Granulocytic MDSC with Deficient CCR5 Alleviates Lipogenesis and Inflammation in Nonalcoholic Fatty Liver Disease.

C-C chemokine receptor type 5 (CCR5) positively contributes to the pathogenesis of nonalcoholic fatty liver disease (NAFLD), a common metabolic liver disease associated with chronic inflammation. CCR5 signaling also facilitates the immunosuppressive activity of a group of immature myeloid cells known as granulocytic myeloid-derived suppressor cells (g-MDSCs). While both hepatocyte and g-MDSC express CCR5, how CCR5 coordinates these two distinct cell types in the hepatic microenvironment remains largely unknown. Here, we used in vivo and ex vivo approaches to define the molecular details of how CCR5 mediates the crosstalk between hepatocytes and g-MDSCs in a mouse model of NAFLD. Global CCR5-deficient mice exhibited more severe steatosis, increased hepatic gene expression of lipogenesis, and exacerbated liver damage in diet-induced obesity. Either NAFLD or CCR5-deficiency per se is causative for the increase of g-MDSCs. Purified g-MDSCs have a higher survival rate in the fatty liver microenvironment, and blockade of CCR5 significantly decreases g-MDSCs' expression of anti-inflammatory factors. On the other hand, the null of CCR5 signaling increases hepatocytes' expression of lipogenic genes in the NAFLD microenvironment. Most importantly, inhibiting g-MDSCs' CCR5 signaling in the fatty liver microenvironment dramatically reduces STAT3 signaling, lipogenic, and pro-inflammatory gene expression in primary hepatocytes. Adoptive cell transfer experiments further demonstrate that CCR5-deficient g-MDSCs mitigate hepatic lipogenic gene expression without facilitating pro-inflammatory cytokine production and liver damage in NAFLD mice. These results suggest that targeting g-MDSCs' CCR5 signaling might serve as a potential therapeutic strategy for NAFLD.

Exosomal microRNAs miR-30d-5p and miR-126a-5p Are Associated with Heart Failure with Preserved Ejection Fraction in STZ-Induced Type 1 Diabetic Rats.

Exosomal microRNAs (EXO-miRNAs) are promising non-invasive diagnostic biomarkers for cardiovascular disease. Heart failure with preserved ejection fraction (HFpEF) is a poorly understood cardiovascular complication of diabetes mellitus (DM). Little is known about whether EXO-miRNAs can be used as biomarkers for HFpEF in DM. We aimed to investigate the relationship between EXO-miRNAs and HFpEF in STZ-induced diabetic rats. We prepared STZ-induced diabetic rats exhibiting a type 1 DM phenotype with low body weight, hyperglycemia, hyperlipidemia and hypoinsulinemia. Histological sections confirmed atrophy and fibrosis of the heart, with collagen accumulation representing diabetic cardiomyopathy. Significant decreases in end-diastolic volume, stroke volume, stroke work, end-systolic elastance and cardiac output indicated impaired cardiac contractility, as well as mRNA conversion of two isoforms of myosin heavy chain (α-MHC and ß-MHC) and increased atrial natriuretic factor (ANF) mRNA indicating heart failure, were consistent with the features of HFpEF. In diabetic HFpEF rats, we examined a selected panel of 12 circulating miRNAs associated with HF (miR-1-3p, miR-21-5p, miR-29a-5p, miR-30d-5p, miR-34a-5p, miR-126a-5p, miR-143-3p, miR-145-5p, miR-195-5p, miR-206-3p, miR-320-3p and miR-378-3p). Although they were all expressed at significantly lower levels in the heart compared to non-diabetic controls, only six miRNAs (miR-21-5p, miR-30d-5p, miR-126a-5p, miR-206-3p, miR-320-3p and miR-378-3p) were also reduced in exosomal content, while one miRNA (miR-34a-5p) was upregulated. Similarly, although all miRNAs were correlated with reduced cardiac output as a measure of cardiovascular performance, only three miRNAs (miR-30d-5p, miR-126a-5p and miR-378-3p) were correlated in exosomal content. We found that miR-30d-5p and miR-126a-5p remained consistently correlated with significant reductions in exosomal expression, cardiac expression and cardiac output. Our findings support their release from the heart and association with diabetic HFpEF. We propose that these two EXO-miRNAs may be important for the development of diagnostic tools for diabetic HFpEF.

Augmented CCL5/CCR5 signaling in brown adipose tissue inhibits adaptive thermogenesis and worsens insulin resistance in obesity.

