHC differentially expressed placental lipid genes that may program the offspring to develop atherosclerosis and associated NAFL in early adulthood.

Lapachol treats non-alcoholic fatty liver disease by modulating the M1 polarization of Kupffer cells via PKM2

AimThis study investigated the mechanism of action of lapachol (LAP) against non-alcoholic fatty liver disease (NAFLD). MethodsPrimary Kupffer cells (KCs) of rats were used for in-vitro experiments. The proportion of M1 cells was assayed by flow cytometry, the levels of M1 inflammatory markers were determined by enzyme-linked immunosorbent assay (ELISA) combined with real-time quantitative fluorescence PCR (RT-qPCR), the expression of p-PKM2 was detected by Western-Blotting. A SD rat model of NAFLD was established with high-fat diet. Following LAP intervention, the changes in blood glucose/lipid, insulin resistance and liver function were detected, and the hepatic histopathologic changes were examined by histological staining. ResultsThe results showed that LAP could inhibit the M1 polarization of KCs, lower the levels of inflammatory cytokines, and suppress the activation of PKM2. The effect of LAP could be counteracted after using PKM2 inhibitor PKM2-IN-1 or knocking out PKM2. Small molecule docking revealed that LAP could inhibit the phosphorylation process of PKM2 by binding to ARG-246, the phosphorylation site of PKM2. In rat experiments, LAP could ameliorate the liver function and lipid metabolism of NAFLD rats, and inhibit the hepatic histopathologic changes. ConclusionOur study found that LAP can inhibit the phosphorylation of PKM2 by binding to PKM2-ARG-246, thereby regulating the M1 polarization of KCs and inhibiting the inflammatory response of liver tissues to treat NAFLD. LAP has potential as a novel pharmaceutical for treating NAFLD.

PFOA exposure induces aberrant glucose and lipid metabolism in the rat liver through the AMPK/mTOR pathway

Perfluorooctanoic acid (PFOA) is the most prominent member of a widely utilized family of compounds named Perfluoroalkyl substances (PFASs). Initially produced for use in both industrial and consumer applications, it has since been recognized that PFASs are extremely persistent in the environment where they have been characterized as persistent organic pollutants (POPs). While previous studies have demonstrated that PFOA may induce disorders of lipid and carbohydrate metabolism, the precise mechanisms by which PFOA produces this phenotype and the involvement of downstream AMPK/mTOR pathways remains unclear. In this study, male rats were exposed to 1.25, 5 and 20 mg PFOA/kg body weight/day for 28 days by oral gavage. After 28 days, blood was collected and tested for serum biochemical indicators and livers were removed and weighed. To investigate aberrant metabolism in rats exposed to PFOA, livers were analyzed by performing LC-MS/MS untargeted metabolomics, quantitative real-time PCR, western blotting, immunohistochemical staining was also performed on exposed tissues. Our results showed that exposure to PFOA induced liver damage, increased the expression of glucose and lipid related biochemical indexes in liver and serum, and altered the expression levels of AMPK/mTOR pathway related genes and proteins. In summary, this study clarifies the mechanisms responsible for PFOA toxicity in the liver of exposed animals.