HFF Mice Research Articles

Antidiabetic actions of GPR55 agonist Abn-CBD and sitagliptin in obese-diabetic high fat fed mice

GPR55 has been recognized as a novel anti-diabetic target exerting positive effects on beta cell function and mass. This study evaluated the metabolic actions and therapeutic efficacy of GPR55 agonist abnormal cannabidiol (Abn-CBD) administered alone and in combination with sitagliptin in diet-induced obese-diabetic mice.Chronic effects of 21-day oral administration of Abn-CBD (0.1 µmol/kg BW) monotherapy and in combination with sitagliptin (50 mg/kg BW) were assessed in obese-diabetic HFF mice (n = 8). Assessments of plasma glucose, circulating insulin, DPP-IV activity, CRP, amylase, lipids, body weight and food intake were undertaken. Glucose tolerance, insulin sensitivity, DEXA scanning and islet morphology analysis were performed at 21-days.Sitagliptin, Abn-CBD alone and in combination with sitagliptin attenuated plasma glucose by 37–53 % (p < 0.01 – p < 0.001) and enhanced circulating insulin concentrations by 23–31 % (p < 0.001). Abn-CBD alone and with sitagliptin reduced bodyweight by 9–10 % (p < 0.05). After 21-days, Abn-CBD in combination with sitagliptin (44 %; p < 0.01) improved glucose tolerance, whilst enhancing insulin sensitivity by 79 % (p < 0.01). Abn-CBD increased islet area (86 %; p < 0.05), beta cell mass (p < 0.05) and beta cell proliferation (164 %; p < 0.001), whilst in combination with sitagliptin islet area was decreased (50 %; p < 0.01). Abn-CBD alone, in combination with sitagliptin or sitagliptin alone decreased triglycerides by 34–65 % (p < 0.001) and total cholesterol concentrations by 15–25 % (p < 0.001). In addition, Abn-CBD in combination with sitagliptin reduced fat mass by 19 % (p < 0.05) and reduced CRP concentrations (39 %; p < 0.05).These findings advocate Abn-CBD monotherapy and in combination with sitagliptin as a novel and effective approach for bodyweight control and the treatment of glucose intolerance and dyslipidaemia in type-2-diabetes.

Pharmacological potential of novel agonists for FFAR4 on islet and enteroendocrine cell function and glucose homeostasis

BackgroundTo investigate the metabolic effects of FFAR4-selective agonists on islet and enteroendocrine cell hormone release and the combined therapeutic effectiveness with DPP-IV inhibitors. MethodsInsulinotropic activity and specificity of FFAR4 agonists were determined in clonal pancreatic BRIN-BD11 cells. Expression of FFAR4 was assessed by qPCR and western blotting following agonist treatment in BRIN-BD11 cells and by immunohistochemistry in mouse islets. Acute in-vivo effects of agonists was investigated after intraperitoneal (i.p.) or oral administration in lean and HFF-obese diabetic mice. ResultsGSK137647 (10–11–10–4 M) and Compound-A (10–10–10–4 M) stimulated insulin secretion at 5.6 mM (p < 0.05-p < 0.001) and 16.7 mM (p < 0.05-p < 0.001) glucose in BRIN-BD11 cells, with no cytotoxicity effects as assessed by MTT. FFAR4 antagonist (AH-7614) abolished the insulintropic effect of GSK137647 (p < 0.05-p < 0.001), whilst FFAR1 antagonist (GW1100) had no effect. Incubation of BRIN-BD11 cells with GSK137647 and Compound-A increased FFAR4 (p < 0.01) gene expression at 16.7 mM glucose, with a corresponding increase in FFAR4 (p < 0.01) protein concentrations. FFAR4 upregulation was attenuated under normoglycaemic conditions. Immunohistochemistry demonstrated co-localisation of FFAR4 and insulin in mouse islets. Orally administered GSK137647 or Compound-A (0.1 µmol/kgBW) improved glucose tolerance (p < 0.001), increased plasma insulin (p < 0.001), GLP-1 (p < 0.05), GIP (p < 0.05) and induced satiety (p < 0.001) in HFF mice, with glucose-lowering effects enhanced in combination with DPP-IV inhibitor (Sitagliptin) (p < 0.05). ConclusionsSpecific FFAR4 agonism improves glucose tolerance through insulin and incretin secretion, with enhanced DPP-IV inhibition in combination with Sitagliptin. General significanceThese findings have for the first time demonstrated that selective FFAR4 activation regulates both islet and enteroendocrine hormone function with agonist combinational therapy, presenting a promising strategy for the treatment of type-2-diabetes.

