High-fat Diet Feeding Research Articles

Abstract 2069: Impact Of Endothelial Stiffening And CD36-mediated Lipid Uptake On Atherosclerotic Lesion Formation

Objective: To determine the impact of endothelial stiffening induced by CD36 (cluster of differentiation 36) lipid uptake in the development of atherosclerosis. Approach and Results: Endothelial stiffening of intact en face monolayers, as assessed by atomic force microscopy, reveal the hyperlipidemic mouse model of atherosclerosis, LDLR -/- (low density lipoprotein receptor knockout) is significantly stiffer, especially in the atheroprone aortic arch, as compared to wild type controls (n=4 male mice with 8-15 tissue measurement sites per condition per mouse, p<0.05). Furthermore, a 4-5 month high fat diet (HFD) feeding in LDLR -/- mice resulted in significantly enhanced stiffening compared to regular chow feeding, an effect that was observed in males but not females (n=6-8 mice, p<0.05). There was a significantly greater plaque development, as analyzed using Oil O Red staining for lipids, in male HFD-fed LDLR -/- mice, as compared to age-matched females (n=5-6, p<0.05). No significant difference between males and females was found in lipid panel parameters (n=4-6) or weight gain (n=10-14). Specific downregulation of CD36 in male hypercholesterolemic Cdh5.CreERT2CD36fl/fl LDLR -/- mice led to both a significant decrease in endothelial stiffening (n=5 mice, p<0.05) and in the areas of atherosclerotic lesion formation (n=5-6 mice, p<0.05). Mechanistically, we show that the saturated fatty acid, palmitic acid, increased endothelial stiffening in the en face monolayers of wild type mice, an effect which is prevented by the pre-treatment of the CD36 irreversible inhibitor, SSO (sulfo-N-succinimidyl oleate, n=5-6 male mice, p<0.05). Conclusions: These results highlight that endothelial stiffness from intact aortas and atherosclerotic plaque formation following HFD feeding is strongly dependent on endothelial CD36.

Microalgal Macular Pigment Alleviated the Pathogenic Process in the Streptozotocin-induced Diabetic ApoE-deficient Mice

Type 2 diabetes mellitus (T2DM) is one of the fastest growing epidemics in human history and characteristic is insulin resistance (IR), and endocrine disorder that promoted to decline in insulin production and inhibit cellular effects. Patients with T2DM would lead to many complications such as diabetic retinopathy, diabetic nephropathy, diabetic neuropathy, diabetic foot, atherosclerosis, vascular inflammation, and hepatogenous diabetes due to hyperglycemia. Even after using medical treatment and lifestyle modifications, it is still hard to achieve the goal of reducing blood glucose to an optimal level. Microalga Chlorella sp., a unicellular green alga that has been used as a health food and nutritional product in the last few decades. Macular pigment (MP) produced by Chlorella sp. consists of rich lutein and zeaxanthin, which are regarded as the antioxidants to hyperglycemia-induced oxidative stress. MP is widely used in eye health care, however, few studies focused on scientifically exploring the protection mechanism of MP on diabetes mellitus. Therefore, this study aimed to explore MP extracted from Chlorella on reducing retinopathy and atherosclerosis damages in the diabetic apolipoprotein E deficient mice (ApoE−/−) with high-fat diet feeding and streptozotocin treatment. The results show that MP supplement could significantly reduce glucose tolerance, IR, inflammatory cytokines TNF-α and IL-6 levels, and the serum level of disease markers, i.e., triglyceride (TG), total cholesterol (TCHO), glutamic-oxaloacetic transaminase (GOT), glutamic-pyruvic transaminase (GPT), total protein (TP), albumin (ALB), and blood urea nitrogen (BUN) in the diabetic ApoE−/− mice. In addition, the expression levels of cellular antioxidant enzymes were significantly increased and the adhesion factors as well as MAPK pathway proteins in renal tissues were significantly reduced after MP supplementation. The pathological changes of pancreatic islet morphology, hepatic lipid accumulation, outer nuclear layer and aortic plaque of the diabetic ApoE−/− mice were significantly alleviated by MP supplement. At present, most MP products are derived from marigold, Calendula offcinalis, in the market. However, microalgae cultivation includes the advantages on no seasonal limit, no competition, high yield and carbon reduction compared with other plants. In summary, the microalgal MP improved IR, ameliorated atherosclerosis, and alleviated retinopathy in the diabetic ApoE−/− mice. Consequently, the microalgal MP is potentially to be a healthy food ingredient for the biological effcacy of scavenging free radicals and stabilizing blood glucose. This work was supported by the grants of NSTC 110-2312-B-A49-001-MY3, NSTC 112-2811-B-A49 -501, NSTC 112-2811-B-A49 -503, NSTC 112-2321-B-A49 -018 and NSTC 112-2321-B-A49-005, from the National Science and Technology Council (NSTC), Taiwan. This work was also financially supported by the “Center for Intelligent Drug Systems and Smart Bio-devices (IDS2B)” from The Featured Areas Research Center Program within the framework of the Higher Education Sprout Project of the National Yang Ming Chiao Tung University and Ministry of Education (MOE), Taiwan. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The type of fatty acid dictates their impact on gut permeability and nutrient absorption

