DIO Rats Research Articles

Differential effects of high-fat diet on endocrine, metabolic and depressive-like behaviors in male and female rats

The complications of obesity extend beyond the periphery to the central nervous system (CNS) and include an increased risk of developing neuropsychiatric co-morbidities like depressive illness. Preclinical studies support this concept, including studies that have examined the effects of a high-fat diet (HFD) on depressive-like behaviors. Although women are approximately two-fold more likely to develop depressive illness compared to men, most preclinical studies have focused on the effects of HFD in male rodents. Accordingly, the goal of this study was to examine depressive-like behaviors in male and female rats provided access to a HFD. In agreement with prior studies, male and female rats provided a HFD segregate into an obesity phenotype (i.e., diet-induced obesity; DIO) or a diet resistant (DR) phenotype. Upon confirmation of the DR and DIO phenotypes, behavioral assays were performed in control chow, DR, and DIO rats. In the sucrose preference test, male DIO rats exhibited significant decreases in sucrose consumption (i.e., anhedonia) compared to male DR and male control rats. In the forced swim test (FST), male DIO rats exhibited increases in immobility and decreases in climbing behaviors in the pre-test sessions. Interestingly, male DR rats exhibited these same changes in both the pre-test and test sessions of the FST, suggesting that consumption of a HFD, even in the absence of the development of an obesity phenotype, has behavioral consequences. Female rats did not exhibit differences in sucrose preference, but female DIO rats exhibited increases in immobility exclusively in the test session of the FST, behavioral changes that were not affected by the stage of the estrous cycle. Collectively, these studies demonstrate that access to a HFD elicits different behavioral outcomes in male and female rats.

Dysfunction of the Brown Adipose Organ in HFD-Obese Rats and Effect of Tart Cherry Supplementation.

Obesity has a great impact on adipose tissue biology, based on its function as a master regulator of energy balance. Brown adipose tissue (BAT) undergoes remodeling, and its activity declines in obese subjects due to a whitening process. The anti-obesity properties of fruit extracts have been reported. The effects of tart cherry against oxidative stress, inflammation, and the whitening process in the BAT of obese rats were investigated. Intrascapular BAT (iBAT) alterations and effects of Prunus cerasus L. were debated in rats fed for 17 weeks with a high-fat diet (DIO), in DIO supplemented with seed powder (DS), and with seed powder plus the juice (DJS) of tart cherry compared to CHOW rats fed with a normo-caloric diet. iBAT histologic observations revealed a whitening process in DIO rats that was reduced in the DS and DJS groups. A modulation of uncoupling protein-1 (UCP-1) protein and gene expression specifically were detected in the obese phenotype. An upregulation of UCP-1 and related thermogenic genes after tart cherry intake was detected compared to the DIO group. Metabolic adjustment, endoplasmic reticulum stress, protein carbonylation, and the inflammatory microenvironment in the iBAT were reported in DIO rats. The analysis demonstrated an iBAT modulation that tart cherry promoted. In addition to our previous results, these data confirm the protective impact of tart cherry consumption on obesity.

1676-P: A Novel GLP1R/GCGR Dual Agonist, DA-1726 Elicits Weight Loss Superior to Semaglutide in Diet-Induced Obese Rats

DA-1726 showed superior weight loss efficacy compared to Semaglutide (SEMA) in DIO mice. In this following study, a dose-response of DA-1726 was evaluated to determine the maximum efficacy in DIO rats that are known to have good translatability to human studies. The half-life of DA-1726 was longer in rats compared to mice, however the in vitro activity of DA-1726 against rat glucagon receptor was less potent than in the mouse, and plasma protein binding was approximately 10-fold higher in rats. Therefore, we set the effective dose for rats higher than for mice. DA-1726 was injected twice a week for about 4 weeks, a dose-dependent and significant weight loss effect was observed. The minimum effective dose in DIO rats was found to be 250 nmol/kg. Then, the maximum body weight loss effects of DA-1726 (500 and 1000 nmole/kg) and SEMA were compared in the DIO rat. In this study, DA-1726 showed an excellent weight loss effect than SEMA (32.6% for DA-1726 at a high dose vs. 24.0% for SEMA, p<0.05). The low-dose DA-1726 group showed a similar effect compared to SEMA even though they consumed more food. The high-dose DA-1726 group showed similar food intake as SEMA, but led to a higher weight loss effect. After repeated dosing, the high-dose DA-1726 significantly increased the expression of energy metabolism-related genes (Ucp1, Ppargc1a) in white adipose tissues (WAT), supporting increased energy expenditure. So then, in order to confirm whether this increased gene change was a direct effect leading to weight loss or an indirect effect caused by weight loss, we assessed the changes in gene expression after single dosing. DA-1726 (250 and 500 nmol/kg) induced small but significant weight loss 3 days after a single injection (-7.6% and -9.5% vs. control). However, the genes expression (Ucp1, Ppargc1a) in WAT was significantly increased only in the 500 nmol/kg-treated group suggesting a direct effect. Therefore, DA-1726 is expected to elicit excellent weight loss effects in humans with a new mechanism. Disclosure Y.Chae: None. T.Kim: None. I.Jung: None. H.Kim: None. M.Kim: Employee; Dong-A ST.