A2A adenosine receptor stimulation regenerates cartilage in osteoarthritis animal model

Purpose: Many studies have been shown that obesity along with joint injury is one of the most common risk factor in the development of osteoarthritis (OA). In a previous study we described that intra-articular injections of liposomal preparations of adenosine completely prevent progression and reverse cartilage loss in post-traumatic OA. TGF-β signaling plays dual and opposing roles in cartilage health and chondrocyte life depending on the signals activated downstream. Activation of downstream signaling pathways for TGF-β leading to localization of phospho-SMAD2/3 associated with maintenance of cartilage. In contrast nuclear localization of phospho-SMAD1/5/8 results in chondrocyte hypertrophy. Here we report that intraarticular injections of liposomal adenosine and A2AR agonist reverses OA in a post-traumatic OA model in rat and in an obesity related mice model. We moreover explore the role of TGF-β signaling in this phenomenon. Methods: Obesity-induced OA model: C57Bl6 mice (5-6 for each group, 12 weeks old) were fed a 60% fat diet (HFF mice) for 3months, after which received intraarticular injections (10 μl) of empty liposomes (Lipo) or liposomes containing the A2AR agonist CGS21680 (Lipo-CGS) or Adenosine (Lipo-Ado) into the knee every 10 days for 4 injections. Post-traumatic OA (PTOA) was induced in Sprague Dawley rats following non-surgical rupture of anterior cruciate ligament (ACL). Four weeks later rats were injected in the knee with 100ul of saline, Lipo or Lipo-CGS every 10 days (6 injections). RNA was isolated from chondrocytes in knee cartilage of rats treated as described above (3 from each group X 3 replicates) and subjected to RNAseq analysis. TC28a2 human chondrocyte cell line was used for in vitro experiments. Results: Lipo-CGS and Lipo-Ado reversed OA in the obesity and post traumatic OA model. Mouse knees had an OARSI score of 5.17±1.84 before treatment. Treatment with LIPO-Ado and lipo-CGS decreased OA severity (OARSI score 1.33±0.81 and 1.83±0.98, respectively, p<0.001 vs pre-treatment; figure 1). In the PTOA model the OARSI score significantly decreases after Lipo-CGS and Lipo-Ado treatment compare to the saline group (OARSI: 1.28±0.39; 1.42±0.69; 2.97±0.0.75 respectively; p<0.05). Analysis of the transcriptome suggests that the treatment of Lipo-CGS promotes the up-regulation of genes involved in proliferation process and downregulations of genes responsible of apoptosis, cartilage catabolism and chondrocyte hypertrophy (Figure 2). TGF-β expression was increased in deep layers of cartilage in the Lipo-CGS-treated rats and there was notable nuclear localization of phospho-SMAD2/3 in these chondrocytes. In contrast, phospho-SMAD1/5/8 was expressed in the nuclei of chondrocytes in the saline and LIPO-treated rats but not in the LIPO-CGS treated rats. Identical changes were observed in the knees of obese mice. To determine whether the effect of A2AR stimulation on TGF-β signaling was direct or indirect we studied the effect of CGS21680 on nuclear phospho-SMAD expression in TC28a2 cells and found that CGS21680 increased nuclear phospho-SMAD2/3 and reduced nuclear phospho-SMAD1/5/8, as detected by immunofluorescence. Conclusions: Administration of an A2AR agonist to established OA knees reverses OA in rats and mice and shifts TGF-β signaling from ALK1/SMAD1/5/8 to ALK5/SMAD2/3 in OA chondrocytes after activation of A2AR in 2 OA animal models.

PGC-1α in hepatic UPR during high-fat high-fructose diet and exercise training in mice.

Diet‐induced obesity is associated with hepatic steatosis, which has been linked with activation of the unfolded protein response (UPR). PGC‐1α is a transcriptional coactivator involved in exercise training‐induced adaptations in muscle and liver. Therefore, the aim of this study was to test the hypothesis that PGC‐1α is required for exercise training‐mediated prevention of diet‐induced steatosis and UPR activation in liver. Male liver‐specific PGC‐1α knockout (LKO) and littermate floxed (lox/lox) mice were divided into two groups receiving either control diet (CON) or high‐fat high‐fructose diet (HFF). After 9 weeks, half of the HFF mice were treadmill exercise trained for 4 weeks (HFF+ExT), while the rest were kept sedentary. HFF resulted in increased body and liver weight, adiposity, hepatic steatosis and whole body glucose intolerance as well as decreased hepatic IRE1α phosphorylation. Exercise training prevented the HFF‐induced weight gain and partially prevented increased liver weight, adiposity and glucose intolerance, but with no effect on liver triglycerides. In addition, BiP protein and CHOP mRNA content increased with exercise training compared with CON and HFF, respectively. Lack of PGC‐1α in the liver only resulted in minor changes in the PERK pathway. In conclusion, this study provides evidence for dissociation between diet‐induced hepatic triglyceride accumulation and hepatic UPR activation. In addition, PGC‐1α was not required for maintenance of basal UPR in the liver and due to only minor exercise training effects on UPR further studies are needed to conclude on the potential role of PGC‐1α in exercise training‐induced adaptations in hepatic UPR.

