Weeks Of High-fat Diet Feeding Research Articles

Resveratrol relieves HFD-induced insulin resistance in skeletal muscle tissue through antioxidant capacity enhancement and the Nrf2-Keap1 signaling pathway.

Resveratrol has received much attention due to its beneficial effects including antioxidant activity. The purpose of this study was to investigate the therapeutic effects of resveratrol treatment on oxidative stress and insulin resistance in the skeletal muscle of high-fat diet (HFD)-fed animals. A total of 30 six-week-old C57BL/6J mice were randomly allocated to three groups (10 animals in each group): The control group in which mice were fed a normal chow diet (NCD); the HFD group in which mice were fed an HFD for 26 weeks; and the HFD-resveratrol group in which HFD was replaced by a resveratrol supplemented-HFD (400mg/kg diet) after 10 weeks of HFD feeding. At the end of this period, gastrocnemius muscle samples were examined to determine insulin resistance and the oxidative status in the presence of HFD and resveratrol. Resveratrol supplementation in HFD-fed mice reduced body and adipose tissue weight, improved insulin sensitivity, and decreased oxidative stress as indicated by lower malonaldehyde (MDA) levels and higher total antioxidant capacity. The supplement also increased the expression and activity of antioxidative enzymes in gastrocnemius muscle and modulated Nrf2 and Keap1 expression levels. These results suggest that resveratrol is effective in improving the antioxidant defense system of the skeletal muscle in HFD-fed mice, indicating its therapeutic potential to combat diseases associated with insulin resistance and oxidative stress.

Sphingosine-1-phosphate receptor 3 regulates the transendothelial transport of high-density lipoproteins and low-density lipoproteins in opposite ways.

The entry of lipoproteins from blood into the arterial wall is a rate-limiting step in atherosclerosis. It is controversial whether this happens by filtration or regulated transendothelial transport.Because sphingosine-1-phosphate (S1P) preserves the endothelial barrier, we investigated in vivo and in vitro, whether S1P and its cognate S1P-receptor 3 (S1P3) regulate the transendothelial transport of lipoproteins. Compared to apoE-haploinsufficient mice (CTRL), apoE-haploinsufficient mice with additional endothelium-specific knock-in of S1P3 (S1P3-iECKI) showed decreased transport of LDL and Evan's Blue but increased transport of HDL from blood into the peritoneal cave. After 30 weeks of high-fat diet feeding, S1P3-iECKI mice had lower levels of non-HDL-cholesterol and less atherosclerosis than CTRL mice. In vitro stimulation with an S1P3 agonist increased the transport of 125I-HDL but decreased the transport of 125I-LDL through human aortic endothelial cells (HAECs). Conversely, inhibition or knock-down of S1P3 decreased the transport of 125I-HDL but increased the transport of 125I-LDL. Silencing of SCARB1 encoding scavenger receptor B1 (SR-BI) abrogated the stimulation of 125I-HDL transport by the S1P3 agonist. The transendothelial transport of 125I-LDL was decreased by silencing of SCARB1 or ACVLR1 encoding activin-like kinase 1 but not by interference with LDLR. None of the three knock-downs prevented the stimulatory effect of S1P3 inhibition on transendothelial 125I-LDL transport. S1P3 regulates the transendothelial transport of HDL and LDL oppositely by SR-BI-dependent and SR-BI-independent mechanisms, respectively. This divergence supports a contention that lipoproteins pass the endothelial barrier by specifically regulated mechanisms rather than passive filtration.

Aldose Reductase (AR) Mediates and Perivascular Adipose Tissue (PVAT) Modulates Endothelial Dysfunction of Short-Term High-Fat Diet Feeding in Mice.

Increased adiposity of both visceral and perivascular adipose tissue (PVAT) depots is associated with an increased risk of diabetes and cardiovascular disease (CVD). Under healthy conditions, PVAT modulates vascular tone via the release of PVAT-derived relaxing factors, including adiponectin and leptin. However, when PVAT expands with high-fat diet (HFD) feeding, it appears to contribute to the development of endothelial dysfunction (ED). Yet, the mechanisms by which PVAT alters vascular health are unclear. Aldose reductase (AR) catalyzes glucose reduction in the first step of the polyol pathway and has been long implicated in diabetic complications including neuropathy, retinopathy, nephropathy, and vascular diseases. To better understand the roles of both PVAT and AR in HFD-induced ED, we studied structural and functional changes in aortic PVAT induced by short-term HFD (60% kcal fat) feeding in wild type (WT) and aldose reductase-null (AR-null) mice. Although 4 weeks of HFD feeding significantly increased body fat and PVAT mass in both WT and AR-null mice, HFD feeding induced ED in the aortas of WT mice but not of AR-null mice. Moreover, HFD feeding augmented endothelial-dependent relaxation in aortas with intact PVAT only in WT and not in AR-null mice. These data indicate that AR mediates ED associated with short-term HFD feeding and that ED appears to provoke 'compensatory changes' in PVAT induced by HFD. As these data support that the ED of HFD feeding is AR-dependent, vascular-localized AR remains a potential target of temporally selective intervention.

