Investigating the role of NPR1 in dilated cardiomyopathy and its potential as a therapeutic target for glucocorticoid therapy.

Background: Dilated cardiomyopathy (DCM), a specific form of cardiomyopathy, frequently presents clinically with either left ventricular or biventricular enlargement, often leading to progressive heart failure. In recent years, the application of bioinformatics technology to scrutinize the onset, progression, and prognosis of DCM has emerged as a fervent area of interest among scholars globally. Methods: In this study, core genes closely related to DCM were identified through bioinformatics analysis, including weighted gene co expression network analysis (WGCNA) and single sample gene set enrichment analysis (ssGSEA) and so on. The correlation was verified through experiments on DCM patients, DCM rat models, and core gene knockout mice. Subsequently, the effects of glucocorticoids on DCM and the regulation of core genes were observed. Result: In the present study, natriuretic peptide receptor 1 (NPR1) was identified as a core gene associated with DCM through WGCNA and ssGSEA. Significant impairment of cardiac and renal function was observed in both DCM patients and rats, concomitant with a notable reduction in NPR1 expression. NPR1 KO mice displayed symptomatic manifestations of DCM, underscoring the pivotal role of NPR1 in its pathogenesis. Notably, glucocorticoid treatment led to substantial improvements in cardiac and renal function, accompanied by an upregulation of NPR1 expression. Discussion: These findings highlight the critical involvement of NPR1 in the pathophysiology of DCM and its potential as a key target for glucocorticoid-based DCM therapy. The study provides a robust theoretical and experimental foundation for further investigations into DCM etiology and therapeutic strategies.

Glucocorticoids ameliorate cardiorenal syndrome through the NPR1/SGK1 pathway in natriuretic peptide receptor A­heterozygous mice.

Natriuretic peptides, which are produced by the heart, bind to natriuretic peptide receptor A (NPR1 encoded by natriuretic peptide receptor 1 gene) and cause vasodilation and natriuresis. Thus, they serve an important role in regulating blood pressure. In the present study, microinjection of CRISPR associated protein 9/single guide RNA into fertilized C57BL/6N mouse eggs was performed to generate filial generation zero (F0) Npr1 knockout homozygous mice (Npr1-/-). F0 mice mated with wild-type (WT) mice to obtain F1 Npr1 knockout heterozygous mice with stable heredity (Npr1+/-). F1 self-hybridization was used to expand the population of heterozygous mice (Npr1+/-). The present study performed echocardiography to investigate the impact of NPR1 gene knockdown on cardiac function. Compared with those in the WT group (C57BL/6N male mice), the left ventricular ejection fraction, myocardial contractility and renal sodium and potassium excretion and creatinine-clearance rates were decreased, indicating that Npr1 knockdown induced cardiac and renal dysfunction. In addition, expression of serum glucocorticoid-regulated kinase 1 (SGK1) increased significantly compared with that in WT mice. However, glucocorticoids (dexamethasone) upregulated NPR1 and inhibited SGK1 and alleviated cardiac and renal dysfunction caused by Npr1 gene heterozygosity. SGK1 inhibitor GSK650394 ameliorate cardiorenal syndrome by suppressing SGK1. Briefly, glucocorticoids inhibited SGK1 by upregulating NPR1, thereby ameliorating cardiorenal impairment caused by Npr1 gene heterozygosity. The present findings provided novel insight into the understanding of cardiorenal syndrome and suggested that glucocorticoids targeting the NPR1/SGK1 pathway may be a potential therapeutic target to treat cardiorenal syndrome.

The PPARα/CYP4A14 bile acid pathway is associated with lipid metabolism disorders caused by low birth weight with high-fat diet.