Increased TASK channel-mediated currents underlie high-fat diet induced vagal afferent dysfunction.

We have previously demonstrated that satiety sensing vagal afferent neurons are less responsive to meal-related stimuli in obesity because of reduced electrical excitability. As leak K+ currents are key determinants of membrane excitability, we hypothesized that leak K+ currents are increased in vagal afferents during obesity. Diet-induced obesity was induced by feeding C57Bl/6J mice a high-fat diet (HFF) (60% energy from fat) for 8-10 wk. In vitro extracellular recordings were performed on jejunal afferent nerves. Whole cell patch-clamp recordings were performed on mouse nodose ganglion neurons. Leak K+ currents were isolated using ion substitution and pharmacological blockers. mRNA for TWIK-related acid-sensitive K+ (TASK) subunits was measured using quantitative real-time PCR. Intestinal afferent responses to nutrient (oleate) and non-nutrient (ATP) stimuli were significantly decreased in HFF mice. Voltage clamp experiments revealed the presence of a voltage-insensitive resting potassium conductance that was increased by external alkaline pH and halothane, known properties of TASK currents. In HFF neurons, leak K+ current was approximately doubled and was reduced by TASK1 and TASK3 inhibitors. The halothane sensitive current was similarly increased. Quantitative PCR revealed the presence of mRNA encoding TASK1 (KCNK3) and TASK3 (KCNK9) channels in nodose neurons. TASK3 transcript was significantly increased in HFF mice. The reduction in vagal afferent excitability in obesity is due in part to an increase of resting (leak) K+ conductance. TASK channels may account for the impairment of satiety signaling in diet-induced obesity and thus is a therapeutic target for obesity treatment. NEW & NOTEWORTHY This study characterized the electrophysiological properties and gene expression of the TWIK-related acid-sensitive K+ (TASK) channel in vagal afferent neurons. TASK conductance was increased and contributed to decreased excitability in diet-induced obesity. TASK channels may account for the impairment of satiety signaling in diet-induced obesity and thus is a promising therapeutic target.

A310 THE INHIBITION OF PTP1B REVERSES IMPAIRED VAGAL AFFERENT SENSITIVITY CAUSED BY DIET-INDUCED OBESITY, REDUCING NITRIC OXIDE AND 2 PORE DOMAIN K+ CONDUCTANCE

AbstractBackgroundWe have demonstrated that diet-induced obesity impairs vagal afferents and that the effects of high concentrations of leptin on vagal afferents in vitro, mimic the effects of obesity. One of downstream molecules in leptin signaling is protein tyrosine phosphatase 1B (PTP1B), which plays a role in negative regulation.AimsWe hypothesized that inhibition of PTP1B would prevent an impairment of vagal afferents caused by diet-induced obesity/hyperleptinemiaMethodsDiet-induced obese (DIO) mice were fed diets composed of 60% or 10% kCal fat for 12–16 weeks. Nodose neurons from DIO mice or standard diet fed mice were incubated overnight with leptin (100nM) and PTP1B inhibitor (10mM). Current and voltage clamp were performed to assess membrane excitability and two-pore domain K+ (K2P) conductance, respectively. NO was measured in culture media using Nitrate/Nitrite fluorometric Assay kit. Media was collected after cell incubation for 24 hrs.ResultsLeptin (100nM) incubation reduced the excitability of vagal afferents. PTP1B inhibitor reversed this inhibitory effect of leptin. PTP1B inhibitor significantly decreased rheobase (100.8 ± 18.6 pA, n=12, leptin) vs. 64.7 ± 5.2 pA (n=15, Leptin + PTP1B inhibitor), *p=0.0497) and significantly increased input resistance (353.8 ± 36.2 MΩ (n=12, leptin) vs. 508.2 ± 52.7 MΩ (n=15, Leptin + PTP1B inhibitor), *p=0.0305). Leptin significantly increased K2P conductance (0.280 ± 0.022 nS (n=17, control) vs. 0.377 ± 0.035 nS (n=16, leptin), *p=0.0108). Inhibition of PTP1B significantly decreased the conductance (0.249 ± 0.013 pA (n=14, Leptin + PTP1B inhibitor), **p=0.0028) in leptin-incubated neuron. In mice fed a HFF diet, there is reduction in vagal excitability, which was reversed by the inhibition of PTP1B. PTP1B inhibitor significantly decreased rheobase (112.1 ± 14.6 pA (n=14, leptin) vs. 70.8 ± 8.1 pA (n=12, HFF + PTP1B inhibitor), *p=0.0268). Rheobase was not significantly different in LFF. Inhibition of PTP1B significantly decreased K2P conductance both in LFF and HFF neurons (LFF; **p=0.0050; HFF; **p=0.0041). PTP1B inhibitor significantly reduced NO fluorescence in leptin-incubated media (33.1 ± 2.2 (Leptin) vs. 12.2 ± 1.0 (Leptin+PTP1B inhibitor), ***<0.0001, n=6) and in neurons from HFF mice (43.4 ± 0.7 (HFF) vs. 29.9 ± 0.6 (HFF+PTP1B inhibitor), ***<0.0001 n=6), but not in LFF (27.8 ± 1.3 (LFF) vs. 25.8 ± 0.8 (LFF+PTP1B inhibitor), NS n=6).ConclusionsIinhibition of PTP1B reverses the effects of HFF and elevated leptin on NO production and K2P conductance in nodose ganglion neurons. Consistent with this, the inhibitory effects of leptin and diet-induced obesity on the excitability of vagal afferent neuron were also reversed by PTP1B blockade. We suggest PTP1B may be a pivotal regulator and potential drug target in obesity.Funding AgenciesCIHR

