Fat-fed Mice Research Articles

Insulin-releasing and glucose-lowering effects of gallic acid in cellular and high fat-fed mice models of type 2 diabetes

Insulin-releasing and glucose-lowering effects of gallic acid in cellular and high fat-fed mice models of type 2 diabetes

In vitro insulinotropic actions of extracts of Gnetum africanum welw and effects on glucose homeostasis in mice with diet-induced obesity-diabetes

IntroductionGnetum africanum leaf is consumed as a vegetable in many parts of Africa. Many ethnobotanical surveys indicate its usage in the management of inflammation-related diseases (such as diabetes). However, mechanisms underlying its antidiabetic actions are poorly understood. MethodsThis study conducted phytochemical analysis and investigated mechanisms underlying anti-diabetic actions of aqueous extracts of G. africanum in cellular and high fat-fed mice models of diabetes. Reversed phase high performance liquid chromatography (HPLC) analysis, qualitative phytochemical screening and the estimation of the total flavonoid and total phenolic content were conducted. Mechanisms underlying insulinotropic actions of the extracts as well as its effects on cytotoxicity, cell viability, intracellular calcium and membrane potential were assessed. In vivo actions were evaluated in mice with diet-induced obesity-diabetes. ResultsG. africanum has total phenolic content of 1.24±0.23GE/g and total flavonoid content of 1.54±0.06QE/g. HPLC analysis revealed the presence of the presence of atropine, vicenin 3, vicenin 2, alpha-chaconine, isoswertisin and solanidine. The extract stimulated non-toxic, concentration-dependent insulin-release from BRIN-BD11 cells (1.3-fold to 3.3-fold, P < 0.05 to P < 0.001). As glucose concentration increased from 1.1 mM to 5.6 mM and 5.6 mM to 16.7 mM, these effects increased by 2.0-fold (P < 0.001) and 1.2-fold (P < 0.05) respectively. Insulinotropic effects increased in cells depolarised with KCl (30 mM, 1.3-fold, P < 0.05). These effects reduced in the presence of diazoxide (300 µM, 67 %, P < 0.001), verapamil (50 nM, 50 %, P < 0.001), and absence of extracellular calcium (55 %, P < 0.001). Membrane potential (48 %, P < 0.05) and intracellular calcium (34 %, P < 0.05) increased in cells incubated with the extract. The extract improved glucose tolerance (22.2 %, P < 0.01 at 150 mg kg-1 bw; 35.1 %, P < 0.001 at 300 mg kg-1 bw) and plasma insulin levels (1.4-fold, P < 0.05 at 150 mg kg-1 bw; 2.9-fold, P < 0.001 at 300 mg kg-1 bw) in high fat fed mice in a dose-dependent manner. ConclusionThese results suggest a role for the KATP-dependent pathway in G. africanum insulinotropic actions and encourage further development of the therapeutic potential of the extract.

Amphibian host-defense peptides with potential for Type 2 diabetes therapy – an updated review

Investigations conducted since 2018 have identified several host-defense peptides present in frog skin secretions whose properties suggest the possibility of their development into a new class of agent for Type 2 diabetes (T2D) therapy. Studies in vitro have described peptides that (a) stimulate insulin release from BRIN-BD11 clonal β-cells and isolated mouse islets, (b) display β-cell proliferative activity and protect against cytokine-mediated apoptosis and (c) stimulate production of the anti-inflammatory cytokine IL-10 and inhibit production of the pro-inflammatory cytokines TNF-α and IL-1β. Rhinophrynin-27, phylloseptin-3.2TR and temporin F are peptides with therapeutic potential. Studies in vivo carried out in db/db and high fat-fed mice have shown that twice-daily administration of [S4K]CPF-AM1 and [A14K]PGLa-AM1, analogs of peptides first isolated from the octoploid frog Xenopus amieti, over 28 days lowers circulating glucose and HbA1c concentrations, increases insulin sensitivity and improves glucose tolerance and lipid profile. Peptide treatment produced potentially beneficial changes in the expression of skeletal muscle genes involved in insulin signaling and islet genes involved in insulin secretion in these murine models of T2D. Lead compounds uncovered by the study of frog HDPs may provide a basis for the design of new types of agents that can be used, alone or in combination with existing therapies, for the treatment of T2D.