Electroacupuncture induces weight loss by regulating tuberous sclerosis complex 1-mammalian target of rapamycin methylation and hypothalamic autophagy in high-fat diet-induced obese rats.

Background: Obesity can be caused by abnormalities of hypothalamic autophagy, which is closely regulated by the epigenetic modification of TSC1-mTOR. However, whether the weight-reducing effect of EA may relate to the modification of TSC1-mTOR methylation and hypothalamic autophagy remain unclear. This study was conducted to reveal the possible mechanism by which EA reduces BW by measuring the levels of TSC1-mTOR methylation and hypothalamic autophagy-related components. Methods: The weight-reducing effect of EA was investigated in high-fat diet (HFD)-induced obese (DIO) rats by monitoring the BW, food consumption, and epididymal white adipose tissue (eWAT)/BW ratio. Hematoxylin and eosin staining was performed for morphological evaluation of eWAT. Immunofluorescence was utilized to observe the localization of LC3 in the hypothalamus. The expressions of autophagy components (Beclin-1, LC3, and p62) and mTOR signaling (mTOR, p-mTOR, p70S6K, and p-p70S6K) were assessed by western blot. The methylation rate of the TSC1 promoter was detected by bisulfite genomic sequencing. Results: Treatment with EA significantly reduced the BW, food consumption, and eWAT/BW ratio; attenuated the morphological alternations in the adipocytes of DIO rats. While HFD downregulated the expression levels of Beclin-1 and LC3 and upregulated those of p62, these changes were normalized by EA treatment. EA markedly decreased the methylation rate of the TSC1 gene promoter and suppressed the protein expressions of mTOR, p-mTOR, p70S6K, and p-p70S6K in the hypothalamus. Conclusion: EA could reduce BW and fat accumulation in DIO rats. This ameliorative effect of EA may be associated with its demethylation effect on TSC1-mTOR and regulation of autophagy in the hypothalamus.

213-OR: Antidiabetic Potential of Novel, Long-Acting Amylin Analogue ZP8396 in ZDF Rats

Amylin improves glucose homeostasis through at least three distinct mechanisms of action, including prevention of the postprandial rise in plasma glucagon, slowing of gastric emptying, and increased satiety, leading to decreased caloric intake and potential weight loss. Clinical efficacy has been demonstrated with short-acting amylin analogue pramlintide. ZP8396, currently in phase 1 clinical testing, is a long-acting, amylin analogue designed to improve solubility, stability and allow for co-formulation with other relevant peptides. We previously reported that ZP8396 provides body weight reduction in DIO rats alone and in combination with semaglutide, a once-weekly GLP-1 agonist (Skarbaliene et al, Obesity Week 2021) . Here we investigated the antidiabetes potential of ZP8396 in Zucker Diabetic Fatty (ZDF) rats. The antidiabetes potential after a single subcutaneous (s.c.) injection of ZP8396 (nmol/kg) or vehicle was measured in ZDF rats (aged 12-13 weeks, n=10) , an animal model of type 2 diabetes, and continuously monitored for 168 h after dosing.ZP8396 induced a significant body weight loss in ZDF rats vs. vehicle group. A significant decrease in body weight was observed in rats treated with ZP8396 from initiation of the dosing and up to 96 h post-dose. In addition, single s.c. injection of ZP8396 showed a significant reduction in blood glucose levels at 24 h post-dose, reaching a 20 % difference in blood glucose levels vs. vehicle group (14,97±0,39 and 18,60±1,18 mmol/L, respectively; p <0.05) . Also, ZP8396 significantly reduced plasma glucagon levels, reaching a 55% difference in ZP8396-treated diabetic rats vs. vehicle group at 24 h post-dose (p <0.05) . In conclusion, in diabetic rat ZP8396 improved glycaemic status, supporting that long-acting amylin analogues may provide future valuable therapeutic options to manage diabetes. Further preclinical characterization of the antidiabetes effects of long-term treatment with ZP8396 is ongoing. Disclosure J.Skarbaliene: Employee; Zealand Pharma A/S, Stock/Shareholder; Zealand Pharma A/S. T.Baader-pagler: Employee; Boehringer Ingelheim International GmbH. P.Eriksson: Employee; Zealand Pharma A/S.