Long-Term Supplementation of Microencapsulated ursodeoxycholic Acid Prevents Hypertension in a Mouse Model of Insulin Resistance.

Hypertension is a significant comorbidity associated with insulin resistance and type-2 diabetes. Limited evidence show that ursodeoxycholic acid (UDCA) has some anti-hypertensive effects. However, the potential effect of UDCA on hypertension induced by type-2 diabetic insulin resistance has not been reported. In C57Bl6 wild-type mice, insulin resistance was induced by the chronic ingestion of diet enriched in fat and fructose (HFF). HFF mice were randomized to treatment with UDCA or candersartan incorporated into the diet to achieve an ingested dose of approximately 70 mg/kg/day of UDCA or 3 mg/kg/day respectively. Systolic and diastolic blood pressure were measured with tail-cuff method. At 4 weeks of dietary treatment systolic and diastolic blood pressure were comparable in HFF and low-fat (LF) control mice. Co-administration of candesartan at 4 weeks significantly decreased systolic and diastolic blood pressure, UDCA showed no anti-hypertensive effect at 4 weeks. At 24 weeks of dietary intervention, HFF fed mice had substantially elevated systolic blood pressure compared to LF controls. The provision of UDCA substantially attenuated the dietary HFF induced increase in systolic blood pressure concomitant with significantly lower plasma angiotensin II. The anti-hypertensive effect of UDCA in HFF mice was comparable to candesartan. The data suggests that long term supplementation of UDCA effectively lowers hypertension in a dietary induced model of type-2 diabetic insulin resistance.

Two novel glucagon receptor antagonists prove effective therapeutic agents in high-fat-fed and obese diabetic mice.

To examine the effect of two novel, enzymatically stable, glucagon receptor peptide antagonists, on metabolic control in two mouse models of obesity/diabetes. The effects of twice daily i.p. administration of desHis(1)Pro(4)Glu(9)-glucagon or desHis(1)Pro(4)Glu(9)Lys(12)FA-glucagon for 10 days on metabolic control in high-fat-fed (HFF; 45% fat) and obese diabetic (ob/ob) mice were compared with saline-treated controls. Neither analogue altered body weight or food intake in either model over 10 days; however, treatment with each peptide restored non-fasting blood glucose towards normal control values in HFF mice. Basal glucose was also reduced (p < 0.01) in desHis(1)Pro(4)Glu(9)Lys(12)FA-glucagon treated ob/ob mice by day 10, coinciding with increases (p < 0.001) in circulating insulin. At the end of the treatment period, both analogues significantly (p < 0.05-0.01) improved oral and i.p. glucose tolerance (p < 0.05) and peripheral insulin sensitivity, increased pancreatic insulin and glucagon content (p < 0.05-0.01) and decreased (p < 0.05) cholesterol levels in HFF mice. Similarly beneficial metabolic effects on oral glucose tolerance (p < 0.01) and pancreatic insulin content (p < 0.05) were observed in ob/ob mice, especially after desHis(1)Pro(4)Glu(9)Lys(12)FA-glucagon treatment. No significant differences in circulating triglycerides or aspects of indirect calorimetry were noted between peptide treatment groups and respective control HFF and ob/ob mice. Finally, glucagon-mediated elevations of glucose and insulin were significantly (p < 0.05-0.01) annulled after 10 days of desHis(1)Pro(4)Glu(9)-glucagon or desHis(1)Pro(4)Glu(9)Lys(12)FA-glucagon treatment in both animal models. These data indicate that peptide-based glucagon receptor antagonists can reverse aspects of genetically and dietary-induced obesity-related diabetes.