Chemokine (C-C motif) ligand 5 (CCL5) and CCR5, one of its receptors have been reported to be highly expressed in white adipose tissue (WAT) and are associated with the progression of inflammation and the development of insulin resistance in obese humans and mice. However, the role of CCL5/CCR5 signaling in obesity-associated dysregulation of energy metabolism remains unclear. Here, we demonstrate that global CCL5/CCR5 double knockout (DKO) mice have higher cold stress-induced energy expenditure and thermogenic function in brown adipose tissue (BAT) than wildtype (WT) mice. DKO mice have higher cold stress-induced energy expenditure and thermogenic function in BAT than WT mice. KEGG pathway analysis indicated that deletion of CCL5/CCR5 further facilitated the cold-induced expression of genes related to oxidative phosphorylation (OxPhos) and lipid metabolic pathways. In primary brown adipocytes of DKO mice, the augmentation of CL-316243-stimulated thermogenic and lipolysis responses was reversed by co-treatment with AMPKα1 and α2 short interfering RNA (siRNA). Overexpression of BAT CCL5/CCR5 genes by local lentivirus injection in WT mice suppressed cold stress-induced lipolytic processes and thermogenic activities. In contrast, knockdown of BAT CCL5/CCR5 signaling further up-regulated AMPK phosphorylation as well as thermogenic and lipolysis responses to chronic adrenergic stimuli and subsequently decreased level of body weight gain. Chronic knockdown of BAT CCL5/CCR5 signaling improved high-fat diet (HFD)-induced insulin resistance in WT mice. It is suggested that obesity-induced augmentation of adipose tissue (AT) CCL5/CCR5 signaling could, at least in part, suppress energy expenditure and adaptive thermogenesis by inhibiting AMPK-mediated lipolysis and oxidative metabolism in thermogenic AT to exacerbate the development of obesity and insulin resistance.

Therapeutic potential of cPLA2 inhibitor to counteract dilated-cardiomyopathy in cholesterol-treated H9C2 cardiomyocyte and MUNO rat.

BACKGROUND AND PURPOSE: The pathogenesis of cardiomyopathy in metabolically unhealthy obesity (MUO) has been well studied. However, the pathogenesis of cardiomyopathy typically associated with high cholesterol levels in metabolically unhealthy nonobesity (MUNO) remains unclear. We investigated whether cholesterol-generated LysoPCs contribute to cardiomyopathy and the role of cytosolic phospholipase A2 (cPLA2) inhibitor in cholesterol-induced MUNO. EXPERIMENTAL APPROACH: Cholesterol diet was performed in Sprague-Dawley rats that were fed either regular chow (C), or high cholesterol chow (HC), or HC diet with 10 % fructose in drinking water (HCF) for 12 weeks. LysoPCs levels were subsequently measured in rats and in MUNO human patients. The effects of cholesterol-mediated LysoPCs on cardiac injury, and the action of cPLA2 inhibitor, AACOCF3, were further assessed in H9C2 cardiomyocytes. KEY RESULTS: HC and HCF rats fed cholesterol diets demonstrated a MUNO-phenotype and cholesterol-induced dilated cardiomyopathy (DCM). Upregulated levels of LysoPCs were found in rat myocardium and the plasma in MUNO human patients. Further testing in H9C2 cardiomyocytes revealed that cholesterol-induced atrophy and death of cardiomyocytes was due to mitochondrial dysfunction and conditions favoring DCM (i.e. reduced mRNA expression of ANF, BNP, DSP, and atrogin-1), and that AACOCF3 counteracted the cholesterol-induced DCM phenotype. CONCLUSION AND IMPLICATIONS: Cholesterol-induced MUNO-DCM phenotype was counteracted by cPLA2 inhibitor, which is potentially useful for the treatment of LysoPCs-associated DCM in MUNO.

Administration of low-dose resveratrol attenuated hepatic inflammation and lipid accumulation in high cholesterol-fructose diet-induced rat model of nonalcoholic fatty liver disease.

Resveratrol (RSV) has been demonstrated to ameliorate nonalcoholic fatty liver disease (NAFLD) in animal studies. However, RSV was given with the dosage that ranged from 7 to 300 mg/kg body weight (BW). Hence, the study aimed to investigate the efficacy of RSV at a lower dosage on high cholesterol-fructose diet (HCFD)-induced rat model of NAFLD. In the study, male Sprague-Dawley rats were fed with HCFD for 15 weeks. RSV was also given at a daily dose of 1 mg/kg BW for 15 days or 15 weeks by oral delivery. At sacrifice, plasma and liver specimens were acquired for detections of alanine and aspartate aminotransferases, proinflammatory cytokines, and lipid contents. Histological examinations and Western blotting analysis were performed using liver tissues. The results showed that RSV administration reduced plasma levels of aminotransferases and proinflammatory cytokines including interleukin-1 beta (IL-1ß), IL-6, and tumor necrosis factor-alpha (TNF-α) in HCFD-induced NAFLD. RSV also mitigated hepatic lipid accumulation and expression of IL-1ß, IL-6, and TNF-α. Besides, phosphorylation of signal transducer and activator of transcription 3 (STAT3) was reduced with RSV supplementation in the liver of HCFD-fed rats. We concluded that low-dose RSV supplementation attenuated hepatic inflammation and lipid accumulation in HCFD-induced NAFLD. The ameliorative effect of RSV on NAFLD could be associated with downregulation of phosphorylated STAT3.