215-LB: Silencing of Fructose 1,6-Bisphosphatase (FBP1) in Liver Improves Glucose Homeostasis in Insulin-Resistant Rodent and Human Models

Insulin resistant individuals display elevated fasting and post-prandial glucose levels which are mainly driven by inadequate inhibition of gluconeogenesis (GNG) and are rarely normalized. The aim of this study was to investigate whether direct inhibition of GNG could be a new therapeutical approach to improve glucose homeostasis using in vitro and in vivo models of insulin resistance. Fructose 1,6-bisphosphatase (FBP1) is key in controlling GNG in liver and kidney and loss of function patients display hypoglycaemia episodes and lactate acidosis. Systemic FBP1 inhibition using small molecules has shown to improve glucose homeostasis. To assess the clinical efficacy and safety potential of silencing FBP1 selectively in the liver we dosed insulin resistant DIO rats with a hepatocyte specific GalXC-FBP1 siRNA entity. GalXC-FBP1 siRNA markedly reduced FBP1 mRNA level in the liver by over 90%. This was associated with a complete lack of glucose excursion following a pyruvate challenge indicating inhibition of GNG. Blood glucose level decreased similarly in rats treated with GalXC-FBP1 siRNA or vehicle during an insulin challenge or a prolonged fast suggesting that hepatic FBP1 silencing is not inducing hypoglycaemia. Insulin sensitivity and hyperinsulinemia were improved. Plasma lactate and liver enzymes were not elevated, but a significant 2-fold increase in liver triglycerides was observed in the GalXC-FBP1 siRNA group. Using human hepatocytes in a Liver-on-Chip in vitro model FBP1 silencing reduced glucose production by 30% supporting the relevance of this approach in humans. Collectively these data suggest that liver specific FBP1 silencing has the potential to improve insulin sensitivity in DIO rats without inducing hypoglycaemia but may be associated with a risk of liver steatosis overtime. This concept highlights the difficulty of blocking liver GNG without re-directing the metabolic intermediates fluxes toward triglycerides accumulation in liver. Disclosure C. Fledelius: Employee; Novo Nordisk A/S. D. Demozay: Employee; Novo Nordisk A/S. H. Iversen: None. R. S. Ingvorsen: None. L. B. Eriksen: None. A. Blois: Employee; Novo Nordisk. Y. Montauban: None. R. Rijnbrand: Employee; Novo Nordisk. W. Han: None. J. F. Jeppesen: Employee; Novo Nordisk A/S.

Obesity-Related Brain Cholinergic System Impairment in High-Fat-Diet-Fed Rats.

A link between obesity and cerebral health is receiving growing recognition. Here, we investigate in the frontal cortex and hippocampus the potential involvement of cholinergic markers in brain alterations previously reported in rats with obesity induced by diet (DIO) after long-term exposure (17 weeks) to a high-fat diet (HFD) in comparison with animals fed with a standard diet (CHOW). The obesity developed after 5 weeks of HFD. Bodyweight, systolic blood pressure, glycemia, and insulin levels were increased in DIO rats compared to the CHOW group. Measurements of malondialdehyde (MDA) provided lipid peroxidation in HFD-fed rats. Western blot and immunohistochemical techniques were performed. Our results showed a higher expression of choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT) in obese rats but not the VAChT expression in the frontal cortex after 17 weeks of HFD. Furthermore, the acetylcholinesterase (AChE) enzyme was downregulated in HFD both in the frontal cortex and hippocampus. In the brain regions analyzed, it was reported a modulation of certain cholinergic receptors expressed pre- and post-synaptically (alpha7 nicotinic receptor and muscarinic receptor subtype 1). Collectively, these findings point out precise changes of cholinergic markers that can be targeted to prevent cerebral injuries related to obesity.

Diet-induced- and genetic-obesity differentially alters male germline histones.

Obesity, an established risk factor for male subfertility or infertility, is primarily due to genetic and environmental causes. Our earlier studies have shown differential effects of high-fat diet-induced- (DIO) and genetically inherited- (GIO) obesity on DNA methylation in male germline and its subsequent effect on fertility. Here, we hypothesized that the effects of DIO and GIO on histone modifications in male germline could also contribute to fertility defects. We observed that DIO affected both active (H3K4me3, H3ac, and H4ac) and repressive (H3K9me3 and H3K27me3) histone marks in testis and their cell types, whereas GIO solely altered acetylated histones. This correlated with the deregulation of histone-modifying enzymes in the testis of both obese groups. Further, we also observed a decrease in chromatin remodelers in the testis of the DIO group, which were increased in the GIO group. Besides, there was an increase in core histones and a decrease in histone marks along with protamine deficiency in spermatozoa of the DIO group, whereas only H3K4me3 levels were increased in spermatozoa of the GIO group. Moreover, we observed alterations in the expression and enrichment patterns of a few developmental genes harbored by the active histone mark in resorbed embryos and spermatozoa of DIO rats. Together these epigenetic defects in the male germline could alter sperm quality and cause fertility defects in these obese groups. Differential changes in two obese groups could also be attributed to differences in their pathophysiological variations. Our study highlights epigenetic differences between DIO and GIO in the male germline and their subsequent impact on male fertility.