Purpose: To investigate possible mechanisms underlying the greater susceptibility of lipid metabolism disorders in low birth weight (LBW) mice fed with high-fat diets (HFDs). Methods: LBW mice model was established by using the pregnancy malnutrition method. Male pups were selected from LBW and normal-birth weight (NBW) offspring at random. After 3 weeks of weaning, all offspring mice were fed with HFD. Serum triglycerides (TGs), cholesterol (TC), low density lipoprotein (LDL-C), total bile acid (TAB), non-esterified fatty acid (NEFA), and mice fecal bile acid profiles were measured. Lipid deposition in liver sections was visualized by Oil Red O staining. The weight ratio of liver, muscle, and adiposity was calculated. Tandem mass tag (TMT) combined with LC-MS/MS was used to determine the differentially expressed proteins (DEPs) of liver tissue in two groups. Bioinformatics was used for further analysis of DEPs to screen key target proteins, and then Western Blot (WB) and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) were performed to validate the expressions of DEPs. Results: LBW mice fed with HFD showed more severe lipid metabolism disorders in the childhood. In contrast to the NBW group, the serum bile acids and fecal ω-muricholic acid (ω-MCA) levels in the LBW group were significantly lower. LC-MS/MS analysis showed that downregulated proteins were associated with lipid metabolism, and further analysis found that these proteins are mainly concentrated in peroxisome proliferation-activated receptor (PPAR) and primary bile acid synthesis signaling pathways and are involved in cellular processes and metabolic processes through binding and catalytic functions. Bioinformatics analysis indicated that the level of Cytochrome P450 Family 46 Subfamily A Member 1 (CYP46A1), PPARα, key factors of cholesterol metabolism and bile acid synthesis, as well as downstream molecules Cytochrome P450 Family 4 Subfamily A Member 14 (CYP4A14), and Acyl-Coenzyme A Oxidase 2 (ACOX2) are markedly different in the liver of LBW individuals fed with HFD, and confirmed by WB and RT-qPCR. Conclusion: LBW mice are more prone to dyslipidemia probably due to downregulated bile acid metabolism-related PPARα/CYP4A14 pathway, resulting in insufficient metabolism of cholesterol to bile acids, which, in turn, leads to elevated blood cholesterol.

Trpc5-regulated AMPKα/mTOR autophagy pathway is associated with glucose metabolism disorders in low birth weight mice under overnutrition.

Previous studies have shown that low birth weight (LBW) individuals are at higher risk of glucose metabolism disorders compared with normal birth weight (NBW) individuals under overnutrition conditions, but the mechanism remains unclear. To explore the underlying mechanism of glucose metabolism disorders induced by LBW under overnutrition in adulthood, the prenatal malnutrition method was applied to ICR mice to establish the LBW mice model and high-fat diets were used to mimic overnutrition conditions. Then the mechanism was further explored on Hepg2 cells treated with nutritional deprivation plus palmitic acid. The results showed that LBW plus high-fat interventions will cause glucose metabolism disorders and inhibit autophagy flux in adulthood. Moreover, the expression of TRPC5-regulated AMPK/mTOR autophagy pathway was downregulated by LBW with high-fat interventions. Collectively, LBW plus high-fat intervention increased the risk of glucose metabolism disorders, which may be related to the alteration of TRPC5 expression level and its regulation of the AMPKα/mTOR autophagy pathway. This study may provide a fundamental basis for the molecular mechanism of glucose metabolism disorders induced by LBW with high-fat diets in adulthood and a new target for the treatment of metabolic diseases in LBW individuals.

Pterostilbene, a Resveratrol Derivative, Improves Ectopic Lipid Deposition in the Kidneys of Mice Induced by a High-Fat Diet.