Resveratrol Mitigates High-Fat Diet-Induced Vascular Dysfunction by Activating the Akt/eNOS/NO and Sirt1/ER Pathway.

We investigated whether resveratrol (RSV) can attenuate obesity and diabetes progression and improve diabetes-induced vascular dysfunction, and we attempted to delineate its underlying mechanisms. Male C57Bl/6 mice were administered a high-fat diet (HFD) for 17 weeks. Mice developed type 2 diabetes with increased body weight, hyperglycemia, hyperinsulinemia, and hyperlipidemia. Oral gavage with RSV significantly reversed the symptoms induced by the HFD. Insulin sensitivity likewise improved after the RSV intervention in these mice. Phenylephrine-induced cremaster arteriolar constriction was impaired, whereas RSV treatment significantly mitigated the vessel responsiveness to phenylephrine. The obese diabetic mice exhibited increased leukocyte rolling, adhesion, and transmigration in the postcapillary venules of the cremaster muscle. By contrast, RSV treatment significantly attenuated HFD-induced extravasation. RSV significantly recovered phosphorylated Akt and eNOS expression in the thoracic aorta. In addition, activated adenosine monophosphate-activated protein kinase in the thoracic aorta was involved in the improvement of epithelial function after RSV intervention. RSV considerably upregulated the plasma NO level in HFD mice. Moreover, RSV-enhanced human umbilical vein endothelial cells healing through Sirt1/ER pathway may be involved in the prevention of leukocyte extravasation. Collectively, RSV attenuates diabetes-induced vascular dysfunction by activating Akt/eNOS/NO and Sirt1/ER pathway. Our mechanistic study provides a potential RSV-based therapeutic strategy against cardiovascular disease.

Early Imaging Biomarker of Myocardial Glucose Adaptations in High-Fat-Diet-Induced Insulin Resistance Model by Using 18F-FDG PET and [U-13C]glucose Nuclear Magnetic Resonance Tracer.

Background: High-fat diet (HFD) induces systemic insulin resistance leading to myocardial dysfunction. We aim to characterize the early adaptations of myocardial glucose utility to HFD-induced insulin resistance. Methods: Male Sprague-Dawley rats were assigned into two groups, fed a regular chow diet or HFD ad libitum for 10 weeks. We used in vivo imaging of cardiac magnetic resonance (CMR), 18F-FDG PET, and ex vivo nuclear magnetic resonance (NMR) metabolomic analysis for the carbon-13-labeled glucose ([U-13C]Glc) perfused myocardium. Results: As compared with controls, HFD rats had a higher ejection fraction and a smaller left ventricular end-systolic volume (P < 0.05), with SUVmax of myocardium on 18F-FDG PET significantly increased in 4 weeks (P < 0.005). The [U-13C]Glc probed the increased glucose uptake being metabolized into pyruvate and acetyl-CoA, undergoing oxidative phosphorylation via the tricarboxylic acid (TCA) cycle, and then synthesized into glutamic acid and glutamine, associated with overexpressed LC3B (P < 0.05). Conclusions: HFD-induced IR associated with increased glucose utility undergoing oxidative phosphorylation via the TCA cycle in the myocardium is supported by overexpression of glucose transporter, acetyl-CoA synthase. Noninvasive imaging biomarker has potentials in detecting the metabolic perturbations prior to the decline of the left ventricular function.

Role of dysfunctional adipocytes in cholesterol-induced nonobese metabolic syndrome.