Potential of Hibiscus sabdariffa Linn. in managing FGF21 resistance in diet-induced-obesity rats via miR-34a regulation.

BackgroundObesity is a cause of FGF21 resistance, which affects the browning and thermogenesis process of the adipose tissue. Decreased receptor expression is influenced by miR‐34a, whose expression is increased in obesity. While FGF21‐based therapies have been widely investigated, the potential activity of Hibiscus sabdariffa Linn. extract (HSE) against FGF21 resistance is unknown.ObjectiveThis study aims to determine the effects of HSE on the expression of miR‐34a and FGF21 receptors in white adipose tissue.MethodsThis experimental study used 24 male Sprague‐Dawley rats and divided into four groups: Control (N); diet‐induced‐obesity rats (DIO); DIO rats with HSE 200 mg/kgBW/day and DIO rats with HSE 400 mg/kgBW/day. Rats were fed a high‐fat diet for 17 weeks. HSE was administered daily for 5 weeks. The administration of HSE 400 mg/kgBW/day resulted in the equivalent expression of miR‐34a to that of the control (p > 0.05).ResultsFGFR1 receptor expression was also similar to controls (p > 0.05). Beta‐klotho expression was significantly lower than that of control (p < 0.05) but equivalent to that of DIO rats (p < 0.05).Conclusions H. sabdariffa has the potential to reduce FGF21 resistance in DIO rats through the suppression of miR‐34a expression and an increase in the number of FGFR1 and beta‐klotho receptors in adipose tissue.

Chronic hindbrain administration of oxytocin elicits weight loss in male diet-induced obese mice.

Previous studies indicate that oxytocin (OT) administration reduces body weight in high-fat diet (HFD)-induced obese (DIO) rodents through both reductions in food intake and increases in energy expenditure. We recently demonstrated that chronic hindbrain [fourth ventricular (4V)] infusions of OT evoke weight loss in DIO rats. Based on these findings, we hypothesized that chronic 4V OT would elicit weight loss in DIO mice. We assessed the effects of 4V infusions of OT (16 nmol/day) or vehicle over 28 days on body weight, food intake, and body composition. OT reduced body weight by approximately 4.5% ± 1.4% in DIO mice relative to OT pretreatment body weight (P < 0.05). These effects were associated with reduced adiposity and adipocyte size [inguinal white adipose tissue (IWAT)] (P < 0.05) and attributed, in part, to reduced energy intake (P < 0.05) at a dose that did not increase kaolin intake (P = NS). OT tended to increase uncoupling protein-1 expression in IWAT (0.05 < P < 0.1) suggesting that OT stimulates browning of WAT. To assess OT-elicited changes in brown adipose tissue (BAT) thermogenesis, we examined the effects of 4V OT on interscapular BAT temperature (TIBAT). 4V OT (1 µg) elevated TIBAT at 0.75 (P = 0.08), 1, and 1.25 h (P < 0.05) postinjection; a higher dose (5 µg) elevated TIBAT at 0.75-, 1-, 1.25-, 1.5-, 1.75- (P < 0.05), and 2-h (0.05 < P < 0.1) postinjection. Together, these findings support the hypothesis that chronic hindbrain OT treatment evokes sustained weight loss in DIO mice by reducing energy intake and increasing BAT thermogenesis at a dose that is not associated with evidence of visceral illness.

Methylation of Hypothalamic Tsc1-mTOR Signaling in Regulation of Obesity and Obesity Resistance.

The Tsc1-mTOR signaling pathway is often related to obesity, and epigenetic modification may lead to expression changes of obesity-related gene. Therefore, we aim to investigate the methylation of the Tsc1-mTOR signaling pathway in regulation of obesity susceptibility. Wistar rats were fed a normal diet or a high-fat diet to develop animal models. Protein and mRNA expression levels of Tsc1-mTOR signaling in the hypothalamus were determined by Western blot and quantitative real-time PCR. Methylation of Tsc1 gene promoter was detected by bisulfite genomic sequence. Both mRNA and protein expression levels of Tsc1 in DIO group hypothalamus were lower; mTOR and its downstream targets S6K1, 4EBP1, and S6 protein expression levels were higher than those of the DIO-R group and the chow group. The Tsc1 gene promoter methylation rate in the hypothalamus was 92.05 ± 3.07% in the DIO group, 87.27 ± 1.91% in the DIO-R group, and 88.18% ± 3.20% in the chow group, respectively, with significantly higher levels in the DIO group. Both the expression levels of Tsc1 gene promoter methylation and Tsc1-mTOR signaling pathway in the hypothalamus of DIO rats and DIO-R rats are different. These findings may shed light on the potential mechanism for the differentiation of obesity susceptibility.