We showed that short- and long-term (3 to 12 weeks) high-fat diet (HFD) feeding reduced protein expression of nutrient transporters in the proximal jejunum of mice. The most abundant fatty acids (FA) in HFD were oleic (OA), palmitic (PA) and stearic (EA) acid. Because PA and EA are generally regarded as pro-inflammatory, we hypothesize that dietary PA and EA alter gut permeability and absorption via inflammatory activation. To test this hypothesis, we orally administered HFD-matched doses of OA, PA, and EA to male C57BL/6 mice, fed on purified diet control (LFD), for 3 weeks. Food intake and body weight were monitored, and composition were also evaluated. We next qPCR-assessed the mRNA expression of cytokines ( TNF-α, IL-6, IL-1β, IFN-γ, IL-10) and inflammatory signaling transducers ( MD-2 and Myd88) as well as the protein content of nutrient transporters (Glut2, Pept1, Fat/cd36 and Npc1l1) and cell-cell adhesion proteins (Occludin) in the gut. Small intestinal permeability was assessed ex vivo and gut morphology was histologically determined. In addition, Caco-2 enterocytes were used to assess the impact of specific cytokines on nutrient transport and gut barrier integrity. All dietary FA reduced food intake without affecting lean or fat mass. In the jejunum, OA increased TNF-α gene expression whereas PA upregulated IFN-γ and MD-2 gene expression. Furthermore, OA reduced the nutrient transporters Glut2, Pept1, and Fat/cd36 without affecting Npc1l1, whereas PA and EA reduced Occludin protein content. PA, but not OA and EA, increased jejunum paracellular permeability ex vivo and increased villus length. In Caco-2 cells, while Tnf-α did not affect Glut2, Pept1 and Fat/cd36 or Npc1l1 content, Ifn-γ downregulated Occludin protein content. We conclude that OA and, more potently, PA exert inflammatory effects which contribute to damage gut barrier integrity and downregulate nutrient absorption. Our data point to IFN-γ as a main player coupling dietary stress from PA to altered gut homeostasis. Funding: FAPESP (grants number: 2014/12871-9, 2019/05086-7, 2020/12201-4, and 2022/14545). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Reoxygenation Mitigates Intermittent Hypoxia-Induced Systemic Inflammation and Gut Microbiota Dysbiosis in High-Fat Diet-Induced Obese Rats.

Obstructive sleep apnea (OSA) is a prevalent sleep breathing disorder characterized by intermittent hypoxia (IH), with continuous positive airway pressure (CPAP) as its standard treatment. However, the effects of intermittent hypoxia/reoxygenation (IH/R) on weight regulation in obesity and its underlying mechanism remain unclear. Gut microbiota has gained attention for its strong association with various diseases. This study aims to explore the combined influence of IH and obesity on gut microbiota and to investigate the impact of reoxygenation on IH-induced alterations. Diet-induced obese (DIO) rats were created by 8-week high-fat diet (HFD) feeding and randomly assigned into three groups (n=15 per group): normoxia (NM), IH (6% O2, 30 cycles/h, 8 h/day, 4 weeks), or hypoxia/reoxygenation (HR, 2-week IH followed by 2-week reoxygenation) management. After modeling and exposure, body weight and biochemical indicators were measured, and fecal samples were collected for 16S rRNA sequencing. DIO rats in the IH group showed increased weight gain (p=0.0016) and elevated systemic inflammation, including IL-6 (p=0.0070) and leptin (p=0.0004). Moreover, IH rats exhibited greater microbial diversity (p<0.0167), and significant alterations in the microbial structure (p=0.014), notably the order Clostridiales, accompanied by an upregulation of bile acid metabolism predicted pathway (p=0.0043). Reoxygenation not only improved IH-exacerbated obesity, systemic inflammation, leptin resistance, and sympathetic activation, but also showed the potential to restore IH-induced microbial alterations. Elevated leptin levels were associated with Ruminococcaceae (p=0.0008) and Clostridiales (p=0.0019), while body weight was linked to Blautia producta (p=0.0377). Additionally, the abundance of Lactobacillus was negatively correlated with leptin levels (p=0.0006) and weight (p=0.0339). IH leads to gut dysbiosis and metabolic disorders, while reoxygenation therapy demonstrates a potentially protective effect by restoring gut homeostasis and mitigating inflammation. It highlights the potential benefits of CPAP in reducing metabolic risk among obese patients with OSA.