Impact of housing temperature on adipose tissue HDAC9 expression and adipogenic differentiation in high fat-fed mice.

Impaired adipogenic differentiation exacerbates metabolic disease in obesity. This study reported that high-fat diet (HFD)-fed mice housed at thermoneutrality exhibited impaired adipogenic differentiation, attributed to increased expression of histone deacetylase 9 (HDAC9). However, the impact of HFD on adipogenic differentiation is reportedly variable, possibly reflecting divergent environmental conditions such as housing temperature. C57BL/6J (wild-type [WT]) mice were housed at either thermoneutral (28-30°C) or ambient (20-22°C) temperature and fed HFD or chow diet (CD) for 12 weeks. For acute exposure experiments, WT or transient receptor potential cation channel subfamily M member 8 (TRPM8) knockout mice housed under thermoneutrality were acutely exposed to ambient temperature for 6 to 24 h. WT mice fed HFD and housed at thermoneutrality, compared with ambient temperature, gained more weight despite reduced food intake. They likewise exhibited increased inguinal adipose tissue HDAC9 expression and reduced adipogenic differentiation in vitro and in vivo compared with CD-fed mice. Conversely, HFD-fed mice housed at ambient temperature exhibited minimal change in adipose HDAC9 expression or adipogenic differentiation. Acute exposure of WT mice to ambient temperature reduced adipose HDAC9 expression independent of sympathetic β-adrenergic signaling via a TRPM8-dependent mechanism. Adipose HDAC9 expression is temperature sensitive, regulating adipogenic differentiation in HFD-fed mice housed under thermoneutrality.

Neprilysin deficiency reduces hepatic gluconeogenesis in high fat-fed mice

Neprilysin is a peptidase that cleaves glucoregulatory peptides, including glucagon-like peptide-1 (GLP-1) and cholecystokinin (CCK). Some studies suggest that its inhibition in diabetes and/or obesity improves glycemia, and that this is associated with enhanced insulin secretion, glucose tolerance and insulin sensitivity. Whether reduced neprilysin activity also improves hepatic glucose metabolism has not been explored. We sought to determine whether genetic deletion of neprilysin suppresses hepatic glucose production (HGP) in high fat-fed mice. Nep+/+ and Nep−/− mice were fed high fat diet for 16 weeks, and then underwent a pyruvate tolerance test (PTT) to assess hepatic gluconeogenesis. Since glycogen breakdown in liver can also yield glucose, we assessed liver glycogen content in fasted and fed mice. In Nep−/− mice, glucose excursion during the PTT was reduced when compared to Nep+/+ mice. Further, liver glycogen levels were significantly greater in fasted but not fed Nep−/− versus Nep+/+ mice. Since gut-derived factors modulate HGP, we tested whether gut-selective inhibition of neprilysin could recapitulate the suppression of hepatic gluconeogenesis observed with whole-body inhibition, and this was indeed the case. Finally, the gut-derived neprilysin substrates, GLP-1 and CCK, are well-known to suppress HGP. Having previously demonstrated elevated plasma GLP-1 levels in Nep−/− mice, we now measured plasma CCK bioactivity and reveal an increase in Nep−/− versus Nep+/+ mice, suggesting GLP-1 and/or CCK may play a role in reducing HGP under conditions of neprilysin deficiency. In sum, neprilysin modulates hepatic gluconeogenesis and strategies to inhibit its activity may reduce HGP in type 2 diabetes and obesity.

Natural emulsifiers lecithins preserve gut microbiota diversity in relation with specific faecal lipids in high fat-fed mice

Synthetic emulsifiers promote metabolic syndrome and considerably alter gut microbiota. Data is lacking regarding natural emulsifiers like plant lecithins, a polar lipid-rich source of 18:3n-3 PUFA (ALA). For 13 weeks, male Swiss mice were fed ALA-replete semi-synthetic high-fat diet (HFD) including lecithin from rapeseed (RL) or soy, vs 2 HFD-controls devoid of lecithin (ALA-replete; low-ALA), vs Chow. Lecithins did not enhance HFD-induced adiposity nor increased inflammation, did not alter gut barrier markers and caecal bile acids, and contributed to n-3 PUFA status. Lecithins improved gut microbiota diversity. RL (10% in fat) even restored α-diversity similar to Chow, increased Lachnospiraceae NK4A136, Lactobacillus and Ruminococcaceae UCG-014 groups, and decreased Blautia genus bacteria. The abundance of most beneficial lecithin-enhanced bacteria was positively correlated to the amount of faecal polar lipid-bound ALA. These findings show that lecithins can beneficially affect the gut microbiota in association with changes in lipid residues in the distal gut.