Chronic intermittent hypobaric hypoxia improves markers of iron metabolism in a model of dietary-induced obesity

BackgroundObesity, a risk factor for many chronic diseases, is a potential independent risk factor for iron deficiency. Evidence has shown that chronic intermittent hypobaric hypoxia (CIHH) has protective or improved effects on cardiovascular, nervous, metabolic and immune systems. We hypothesized that CIHH may ameliorate the abnormal iron metabolism in obesity. This study was aimed to investigate the effect and the underlying mechanisms of CIHH on iron metabolism in high-fat-high-fructose-induced obese rats.MethodsSix to seven weeks old male Sprague-Dawley rats were fed with different diet for 16 weeks, and according to body weight divided into four groups: control (CON), CIHH (28-day, 6-h daily hypobaric hypoxia treatment simulating an altitude of 5000 m), dietary-induced obesity (DIO; induced by high fat diet and 10% fructose water feeding), and DIO + CIHH groups. The body weight, systolic arterial pressure (SAP), Lee index, fat coefficient, blood lipids, blood routine, iron metabolism parameters, interleukin6 (IL-6) and erythropoietin (Epo) were measured. The morphological changes of the liver, kidney and spleen were examined. Additionally, hepcidin mRNA expression in liver was analyzed.ResultsThe DIO rats displayed obesity, increased SAP, lipids metabolism disorders, damaged morphology of liver, kidney and spleen, disturbed iron metabolism, increased IL-6 level and hepcidin mRNA expression, and decreased Epo compared to CON rats. But all the aforementioned abnormalities in DIO rats were improved in DIO + CIHH rats.ConclusionsCIHH improves iron metabolism disorder in obese rats possibly through the down-regulation of hepcidin by decreasing IL-6 and increasing Epo.

Metformin effectively restores the HPA axis function in diet-induced obese rats.

IntroductionThe hypothalamo-pituitary-adrenal (HPA) axis is perturbed in obesity. We previously reported presence of leptin resistance in the brainstem and uncoupling between central noradrenergic tone and the HPA axis in obesity-prone (DIO) rats. Metformin is shown to lower body weight and adiposity, but the underlying mechanism is unclear. We hypothesized that this is associated with restored HPA axis function.MethodsAdult male DIO rats were placed on either a regular chow or HF diet for 7 weeks. Starting week 4, the animals were given either a low dose (60mg/kg) or high dose (300mg/kg) of metformin in drinking water. In addition to body weight and feeding, we examined different arms of the HPA axis to test if metformin can reinstate its function and coupling. To understand potential mechanisms, leptin signaling in the brainstem and circulating free fatty acid levels were also assessed.ResultsMetformin treatment lowered weight gain, fat mass, caloric intake, and serum leptin levels. HPA axis activity as determined by corticotropin-releasing hormone in the median eminence and serum corticosterone was decreased by metformin in a dose-dependent manner, and so was norepinephrine (NE) in the paraventricular nucleus. Importantly, metformin completely normalized the NE-HPA axis uncoupling. While brainstem pSTAT-3 and SOCS-3, key markers of leptin signaling, were not different between groups, circulating saturated and unsaturated free fatty acids were reduced in HF-fed, metformin-treated animals.ConclusionsThese findings suggest that oral metformin can successfully correct HPA axis dysfunction that is associated with lowered circulating free fatty acids in DIO rats, thereby uncovering a novel effect of metformin in the treatment of obesity.

Effects of combined oxytocin and beta‐3 receptor agonist (CL 316243) treatment on body weight and adiposity in high fat diet‐induced obese rats