High-fat diet-induced obesity accelerates the progression of spontaneous osteoarthritis in Senescence-Accelerated Mouse Prone 8 (SAMP8).

Aging and obesity are major risk factors for osteoarthritis (OA), a widespread disease currently lacking efficient treatments. Senescence-accelerated mouse prone 8 (SAMP8) display early-onset aging phenotypes, including OA. This study investigates the impacts of high-fat diet (HFD)-induced obesity on OA development in SAMP8. SAMP8 at five weeks were fed either a normal chow diet or an HFD for ten weeks to induce obesity. Parameters related to obesity, liver function, and lipid and glucose metabolism were analyzed. At 14 weeks of age, knee joint pathology, bone mineral density, and muscle strength were assessed. Immunohistochemistry and TUNEL staining were performed to evaluate markers for cartilage degeneration and chondrocyte apoptosis. At 14 weeks of age, HFD-induced obesity increased liver and adipose tissue inflammation in SAMP8 without further exacerbating diabetes. Histological scoring revealed aggravated cartilage, menisci deterioration, and synovitis, while no further loss of bone mineral density or muscle strength was observed. Increased chondrocyte apoptosis was detected in knee joints following HFD feeding. Ten weeks of HFD feeding promotes spontaneous OA progression in 14-week-old SAMP8, potentially via liver damage subsequent chondrocyte apoptosis. This aging-obese mouse model may prove valuable for further exploration of spontaneous OA pathophysiology.

1577-P: Hepatic Steatosis and Glucose Intolerance in Liver-Specific Estrogen Receptor Alpha Knockout Mice

One of the consequences of sedentary lifestyle and consumption of a high-fat diet is Nonalcoholic Fatty Liver Disease (NAFLD), which affects approximately 30% of adults and up to 10% of children in developed countries. Interestingly, metabolic complications, such as insulin resistance and obesity, are less frequent in young women than in men of the same age or in postmenopausal women. In fact, reduced estrogen concentration during menopause is associated with increased visceral fat, metabolic diseases, insulin resistance, and T2DM. However, how estradiol is related to the development and progression of NAFLD remains unknown. Therefore, the general objective of this project was to study (in vivo) the effects of estrogen receptor alpha (ERα) ablation on hepatic energy metabolism. To this end, C57BL6 mice with ERα deletion specifically in the liver were generated using the Cre-Lox system (LERaKO). Sixth-week-old mice (Control and LERaKO mice) were fed a high-fat diet (HFD) with 45% fat. LERaKO mice displayed no difference in final body weight compared with control mice after four weeks of HFD feeding. A glucose tolerance test (GTT) was also performed after four weeks of HFD feeding. LERaKO mice displayed an increase in glucose intolerance by ~21% (P<0.05) compared with control mice, despite showing no difference in insulin secretion over the course of the GTT. Interestingly, we observed an increase in hepatic triglyceride (TAG) content by ~157% (P<0.05) in LERaKO mice compared with control mice and a decrease in plasma triglyceride concentration. We did not observe a significant difference in the expression of pro- and anti-inflammatory genes, but observed a significant increase in the expression of genes related to lipid synthesis and gluconeogenesis (P<0.05). Taken together, these data confirm the hypothesis that ERα plays a pivotal role in hepatic metabolism in mice, as liver-specific ERα ablation leads to glucose intolerance and hepatic steatosis. Disclosure F. Sucupira: None. L. Araujo: None. J. Camporez: None. Funding FAPESP (2021/02638-9)

345-OR: Exercise and Liraglutide Synergistically Attenuate Vascular Inflammation and Enhance Metabolic Insulin Action in Diet-Induced Obesity

Endothelial oxidative stress and vascular inflammation induce microvascular insulin resistance which is an early event in diet-induced obesity and contributes to metabolic insulin resistance. To examine whether exercise and glucagon-like peptide 1 (GLP-1) receptor agonism, alone or in combination, modulate vascular and metabolic insulin actions during obesity development, we performed an euglycemic insulin clamp, in conjunction with contrast enhanced ultrasound, in adult male rats after 2 weeks of high-fat diet feeding with either access to running wheel (exercise), liraglutide (200 mg/kg subcutaneously twice daily), or the combination of both. Rats exhibited increased visceral adiposity and blunted microvascular responses (microvascular blood volume, flow velocity, and total flow) and metabolic insulin actions (glucose infusion rates). Exercise and liraglutide alone each improved muscle insulin sensitivity but their combination fully restored insulin-mediated glucose infusion rates. The combined exercise and liraglutide intervention enhanced insulin-mediated muscle microvascular perfusion, reduced perivascular macrophage accumulation and oxidative stress in the muscle, attenuated blood vessel inflammation and improved endothelial function, along with increased nucleus translocation of nuclear factor erythroid 2-related factor 2 (NRF2) and phosphorylation of AMP-activated protein kinase (AMPK) and nitric oxide synthase (eNOS) in endothelial cells. We conclude that exercise and liraglutide synergistically enhance the metabolic actions of insulin and reduce vascular oxidative stress and inflammation in the early stage of obesity development. Our data suggest that early combination use of exercise and GLP-1 receptor agonism might be an effective strategy in preventing microvascular and metabolic insulin resistance and associated complications during the development of obesity. Disclosure J.Liu: None. K.W.Aylor: None. Z.Liu: None. Funding National Institutes of Health (R01DK125330), (R01DK1124344 to Z.L.)