Black Seed Oil Improves Beef Fat-induced Blood-biochemical Abnormalities in Swiss Albino Mice

Background: The high saturated fatty acid content of beef has been linked to obesity and cardiovascular diseases. The study investigated the impact of black seed oil (BSO) on beef fat-altered hemato-biochemical and pathophysiological alterations in albino mice. Methods: Twenty four Swiss Albino mice (Mus musculus) of 25-28 days old were equally divided into three groups (n=8) namely A, B and C. Mice of group A was designated as the untreated control group. Groups B and C were fed pellets containing 10% beef fat and 10% BSO, respectively for 9 weeks. Result: The results revealed that beef fat-fed mice gained weight and BSO normalized the weight gain. The hematological values of beef fat supplemented mice improved after being supplemented with 10% BSO. LDL-c, Triglycerides and total cholesterol levels were all significantly lower in mice fed BSO plus beef fat. HDL-c levels were significantly higher in BSO plus beef fat-fed mice. Spleens in BSO plus beef fat-fed mice were slightly enlarged without gross abnormalities. 10% beef fat caused minor changes in the histostructures of the kidney, heart and liver. There were significant changes in cardiac muscle and massive fatty changes in kidney tissue. This study concluded that BSO has beneficial effects on the body and can prevent beef fat-induced abnormalities.

Identification of biomarkers for glycaemic deterioration in type 2 diabetes

We identify biomarkers for disease progression in three type 2 diabetes cohorts encompassing 2,973 individuals across three molecular classes, metabolites, lipids and proteins. Homocitrulline, isoleucine and 2-aminoadipic acid, eight triacylglycerol species, and lowered sphingomyelin 42:2;2 levels are predictive of faster progression towards insulin requirement. Of ~1,300 proteins examined in two cohorts, levels of GDF15/MIC-1, IL-18Ra, CRELD1, NogoR, FAS, and ENPP7 are associated with faster progression, whilst SMAC/DIABLO, SPOCK1 and HEMK2 predict lower progression rates. In an external replication, proteins and lipids are associated with diabetes incidence and prevalence. NogoR/RTN4R injection improved glucose tolerance in high fat-fed male mice but impaired it in male db/db mice. High NogoR levels led to islet cell apoptosis, and IL-18R antagonised inflammatory IL-18 signalling towards nuclear factor kappa-B in vitro. This comprehensive, multi-disciplinary approach thus identifies biomarkers with potential prognostic utility, provides evidence for possible disease mechanisms, and identifies potential therapeutic avenues to slow diabetes progression.

Peroxisome proliferator-activated receptors-alpha and gamma synergism modulate the gut-adipose tissue axis and mitigate obesity

AimTo evaluate the effects of single PPARα or PPARγ activation, and their synergism (combined PPARα/γ activation) upon the gut-adipose tissue axis, focusing on the endotoxemia and upstream interscapular brown adipose tissue (iBAT) function in high-saturated fat-fed mice. Methods: Male C57BL/6 mice received a control diet (C, 10% lipids) or a high-fat diet (HF, 50% lipids) for 12 weeks. Then, the HF group was divided to receive the treatments for four weeks: HFγ (pioglitazone, 10 mg/kg), HFα (WY-14643, 3.5 mg/kg), and HFα/γ (tesaglitazar, 4 mg/kg). Results: The HF group exhibited overweight, oral glucose intolerance, gut dysbiosis, altered gut permeability, and endotoxemia, culminating in iBAT whitening. The downregulation of LPS-Tlr4 signaling underpinned reduced inflammation and improved lipid metabolism in iBAT in the HFα/γ group, the unique to show normalized body mass and increased energy expenditure. Conclusion: PPARα/γ synergism treated obesity by ameliorating the gut-adipose tissue axis, where restored gut microbiota and permeability controlled endotoxemia and rescued iBAT whitening through favored thermogenesis.