A novel GIP-oxyntomodulin hybrid peptide acting through GIP, glucagon and GLP-1 receptors exhibits weight reducing and anti-diabetic properties

Oxyntomodulin (Oxm) is a 37-amino acid peptide linked to alleviation of obesity-diabetes through a dual mode of action mediated at both glucagon and GLP-1 receptors. GIP is the principle physiological regulator of postprandial insulin secretion. Therefore, the primary aim was to design a novel GIP-Oxm peptide incorporating the actions of GIP, GLP-1 and glucagon in a single molecule. The first 11 N-terminal residues of Oxm were substituted with the sequence of stable dA2GIP molecule to generate a novel GIP-Oxm peptide (dA2GIP-Oxm). dA2GIP-Oxm was resistant to DPP-IV and significantly stimulated in vitro insulin release. dA2GIP-Oxm stimulated cAMP production in GIP-R, glucagon-R and GLP-1-R transfected cells by up to 95%, 83% and 77% of that elicited by respective native ligands. Acute administration of dA2GIP-Oxm to HFF mice resulted in reduced plasma glucose (45% reduction) and increased insulin concentrations (1.7-fold increase). Furthermore, dA2GIP-Oxm lowered plasma glucose (42% reduction) and increased plasma insulin (1.6-fold increase) when administered to HFF mice four hours prior to a glucose load. Once-daily administration of dA2GIP-Oxm for 15 days in HFF mice lowered body weight (13% reduction), reduced plasma glucose (40% reduction) and increased plasma insulin (1.7-fold increase). Furthermore, glycemic responses were improved (38% reduction) and glucose-mediated plasma insulin concentrations enhanced (2-fold increase). These improvements in metabolic control were independent of changes in food intake and insulin sensitivity. dA2GIP-Oxm exerts positive beneficial actions on glucose homeostasis, beta-cell insulin secretion and body weight, mediated through GIP, glucagon and GLP-1 receptors. Such multiple-acting peptides may hold promise as novel therapies for obesity-diabetes.

Modulatory effects of mesenchymal stem cells on hepatic mitochondrial reactive oxygen species production in dietary‐induced insulin resistance

Mesenchymal stem cells (MSCs) are thought to exert their effects in part by limiting oxidative stress. Of therapeutic interest are the effects of MSCs in insulin resistance, a cause of elevated reactive oxygen species (ROS). In this study, we analyzed the antioxidant activity of MSCs in the dietary‐induced insulin resistant C57BL6 model. Male mice were placed on either a chow (CH) or high fat (HFF, 60% kcal) diet for 18wk. Over a 6d period, mice received two serial injections of either murine serum or MSC enriched serum (2 million cells/injection, separated by 2d) via the tail vein. Analyses included cell survival, glucose tolerance, insulin resistance, respirometry and levels of hepatic mitochondrial ROS production. HFF mice were obese (31±2g vs. 46±3g) and hyperglycemic. Results demonstrated no difference in glucose tolerance or insulin resistance with MSCs. Interestingly, MSCs did not alter the rate of ROS production in CH (H2O2 production: 0.68±0.14 pmol.min−1μg−1 in CH‐MSC vs. 0.63±0.11 pmol.min−1μg−1 in CH‐Serum) but significantly reduced mitochondrial ROS production in HFF (4.85±0.39 vs. 0.72±0.17 pmol.min−1μg−1). In conclusion, short‐term MSC treatment does not impact hyperglycemia but significantly reduces rates of hepatic mitochondrial ROS production. Results invite further exploration into the specific mechanisms by which MSCs reduce ROS in insulin resistant states.Support: HSF, CDA, AIHS

Impaired intestinal afferent nerve satiety signalling and vagal afferent excitability in diet induced obesity in the mouse