Previous studies have indicated that oxytocin (OXT) reduces body weight in high fat diet (HFD)‐induced obese (DIO) rodents, nonhuman primates and obese humans through reductions in energy intake and increases in energy expenditure. Recent findings support a role for brown adipose tissue (BAT) thermogenesis in OXT‐elicited weight loss as chronic administration 1) increases interscapular brown adipose tissue (IBAT) temperature (TIBAT) in DIO rats during a period that coincides with the OXT’s initial weight‐lowering effect and 2) upregulates beta‐3 receptor mRNA expression in IBAT. Based on these findings, we hypothesized that OXT would enhance the effectiveness of the beta‐3 receptor agonist, CL 316243, to evoke weight loss in DIO rats. To test this hypothesis, rats were fed a HFD for approximately 7 months prior to being implanted with a fourth ventricular (4V) cannula (as a strategy to target hindbrain OXT receptors), 28‐day minipump that infused OXT (16 nmol/day) or vehicle (VEH; saline) and temperature transponder underneath an IBAT depot. DIO rats were subsequently matched for body weight and adiposity, as well as OXT‐elicited reductions in body weight during the first week of the infusion period, prior to receiving 1x daily injections of VEH (sterile water, IP) or CL 316243 at a dose (0.5 mg/kg, IP) found to stimulate TIBAT in a separate group of DIO rats (P&lt;0.05; N=4–5/group). Daily energy intake, TIBAT and body weight were tracked for 28 days. Body composition was assessed immediately prior to VEH or OXT treatment and near treatment completion using Quantitative Magnetic Resonance (QMR; EchoMRI 4‐in‐1). Preliminary data indicate that OXT (16 nmol/day) alone and CL 316243 alone tended to reduce body weight by approximately 6.5±2.2% (0.05&lt;P&lt;0.1) and 4.7±0.9% (P&lt;0.05), respectively (N=4–5/group), relative to pre‐infusion baseline. However, the combination of OXT and CL 316243 produced a more pronounced reduction in body weight (13.9±1.6%; P&lt;0.05) compared with either treatment alone (N=4–5/group). Weight loss in response to the combination treatment (OXT and CL 316243) was the result of a sustained reduction of body fat adiposity stores (P&lt;0.05) without any change in lean mass (P=NS). These effects were associated with reductions in energy intake (P&lt;0.05) suggesting that decreased energy intake contributes, in part, to the effectiveness of OXT and CL 316243 to reduce body weight. Furthermore, repeated 1x daily administration of CL 316243 alone (0.05&lt;P&lt;0.1), and in combination with OXT (P=0.1), tended to elevate TIBAT at 0.5‐h post‐injection relative to vehicle treatment throughout the entire treatment period (N=4–5/group). Together, these findings support the hypothesis that the combination of OXT and a beta‐3 agonist produces an additive effect to evoke weight loss and body adiposity in DIO rats by reducing energy intake and increasing BAT thermogenesis.Support or Funding InformationNational Institutes of Health R01 DK115976 and VA Merit Review Award BX004102

Chronic hindbrain administration of oxytocin elicits weight loss in high fat diet‐induced obese mice

Previous studies indicate that oxytocin (OXT) reduces body weight (BW) in high fat diet (HFD)‐induced obese (DIO) rodents through mechanisms that may involve an enhanced ability of OXT to reduce food intake as well as increase energy expenditure. We have previously demonstrated that chronic fourth ventricular (4V) infusions of OXT (as a strategy to target hindbrain OXT receptors) evokes weight loss in DIO rats. Based on these findings, we hypothesized that increased hindbrain OXT signaling would elicit BW loss in DIO mice maintained on a HFD (60% kcal from fat). To test this hypothesis, age‐matched mice were fed HFD or chow for 4 months prior to implantation of 4V cannulas and 28‐day minipumps that infused OXT (16 nmol/day) or vehicle (VEH; saline). Food intake and BW were tracked for 28 days. Body composition was assessed immediately prior to VEH or OXT treatment and near treatment completion using Quantitative Magnetic Resonance (QMR; EchoMRI 4‐in‐1). OXT preferentially reduced BW by approximately 4.5±1.4% in DIO mice relative to chow‐fed mice (P&lt;0.05; N=9–11/group). OXT‐elicited BW loss was associated with reductions in body adiposity, adipocyte size [inguinal white adipose tissue (IWAT)] and serum leptin (P&lt;0.05). These effects were attributed, in part, to an enhanced effect of OXT to reduce energy intake in DIO mice (P&lt;0.05) at a dose that was not associated with increased kaolin intake (pica behavior; P=NS). While reductions in energy intake contribute to OXT’s effects on BW, chronic 4V OXT appeared to increase uncoupling protein‐1 (UCP‐1) expression in IWAT (0.05&lt;P&lt;0.1; N=9–10/group) suggesting that OXT may also stimulate beiging of WAT. To assess OXT‐elicited changes in brown adipose tissue (BAT) thermogenesis, we examined the effects of acute 4V injections of OXT on interscapular BAT temperature (TIBAT) in mice. Consistent with our previous findings in rats, preliminary data in mice indicate that acute 4V injections of a lower dose (1 μg) appeared to elevate TIBAT at 0.5‐ (P=0.1) and 1.25‐h post‐injection (P=0.15) while a higher dose (5 μg) appeared to elevate TIBAT at 1.5‐ (P&lt;0.05) and 4‐h (0.05&lt;P&lt;0.1) post‐injection (N=4/group). Together, these findings support the hypothesis that chronic hindbrain OXT treatment evokes sustained weight loss in DIO mice by reducing energy intake and increasing BAT thermogenesis and beiging of WAT at a dose that is not associated with visceral illness.Support or Funding InformationNational Institutes of Health R01 DK115976 and VA Merit Review Award BX004102

Liraglutide Lowers Body Weight Set Point in DIO Rats and its Relationship with Hypothalamic Microglia Activation.