BACKGROUND: Diabetic kidney disease is a major cause of global end-stage renal diseases. Ectopic lipid deposition in the renal tissues of diabetic kidney disease is one major factor leading to renal fibrosis and chronic kidney disease. Pterostilbene has been reported to display lipid-lowing activity and participate in many kidney diseases. However, the influence of pterostilbene on the ectopic lipid deposition is unclear. We intend to explore the influence of pterostilbene on the ectopic lipid deposition in the kidneys of mice induced by high fat. METHODS: A high-fat diet-induced diabetic mouse model was established to detect the alleviative effect of pterostilbene on the ectopic lipid deposition in the kidneys of diabetic mice. A biochemical analysis was performed to examine the levels of urine albumin, urine creatinine, serum creatinine, and blood urea nitrogen in mice after pterostilbene treatment. Histological analysis was conducted to detect the degree of renal injury and fibrosis. Oil red O staining and immunohistochemical staining were carried out to evaluate lipid droplets and the expression of adipose differentiation-related protein in renal tissues of the mice treated by pterostilbene. The protein levels were assessed by Western blotting. RESULTS: Pterostilbene inhibits the expression of the TGF-ß1 and p-smad3 and suppresses the protein levels of SREBP-1 and FAS, and it ultimately reduces the ectopic lipid deposition, alleviates the renal tubular damage and renal fibrosis in the kidneys of diabetic mice induced by high fat, and improves kidney function. CONCLUSION: Pterostilbene alleviates renal fibrosis and ectopic lipid deposition in the kidneys of diabetic mice induced by high-fat diet by inhibiting the TGF-ß1/smad3 signaling.

Adipose Dysfunction in Adulthood Insulin Resistance of Low-Birth Weight Mice: A Proteomics Study.

Purpose: To investigate changes in the protein expression profile of white adipose tissue in low-birth weight (LBW) mice with high-fat diets using tandem mass tag (TMT) and liquid chromatography-mass spectrometry (LC-MS/MS) and parallel reaction monitoring (PRM). Methods: Institute of Cancer Research (ICR) mice were used to establish an LBW model using malnutrition during pregnancy. Male pups were randomly selected from LBW and normal-birth weight (NBW) offspring, then all given a high-fat diet. Blood glucose, serum insulin, total cholesterol (TC) and triglyceride (TG) levels were measured. The weight ratio of liver, muscle, and adiposity index were calculated. Hematoxylin and eosin staining was used to visualize adipose tissue morphology. Oil red O staining of liver and TG content of muscle were used to determine ectopic lipid deposition. TMT combined with LC-MS/MS was used to analyze protein expression in white adipose tissue. PRM and Western blot were used to verify the expression of CD36, SCD1, PCK1 and PPARγ. Results: Compared with NBW mice, fasting blood glucose, insulin and HOMA-IR significantly increased in LBW mice, indicating insulin resistance and impaired glucose regulation; TC, TG, adipocyte size, and adiposity index were increased in LBW mice, suggesting obesity and disorder of lipid metabolism. We observed ectopic lipid deposition in liver and muscle. There were 996 differentially expressed proteins (DEPs) in the LBW/NBW groups. Peroxisome proliferator-activated receptor (PPAR) was a relatively important signaling pathway regulating metabolic process in functional enrichment analysis of DEPs. Up-regulated expression of CD36, SCD1, and PCK1 in the adipose tissue of LBW mice was observed through PPAR pathways cluster analysis. And PRM and Western blot assay validated the proteomics findings. Conclusion: When exposed to high-fat diets, LBW mice exhibited insulin resistance and disorder of lipid metabolism compared with NBW mice. The expression of PPARγ was elevated, as well as upstream CD36, downstream SCD1 and PCK1 of the PPARγ in the adipose tissue of LBW mice. It was suggested that the activation in CD36/PPARγ/SCD1 and CD36/PPARγ/PCK1 pathways may induce adipose dysfunction, thereby increasing susceptibility to insulin resistance.

The role of CD36-Fabp4-PPARγ in skeletal muscle involves insulin resistance in intrauterine growth retardation mice with catch-up growth.