Progesterone Has No Impact on the Beneficial Effects of Estradiol Treatment in High-Fat-Fed Ovariectomized Mice

In recent decades, clinical and experimental studies have revealed that estradiol contributes enormously to glycemic homeostasis. However, the same consensus does not exist in women during menopause who undergo replacement with progesterone or conjugated estradiol and progesterone. Since most hormone replacement treatments in menopausal women are performed with estradiol (E2) and progesterone (P4) combined, this work aimed to investigate the effects of progesterone on energy metabolism and insulin resistance in an experimental model of menopause (ovariectomized female mice-OVX mice) fed a high-fat diet (HFD). OVX mice were treated with E2 or P4 (or both combined). OVX mice treated with E2 alone or combined with P4 displayed reduced body weight after six weeks of HFD feeding compared to OVX mice and OVX mice treated with P4 alone. These data were associated with improved glucose tolerance and insulin sensitivity in OVX mice treated with E2 (alone or combined with P4) compared to OVX and P4-treated mice. Additionally, E2 treatment (alone or combined with P4) reduced both hepatic and muscle triglyceride content compared with OVX control mice and OVX + P4 mice. There were no differences between groups regarding hepatic enzymes in plasma and inflammatory markers. Therefore, our results revealed that progesterone replacement alone does not seem to influence glucose homeostasis and ectopic lipid accumulation in OVX mice. These results will help expand knowledge about hormone replacement in postmenopausal women associated with metabolic syndrome and non-alcoholic fatty liver disease.

Effects of Atorvastatin on Bile Acid Metabolism in High-fat Diet-fed ApoE -/- Mice.

Statins are considered as the cornerstone of the prevention and treatment of atherosclerotic cardiovascular disease, where pleiotropic effects are thought to contribute greatly in addition to the lipid-lowering effect. Bile acid metabolism has been gradually reported to be involved in the antihyperlipidemic and antiatherosclerotic effects of statins, but with inconsistent results and few studies carried out on animal models of atherosclerosis. The study aimed to examine the possible role of bile acid metabolism in the lipid-lowering and antiatherosclerotic effects of atorvastatin (ATO) in high-fat diet-fed ApoE -/- mice. The results showed that the levels of liver and faecal TC as well as ileal and faecal TBA were significantly increased in mice of the model group after 20 weeks of high-fat diet feeding compared with the control group, with significantly downregulated mRNA expression of liver LXR-α, CYP7A1, BSEP, and NTCP. ATO treatment further increased the levels of ileal and faecal TBA and faecal TC, but no obvious effect was observed on serum and liver TBA. In addition, ATO significantly reversed the mRNA levels of liver CYP7A1 and NTCP, and no obvious changes were observed in the expression of LXR-α and BSEP. Our study suggested that statins may enhance the synthesis of bile acids and facilitate the reabsorption of bile acids from the ileum via portal into the liver, possibly through the upregulation of the expression of CYP7A1 and NTCP. The results are helpful in enriching the theoretical basis for the clinical use of statins and have good translational value.

Diet Supplementation of Luteolin before Fatty Liver Formation Improves Hepatic Steatosis in Obese Mice by Inhibiting Visceral Adipose Tissue Lipolysis.

Serotonin (5-HT)-induced visceral adipocyte lipolysis is essential for the development of obesity-related complications. Diet supplementation of luteolin prevents high-fat diet (HFD)-fed mice against obesity and associated fatty liver. However, independent of the body weight loss, whether dietary luteolin can substantially reduce hepatic steatosis remains unclear. In differentiated 3T3-L1 cells, 5-HT treatment promotes adipocyte lipolysis, while luteolin significantly inhibits 5-HT-induced lipolysis, Ca2+ -PKG cascade, and SIRT1/FoxO1/AMPKα signaling through binding to 5-HT receptor HTR2B. Further, 5-week-old mice are fed with an HFD for 16 weeks. At the 6th, 8th, or 10th weeks of HFD feeding, some mice are switched to a luteolin-containing HFD, respectively. In all HFD-fed mice, body weight gain and body component are unaffected by dietary luteolin. However, diet supplementation of luteolin at the 6th or 8th, rather than at the 10th weeks, alleviates hepatic steatosis. Meanwhile, dietary luteolin reduces epididymal adipose tissue (EAT) lipolysis, and represses the level of lipolytic enzyme, the expression of Htr2b, and the activation of PKG and SIRT1/FoxO1/AMPKα signaling in EAT. Diet supplementation of luteolin before the formation of fatty liver protects HFD-fed mice against ectopic lipid deposition in liver by inhibiting visceral adipocyte lipolysis.