Many studies have reported the causes of obese metabolic syndrome (MS); however, the causes of nonobese MS (NMS) remain unknown. In this study, we demonstrated that inflamed dysfunctional adipose tissue plays a crucial role in cholesterol-induced NMS. Control (C), high cholesterol (HC) and HC with 10% fructose in drinking water (HCF) diets were fed to Sprague-Dawley rats for 12 weeks. After 12 weeks, the body weights of the C- and HC-fed rats were comparable, but the weights of the HCF-fed rats were relatively low. Cholesterol caused metabolic problems such as high blood pressure, hypercholesterolemia and hypoinsulinemia. The HCF-fed rats exhibited whole-body insulin resistance with low circulating high-density lipoprotein levels. Increases in the tumor necrosis factor α level in the plasma, the number of CD68+ macrophages and the free nuclear factor-κB level in gonadal white adipose tissue (gWAT) resulted in local inflammation, which appeared as inflamed dysfunctional gWAT. Reduced superoxide dismutases (SODs) deteriorate natural antioxidant defense systems and induce reactive oxygen species in gWAT. Dysregulation of plasma levels of catecholamine, adipokines (leptin and adiponectin), hormone-sensitive lipase and perilipin in cholesterol-induced inflamed adipose tissue contributed to increased lipolysis and increased circulating nonesterified fatty acids. Cholesterol activated inflammation, lipolysis and cell death in 3T3-L1 adipocytes. Moreover, Chol-3T3-CM reduced the population of M2-type Raw264.7 macrophages, indicating that the macrophage polarization is mediated by cholesterol. Together, our findings indicate that inflamed dysfunctional adipocytes are critical in NMS, supporting the development of anti-inflammatory agents as potential therapeutic drugs for treating NMS.

Docosapentaenoic acid and docosahexaenoic acid are positively associated with insulin sensitivity in rats fed high-fat and high-fructose diets.

BACKGROUND: The aim of the present study was to compare insulin resistance and metabolic changes using a global lipidomic approach. METHODS: Rats were fed a high-fat diet (HFD) or a high-fructose diet (HFrD) for 12 weeks to induce insulin resistance (IR) syndrome. After 12 weeks feeding, physiological and biochemical parameters were examined. Insulin sensitivity and plasma metabolites were evaluated using a euglycemic-hyperinsulinemic clamp and mass spectrometry, respectively. Pearson's correlation coefficient was used to investigate the strength of correlations. RESULTS: Rats on both diets developed IR syndrome, characterized by hypertension, hyperlipidemia, hyperinsulinemia, impaired fasting glucose, and IR. Compared with HFrD-fed rats, non-esterified fatty acids were lower and body weight and plasma insulin levels were markedly higher in HFD-fed rats. Adiposity and plasma leptin levels were increased in both groups. However, the size of adipocytes was greater in HFD- than HFrD-fed rats. Notably, the lipidomic heat map revealed metabolites exhibiting greater differences in HFD- and HFrD-fed rats compared with controls. Plasma adrenic acid levels were higher in HFD- than HFrD-fed rats. Nevertheless, linoleic and arachidonic acid levels decreased in HFrD-fed rats compared with controls. Plasma concentrations of docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA) were significantly reduced after feeding of both diets, particularly the HFrD. There was a strong positive correlation between these two fatty acids and the insulin sensitivity index. CONCLUSIONS: The systemic lipidomic analysis indicated that a reduction in DHA and DPA was strongly correlated with IR in rats under long-term overnutrition. These results provide a potential therapeutic target for IR and metabolic syndrome.

High-fructose and high-fat feeding correspondingly lead to the development of lysoPC-associated apoptotic cardiomyopathy and adrenergic signaling-related cardiac hypertrophy.

BACKGROUND: The heart is a highly adaptive organ that demonstrates remarkable structural, functional, and metabolic remodeling in response to physiological and pathological stimuli. We hypothesize that the heart undergoes differential adaptations in high-fat and high-fructose diet, resulting in a distinct phenotype. METHODS: High-fat and high-fructose diet-induced obese and non-obese insulin resistance (IR) rat models were used to understand how the heart adapts to long-term (12-week) overnutrition. RESULTS: Rats fed the high-fat diet developed obese IR, whereas high-fructose diet developed non-obese IR. Obese IR rats developed fibrotic hypertrophy with impairment of preload-independent contractility. The sympathetic and renin-angiotensin-aldosterone (RAA) systems and myocardial adrenergic signaling were activated in obese IR rats. Non-obese IR rats developed apoptotic cardiomyopathy with severe systolic dysfunction. Myocardial calcium cycling regulatory proteins (CCRPs) were dysregulated in non-obese IR rats; specifically, troponin I protein expression was downregulated. Moreover, compared with the controls, lipidomics analysis revealed substantial differences in lipid metabolites in non-obese IR and obese IR rats. The overproduction of lysophosphatidylcholine (lysoPC) and fatty acids was observed in non-obese IR rat hearts. A strong correlation was observed between the myocardial lysoPC and plasma troponin I levels. Treatment of cardiomyocytes with lysoPC resulted in cell death in a dose- and time-dependent manner. The overproduction of myocardial lysoPCs was associated with circulating sPLA2 levels. CONCLUSION: Obese IR rats developed severe fibrotic hypertrophy with the activation of adrenergic signaling and sympathetic and RAA systems. The sPLA2-lysoPC may play a crucial role in the induction of apoptotic cardiomyopathy in high fructose-induced non-obese IR rats.