High Fat Feeding Blunts Skeletal Muscle Oxygen Mediated Blood Flow Responses in Healthy and Type 2 Diabetic Rats

Hypothesis: High fat diet feeding impairs microvascular blood flow responses to dynamic changes in local oxygen concentration ([O2]) in Sprague-Dawley (SD) and Zucker Diabetic Sprague-Dawley (ZDSD) rats. Methods: Seven-week-old male SD and ZDSD rats were fed either a standard chow (N = 7 SD, 5 ZDSD) or a high saturated fat diet (N = 8 SD, 6 ZDSD) for 7 weeks. At 14 weeks of age animals were anaesthetized, instrumented for systemic monitoring, and mechanically ventilated. The extensor digitorum longus muscle was blunt dissected and reflected over a gas exchange chamber imbedded in the stage of an inverted microscope. Microvascular blood flow responses to high and low [O2] challenges (7 – 12 %, and 7 – 2 %) and sequential changes in [O2] (7 – 12 – 2 – 7 %) were recorded at baseline and during hyperinsulinemic-euglycemic clamp. Gas conditions within the chamber were set at 7 % for a 1-minute baseline followed by 2 minutes at either 2 or 12 % O2. The 4-minute square wave oscillation consisted of 1 minute at each [O2] (7 – 12 – 2 – 7 %). Hyperinsulinemic-euglycemic clamp was achieved by simultaneous infusion of insulin (2 U/hr/kg) and variable rate of 50 % glucose until euglycemia was reached (5 - 7 mM blood glucose). Analysis of intravital videos was completed using custom MATLAB software. All animal protocols were approved by Memorial University’s Animal Care Committee. Results: Chow fed SD had significantly higher glucose infusion rates compared to high fat fed SD, chow fed ZDSD, and high fat fed ZDSD (28.11 ± 1.807 vs. 25.14 ± 1.18, 24.56 ± 2.78, 24.77 ± 2.43 mg/kg/min, p = 0.0429, 0.0305, 0.0323, respectively). Red blood cell (RBC) saturation significantly increased and decreased in all groups within 4 s following the increase and decrease in [O2], respectively. Chow fed SD had significant decrease in RBC supply rate (SR) in response to 12 % [O2] (10.41 ± 11.31 vs 7.44 ± 8.40 cells/s, p &lt; 0.0001, τ = 15.85 s). In response to low [O2], significant increases in SR were measured in the chow fed SD and ZDSD groups (7.10 ± 8.76 vs 11.65 ± 11.36 cells/s, p &lt; 0.0001, τ = 17.65 s, and 10.86 ± 13.62 vs 13.86 ± 14.69 cells/s, p &lt; 0.0001, τ = 85.74 s, respectively). High fat fed SD and ZDSD had significant increases in RBC SR during 2 % [O2] (8.47 ± 9.75 vs 11.84 ± 12.11 cells/s, p &lt; 0.0001, τ = 68.49 s, and 16.09 ± 18.12 vs 20.73 ± 21.92 cells/s, p &lt; 0.0001, τ = 23.98 s). High fat fed ZDSD had higher, but not significantly different, baseline RBC SR compared to the chow fed ZDSD group. Chow fed SD were the only group to have an increase in RBC SR at low [O2] during hyperinsulinemic-euglycemic clamp (13.80 ± 17.78 vs 18.07 ± 20.25 cells/s, p = 0.0003, τ = 10.88 s). Conclusions: High fat feeding impairs the capillary blood flow response to local increases in [O2] in healthy and high fat fed T2D model rats. Chow fed ZDSD rats had slower capillary blood flow dynamics than the SD control group. Hyperinsulinemic-euglycemic clamp diminishes the O2 mediated blood flow response in 14-week-old SD and ZDSD rats fed a high fat diet. This project was supported by the Canadian Institute of Health Research through a project grant to GMF. Studentships were supported by Memorial University's School of Graduate Studies and the Faculty of Medicine. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Muscle-specific Asah1 knockout mice develop whole-body insulin resistance

Skeletal muscle is a major tissue for glucose and lipid metabolism. Increasing ectopic deposition of lipid species is often found in obese populations, and this has been associated with the development of insulin resistance. Sphingolipids, particularly ceramides (Cer), are bioactive lipid species, and their accumulation in metabolically active tissues has been shown to desensitize insulin action. For example, using palmitate or Cer analogues in C2C12 or L6 myotubes increases long chain Cer accumulation in the myotubes and inhibits insulin-stimulated Akt phosphorylation. Moreover, inhibiting Cer synthesis improves insulin stimulated glucose uptake in obese rats and mice. However, genetic approaches to evaluating ceramide's effects have been limited to in vitro system and the effects of Cer in skeletal muscle on energy metabolism remain unprobed. Here, we developed and utilized a genetically modified mouse model to induce endogenous Cer accumulation in selectively in skeletal muscle to determine the effects of Cer accrual in skeletal muscle metabolism. Mice were fed high-fat diet (HFD) for 30-day or 60-day. Thirty-day diet intervention significantly increased muscle Cer, which returned to the baseline after 60-days of HFD. We found the expression of acid ceramidase ( Asah1) gene, which is responsible for degrading Cer, was highly elevated only in skeletal muscle from mice fed 30- and 60-day HFD, indicating Asah1 may be an important regulator of muscle ceramides. In order to probe the effect of Asah1 expression on muscle metabolism, we generated tamoxifen-inducible muscle-specific Asah1 knockout mice (Asah1MKO) and provided HFD for 20 weeks. There was no difference in bodyweight between control littermates (Asah1fl/fl) and Asah1MKO during the intervention. We confirmed that Asah1MKO mice significantly increased muscle Cer contents compared to Asah1fl/fl controls. At 18-week of HFD feeding, mice from both groups were tested for insulin and glucose tolerance, but there was no change between the groups. Finally, the mice were tested by hyperinsulinemic-euglycemic clamp to determine whether increased muscle Cer affects whole-body insulin sensitivity. Interestingly, we found that HFD-fed Asah1MKO mice dramatically reduced glucose infusion rate required to maintain euglycemia and blunt 2-deoxyglucose uptake into skeletal muscle compared to controls. These data suggest that Cer accrual in skeletal muscle can be a major contributor to whole-body insulin resistance. In summary, we found that Asah1 is increased only in skeletal muscle in response to HFD, and knocking out Asah1 in mouse skeletal muscle impairs whole-body insulin sensitivity, suggesting that the levels of muscle Cer are potentially critical in the development of insulin resistance. This research work was supported by the National Institute of Health grant (R01DK115824). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The prostaglandin E2 EP3 receptor has disparate effects on islet insulin secretion and content in β-cells in a high-fat diet-induced mouse model of obesity.