Consumption of Antioxidant-Rich "Cerrado" Cashew Pseudofruit Affects Hepatic Gene Expression in Obese C57BL/6J High Fat-Fed Mice.

The pseudofruit of A. othonianum Rizzini, “Cerrado” cashew pulp, has been described as rich in flavonoids, phenolic compounds, and vitamin C. The objective of this work was to evaluate the beneficial health effects seen with the addition of “Cerrado” cashew pulp (CP) to an obesogenic high fat diet provided to C57BL/6J male mice. In week 9, the HF-fed group had a significantly higher baseline glucose concentration than the LF- or HF+CP-fed groups. In RNAseq analysis, 4669 of 5520 genes were found to be differentially expressed. Among the genes most upregulated with the ingestion of the CP compared to HF were Ph1da1, SLc6a9, Clec4f, and Ica1 which are related to glucose homeostasis; Mt2 that may be involved steroid biosynthetic process; and Ciart which has a role in the regulation of circadian rhythm. Although “Cerrado” CP intake did not cause changes in the food intake or body weight of fed mice with HF diet, carbohydrate metabolism appeared to be improved based on the observed changes in gene expression.

Camellia (Camellia oleifera Abel.) Seed Oil Regulating of Metabolic Phenotype and Alleviates Dyslipidemia in High Fat-Fed Mice through Serum Branch-Chain Amino Acids.

Camellia (Camellia oleifera Abel.) seed oil (CO) has been shown to effectively reduce the blood lipid level of its host due to its fatty acid content, but the specific molecular mechanism associated with the metabolic phenotype after digestion is not clear. Here, we further investigated the relationship between branched-chain amino acids (BCAA) and the metabolic phenotype that may exhibit the anti-dyslipidemia effect of CO on mice fed a high-fat diet for 30 day C57BL/6J male mice were allocated to three groups: the control group (Cont), the high-fat feed group (HFD), and a high-fat feed group with CO treatment (CO). A serum sample was collected to detect lipid biomarkers and BCAA concentration. Notably, Low-density lipoprotein (LDL), Total Cholesterol (TC), and Triglycerides (TG) showed a significant decrease, whereas High-density lipoprotein (HDL) increased in CO mice but not in the HFD group. The concentration of Isoleucine (Ile), leucine (Leu), and valine (Val) was similar between the Cont and CO groups compared with the HFD group, exhibiting an inhibition induced by CO in mice fed with a high-fat diet. A metabolic phenotype from serum examined by non-targeted metabolite analysis using UHPLC/MS showed most metabolites exhibited lipid and BCAA metabolism. The results indicated that CO treatment notably regulated the metabolism of arachidonic acid and steroid biosynthesis in response to HFD-induced dyslipidemia. In addition, the expression of PPARγ genes that correlated with the BCAA and serum lipid biomarkers were compared, and significant inhibition was noticed, which might lead to the potential exposure of the anti-dyslipidemia mechanism of CO in HFD-fed mice. In conclusion, the expression of PPARγ genes, serum lipid level, BCAA concentration, and the metabolic phenotype was significantly positive in correlation with a high-fat diet, whereas oral CO improved the biomarkers and metabolism of some specific serum metabolites in HFD-fed mice.

Metabolic Effects of Dietary pH, Protein Source and Fat Content in Diet-induced Obese Mice