Lactobacillus rhamnosus Sex-Specifically Attenuates Depressive-like Behavior and Mitigates Metabolic Consequences in Obesity

BackgroundPatients with diabetes exhibit an increased prevalence for emotional disorders compared with healthy humans, partially due to a shared pathogenesis including hormone resistance and inflammation, which is also linked to intestinal dysbiosis. The preventive intake of probiotic lactobacilli has been shown to improve dysbiosis along with mood and metabolism. Yet, a potential role of Lactobacillus rhamnosus (Lacticaseibacillus rhamnosus 0030) (LR) in improving emotional behavior in established obesity and the underlying mechanisms are unknown. MethodsFemale and male C57BL/6N mice were fed a low-fat diet (10% kcal from fat) or high-fat diet (HFD) (45% kcal from fat) for 6 weeks, followed by daily oral gavage of vehicle or 1 × 108 colony-forming units of LR, and assessment of anxiety- and depressive-like behavior. Cecal microbiota composition was analyzed using 16S ribosomal RNA sequencing, plasma and cerebrospinal fluid were collected for metabolomic analysis, and gene expression of different brain areas was assessed using reverse transcriptase quantitative polymerase chain reaction. ResultsWe observed that 12 weeks of HFD feeding induced hyperinsulinemia, which was attenuated by LR application only in female mice. On the contrary, HFD-fed male mice exhibited increased anxiety- and depressive-like behavior, where the latter was specifically attenuated by LR application, which was independent of metabolic changes. Furthermore, LR application restored the HFD-induced decrease of tyrosine hydroxylase, along with normalizing cholecystokinin gene expression in dopaminergic brain regions; both tyrosine hydroxylase and cholecystokinin are involved in signaling pathways impacting emotional disorders. ConclusionsOur data show that LR attenuates depressive-like behavior after established obesity, with changes in the dopaminergic system in male mice, and mitigates hyperinsulinemia in obese female mice.

Nitrative Modification of Caveolin-3: A Novel Mechanism of Cardiac Insulin Resistance and a Potential Therapeutic Target Against Ischemic Heart Failure in Prediabetic Animals.

Myocardial insulin resistance is a hallmark of diabetic cardiac injury. However, the underlying molecular mechanisms remain unclear. Recent studies demonstrate that the diabetic heart is resistant to other cardioprotective interventions, including adiponectin and preconditioning. The "universal" resistance to multiple therapeutic interventions suggests impairment of the requisite molecule(s) involved in broad prosurvival signaling cascades. Cav (Caveolin) is a scaffolding protein coordinating transmembrane signaling transduction. However, the role of Cav3 in diabetic impairment of cardiac protective signaling and diabetic ischemic heart failure is unknown. Wild-type and gene-manipulated mice were fed a normal diet or high-fat diet for 2 to 12 weeks and subjected to myocardial ischemia and reperfusion. Insulin cardioprotection was determined. Compared with the normal diet group, the cardioprotective effect of insulin was significantly blunted as early as 4 weeks of high-fat diet feeding (prediabetes), a time point where expression levels of insulin-signaling molecules remained unchanged. However, Cav3/insulin receptor-β complex formation was significantly reduced. Among multiple posttranslational modifications altering protein/protein interaction, Cav3 (not insulin receptor-β) tyrosine nitration is prominent in the prediabetic heart. Treatment of cardiomyocytes with 5-amino-3-(4-morpholinyl)-1,2,3-oxadiazolium chloride reduced the signalsome complex and blocked insulin transmembrane signaling. Mass spectrometry identified Tyr73 as the Cav3 nitration site. Phenylalanine substitution of Tyr73 (Cav3Y73F) abolished 5-amino-3-(4-morpholinyl)-1,2,3-oxadiazolium chloride-induced Cav3 nitration, restored Cav3/insulin receptor-β complex, and rescued insulin transmembrane signaling. It is most important that adeno-associated virus 9-mediated cardiomyocyte-specific Cav3Y73F reexpression blocked high-fat diet-induced Cav3 nitration, preserved Cav3 signalsome integrity, restored transmembrane signaling, and rescued insulin-protective action against ischemic heart failure. Last, diabetic nitrative modification of Cav3 at Tyr73 also reduced Cav3/AdipoR1 complex formation and blocked adiponectin cardioprotective signaling. Nitration of Cav3 at Tyr73 and resultant signal complex dissociation results in cardiac insulin/adiponectin resistance in the prediabetic heart, contributing to ischemic heart failure progression. Early interventions preserving Cav3-centered signalsome integrity is an effective novel strategy against diabetic exacerbation of ischemic heart failure.