Signaling through prostaglandin E2 EP3 receptor (EP3) actively contributes to the β-cell dysfunction of type 2 diabetes (T2D). In T2D models, full-body EP3 knockout mice have a significantly worse metabolic phenotype than wild-type controls due to hyperphagia and severe insulin resistance resulting from loss of EP3 in extra-pancreatic tissues, masking any potential beneficial effects of EP3 loss in the β cell. We hypothesized β-cell-specific EP3 knockout (EP3 βKO) mice would be protected from high-fat diet (HFD)-induced glucose intolerance, phenocopying mice lacking the EP3 effector, Gαz, which is much more limited in its tissue distribution. When fed a HFD for 16 wk, though, EP3 βKO mice were partially, but not fully, protected from glucose intolerance. In addition, exendin-4, an analog of the incretin hormone, glucagon-like peptide 1, more strongly potentiated glucose-stimulated insulin secretion in islets from both control diet- and HFD-fed EP3 βKO mice as compared with wild-type controls, with no effect of β-cell-specific EP3 loss on islet insulin content or markers of replication and survival. However, after 26 wk of diet feeding, islets from both control diet- and HFD-fed EP3 βKO mice secreted significantly less insulin as a percent of content in response to stimulatory glucose, with or without exendin-4, with elevated total insulin content unrelated to markers of β-cell replication and survival, revealing severe β-cell dysfunction. Our results suggest that EP3 serves a critical role in temporally regulating β-cell function along the progression to T2D and that there exist Gαz-independent mechanisms behind its effects.NEW & NOTEWORTHY The EP3 receptor is a strong inhibitor of β-cell function and replication, suggesting it as a potential therapeutic target for the disease. Yet, EP3 has protective roles in extrapancreatic tissues. To address this, we designed β-cell-specific EP3 knockout mice and subjected them to high-fat diet feeding to induce glucose intolerance. The negative metabolic phenotype of full-body knockout mice was ablated, and EP3 loss improved glucose tolerance, with converse effects on islet insulin secretion and content.

The aging of neutrophils is actively involved in the metabolic consequences of high fat diet

Aim: The epigenetic modifications induced by High Fat Diets (HFDs) in long-living hematopoietic cells have been well described, but whether they affect the “aging” of neutrophils, characterized by far shorter half-life, is less clear. Neutrophils "age" through a reciprocal regulation of CXCR4, promoting a "fresh" status when leaving the BM, and CXCR2, accelerating their aging in the circulation. We study whether derailed aging exacerbates the metabolic and inflammatory consequences of HFD. Methods: We immunophenotyped neutrophils and characterized the metabolic responses in physiology (wild-type mice, WT) and in mice with either constitutively aged neutrophils (MRP8 driven conditional deletion of CXCR4; herein CXCR4fl/flCre+) or with constitutively fresh neutrophils (MRP8 driven conditional deletion of CXCR2; CXCR2fl/flCre+), following 20 weeks of HFD feeding (45% Kcal from fat). Results: CXCR4fl/flCre+ mice display higher plasma triglycerides levels versus WT, despite comparable glucose levels, when monitored during standard feeding. This metabolic difference was exacerbated by feeding mice a HFD for 20 weeks. Indeed, despite a comparable gluco-metabolic profile between CXCR4fl/flCre+ and WT mice, liver damage was increased in CXCR4fl/flCre+, linked to the higher accumulation of CXCR4fl/flCre+ neutrophils in the liver after 20 weeks and two hours after intragastric gavage with olive oil versus fasting. As this finding was not observed after 20 weeks of standard fat diet, these results suggest that HFD feeding redirects aged neutrophil to the liver, resulting in enriched oxidative metabolism and NETosis- and inflammation-related pathways in the liver of CXCR4fl/flCre+ mice. Conversely, CXCR2fl/flCre+ mice were protected from obesity and insulin resistance, exhibiting a proresolutive phenotype. In humans, increased plasma levels of Cxcl1 (ligand of CXCR2) correlated with visceral obesity and metabolic syndrome. Conclusions: Neutrophil aging might contribute to the cardio-metabolic consequences of HFD. This aging could represent a new therapeutic target beyond the current anti-inflammatory therapies approved for the treatment of cardiovascular diseases.