Abstract Objectives Higher intake of saturated fats, salts, and fatty proteins has been linked to metabolic diseases (e.g., obesity), in part through low-grade metabolic acidosis and inflammation. Hence, our objective was to test the effects of protein sources and preparations in diet induced obese B6 male and female mice, compared to low fat-fed mice. We hypothesized that metabolic health will be improved by consuming a diet containing pH-enhanced beef or casein diets, compared to a non-pH-enhanced diets. Methods B6 male and female mice were randomized (n = 10) into 8 diets that differ in protein source, pH enhancement, and fat content: low fat casein (LFC), pH-enhanced (with ammonia) LFC (LFCN), LF lean beef (LFB), LFBN, high fat casein (HFC), HFCN, HF beef (HFB), HFBN. Body weight and food intake were measured weekly for 12 weeks. White adipose tissue (WAT) was collected at study termination and used for histology, RNA and protein isolation for analyses of fat cell size, and gene (qRT-PCR) and protein (Western blotting) expression. Three-way ANOVA was performed to identify main effects as well as interactions of dietary pH x protein source x fat content, separately in males, and females. Results We identified a significant main effect of diet (LF/HF) on fat pad weights in males (F (1,71) = 91.94, p &amp;lt; 0.0001) and females (F (1,72) = 5.111, p = 0.0268). No effect of protein source or change of dietary pH was observed on fat pad weights. However, WAT histology indicated a substantial change in adipose tissue quality, with reduced adipocyte area (p &amp;lt; 0.05) in LF, LFN, HFCN, HFB, HFBN groups compared to HFC in males. Additionally, in both males and females, protein levels of pAkt and pAMPK were upregulated in adipose tissue of mice fed HF diets with increased pH (HFCN, HFBN), compared to non-pH enhanced HF diets. Conclusions Our findings indicate potential metabolic benefits of increasing dietary pH, as shown by improved adipose tissue cellularity and improved insulin signaling and energy sensing/fat oxidation-related markers. Thus, additional research is warranted to determine mechanisms underlying the metabolic effects of pH enhancement. These findings also merit further mechanistic studies in animals, as well as future clinical research to translate our results into humans and dissect interactions between protein source, pH and fat content on metabolic diseases. Funding Sources Empirical Foods.

Estrogen Sulfotransferase is Highly Expressed in Vascular Endothelial Cells Overlying Atherosclerotic Plaques.

Cytosolic estrogen sulfotransferase (SULT1E1) mainly catalyzes the sulfoconjugation and deactivation of estrogens that are known to exert potent anti-atherogenic effects. However, it remains unknown about the connection between SULT1E1 andatherosclerosis. Recently, we reported that SULT1E1 is highly expressed in the aorta with plaques of high fat-fed ApoE knockout (KO) mice (mouse model of atherosclerosis), and interacts with oxidized low-density lipoprotein (Ox-LDL) known as a major component of atherosclerotic lesions. In this study, immunohistochemical staining for SULT1E1 in the aorta of high fat-fed ApoE KO mice showed that SULT1E1 is detected in vascular endothelial cells overlying atherosclerotic plaques. Results from Western blotting showed that Ox-LDL induces the protein expression of both SULT1E1 and peroxisome proliferator-activated receptor (PPAR) γ in human umbilical vein endothelial cells (HUVECs), and then that a PPARγ antagonist GW9662, but not a PPARα antagonist GW6471, inhibited the protein expression of SULT1E1 induced by Ox-LDL. Moreover, GW9662 significantly increased the proliferation of HUVECs induced by Ox-LDL. Our results suggest that SULT1E1 and PPARγ, both of which are increased by Ox-LDL, may interact with each other, and then may reduce cooperatively Ox-LDL-induced proliferation of vascular endothelial cells overlying atherosclerotic plaques, leading to against atherosclerosis.

An oral GLP-1 and GIP dual receptor agonist improves metabolic disorders in high fat-fed mice

Dual activation of the glucagon-like peptide 1 (GLP-1) receptor and the glucose-dependent insulinotropic polypeptide (GIP) receptor has potential as a novel strategy for treatment of diabesity. Here, we created a hybrid peptide which we named 19W, and show that it is more stable in presence of murine plasma than exendin-4 is. In vitro studies were performed to reveal that 19W could stimulate insulin secretion from INS-1 cells in a dose-dependent manner, just like the native peptide GIP and exendin-4 do. 19W effectively evoked dose-dependent cAMP production in cells targeting both GLP-1R and GIPR. In healthy C57BL/6J mice, the single administration of 19W significantly improved glucose tolerance. When administered in combination with sodium deoxycholate (SDC), its oral hypoglycemic activity was enhanced. Pharmacokinetics studies in Wistar rats revealed that 19W was absorbed following oral uptake, while SDC increased its bioavailability. A long-term (28 days) exposure study of twice-daily oral administration to high fat-fed (HFF) mice showed that 19W significantly reduced animal food intake, body weight, fasting blood glucose, total serum cholesterol (T-CHO), non-esterified free fatty acids (NEFA), and low-density lipoprotein cholesterol (LDL-C) levels. It also significantly improved glucose tolerance and the pancreatic β/α cell ratio, and decreased the area of liver fibrosis. These results clearly demonstrate the beneficial action of this novel oral GLP-1/GIP dual receptor agonist to reduce adiposity and hyperglycemia in diabetic mice and to ameliorate liver fibrosis associated with obesity. This dual-acting peptide can be considered a good candidate for novel oral therapy to treat obesity and diabetes.