This study aimed to investigate the effects of liraglutide on thebody weight set point (BWSP) in diet-induced obese rats and to determine the relationship between BWSP and hypothalamic arcuate nucleus (ARC) microglial activation. Diet-induced obesity (DIO) rats were divided into three groups: continuous high-fat diet (HFD) plus saline, HFD with liraglutide, and HFD with liraglutide pair feeding. Body weight, BWSP, inflammatory cytokines, suppressor of cytokine signaling 3, orexigenic/anorexigenic proteins, apoptosis, and microglia in the ARC were assessed. The effect of liraglutide on the Notch-1 signaling pathway and its relationships with nuclear factor-κB and p38 mitogen-activated protein kinasewere also investigated in a lipopolysaccharide (LPS)-induced microglia activation model. Liraglutide reduced BWSP; reversed adverse changes in hypothalamic inflammation, suppressor of cytokine signaling 3, and apoptosis; and diminished microgliosis in DIO rats. The BWSP showed a linear correlation with ARC microglial density. Liraglutide inhibited LPS-induced M1 microglial polarization and promoted microglial polarization to theM2 phenotype, diminishing inflammatory cytokine expression. Liraglutide inhibited Notch-1 signaling pathway activation and decreased nuclear factor-κB and p38 mitogen-activated protein kinase pathway activation in LPS-stimulated microglia. Liraglutide can reduce BWSP in DIO rats. There is a linear correlation between hypothalamic microgliosis and BWSP. Liraglutide reduces excessive microglial activation and inflammation, which may contribute to BWSP reduction.

Taste Responses in the Nucleus of the Solitary Tract of Awake Obese Rats Are Blunted Compared With Those in Lean Rats.

Taste perception changes with obesity but the underlying neural changes remain poorly understood. To address this issue, we recorded taste responses from single cells in the nucleus tractus solitarius (NTS, the first synapse in the central gustatory circuit) in awake, diet-induced obese [(DIO; ≥ 8 weeks on a high-energy diet (45%fat, 17% sugar; HED)], and lean rats. Rats were implanted with a bundle of microelectrodes in the NTS and allowed to recover. Water-deprived rats were allowed to freely lick various tastants in an experimental chamber. Taste stimuli included an array of sapid stimuli dissolved in artificial saliva (AS). Each taste trial consisted of five consecutive licks followed by five AS licks presented on a VR5 schedule. Results showed that taste responses (n = 49 for DIO; n = 74 for lean rats) in NTS cells in DIO rats were smaller in magnitude, shorter in duration, and longer in latency that those in lean rats. However, there were proportionately more taste-responsive cells in DIO than in lean rats. Lick coherence in DIO rats was significantly lower than in lean rats, both in taste-responsive, and lick-related cells (n = 172 in lean; n = 65 in DIO). Analyses of temporal coding showed that taste cells in DIO rats conveyed less information about taste quality than cells in lean rats. Collectively, results suggest that a HED produces blunted, but more prevalent, responses to taste in the NTS, and a weakened association of taste responses with ingestive behavior. These neural adaptations may represent both negative effects and compensatory mechanisms of a HED that may underlie deficits in taste-related behavior associated with obesity.

108-LB: Sustained Glucagon Effect on Blood Glucose and Improvements of Insulin Resistance Mediated by a Novel Long-Acting Glucagon Analog, HM15136, in Animal Models

HM15136 is a novel long-acting glucagon (GCG) analog conjugated with human IgG FC fragment via a short PEG linker. Previously, HM15136 treatment led to body weight loss (BWL) in obese animal models while showing transient effect on blood glucose (BG). Insulin resistance (IR) can lead to imbalances in BG homeostasis in obesity, representing a key risk factor for T2DM. Considering the potential benefit of BWL in IR improvement, HM15136 could be a novel therapeutic option for obesity treatment. To assess this concept, the present study evaluated the effect of HM15136 on BG as well as BW, and investigated the mechanism leading to IR improvement in vitro and in vivo. In DIO rats, 4 weeks treatment of HM15136 led to remarkable BWL (-17 ∼ -42% vs. Veh). BG monitoring showed that an initial BG elevation was rapidly normalized and maintained normal thereafter, with no HbA1c increase (3.4 and 3.7% for Veh and HM15136). Similar results were also observed in DIO mice, showing no abnormal glycemic control by HM15136. Interestingly, BWL effect were correlated with free-fatty acid (FFA) and HOMA-IR reduction. In vitro studies in 3T3-L1 adipocytes further confirmed the deteriorating effect of FFA on insulin signaling. These results suggested that improvements in the lipotoxic milieu explains, at least in part, the IR improvement seen with HM15136. To further explore additional mechanism, inflammatory markers and adipokines affecting insulin signaling were evaluated. HM15136 not only inhibited F4/80 expression, but also increased FGF-21 and adiponectin expression in white adipose tissue and 3T3-L1 adipocytes. Our results demonstrated that BWL under by HM15136 therapy is accompanied by the reduction of 1) lipotoxicity, 2) inflammatory biomarkers, and the expression of 3) insulin-sensitizing adipokines, eventually leading to IR reversal and normal BG maintenance. Human studies are needed to confirm whether these findings will translate into humans. Disclosure J. Choi: None. S. Lee: None. J. Kim: None. J. Lee: None. Y. Kim: None. I. Choi: Employee; Self; Hanmi Pharm. Co., Ltd.