Visceral adipose tissue-directed human kallistatin gene therapy improves adipose tissue remodeling and metabolic health in obese mice

ObjectiveAdipose tissue remodeling is a dynamic process that is pathologically expedited in the obese state and is closely related to obesity-associated disease progression. This study aimed to explore the effects of human kallistatin (HKS) on adipose tissue remodeling and obesity-related metabolic disorders in mice fed with a high-fat diet (HFD). MethodsAdenovirus-mediated HKS cDNA (Ad.HKS) and a blank adenovirus (Ad.Null) were constructed and injected into the epididymal white adipose tissue (eWAT) of 8-weeks-old male C57B/L mice. The mice were fed normal or HFD for 28 days. The body weight and circulating lipids levels were assessed. Intraperitoneal glucose tolerance test (IGTT) and insulin tolerance test (ITT) were also performed. Oil-red O staining was used to assess the extent of lipid deposition in the liver. Immunohistochemistry and HE staining were used to measure HKS expression, adipose tissue morphology, and macrophage infiltration. Western blot and qRT-PCR were used to evaluate the expression of adipose function-related factors. ResultsAt the end of the experiment, the expression of HKS in the serum and eWAT of the Ad.HKS group was higher than in the Ad.Null group. Furthermore, Ad.HKS mice had lower body weight and decreased serum and liver lipid levels after four weeks of HFD feeding. IGTT and ITT showed that HKS treatment maintained balanced glucose homeostasis. Additionally, inguinal white adipose tissue (iWAT) and eWAT in Ad.HKS mice had a higher number of smaller-size adipocytes and had less macrophage infiltration than Ad.Null group. HKS significantly increased the mRNA levels of adiponectin, vaspin, and eNOS. In contrast, HKS decreased RBP4 and TNFα levels in the adipose tissues. Western blot results showed that local injection of HKS significantly upregulated the protein expressions of SIRT1, p-AMPK, IRS1, p-AKT, and GLUT4 in eWAT. ConclusionsHKS injection in eWAT improves HFD-induced adipose tissue remodeling and function, thus significantly improving weight gain and dysregulation of glucose and lipid homeostasis in mice.

GPS2-mediated regulation of the adipocyte secretome modulates adipose tissue remodeling at the onset of diet-induced obesity

ObjectiveDysfunctional, unhealthy expansion of white adipose tissue due to excess dietary intake is a process at the root of obesity and Type 2 Diabetes development. The objective of this study is to contribute to a better understanding of the underlying mechanism(s) regulating the early stages of adipose tissue expansion and adaptation to dietary stress due to an acute, high-fat diet (HFD) challenge, with a focus on the communication between adipocytes and other stromal cells. MethodsWe profiled the early response to high-fat diet exposure in wildtype and adipocyte-specific GPS2-KO (GPS2-AKO) mice at the cellular, tissue and organismal level. A multi-pronged approach was employed to disentangle the complex cellular interactions dictating tissue remodeling, via single-cell RNA sequencing and FACS profiling of the stromal fraction, and semi-quantitative proteomics of the adipocyte-derived exosomal cargo after 5 weeks of HFD feeding. ResultsOur results indicate that loss of GPS2 in mature adipocytes leads to impaired adaptation to the metabolic stress imposed by HFD feeding. GPS2-AKO mice are significantly more inflamed, insulin resistant, and obese, compared to the WT counterparts. At the cellular level, lack of GPS2 in adipocytes impacts upon other stromal populations, with both the eWAT and scWAT depots exhibiting changes in the immune and non-immune compartments that contribute to an increase in inflammatory and anti-adipogenic cell types. Our studies also revealed that adipocyte to stromal cell communication is facilitated by exosomes, and that transcriptional rewiring of the exosomal cargo is crucial for tissue remodeling. Loss of GPS2 results in increased expression of secreted factors promoting a TGFβ-driven fibrotic microenvironment favoring unhealthy tissue remodeling and expansion. ConclusionsAdipocytes serve as an intercellular signaling hub, communicating with the stromal compartment via paracrine signaling. Our study highlights the importance of proper regulation of the ‘secretome’ released by energetically stressed adipocytes at the onset of obesity. Altered transcriptional regulation of factors secreted via adipocyte-derived exosomes (AdExos), in the absence of GPS2, contributes to the establishment of an anti-adipogenic, pro-fibrotic adipose tissue environment, and to hastened progression towards a metabolically dysfunctional phenotype.