Sotagliflozin, a Dual SGLT1/2 Inhibitor, Improves Cardiac Outcomes in a Normoglycemic Mouse Model of Cardiac Pressure Overload.

Selective SGLT2 inhibition reduces the risk of worsening heart failure and cardiovascular death in patients with existing heart failure, irrespective of diabetic status. We aimed to investigate the effects of dual SGLT1/2 inhibition, using sotagliflozin, on cardiac outcomes in normal diet (ND) and high fat diet (HFD) mice with cardiac pressure overload. Five-week-old male C57BL/6J mice were randomized to receive a HFD (60% of calories from fat) or remain on ND for 12 weeks. One week later, transverse aortic constriction (TAC) was employed to induce cardiac pressure-overload (50% increase in right:left carotid pressure versus sham surgery), resulting in left ventricular hypertrophic remodeling and cardiac fibrosis, albeit preserved ejection fraction. At 4 weeks post-TAC, mice were treated for 7 weeks by oral gavage once daily with sotagliflozin (10 mg/kg body weight) or vehicle (0.1% tween 80). In ND mice, treatment with sotagliflozin attenuated cardiac hypertrophy and histological markers of cardiac fibrosis induced by TAC. These benefits were associated with profound diuresis and glucosuria, without shifts toward whole-body fatty acid utilization, increased circulating ketones, nor increased cardiac ketolysis. In HFD mice, sotagliflozin reduced the mildly elevated glucose and insulin levels but did not attenuate cardiac injury induced by TAC. HFD mice had vacuolation of proximal tubular cells, associated with less profound sotagliflozin-induced diuresis and glucosuria, which suggests dampened drug action. We demonstrate the utility of dual SGLT1/2 inhibition in treating cardiac injury induced by pressure overload in normoglycemic mice. Its efficacy in high fat-fed mice with mild hyperglycemia and compromised renal morphology requires further study.

Safety assessment of two strains and anti-obese effects on mice fed a high-cholesterol diet

Previous study documented that Lactobacillus paracasei S0940 and Streptococcus thermophilus ldbm1 have obvious cholesterol-lowering abilities in vitro. In this study, the safety of two strains were evaluated by nitroreductase test, hemolysis test and antibiotic sensitivity test and to evaluate the cholesterol-reducing abilities in vivo. The results indicated that two strains did not exhibit nitroreductase activities and were ɤ-hemolytic on blood agar plates. Further, both strains did not represent a health risk by antibiotic sensitivity test, and significantly reduced serum and liver cholesterol and triglyceride levels of high fat-fed mice. Compared with the high-fat model group, administration of the strains to mice fed a high-cholesterol diet increased fecal water content and fecal cholesterol and significantly improved the intestinal microbiota, which indicating that Lactobacillus paracasei S0940 and Streptococcus thermophilus ldbm1 have a positive effect on reducing cholesterol levels and may be used in functional food.

Impact of Processing on the Anti-inflammatory and Gastrointestinal Protective Effects of Cocoa in High Fat-Fed Obese Mice