991-P: Potential of a Novel Long-Acting Glucagon Analog, HM15136, for the Treatment of Obesity

Obesity is considered as a main risk factor for metabolic syndrome. Although there are several anti-obesity drugs, their main mode of action (MoA) is appetite suppression. Thus, body weight loss (BWL) efficacy is limited with benefits fading when the drugs stopped. Glucagon (GCG) has been used to treat hypoglycemia. Recent studies indicated a stimulatory effect of GCG on energy expenditure, suggesting its utilization as an anti-obese drug. Since BWL is a key for insulin resistance (IR) reversal and blood glucose (BG) normalization, we hypothesized that sustained GCG treatment may reduce hyperglycemic risk via IR improvement following efficient BWL. To investigate this hypothesis, we developed a novel long-acting GCG analog, HM15136, and evaluated the effect of HM15136 on BW and BG in vivo. In obese animals, HM15136 demonstrated dose-dependent BWL (-21 ∼ -53% vs. Veh. for DIO mice, -10 ∼ -64% vs. Veh. for DIO rats). Of note, despite an initial increase, BG level rapidly normalized. As to the responsible MoA, a reduced blood lipid and HOMA-IR were observed. In vitro studies demonstrated the increased FGF-21 and adiponectin expression by HM15136 in 3T3-L1 adipocytes. These results suggest that HM15136 could improve insulin sensitivity via multiple mechanisms, leading to normal BG maintenance under chronic GCG receptor stimulation. To further investigate the therapeutic potential of HM15136, BWL efficacy was compared to liraglutide: Greater BWL was observed with HM15136 (-34% and -21% vs. Veh. for HM15136 and liraglutide, respectively). Indirect calorimetry demonstrated enhanced energy expenditure by HM15136. Most interestingly, efficient BWL achieved by HM15136 was maintained during 4 weeks drug-holiday, compared to liraglutide. In conclusion, our results demonstrate the potential for prolonged GCG receptor stimulation with agent like HM15136 as a novel therapeutic option for obesity treatment. Human studies are needed to confirm these findings and to evaluate the potential safety of HM15136. Disclosure J. Kim: None. J. Kim: None. S. Lee: None. J. Lee: None. S. Lee: Employee; Self; Hanmi Pharm. Co., Ltd. I. Choi: Employee; Self; Hanmi Pharm. Co., Ltd.

Antiobesity Activity of Flavanoids Isolated from Solanum macrocarpum in Wistar Rats

Aims: To correlate obesity/atherosclerosis with body mass index, serum cholesterol, serum triglyceride, serum low density lipoprotein and serum high density lipoprotein of diet induced obese wistar rats.&#x0D; Study Design: Department of Home Science, Nutrition and Dietetics (Animal research house) and Department of veterinary science both in University of Nigeria Nsukka. The study was conducted between January to March 2012.&#x0D; Methodology: Four groups of twenty male Wistar rats were fed a highly palatable diet for 2 weeks to induce obesity resembling mild obesity condition in human population after one week acclimatization period. DIO rats received rat chow and flavonoids extract daily for 6 weeks. Group 1 received rat chow alone; Group 2- 0.05% of flavonoids extract and rat chow; Group 3- 0.15% of flavonoid extract and rat chow; and Group 4- 0.25% of flavonoid extract and rat chow. BMI, Total cholesterol, HDL, LDL and Triglyceride were evaluated using standard assay technique. The data were statistically analyzed using ANOVA and mean separated using LSD.&#x0D; Results: Feeding the rats with palatable diet showed increased in BMI (from 0.35-0.40 to 0.60-0.65), total cholesterol, LDL and triglyceride levels along with decrease in HDL (p&lt;0.05). Consumption of flavonoids resulted in the significant reduction in BMI, LDL, total cholesterol and triglyceride level and exhibit significant elevation in HDL cholesterol compared to the rats fed only rat chow (p&lt;0.05). It was observed that the decrease in BMI, Triglyceride, total cholesterol and LDL cholesterol level of rats fed 0.25% of flavonoids were significantly different (p&lt;0.05) from those fed 0.15% and 0.05% flavonoids.&#x0D; Conclusions: The results suggest that flavonoids extract from Solanum macranthum has atherogenic effect which can help to reduce obesity.