Hepatocyte-Specific PEX16 Abrogation in Mice Leads to Hepatocyte Proliferation, Alteration of Hepatic Lipid Metabolism, and Resistance to High-Fat Diet (HFD)-Induced Hepatic Steatosis and Obesity.

Obesity results in hepatic fat accumulation, i.e., steatosis. In addition to fat overload, impaired fatty acid β-oxidation also promotes steatosis. Fatty acid β-oxidation takes place in the mitochondria and peroxisomes. Usually, very long-chain and branched-chain fatty acids are the first to be oxidized in peroxisomes, and the resultant short chain fatty acids are further oxidized in the mitochondria. Peroxisome biogenesis is regulated by peroxin 16 (PEX16). In liver-specific PEX16 knockout (Pex16Alb-Cre) mice, hepatocyte peroxisomes were absent, but hepatocytes proliferated, and liver mass was enlarged. These results suggest that normal liver peroxisomes restrain hepatocyte proliferation and liver sizes. After high-fat diet (HFD) feeding, body weights were increased in PEX16 floxed (Pex16fl/fl) mice and adipose-specific PEX16 knockout (Pex16AdipoQ-Cre) mice, but not in the Pex16Alb-Cre mice, suggesting that the development of obesity is regulated by liver PEX16 but not by adipose PEX16. HFD increased liver mass in the Pex16fl/fl mice but somehow reduced the already enlarged liver mass in the Pex16Alb-Cre mice. The basal levels of serum triglyceride, free fatty acids, and cholesterol were decreased, whereas serum bile acids were increased in the Pex16Alb-Cre mice, and HFD-induced steatosis was not observed in the Pex16Alb-Cre mice. These results suggest that normal liver peroxisomes contribute to the development of liver steatosis and obesity.

Single and combined effects of high-fat diet and ammonia nitrogen on oxidative stress, lipid accumulation and inflammation in liver of grass carp Ctenopharyngodon idella

The current study aims to investigate the role of high-fat diet (HFD) and ammonia nitrogen (AN) in the fatty liver disease progression in farmed fish. In the present study, grass carp were fed the normal fat diet or HFD for 4 weeks to set up the Control group, HFD group, AN group and HFD + AN group. In the AN group and HFD + AN group, grass carp were exposed to 0.52 mg/L NH3-N for the last week. The results show that HFD feeding had no effect on the growth of grass carp but AN stress significantly inhibited it. Further, the HFD feeding and AN challenge both significantly increased the level of alanine aminotransferase (ALT) and reduced the total antioxidant capacity (T-AOC) significantly. Moreover, a further aggravated liver injury appeared in HFD-fed grass carp after AN challenge, as evidenced by the significant increasing of the level of ALT, AST and malondialdehyde (MDA) and marked reducing of glutathione (GSH) and T-AOC. Moreover, the results of Oil Red O and TG contents in liver all indicate that HFD and AN challenge both promoted the lipid accumulation in grass carp liver. Corresponding to this, the results of genes and protein related to lipid metabolism reveal that AN challenge dramatically inhibited lipid hydrolysis, lipophagy and fatty acid oxidation in HFD-fed grass carp liver but the single HFD feeding or AN challenge did not have this effect. Moreover, the single HFD feeding and AN challenge both promoted fatty acid transport to the liver and lipid synthesis in grass carp liver. In summary, the HFD feeding and AN challenge both can disturb the lipid metabolism in grass carp liver. In parallel, the results of expression levels of genes and protein related to inflammation indicate that the single HFD feeding might not significantly contribute to the inflammation in grass carp liver, but the single AN challenge apparently promoted the inflammatory process. Most importantly, inflammatory progression in liver was further aggravated in the HFD-fed grass carp after AN challenge compared to grass carp with only AN stress, mostly evidenced by the increased levels of inflammatory factors (il1β, il2, TNF-α, etc.). In summary, AN challenge significantly promoted the lipid accumulation and inflammatory process in the liver of HFD-fed grass carp, which might have further aggravated liver injury.

Deficiency of neutral cholesterol ester hydrolase 1 (NCEH1) impairs endothelial function in diet-induced diabetic mice