Gastrointestinal vagal afferents transmit satiety signals to the brain via both chemical and mechanical mechanisms. There is indirect evidence that these signals may be attenuated in obesity. We hypothesized that responses to satiety mediators and distension of the gut would be attenuated after induction of diet induced obesity. Obesity was induced by feeding a high fat diet (60% kcal from fat). Low fat fed mice (10% kcal from fat) served as a control. High fat fed mice were obese, with increased visceral fat, but were not hyperglycaemic. Recordings from jejunal afferents demonstrated attenuated responses to the satiety mediators cholecystokinin (CCK, 100 nm) and 5-hydroxytryptamine (5-HT, 10 μm), as was the response to low intensity jejunal distension, while responses to higher distension pressures were preserved. We performed whole cell patch clamp recordings on nodose ganglion neurons, both unlabelled, and those labelled by fast blue injection into the wall of the jejunum. The cell membrane of both labelled and unlabelled nodose ganglion neurons was less excitable in HFF mice, with an elevated rheobase and decreased number of action potentials at twice rheobase. Input resistance of HFF neurons was also significantly decreased. Calcium imaging experiments revealed reduced proportion of nodose ganglion neurons responding to CCK and 5-HT in obese mice. These results demonstrate a marked reduction in afferent sensitivity to satiety related stimuli after a chronic high fat diet. A major mechanism underlying this change is reduced excitability of the neuronal cell membrane. This may explain the development of hyperphagia when a high fat diet is consumed. Improving sensitivity of gastrointestinal afferent nerves may prove useful to limit food intake in obesity.

W1361 Colon Electrical Stimulation: A Potential Treatment for Obesity

In Vitro extracellular recordings were performed from jejunal afferents. Nodose ganglion neurons (NGNs) were dissociated and plated onto coverslips. Whole cell patch clamp recordings or Fura-2AM calcium imaging experiments on NGNs were performed 18-24 h post-dissociation. Results: In recordings from jejunal afferents, responses to 100nM CCK (11.5 ± 3.2 vs. 3.35 ± 1.0 spikes/s n=10, p<0.05) and 10μM 5-HT (4.85 ± 1.4 vs. 1.75 ± 0.25, n=10, p<0.05) were decreased in HFF mice compared to LFF controls. However responses to non satiety related compounds such as nicotine, and baseline firing rate were also decreased, suggesting a global decrease in excitability. Current clamp experiments were performed on NGNs to assess the membrane properties in LFF and HFF neurons. Resting membrane potentials were similar -46.6 ± 3.5 mV (LFF, n=5) and -43.1 ± 2.5mV (HFF, n=5). Rheobase was significantly increased (34.0 ± 9.3 vs. 128.0 ± 24.6 pA, n=5, P < 0.05) and the number of action potentials at 2X rheobase was decreased (3.0 ± 0.8 vs. 1 ± 0, n= 5 p<0.05) in HFF compared to LFF, and there was a large, but non statistically significant reduction in input resistance (LFF, 725.6 ± 266.9 MΩ vs. HFF, 141 ± 15.6 MΩ). In Calcium imaging studies on HFF neurons, 0.5% (1/21) and 9.5% (2/21) of the neurons responded to 100nM CCK-8s and 1μM 5-HT respectively. The number of responsive neurons was higher in LFF mice CCK; 18.9 % (7/37), 5-HT; 21.6% (8/37). Conclusions: Responses to the satiety mediators CCK and 5-HT are significantly attenuated in gut vagal afferents. This reduction in response appears to be due at least in part to a global reduction in excitability of vagal afferents in diet induced obese mice, raising the possibility that high fat diet can cause vagal hyposensitivity, impairing normal satiety signals from the GI tract.

W1360 Effects of Rimonabant on Gastrointestinal Transit in Dogs

In Vitro extracellular recordings were performed from jejunal afferents. Nodose ganglion neurons (NGNs) were dissociated and plated onto coverslips. Whole cell patch clamp recordings or Fura-2AM calcium imaging experiments on NGNs were performed 18-24 h post-dissociation. Results: In recordings from jejunal afferents, responses to 100nM CCK (11.5 ± 3.2 vs. 3.35 ± 1.0 spikes/s n=10, p<0.05) and 10μM 5-HT (4.85 ± 1.4 vs. 1.75 ± 0.25, n=10, p<0.05) were decreased in HFF mice compared to LFF controls. However responses to non satiety related compounds such as nicotine, and baseline firing rate were also decreased, suggesting a global decrease in excitability. Current clamp experiments were performed on NGNs to assess the membrane properties in LFF and HFF neurons. Resting membrane potentials were similar -46.6 ± 3.5 mV (LFF, n=5) and -43.1 ± 2.5mV (HFF, n=5). Rheobase was significantly increased (34.0 ± 9.3 vs. 128.0 ± 24.6 pA, n=5, P < 0.05) and the number of action potentials at 2X rheobase was decreased (3.0 ± 0.8 vs. 1 ± 0, n= 5 p<0.05) in HFF compared to LFF, and there was a large, but non statistically significant reduction in input resistance (LFF, 725.6 ± 266.9 MΩ vs. HFF, 141 ± 15.6 MΩ). In Calcium imaging studies on HFF neurons, 0.5% (1/21) and 9.5% (2/21) of the neurons responded to 100nM CCK-8s and 1μM 5-HT respectively. The number of responsive neurons was higher in LFF mice CCK; 18.9 % (7/37), 5-HT; 21.6% (8/37). Conclusions: Responses to the satiety mediators CCK and 5-HT are significantly attenuated in gut vagal afferents. This reduction in response appears to be due at least in part to a global reduction in excitability of vagal afferents in diet induced obese mice, raising the possibility that high fat diet can cause vagal hyposensitivity, impairing normal satiety signals from the GI tract.