Abstract Objectives The objective of this study was to determine the impact of fermentation and roasting steps on the anti-inflammatory and gastrointestinal (GI) protective efficacy of dietary cocoa in high fat-fed obese mice. Fermentation and roasting are important for flavor development, but have been shown to reduce total phenolic content in cocoa and are hypothesized to be detrimental for the health beneficial effects of cocoa. To date, however, the available data demonstrating the effect of fermentation and roasting on the anti-inflammatory and GI protective effects of cocoa are limited. Methods Obese male and female C57BL/6J mice were fed a high fat diet (HF) supplemented with one of seven cocoa powders (80 mg/g) that had been processed using different fermentation and roasting parameters. After 9 weeks, GI permeability was determined by gavaging mice with fluorescein isothiocyanate-conjugated dextran and measuring fluorescence in the blood. Markers of colonic inflammation were measured by quantitative reverse-transcriptase PCR. Metabolic endotoxemia was determined by measuring plasma lipopolysaccharide (LPS) and LPS binding protein (LBP). Results Cocoa treatment reduced GI permeability by 48 – 80% in male and female mice compared to HF-fed controls. These differences were paralleled by decreased plasma levels of LPS (25 – 70% lower) and LBP (25 – 57% lower) compared to HF-fed controls. Cocoa-treated mice had reduced colonic mRNA expression of interleukin (IL)1β, IL4, IL6, tumor necrosis factor (TNF)α, and inducible nitric oxide synthase (NOS2) compared HF-fed controls. In contrast to the hypothesis that fermentation and roasting are detrimental to the anti-inflammatory efficacy of cocoa, we found that unfermented/unroasted cocoa and more aggressively fermented/roasted cocoa were equally efficacious at reducing metabolic endotoxemia and colonic expression of IL1β, TNFα, and NOS2. Conclusions In summary, we found that dietary supplementation with cocoa reduces GI permeability, inflammation, and metabolic endotoxemia in HF-fed obese mice, and that the efficacy of cocoa was resilient to fermentation and roasting. Our on-going studies are focused on determining the phytochemical drivers of cocoa bioactivity, and identifying the role of the colonic microbiota as a mediator of the effects of cocoa. Funding Sources The current studies were funded by USDA NIFA.

The Impact of Omega‐3 Fatty Acids on Cardiac Autonomic Nerve Fiber Innervation in High Fat‐Fed Mice

Peripheral neuropathy and cardiac autonomic neuropathy (CAN) are common and serious complications of diabetes, characterized by loss of peripheral nerves that innervate the skin and loss of parasympathetic and sympathetic nerves that innervate the heart, respectively. CAN is associated with increased risk of myocardial infarction and mortality, yet often goes undetected in patients. Both prediabetic and diabetic patients develop peripheral neuropathy and CAN, suggesting that the level of hyperglycemia at which peripheral neuropathy and CAN develop precedes the onset of diabetes. Although evidence from human and rodent studies indicate that omega-3-fatty acids prevent and/or reverse the development of peripheral neuropathy, the effects of omega-3-fatty acids on CAN has not been investigated. PURPOSE Assess the effects of omega-3-fatty acids on cardiac autonomic nerve fiber innervation in high fat diet-induced prediabetic mice. We hypothesize that omega-3 fatty acids will attenuate a reduction in cardiac autonomic nerve fiber innervation in high fat-fed mice. METHODS Male C57BL/6 mice were randomized into four diet groups (n = 12-14/group) for 14 weeks: 10% low fat fish oil (LFFO), 54% high fat-fish oil (HFFO), 10% low fat-lard (LFL), or 54% high fat-lard (HFL). The von Frey behavioral test for hindpaw mechanical sensitivity was used to characterize peripheral neuropathy at baseline then biweekly for 14 weeks. At study completion, fasting glucose and insulin values were used to calculate the homeostatic model assessment of insulin resistance (HOMA-IR). Mice were anesthetized, transcardially perfused with 1% PBS, and heart tissue was harvested and frozen in O.C.T. Heart was sectioned in three planes (anterior coronal, posterior coronal, and sagittal) and mounted on slides. Fluorescence immunohistochemistry was performed on heart cryosections using anti-choline acetyl-transferase and anti-tyrosine hydroxylase followed by Alexa Fluor secondary antibodies to visualize parasympathetic and sympathetic nerve fibers. For each mouse, nerve fibers were quantified on three slides (1 from each plane) and at least 3 areas with the highest nerve fiber density for each slide were examined. Nerve fiber density was determined using Nikon Elements and was calculated as the total length of all counted nerve fibers divided by the total area examined (µm/mm2). RESULTS Body weight, fasting insulin and glucose, and HOMA-IR were higher in HFL compared to all other groups. Fasting glucose was also higher in LFL compared to LFFO. Hindpaw withdrawal threshold was lower in HFL compared to HFFO, indicative of hindpaw hypersensitivity in HFL. CONCLUSIONS Omega-3 fatty acids prevented excess body weight, insulin resistance, elevated glucose, and hindpaw hypersensitivity in mice fed a high fat diet. Based on this data, we expect a diet rich in omega-3 fatty acids will attenuate cardiac autonomic nerve fiber denervation. Taken together, these findings would suggest that omega-3 fatty acids might hold therapeutic potential in the prevention of metabolic derangements as well as peripheral and CAN in individuals who consume a high fat, high calorie diet.