BackgroundNeutral cholesterol ester hydrolase 1 (NCEH1) plays a critical role in the regulation of cholesterol ester metabolism. Deficiency of NCHE1 accelerated atherosclerotic lesion formation in mice. Nonetheless, the role of NCEH1 in endothelial dysfunction associated with diabetes has not been explored. The present study sought to investigate whether NCEH1 improved endothelial function in diabetes, and the underlying mechanisms were explored.MethodsThe expression and activity of NCEH1 were determined in obese mice with high-fat diet (HFD) feeding, high glucose (HG)-induced mouse aortae or primary endothelial cells (ECs). Endothelium-dependent relaxation (EDR) in aortae response to acetylcholine (Ach) was measured.ResultsResults showed that the expression and activity of NCEH1 were lower in HFD-induced mouse aortae, HG-exposed mouse aortae ex vivo, and HG-incubated primary ECs. HG exposure reduced EDR in mouse aortae, which was exaggerated by endothelial-specific deficiency of NCEH1, whereas NCEH1 overexpression restored the impaired EDR. Similar results were observed in HFD mice. Mechanically, NCEH1 ameliorated the disrupted EDR by dissociating endothelial nitric oxide synthase (eNOS) from caveolin-1 (Cav-1), leading to eNOS activation and nitric oxide (NO) release. Moreover, interaction of NCEH1 with the E3 ubiquitin-protein ligase ZNRF1 led to the degradation of Cav-1 through the ubiquitination pathway. Silencing Cav-1 and upregulating ZNRF1 were sufficient to improve EDR of diabetic aortas, while overexpression of Cav-1 and downregulation of ZNRF1 abolished the effects of NCEH1 on endothelial function in diabetes. Thus, NCEH1 preserves endothelial function through increasing NO bioavailability secondary to the disruption of the Cav-1/eNOS complex in the endothelium of diabetic mice, depending on ZNRF1-induced ubiquitination of Cav-1.ConclusionsNCEH1 may be a promising candidate for the prevention and treatment of vascular complications of diabetes.

“Qi Nan” agarwood restores podocyte autophagy in diabetic kidney disease by targeting EGFR signaling pathway

BackgroundDiabetic kidney disease (DKD) is a microvascular complication of diabetes mellitus, contributing to end-stage renal disease with limited treatment options. The development of DKD is attributed to podocyte injury resulting from abnormal podocyte autophagy. Consequently, the restoration of podocyte autophagy is deemed a practicable approach in the treatment of DKD.MethodsDiabetic mice were induced by streptozotocin and high-fat diet feeding. Following 8 weeks of “QN” agarwood treatment, metrics such as albuminuria, serum creatinine (Scr), and blood urea nitrogen (BUN) were evaluated. Renal histological lesions were evaluated by H&E, PAS, Masson, and Sirius red staining. Evaluation of the effects of “QN” agarwood on renal inflammation and fibrosis in DKD mice through WB, q-PCR, and IHC staining analysis. Cytoscape 3.7.1 was used to construct a PPI network. With the DAVID server, the gene ontology (GO) functional annotation and the Kyoto encyclopedia of genes and genomes (KEGG) signaling pathways of the target enrichment were performed. Molecular docking and binding affinity calculations were conducted using AutoDock, while PyMOL software was employed for visualizing the docking results of active compounds and protein targets.ResultsThe results of this study show that “QN” agarwood reduced albuminuria, Scr, and BUN in DKD mice, and improved the renal pathological process. Additionally, “QN” agarwood was observed to downregulate the mRNA and protein expression levels of pro-inflammatory and pro-fibrotic factors in the kidneys of DKD mice. Network pharmacology predicts that “QN” agarwood modulates the epidermal growth factor receptor (EGFR) signaling pathway. “QN” agarwood can increase the expression of LC3B and Nphs1 in DKD mice while reducing the expression of EGFR.ConclusionThe present study demonstrated that “QN” agarwood ameliorated renal injury in DKD by targeting EGFR and restoring podocyte autophagy.Graphical

Dapagliflozin promotes browning of white adipose tissue through the FGFR1-LKB1-AMPK signaling pathway

BackgroundObesity is associated with a wide variety of metabolic disorders that impose significant burdens on patients and society. The “browning” phenomenon in white adipose tissue (WAT) has emerged as a promising therapeutic strategy to combat metabolic disturbances. However, though the anti-diabetic drug dapagliflozin (DAPA) is thought to promote “browning,” the specific mechanism of this was previously unclear.MethodsIn this study, C57BL/6 J male mice were used to establish an obesity model by high-fat diet feeding, and 3T3-L1 cells were used to induce mature adipocytes and to explore the role and mechanism of DAPA in “browning” through a combination of in vitro and in vivo experiments.ResultsThe results show that DAPA promotes WAT "browning" and improves metabolic disorders. Furthermore, we discovered that DAPA activated "browning" through the fibroblast growth factor receptors 1-liver kinase B1-adenosine monophosphate-activated protein kinase signaling pathway.ConclusionThese findings provide a rational basis for the use of DAPA in treating obesity by promoting the browning of white adipose tissue.

Risk assessment of di-(2-ethylhexyl) phthalate plus a high-fat diet in the rat kidney

Di-(2-ethylhexyl) phthalate (DEHP) is used in food packaging and medical products, resulting in unintentional human ingestion. Concurrently, a high fat (HF)-oriented diet is very popular for many people worldwide. Here we investigated the regulatory role of renin-angiotensin system in kidney dysfunction. The HF diet-fed rats were fed a HF diet plus different doses of 10, 100 and 300 mg/kg bw DEHP for 35 days by gavage. Serum lipid contents and protein activities of renin-angiotensin system (RAS) components were measured using biochemical methods and ELISA; key metabolic genes and proteins of the kidney were determined by RT-PCR and western blotting techniques. Kidney morphology was examined through hematoxylin-eosin (HE) staining. Kidney weight to body weight ratio and mRNA expression of kidney damage markers were significantly increased following DEHP plus HF administration, which corresponded histologically with glomerular basement membrane thickening and glomerular swelling. Furthermore, DEHP treatment plus a HF diet induced dyslipidemia, accompanied by increased levels of renin-angiotensin system components, such as ACE protein, MAS-1 protein, serum renin and aldosterone. In addition, mRNA expression levels of pro-inflammatory cytokines were significantly upregulated in the kidneys. Pearson correlation analysis revealed that higher kidney ACE expression and ACE/ACE-2 ratio were positively associated with TG, TC and LDL-C levels. These data suggest that combined exposure to DEHP exposure plus a HF diet feeding may induce renal damage through overactivation of RAS.