Coenzyme Q10 supplementation lowers hepatic oxidative stress and inflammation associated with diet-induced obesity in mice

Background Diabetes and obesity are metabolic disorders induced by an excessive dietary intake of fat, usually related to inflammation and oxidative stress. Aims The aim of the study is to investigate the effect of the antioxidant coenzyme Q10 (CoQ10) on hepatic metabolic and inflammatory disorders associated with diet-induced obesity and glucose intolerance. Methods C57bl6/j mice were fed for 8 weeks, either a control diet (CT) or a high-fat diet plus 21% fructose in the drinking water (HFF). CoQ10 supplementation was performed in this later condition (HFFQ). Results HFF mice exhibit increased energy consumption, fat mass development, fasting glycaemia and insulinemia and impaired glucose tolerance. HFF treatment promoted the expression of genes involved in reactive oxygen species production (NADPH oxidase), inflammation (CRP, STAMP2) and metabolism (CPT1α) in the liver. CoQ10 supplementation decreased the global hepatic mRNA expression of inflammatory and metabolic stresses markers without changing obesity and tissue lipid peroxides compared to HFF mice. HFF diets paradoxically decreased TBARS (reflecting lipid peroxides) levels in liver, muscle and adipose tissue versus CT group, an effect related to vitamin E content of the diet. Conclusion In conclusion, HFF model promotes glucose intolerance and obesity by a mechanism independent on the level of tissue peroxides. CoQ10 tends to decrease hepatic stress gene expression, independently of any modulation of lipid peroxidation, which is classically considered as its most relevant effect.

Diet‐induced obesity and cardiovascular regulation in C57BL/6J mice

1. In the present study, we determined the effect of diet-induced obesity on cardiovascular and metabolic regulation in mice at standard laboratory temperatures (ambient temperature (Ta) = 22 degrees C) and during exposure to thermoneutrality (Ta = 30 degrees C). 2. Male C57BL/6J (B6) mice fed a high-fat diet (HFF; n = 17) or chow (CHW; n = 14) for 15 weeks were surgically instrumented with telemetry devices, housed in metabolic chambers and assigned to either control or atenolol treatment (25 mg/kg per day in drinking water) to determine the effects of obesity on baseline cardiovascular function and on the responses to thermoneutrality and 24 h fasting. Mean arterial pressure (MAP), heart rate (HR), arterial pressure and HR variability (time and frequency domain), oxygen consumption (VO2) and locomotor activity were determined. 3. The HFF mice exhibited increased bodyweight (+10.6 +/- 4.1 g), moderate light period hypertension (+8.6 +/- 2.6 mmHg), no difference in HR and increased HR variability at standard laboratory temperature compared with CHW controls. Atenolol produced less of a decrease in HR in HFF mice (-42 +/- 10 b.p.m.) compared with CHW controls (-73 +/- 15 b.p.m.). Acute exposure to thermoneutrality (Ta = 30 degrees C) reduced HR similarly in both HFF and CHW mice (approximately 175 b.p.m.), but reduced MAP less in HFF than in CHW mice (-7.3 +/- 2.5 and -15.2 +/- 1.0 mmHg), respectively. Atenolol treatment had only minor effects on the HR response to thermonuetrality (-114 +/- 13 and -129 +/- 8 b.p.m. in HFF and CHW mice, respectively). The HFF mice displayed greater fasting-induced reductions in light period MAP than did CHW mice (-10.0 +/- 1.1 vs-3.1 +/- 3.5 mmHg, respectively), whereas HR was decreased equally in both groups. Fasting-induced increases in HR variability were attenuated in HFF mice. 4. We conclude that diet-induced obesity produced generally minor changes in cardiovascular regulation in B6 mice at baseline, some of which are distinct from the effects of diet-induced obesity in larger animal models. In contrast, acute variations in Ta or caloric availability produce pronounced alterations in cardiovascular function in either lean or obese mice, which are generally evident after atenolol and, thus, presumably not due exclusively to variation in cardiac sympathetic activity. Interestingly, the degree of obesity induced hypertension was augmented when mice were studied at thermonuetrality. The results suggest an important unrecognized role for vagal tone in the regulation of cardiovascular function in mice and support the need for considerable caution when using mouse models of obesity to examine regulation of cardiovascular function. We argue that mouse physiology studies should be performed in thermoneutral conditions.