Effect of local unstable atherosclerotic plaque on plaque formation in the carotid artery and abdominal aorta of rabbits

To analyze the effect of local unstable atherosclerotic plaque on plaque formation in the carotid and aortic arteries of rabbits. Thirty New Zealand white rabbits were randomly divided into atherosclerosis model group, highfat diet feeding group, and normal chow feeding group (blank control group). In the model group, carotid artery balloon injury was induced after 4 weeks of high-fat diet feeding. Eight weeks later, all the rabbits were euthanized for histopathological examination of the carotid artery and abdominal aorta, and the mean intimal thickness and plaque to lumen area ratio were measured using image analysis software. Venous blood samples were collected from the rabbits for blood lipid analysis. At the ends of 4, 8 and 12 weeks, the rabbits in the model group and high-fat feeding group, but not those in the blank control group, all showed significant weight gain compared with their body weight at 0 week (P < 0.05). The mean intimal thickness was significantly greater in atherosclerosis model group than in the other two groups (P < 0.05). In atherosclerosis model group, the mean intimal thickness and plaque to lumen area ratio in the injured carotid artery were significantly greater than those in the contralateral carotid artery and abdominal aorta (P < 0.05). At the end of the 12 weeks, the levels of triglyceride (TG), total cholesterol (TC), low-density cholesterol (LDL-C), high-density cholesterol (HDL-C) and highsensitivity C-reactive protein (CRP) were all significantly higher in the model group and high-fat feeding group than in the blank control group (P < 0.05); the levels of TG, TC, LDL-C, or HDL-C did not differ significantly between the model group and high-fat feeding group (P>0.05), but the level of CRP was significantly higher in arteriosclerosis model group (P < 0.05). Local unstable atherosclerotic plaque can increase the level of CRP and promote the formation of atherosclerotic plaques in the carotid artery and abdominal aorta in rabbits.

S-12-3: BENEFICIAL EFFECTS OF EMPAGLIFLOZIN ON METABOLIC PARAMETERS AND DIASTOLIC FUNCTION IN SPONTANEOUSLY HYPERTENSIVE RATS FED A HIGH-FAT DIET

Objective: Non-alcoholic fatty liver disease (NAFLD) is a multifactorial pathological syndrome characterized by lipid accumulation in hepatocytes. This can lead to non-alcoholic steatohepatitis (NASH) which can progress to liver fibrosis, cirrhosis and liver cancer. Unfortunately, the pathogenesis of NAFLD is still unknown. However, current studies suggest that gliflozins, primarily antidiabetics, could have beneficial effects on liver metabolism. The aim of this study was therefore to examine whether empagliflozin (10 mg.kg-1.day-1 in drinking fluid) will have beneficial effects. Design and methods: NAFLD was induced by eight weeks of high-fat diet feeding (60% protein) in adult (6-month-old) spontaneously hypertensive rats (SHR). Control SHR were fed a standard diet. Then, the animals were either untreated or treated with empagliflozin for eight weeks. The evaluation of renal function (metabolic cages) and oral glucose tolerance (OGT) test were performed before the start of the treatment and at the end of the study. Cardiac function evaluated by echocardiography as well as metabolic parameters in serum and in liver were determined at the end of the experiment. Results: High-fat diet feeding increased body weight and visceral adiposity. Moreover, it worsened insulin resistance (evaluated as OGT test). In addition, it induced increase in triglycerides and cholesterol levels. Although it had no effect on renal function, it substantially deteriorated not only systolic but also diastolic function of the heart in SHR rats. Empagliflozin had no effect on body weight or visceral fat deposition but improved insulin sensitivity (OGT test) and several metabolic parameters (plasma insulin, non-esterified fatty acids, uric acid and HDL-cholesterol). Moreover, it had beneficial effects on systolic (fractional shortening) and especially diastolic (PWTd, RWT, and E/A ratio) function. Conclusions: Our results demonstrated that the treatment with empagliflozin had beneficial hepatoprotective and cardioprotective actions in hypertensive rats with experimentally induced non-alcoholic liver fatty disease.

Ruminococcaceae_UCG-013 Promotes Obesity Resistance in Mice.