Impact of Ghrelin on Islet Size in Nonpregnant and Pregnant Female Mice.

Reducing ghrelin by ghrelin gene knockout (GKO), ghrelin-cell ablation, or high-fat diet feeding increases islet size and β-cell mass in male mice. Here we determined if reducing ghrelin also enlarges islets in females and if pregnancy-associated changes in islet size are related to reduced ghrelin. Islet size and β-cell mass were larger (P = .057 for β-cell mass) in female GKO mice. Pregnancy was associated with reduced ghrelin and increased liver-expressed antimicrobial peptide-2 (LEAP2; a ghrelin receptor antagonist) in wild-type mice. Ghrelin deletion and pregnancy each increased islet size (by ∼19.9-30.2% and ∼34.9-46.4%, respectively), percentage of large islets (>25 µm2×103, by ∼21.8-42% and ∼21.2-41.2%, respectively), and β-cell mass (by ∼15.7-23.8% and ∼65.2-76.8%, respectively). Neither islet cross-sectional area, β-cell cross-sectional area, nor β-cell mass correlated with plasma ghrelin, although all positively correlated with LEAP2 (P = .081 for islet cross-sectional area). In ad lib-fed mice, there was an effect of pregnancy, but not ghrelin deletion, to change (raise) plasma insulin without impacting blood glucose. Similarly, there was an effect of pregnancy, but not ghrelin deletion, to change (lower) blood glucose area under the curve during a glucose tolerance test. Thus, genetic deletion of ghrelin increases islet size and β-cell cross-sectional area in female mice, similar to males. Yet, despite pregnancy-associated reductions in ghrelin, other factors appear to govern islet enlargement and changes to insulin sensitivity and glucose tolerance in the setting of pregnancy. In the case of islet size and β-cell mass, one of those factors may be the pregnancy-associated increase in LEAP2.

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.

Interindividual differences of dietary fat-inducible Mest in white adipose tissue of C57BL/6J mice are not heritable.

Differences in white adipose tissue (WAT) expression of mesoderm-specific transcript (Mest) in C57BL6/J mice fed a high-fat diet (HFD) are concomitant with and predictive for the development of obesity. However, the basis for differences in WAT Mest among mice is unknown. This study investigated whether HFD-inducible WAT Mest, as well as susceptibility to obesity, is transmissible from parents to offspring. WAT biopsies of mice fed an HFD for 2 weeks identified parents with low and high WAT Mest for breeding. Obesity phenotypes, WAT Mest, hepatic gene expression, and serum metabolites were determined in offspring fed an HFD for 2 weeks. Offspring showed no heritability of obesity or WAT Mest phenotypes from parents but did show hepatic and serum metabolite changes consistent with their WAT Mest. Importantly, retired male breeders showed WAT Mest expression congruent with initial WAT biopsies even though HFD exposure occurred early in life. Disparity of HFD-induced Mest in mice is not heritable but, rather, is reestablished during each generation and remains fixed from an early age to adulthood. Short-term HFD feeding reveals variation of WAT Mest expression within isogenic mice that is positively associated with the development of obesity.

Effects of moxibustion on intestinal barrier function and TLR4/NF-κB p65 signaling pathway in obese rats.