Leptin receptor, NPY, POMC mRNA expression in the diet-induced obese mouse brain

A high fat diet leads to progressive development of obesity and leptin resistance in C57 mice with a middle stage of peripheral, but not central, leptin resistance. This stage is characterized by increased fat accumulation despite relative hypophagia. At a later stage central leptin resistance ensues along with hyperphagia, rapid weight and fat gain. The aim of this study is to characterize the mRNA levels of leptin receptor (LR), neuropeptide Y (NPY) and pro-opiomelanocortin (POMC) in high fat (HFF) and low fat (LFF) fed groups of mice. The hypothalamic arcuate nucleus (Arc) was investigated, as was the choroid plexus (ChP) in the case of the leptin receptor. No differences between groups were seen in LR, NPY or POMC mRNA levels after 1 week of feeding. After 8 and 19 weeks, the HFF mice, compared to LFF controls, demonstrated a +45% ( P<0.003) and +84% ( P<0.0001) increase in the ratio of visceral fat to body weight and +223% ( P<0.0001) and +468% ( P<0.0001) elevation in plasma leptin levels, respectively. At 8 weeks, LR mRNA expression showed a +98% ( P<0.016) and +66% ( P<0.0001) increase in ChP and Arc, respectively, while Arc NPY mRNA showed down-regulation by −45% ( P<0.006). Arc POMC mRNA showed no significant changes between groups at 8 weeks. However, after long-term (19 weeks) feeding, the HFF mice displayed significantly −26% ( P<0.039) and −33% ( P<0.0015) reduced LR mRNA in the ChP and Arc, respectively, with Arc POMC and NPY mRNAs down by −55% ( P<0.004) and −32% ( P<0.009), respectively. The present results suggest that in the middle stage of development of high fat-induced obesity, when central leptin sensitivity is maintained, the increased leptin receptor expression may play a role to defend against obesity which is overwhelmed as central leptin insensitivity develops. In this later stage the down-regulation of the POMC system may be important in the final breakdown of weight homeostasis.

Recombinant DNA transfer to Escherichia coli of human faecal origin in vitro and in digestive tract of gnotobiotic mice

The role of helper elements in the mobilisation of pBR recombinant plasmids (tra−, mob−, oriT+ and tra−, mob−, oriT−) from genetically engineered Escherichia coli K12 strains to other K12-strains and to wild-type E. coli strains of human faecal origin was examined. Transfer experiments were done in the digestive tract of axenic (germ free) and gnotobiotic mice, associated with human faecal flora, HFF. The kinetics of implantation of donors, recipients and transconjugants were determined. Mobilisation of oriT+ pBR-type plasmids, by trans-complementation with the products of tra and mob genes was obtained with E. coli K12, in the digestive tract of axenic mice and the resulting transconjugants became established together with the recipient and donor strains. Such mobilisation was only observed sporadically with one E. coli of human origin in axenic mice, but did not occur in gnotobiotic HFF mice. The E. coli strains of human origin were able to promote transfer of an oriT− pBR-type plasmid in vitro but not in axenic or gnotobiotic mice. Transconjugants of wild-type strains obtained in in vitro mating experiments and inoculated into gnotobiotic HFF mice were eliminated as rapidly as the recombinant K12 strains. This work indicates that ≥ 50% of wild-type E. coli strains were able to promote transfer of pBR oriT− plasmids in vitro.

Recombinant DNA transfer to Escherichia coli of human faecal origin in vitro and in digestive tract of gnotobiotic mice

The role of helper elements in the mobilisation of pBR recombinant plasmids (tra−, mob−, oriT+ and tra−, mob−, oriT−) from genetically engineered Escherichia coli K12 strains to other K12-strains and to wild-type E. coli strains of human faecal origin was examined. Transfer experiments were done in the digestive tract of axenic (germ free) and gnotobiotic mice, associated with human faecal flora, HFF. The kinetics of implantation of donors, recipients and transconjugants were determined. Mobilisation of oriT+ pBR-type plasmids, by trans-complementation with the products of tra and mob genes was obtained with E. coli K12, in the digestive tract of axenic mice and the resulting transconjugants became established together with the recipient and donor strains. Such mobilisation was only observed sporadically with one E. coli of human origin in axenic mice, but did not occur in gnotobiotic HFF mice. The E. coli strains of human origin were able to promote transfer of an oriT− pBR-type plasmid in vitro but not in axenic or gnotobiotic mice. Transconjugants of wild-type strains obtained in in vitro mating experiments and inoculated into gnotobiotic HFF mice were eliminated as rapidly as the recombinant K12 strains. This work indicates that ≥ 50% of wild-type E. coli strains were able to promote transfer of pBR oriT− plasmids in vitro.