BACKGROUND: Studies have shown that the high incidence of type 2 diabetes in China is associated with low birth weight and excessive nutrition in adulthood, which occurred during the famine years of the 1950s and 1960s, though the specific molecular mechanisms are unclear. In this study, we proposed a severe maternal caloric restriction during late pregnancy, followed by a post weaning high-fat diet in mice. After weaning, normal and high-fat diets were provided to mice to simulate the dietary pattern of modern society. METHODS: The pregnant mice were divided into two groups: normal birth weight (NBW) group and low birth weight (LBW) group. After 3 weeks for weaning, the male offspring mice in the NBW and LBW groups were then randomly divided into four subgroups: NC, NH, LC and LC groups. The offspring mice in the NC, NH, LC and LC groups were respectively fed with normal diet, normal diet, high-fat diet and high-fat diet for 18 weeks. After 18 weeks of dietary intervention, detailed analyses of mRNA and protein expression patterns, signaling pathway activities, and promoter methylation states were conducted for all relevant genes. RESULTS: After dietary intervention for 18 weeks, the expressions of CD36, Fabp4, PPARγ, FAS, and ACC1 in the skeletal muscle tissue of the LH group were significantly increased compared with the LC and NH groups (P < 0.05). The level of p-AMPK/AMPK in the skeletal muscle tissue of the LH group was significantly decreased compared with the LC and NH groups (P < 0.05). CPT1 and PGC-1α protein expressions were up-regulated in the LH group (P < 0.05) compared to the LC group. Additionally, the DNA methylation levels of the PGC-1α and GLUT4 gene promoters in the skeletal muscle of the LH groups were higher than those of the LC and NH groups (P < 0.05). However, PPARγ DNA methylation level in the LH group was lower than those of the LC and NH groups (P < 0.05). CONCLUSIONS: LBW combined with high-fat diets may increase insulin resistance and diabetes through regulating the CD36-related Fabp4-PPARγ and AMPK/ACC signaling pathways.

Resveratrol Ameliorates High-Fat-Diet-Induced Abnormalities in Hepatic Glucose Metabolism in Mice via the AMP-Activated Protein Kinase Pathway.

Diabetes mellitus is highly prevalent worldwide. High-fat-diet (HFD) consumption can lead to liver fat accumulation, impair hepatic glycometabolism, and cause insulin resistance and the development of diabetes. Resveratrol has been shown to improve the blood glucose concentration of diabetic mice, but its effect on the abnormal hepatic glycometabolism induced by HFD-feeding and the mechanism involved are unknown. In this study, we determined the effects of resveratrol on the insulin resistance of high-fat-diet-fed mice and a hepatocyte model by measuring serum biochemical indexes, key indicators of glycometabolism, glucose uptake, and glycogen synthesis in hepatocytes. We found that resveratrol treatment significantly ameliorated the HFD-induced abnormalities in glucose metabolism in mice, increased glucose absorption and glycogen synthesis, downregulated protein phosphatase 2A (PP2A) and activated Ca2+/CaM-dependent protein kinase kinase ß (CaMKKß), and increased the phosphorylation of AMP-activated protein kinase (AMPK). In insulin-resistant HepG2 cells, the administration of a PP2A activator or CaMKKß inhibitor attenuated the effects of resveratrol, but the administration of an AMPK inhibitor abolished the effects of resveratrol. Resveratrol significantly ameliorates abnormalities in glycometabolism induced by HFD-feeding and increases glucose uptake and glycogen synthesis in hepatocytes. These effects are mediated through the activation of AMPK by PP2A and CaMKKß.

Exploring the Multi-Tissue Crosstalk Relevant to Insulin Resistance Through Network-Based Analysis.

Insulin resistance (IR) is a precursor event that occurs in multiple organs and underpins many metabolic disorders. However, due to the lack of effective means to systematically explore and interpret disease-related tissue crosstalk, the tissue communication mechanism in pathogenesis of IR has not been elucidated yet. To solve this issue, we profiled all proteins in white adipose tissue (WAT), liver, and skeletal muscle of a high fat diet induced IR mouse model via proteomics. A network-based approach was proposed to explore IR related tissue communications. The cross-tissue interface was constructed, in which the inter-tissue connections and also their up and downstream processes were particularly inspected. By functional quantification, liver was recognized as the only organ that can output abnormal carbohydrate metabolic signals, clearly highlighting its central role in regulation of glucose homeostasis. Especially, the CD36-PPAR axis in liver and WAT was identified and verified as a potential bridge that links cross-tissue signals with intracellular metabolism, thereby promoting the progression of IR through a PCK1-mediated lipotoxicity mechanism. The cross-tissue mechanism unraveled in this study not only provides novel insights into the pathogenesis of IR, but also is conducive to development of precision therapies against various IR associated diseases. With further improvement, our network-based cross-tissue analytic method would facilitate other disease-related tissue crosstalk study in the near future.