Alterations in the gut microbiome have been linked to obesity and type 2 diabetes, in epidemiologic studies and studies of fecal transfer effects in germ-free mice. Here, we aimed to identify the effects of specific gut microbes on the phenotype of mice fed a high-fat diet (HFD). After eight weeks of HFD feeding, male C57BL/6J mice in the HFD group ranking in the upper and lower quartiles for body weight gain were considered obese prone and obese resistant, respectively. 16S rRNA gene sequencing was used to determine the composition of the intestinal microbiota, and fecal transplantation (FMT) was conducted to determine whether the microbiota plays a causal role in phenotypic variation. Ruminococcaceae_UCG-013 was more abundant in the gut microbes of mice with a lean phenotype than in those with an obese phenotype. Ruminococcaceae_UCG-013 was identified as the most significant biomarker for alleviating obesity by random forest analysis. In a correlation analysis of serum parameters and body weight, Ruminococcaceae_UCG-013 was positively associated with serum HDL-C levels and negatively associated with serum TC, TG, and LDL-C levels. To conclude, Ruminococcaceae_UCG-013 was identified as a novel microbiome biomarker for obesity resistance, which may serve as a basis for understanding the critical gut microbes responsible for obesity resistance. Ruminococcaceae_UCG-013 may serve as a target for microbiome-based diagnoses and treatments in the future.

Abstract 15194: Nitrative Modification of Caveolin-3, a Novel Mechanism Revealing Cardiac Insulin Resistance in Ischemic Heart Failure

Introduction: Myocardial insulin resistant is a hallmark of diabetic cardiac injury. However, underlying molecular mechanisms remain unclear. Recent studies show that diabetic heart is resistant to other cardioprotective interventions, including adiponectin and pre-conditioning. The “universal” resistance to multiple therapeutic interventions suggests impairment of the requisite molecule(s) involved in broad pro-survival signaling cascades. Caveolin (Cav) is a scaffolding protein coordinating transmembrane signaling transduction. However, role of Cav3 in diabetic impairment of cardiac protective signaling and diabetic ischemic heart failure (HF) is unknown. Methods and Results: Mice were fed normal diet (ND) or high-fat-diet (HFD) and subjected to myocardial ischemia and reperfusion. The cardioprotective effect of insulin was significantly blunted as early as 4 weeks of HFD feeding (pre-diabetes), when insulin signals remain unchanged. However, Cav3/IRβ complex formation, requisite for insulin transmembrane signaling, was significantly reduced. Among multiple post-translational modifications altering proteins interaction, Cav3 (not IRβ or AdipoR1) tyrosine nitration is prominent in the pre-diabetic heart. SIN-1 treatment reduced the signalsome formation and blocked insulin transmembrane signaling. Mass spectrometry identified Tyr 73 is the Cav3 nitration site. Phenylalanine substitution of Tyr 73 (Cav3 Y73F ) abolished SIN-1 induced Cav3 nitration, restored Cav3/IRβ complex, and rescued insulin transmembrane signaling. AAV9-mediated cardiac Cav3 Y73F re-expression blocked HFD-induced Cav3 nitration, preserved Cav3 signalsome integrity, and rescued insulin protective action against ischemic HF. Finally, diabetic nitrative modification of Cav3 at Tyr 73 also reduced Cav3/AdipoR1 complex formation and blocked adiponectin cardioprotective signaling. Conclusion: Nitration of Cav3 at Tyr 73 and resultant signal complex dissociation is responsible for cardiac insulin/adiponectin resistance in the pre-diabetic heart, contributing to ischemic HF progression. Early interventions preserving Cav3-centered signalsome integrity is an effective novel strategy against diabetic exacerbation of ischemic HF.

Ginger Root Extract Improves GI Health in Diabetic Rats by Improving Intestinal Integrity and Mitochondrial Function.

Background Emerging research suggests hyperglycemia can increase intestinal permeability. Ginger and its bioactive compounds have been reported to benefit diabetic animals due to their anti-inflammatory and antioxidant properties. In this study, we revealed the beneficial effect of gingerol-enriched ginger (GEG) on intestinal health (i.e., barrier function, mitochondrial function, and anti-inflammation) in diabetic rats. Methods Thirty-three male Sprague Dawley rats were assigned to three groups: low-fat diet (control group), high-fat-diet (HFD) + streptozotocin (single low dose 35 mg/kg body weight (BW) after 2 weeks of HFD feeding) (DM group), and HFD + streptozotocin + 0.75% GEG in diet (GEG group) for 42 days. Glucose tolerance tests (GTT) and insulin tolerance tests (ITT) were conducted at baseline and prior to sample collection. Total pancreatic insulin content was determined by ELISA. Total RNA of intestinal tissues was extracted for mRNA expression using qRT-PCR. Results Compared to the DM group, the GEG group had improved glucose tolerance and increased pancreatic insulin content. Compared to those without GEG (DM group), GEG supplementation (GEG group) increased the gene expression of tight junction (Claudin-3) and antioxidant capacity (SOD1), while it decreased the gene expression for mitochondrial fusion (MFN1), fission (FIS1), biogenesis (PGC-1α, TFAM), mitophagy (LC3B, P62, PINK1), and inflammation (NF-κB). Conclusions Ginger root extract improved glucose homeostasis in diabetic rats, in part, via improving intestinal integrity and mitochondrial dysfunction of GI health.