To observe the effects of moxibustion on intestinal barrier function and Toll-like receptor 4 (TLR4)/nuclear factor-κB p65 (NF-κB p65) signaling pathway in obese rats and explore the mechanism of moxibustion in the intervention of obesity. Fifty-five Wistar rats of SPF grade were randomly divided into a normal group (10 rats) and a modeling group (45 rats). In the modeling group, the obesity model was established by feeding high-fat diet. Thirty successfully-modeled rats were randomized into a model group, a moxibustion group, and a placebo-control group, with 10 rats in each one. In the moxibustion group, moxibustion was applied at the site 3 cm to 5 cm far from the surface of "Zhongwan" (CV 12), with the temperature maintained at (46±1 ) ℃. In the placebo-control group, moxibustion was applied at the site 8 cm to 10 cm far from "Zhongwan" (CV 12), with the temperature maintained at (38±1) ℃. The intervention was delivered once daily for 8 weeks in the above two groups. The body mass and food intake of the rats were observed before and after intervention in each group. Using ELISA methool, the levels of serum triacylglycerol (TG), total cholesterol (TC) and lipopolysaccharide (LPS) were detected and the insulin resistance index (HOMA-IR) was calculated. HE staining was used to observe the morphology of colon tissue. The mRNA expression of zonula occludens-1 (ZO-1), Occludin, Claudin-1, TLR4 and NF-κB p65 in the colon tissue was detected by quantitative real-time PCR; and the protein expression of ZO-1, Occludin, Claudin-1, TLR4 and NF-κB p65 was detected by Western blot in the rats of each group. Compared with the normal group, the body mass, food intake, the level of HOMA-IR, and the serum levels of TC, TG and LPS were increased in the rats of the model group (P<0.01); those indexes in the moxibustion group were all reduced when compared with the model group and the placebo-control group respectively (P<0.01, P<0.05). Compared with the normal group, a large number of epithelial cells in the mucosa of colon tissue was damaged, shed, and the inflammatory cells were infiltrated obviously in the interstitium in the rats of the model group. When compared with the model group, in the moxibustion group, the damage of the colon tissue was recovered to various degrees and there were few infiltrated inflammatory cells in the interstitium, while, the epithelial injury of the colon tissue was slightly recovered and the infiltrated inflammatory cells in the interstitium were still seen in the placebo-control group. The mRNA and protein expressions of ZO-1, Occludin and Caudin-1 were decreased in the model group compared with those in the normal group (P<0.01). When compared with the model group and the placebo-control group, the mRNA and protein expressions of these indexes were increased in the moxibustion group (P<0.01, P<0.05). In the model group, the mRNA and protein expressions of TLR4 and NF-κB p65 were increased when compared with those in the normal group (P<0.01), and the mRNA and protein expressions of these indexes were reduced in the moxibustion group when compared with those in the model group and the placebo-control group (P<0.01). Moxibustion can reduce the body mass and food intake, regulate the blood lipid and improve insulin resistance in the rats of obesity. It may be related to alleviating inflammatory response through improving intestinal barrier function and modulating the intestinal TLR4/NF-κB p65 signaling pathway.

High-fat diet consumption promotes adolescent neurobehavioral abnormalities and hippocampal structural alterations via microglial overactivation accompanied by an elevated serum free fatty acid concentration

Mounting evidence suggests that high-fat diet (HFD) consumption increases the risk for depression, but the neurophysiological mechanisms involved remain to be elucidated. Here, we demonstrated that HFD feeding of C57BL/6J mice during the adolescent period (from 4 to 8 weeks of age) resulted in increased depression- and anxiety-like behaviors concurrent with changes in neuronal and myelin structure in the hippocampus. Additionally, we showed that hippocampal microglia in HFD-fed mice assumed a hyperactive state concomitant with increased PSD95-positive and myelin basic protein (MBP)-positive inclusions, implicating microglia in hippocampal structural alterations induced by HFD consumption. Along with increased levels of serum free fatty acids (FFAs), abnormal deposition of lipid droplets and increased levels of HIF-1α protein (a transcription factor that has been reported to facilitate cellular lipid accumulation) within hippocampal microglia were observed in HFD-fed mice. The use of minocycline, a pharmacological suppressor of microglial overactivation, effectively attenuated neurobehavioral abnormalities and hippocampal structural alterations but barely altered lipid droplet accumulation in the hippocampal microglia of HFD-fed mice. Coadministration of triacsin C abolished the increases in lipid droplet formation, phagocytic activity, and ROS levels in primary microglia treated with serum from HFD-fed mice. In conclusion, our studies demonstrate that the adverse influence of early-life HFD consumption on behavior and hippocampal structure is attributed at least in part to microglial overactivation that is accompanied by an elevated serum FFA concentration and microglial aberrations represent a potential preventive and therapeutic target for HFD-related emotional disorders.

Hepatoprotective and cardioprotective effects of empagliflozin in spontaneously hypertensive rats fed a high-fat diet

A combination of liver and heart dysfunction worsens the prognosis of human survival. The aim of this study was to investigate whether empagliflozin (a sodium-glucose transporter-2 inhibitor) has beneficial effects not only on cardiac and renal function but also on hepatic function. Adult (6-month-old) male spontaneously hypertensive rats (SHR) were fed a high-fat diet (60% fat) for four months to induce hepatic steatosis and mild heart failure. For the last two months, the rats were treated with empagliflozin (empa, 10 mg.kg-1.day-1 in the drinking water). Renal function and oral glucose tolerance test were analyzed in control (n=8), high-fat diet (SHR+HF, n=10), and empagliflozin-treated (SHR+HF+empa, n=9) SHR throughout the study. Metabolic parameters and echocardiography were evaluated at the end of the experiment. High-fat diet feeding increased body weight and visceral adiposity, liver triglyceride and cholesterol concentrations, and worsened glucose tolerance. Although the high-fat diet did not affect renal function, it significantly worsened cardiac function in a subset of SHR rats. Empagliflozin reduced body weight gain but not visceral fat deposition. It also improved glucose sensitivity and several metabolic parameters (plasma insulin, uric acid, and HDL cholesterol). In the liver, empagliflozin reduced ectopic lipid accumulation, lipoperoxidation, inflammation and pro-inflammatory HETEs, while increasing anti-inflammatory EETs. In addition, empagliflozin improved cardiac function (systolic, diastolic and pumping) independent of blood pressure. The results of our study suggest that hepatoprotection plays a decisive role in the beneficial effects of empagliflozin in preventing the progression of cardiac dysfunction induced by high-fat diet feeding.