Liraglutide ameliorates myocardial damage in experimental diabetic rats by inhibiting pyroptosis via Sirt1/AMPK signaling.

OBJECTIVES: Liraglutide, a well-established drug for treating diabetes mellitus (DM), has recently gained attention for its cardiovascular benefits in diabetes via multiple cellular activities; however, whether liraglutide improves myocardial damage by inhibiting pyroptosis and the mechanisms of these potential effects remain unknown. MATERIALS AND METHODS: In this study, high-fat diet feeding and low-dose streptozotocin (STZ) injection were used to construct a rat DM model. Rats with fasting blood glucose (FBG) levels >16.7 mmol/l received subcutaneous injections of liraglutide (0.2 mg/kg) for 4 weeks. Metabolic parameters, the heart weight/body weight (HW/BW) ratio, and histopathology were examined. Protein levels of inflammatory, pyroptosis, and NOD-like receptor protein 3 (NLRP3) inflammasome markers were assessed via Western blotting. In in vitro studies, a sirtuin 1 (Sirt1) inhibitor (EX 527, 200 nM) and an AMP-activated protein kinase (AMPK) inhibitor (compound C, 20 µM) were used to inhibit Sirt1 and AMPK pathways, respectively. RESULTS: Liraglutide significantly attenuated cardiac hypertrophy, pathological changes, inflammation, pyroptosis, and NLRP3 inflammasome activation, accompanied by increased Sirt1 and AMPK activation. Consistent with the in vivo results, liraglutide attenuated high glucose (HG)-induced pyroptosis and NLRP3 inflammasome activation along with enhanced Sirt1 and AMPK activation. After blockade of Sirt1 and AMPK signaling, the protective effect of liraglutide was restrained. Notably, EX 527 abolished the stimulatory effect of liraglutide on Sirt1 and AMPK signaling, whereas compound C blunted AMPK signaling without affecting Sirt1 signaling. CONCLUSION: Liraglutide may protect against myocardial damage by activating the Sirt1/AMPK signaling pathways to inhibit cellular pyroptosis in DM.

A high-fructose diet in rats induces systemic iron deficiency and hepatic iron overload by an inflammation mechanism.

Nonalcoholic fatty liver disease (NAFLD) correlates with the high intake of fructose-rich soft drinks. Both inflammation and dysregulated iron metabolism are pathogenic factors in the development of NAFLD. The present investigation assessed the effects of a high-fructose diet (HF diet) on inflammation and iron metabolism. In this study, rats were fed a control or HF diet for 4, 8, or 12 weeks, after which insulin resistance, transaminases levels, serum and liver lipid profiles, inflammatory factors, and iron metabolism-related molecules were evaluated. The activities of the hepatic inflammation-associated pathways, IKKß/NF-κB, and JAK2/STAT3, were detected by western blot. Result showed that the HF diet-fed animals developed a time-dependent serum lipid increase and hepatic lipid accumulation as well as insulin resistance. Serum iron (SI), serum ferritin (SF), and transferrin saturation (TS) decreased while total iron-binding capacity (TIBC) and serum transferrin (s-TF) increased at 8 and 12 weeks in the HF diet group. The HF diet led to increased transaminases levels at 8 and 12 weeks, and iron deposition was observed in the liver, accompanied by an upregulation of ferritin light chain (FTL), hepcidin (HEPC), transferrin (TF), transferrin receptor 1 (TfR1), iron regulatory protein 1 (IRP1), hemojuvelin (HJV), and divalent metal transporter 1 (DMT1). Moreover, ferroportin (FPN1) levels were downregulated, as expected from the increased HEPC. A progressive inflammation phenotype was apparent, with increased inflammatory factors, MDA, IL-1ß, IL-6, and TNF-α, in the serum and liver tissue. Concomitantly, the hepatic IKKß/NF-κB and JAK2/STAT3 pathways were activated. In summary, we verified that HF diet induces systemic iron deficiency and hepatic iron accumulation, likely due to the activation of inflammation via the NF-κB and JAK2/STAT3 pathways. PRACTICAL APPLICATIONS: As increasing numbers of individuals consume HF diets, the health implications of this type of over nutrition become globally relevant. Using a high-fructose diet rat model, our present study reveals inflammation as the link between a HF diet and dysregulated iron metabolism. Importantly, both inflammation and disrupted iron metabolism have been shown to be pathogenic factors in nonalcoholic fatty liver disease (NAFLD). The iron regulatory hormone, HEPC, is a link between the liver, inflammation, and iron metabolism. As fructose-rich foods become increasingly abundant and people's fructose intake increases, the impact of high fructose on health requires increased attention. Little research has been conducted on the effects of fructose on iron metabolism. Our study provides useful insights into the prevention and treatment of iron metabolism disorders arising from metabolic syndrome.