Dicer prevents activation of the type I interferon pathway in lipid-loaded macrophages

Abstract Background The expression of the endonuclease Dicer in macrophages decreases atherosclerosis and necrotic core formation by producing microRNAs such as miR-10a. This effect of Dicer is associated with enhanced mitochondrial respiration in lipid-loaded macrophages. However, the mechanism by which Dicer-dependent production of miRNAs in lipid-loaded macrophages regulates mitochondrial function is unclear. Purpose We aimed to determine the effect of Dicer on lipid-loaded macrophages in the context of atherosclerosis. Methods Mice with a myeloid cell-specific knockout of Dicer (Lys-Cre/Dicerflox/flox/Apoe−/− mice [M-Dicer−/−]) and control mice (Lys-Cre/DicerWT/WT/Apoe−/− mice [M-Dicer+/+]) were fed a high-fat diet (HFD) for 24 weeks. The oxygen consumption rate (OCR) in aortic arch plaques was studied ex vivo by Seahorse Flux XF 24 Analyzer. Bone marrow-derived macrophages (BMDMs) were stimulated with oxLDL (100 μg/mL) for 72 h. Lipid-loaded macrophages were used for proteomic analysis by mass spectrometry. RIP-prime- seq was performed in tAgo2 immunoprecipitates (IP) from Dicer+/+ and Dicer−/− BMDMs. The miRNA expression profile was determined in lipid-loaded BMDMs by NanoString technology. Stat1 phosphorylation was determined in lipid-loaded macrophages by Jess automated western blot system (ProteinSimple). Result The OCR in aortic arch tissue with plaques was higher than in those without plaques in M-Dicer+/+ mice after 24 weeks of HFD feeding. Dicer knockout in macrophages decreased the OCR in aortic arch tissues with plaques but not in aortic arch tissues without plaques (n=3–4, p&amp;lt;0.05). The proteomic analysis of oxLDL-treated BMDMs indicated that Dicer knockout activated the type I interferon signaling pathway by up-regulating the expression of STAT1/2 and interferon-stimulated genes, such as ISG15. Proteins related to mitochondrial DNA were upregulated (e.g., Dnmt3a) or downregulated (e.g., Tfam) by Dicer knockout (n=5, adj.p&amp;lt;0.05). RIP-prime-seq from BMDMs showed that 1376 transcripts were significantly enriched in the tAgo IP from Dicer+/+ compared with that from Dicer−/− BMDMs (n=3–5, adj.p&amp;lt;0.05) including Dnmt3a and Stat2. STAT1 phosphorylation was increased in Dicer−/− compared with Dicer+/+ BMDMs. Among the 299 miRNAs downregulated by Dicer knockout in lipid-loaded macrophages (n=6, adj.p&amp;lt;0.05), miR-29 and miR-30 have highly conserved binding sites (predicted by TargetScan) for Dnmt3a. Conclusion Our results indicate that Dicer expression in lipid-loaded macrophages limits Stat1/2-driven type I interferon response due to mitochondrial damage. This effect may be mediated by the suppression of Dnmt3a by miRNAs such as miR-29 and miR-30. This suggests that targeting Dnmt3a-mediated mitochondrial damage in lipid-loaded macrophages by miRNAs may be therapeutic strategy to limit atherosclerosis. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): DFG

Tregs in visceral adipose tissue up-regulate circadian-clock expression to promote fitness and enforce a diurnal rhythm of lipolysis.

Regulatory T cells (Tregs) in nonlymphoid organs provide critical brakes on inflammation and regulate tissue homeostasis. Although so-called "tissue Tregs" are phenotypically and functionally diverse, serving to optimize their performance and survival, up-regulation of pathways related to circadian rhythms is a feature they share. Yet the diurnal regulation of Tregs and its consequences are controversial and poorly understood. Here, we profiled diurnal variations in visceral adipose tissue (VAT) and splenic Tregs in the presence and absence of core-clock genes. VAT, but not splenic, Tregs up-regulated their cell-intrinsic circadian program and exhibited diurnal variations in their activation and metabolic state. BMAL1 deficiency specifically in Tregs led to constitutive activation and poor oxidative metabolism in VAT, but not splenic, Tregs. Disruption of core-clock components resulted in loss of fitness: BMAL1-deficient VAT Tregs were preferentially lost during competitive transfers and in heterozygous TregBmal1Δ females. After 16 weeks of high-fat diet feeding, VAT inflammation was increased in mice harboring BMAL1-deficient Tregs, and the remaining cells lost the transcriptomic signature of bona fide VAT Tregs. Unexpectedly, VAT Tregs suppressed adipocyte lipolysis, and BMAL1 deficiency specifically in Tregs abrogated the characteristic diurnal variation in adipose tissue lipolysis, resulting in enhanced suppression of lipolysis throughout the day. These findings argue for the importance of the cell-intrinsic clock program in optimizing VAT Treg function and fitness.