Dietary cocoa ameliorates non-alcoholic fatty liver disease and increases markers of antioxidant response and mitochondrial biogenesis in high fat-fed mice

Cocoa powder, derived Theobroma cacao, is a popular food ingredient that is commonly consumed in chocolate. Epidemiological and human intervention studies have reported that chocolate consumption is associated with reduced risk of cardiometabolic diseases. Laboratory studies have reported the dietary supplementation with cocoa or cocoa polyphenols can improve obesity and obesity-related comorbidities in preclinical models. Non-alcoholic fatty liver disease (NAFLD), one such comorbidity, is a risk factor for cirrhosis and hepatocellular carcinoma. Limited studies have examined the effect of cocoa/chocolate on NAFLD and underlying hepatoprotective mechanisms. Here, we examined the hepatoprotective effects of dietary supplementation with 80 mg/g cocoa powder for 10 wks in high fat (HF)-fed obese male C57BL/6J mice. We found that cocoa-supplemented mice had lower rate of body weight gain (22%), hepatic triacylglycerols (28%), lipid peroxides (57%), and mitochondrial DNA damage (75%) than HF-fed controls. These changes were associated with higher hepatic superoxide dismutase and glutathione peroxidase enzyme activity and increased expression of markers of hepatic mitochondrial biogenesis. We also found that the hepatic protein expression of sirtuin 3 (SIRT3), and mRNA expression of peroxisome proliferator activated receptor g coactivator (PGC) 1a, nuclear respiratory factor 1, and forkhead box O3 were higher in cocoa-treated mice compared to HF-fed controls. These factors play a role in coordinating mitochondrial biogenesis and expression of mitochondrial antioxidant response factors. Our results indicate that cocoa supplementation can mitigate the severity of NAFLD in obese mice and that these effects are related to SIRT3/PGC1a-mediated increases in antioxidant response and mitochondrial biogenesis.

Supplementation with anthocyanins reverts established endotoxemia in high fat-fed mice through the regulation of colonic physiology by modulating redox signaling

As an efficient method for inducing tumor cell apoptosis, ROS can be constantly formed and accumulated in NADPH oxidase overactivated-cells, resulting in further mitochondrial membrane damage and mitochondria-dependent apoptosis. In addition, JNK mitogen-activated protein kinase (JNK MAPK) signal also acts as a vital candidate pathway for inducing tumor cell apoptosis by targeting mitochondrial death pathway. However, the relationship between NADPH oxidase-ROS and JNK MAPK signal still remains unclear. Here, we discovered a novel self-driven signal circuit between NADPH oxidase-ROS and JNK MAPK, which was induced by a cytotoxic steroidal saponin (ASC) in hepatoma carcinoma cells. NADPH oxidase-dependent ROS production was markedly activated by ASC and directly led to JNK MAPK activation. Moreover, antioxidant, NADPH oxidase inhibitor and specific knock-out for p47 subunit of NADPH oxidase could effectively block NADPH oxidase–ROS-dependent JNK activation, suggesting that NADPH oxidase is an upstream regulator of JNK MAPK. Conversely, a specific JNK inhibitor could inhibit ASC-induced NADPH oxidase activation and down-regulate ROS levels as well, indicating that JNK might also regulate NADPH oxidase activity to some extent. These observations indicate that NADPH oxidase and JNK MAPK activate each other as a signal circuit. Furthermore, drug pretreatment experiments with ASC showed this signal circuit operated continuously via a self-driven mode and finally induced apoptosis in hepatoma carcinoma cells. Taken together, we provide a proof for inducing hepatoma carcinoma cell apoptosis by activating the JNK–NADPH oxidase–ROS-dependent self-driven signal circuit pathway.