MicroRNA­802 increases hepatic oxidative stress and induces insulin resistance in high­fat fed mice.

The expression of microRNA­802 (miR­802) is known to be associated with insulin resistance (IR); however, the mechanism remains unclear. The present study investigated how miR­802 contributes to the development of IR using C57BL/6J mice fed a high­fat diet (HFD) to establish a model of IR. Adeno­associated virus overexpressing miR­802 was administered to the mice via tail vein injection. The effects of miR­802 on reactive oxygen species (ROS), lipid peroxidation (LPO) and the activities of multiple ROS­related enzymes were investigated. Western blot analysis was used to estimate the protein levels of extracellular signal regulated kinase (ERK), p38mitogen­activated protein kinases (p38MAPK), c­Jun N­terminal kinase (JNK), insulin receptor substrate 1 (IRS­1) and protein kinase B (AKT1). The results demonstrated that the levels of ROS and LPO production were increased in the livers of the miR­802­treated group compared with the control group. The activities of the ROS­related enzymes were reduced. Furthermore, the expression of phosphorylated (phosphor)­p38MAPK and phosphor­JNK were upregulated in the miR­802 overexpression group, whereas there was no difference in the expression levels of phosphor­ERK. The expression levels of phosphor­AKT1 were reduced in the miR­802­treated group and these effects were reversed by miR­802 knockdown. In conclusion, the results demonstrate that miR­802 may cause IR by activating the JNK and p38MAPK pathways to increase hepatic oxidative stress.

Farnesoid X receptor agonist decreases lipid accumulation by promoting hepatic fatty acid oxidation in db/db mice.

The development of type­2 diabetes and its complications is associated with lipid metabolism disorder. Farnesoid X receptor (FXR) has an important role in regulating lipid and glucose metabolism. However, the underlying mechanism of this remains unclear. The present study investigated the role of fexaramine (Fex), an FXR agonist, on lipid metabolism. For this purpose, 6­week­old db/db mice were treated with Fex for 8 weeks via oral gavage and db/db mice treated with corn oil were used as controls. Body weight and food intake were monitored daily and bi­weekly, respectively. A glucose tolerance test was performed during the final week of feeding. Blood samples were obtained for the analysis of lipids and enzymes related to hepatic function, and liver tissues were analyzed by histology and molecular examination. The results indicated that serum and liver triglyceride levels were decreased in db/db mice administered with Fex. Fewer small lipid droplets were observed in the liver. Small heterodimer partner (SHP), a downstream gene of FXR, was upregulated following Fex treatment. The mRNA and protein expression of genes associated with fatty acid oxidation [acetyl coenzyme A carboxylase (ACC), carnitine palmitoyl transferase 1α (CPT1­α) and peroxisome proliferator­activated receptor­coactivator­1α] was also increased. Additionally, the expression of AMP­activated protein kinase (AMPK) was also increased. However, the expression of sterol­regulatory element binding protein­1c and fatty acid synthase, which are associated with fatty acid synthesis, was not significantly different. Taken together, the results of the present study suggested that activation of FXR and its downstream gene SHP may induce the AMPK­ACC­CPT1­α signaling pathway, which promotes fatty acids oxidation, ultimately achieving its lipid­lowering effect.