High-fat Diet Rabbits Research Articles

The Anti-Inflammatory Effect of Lactococcus lactis-Ling-Zhi 8 on Ameliorating Atherosclerosis and Nonalcoholic Fatty Liver in High-Fat Diet Rabbits

Inflammation plays a crucial role in atherosclerosis and nonalcoholic fatty liver disease (NAFLD). We previously engineered a recombinant Lactococcus lactis strain expressing the Ling-Zhi immunomodulatory protein (L. lactis-LZ8). This study investigated the anti-atherosclerotic effects of L. lactis-LZ8 in rabbits fed a high-fat diet (HFD). Changes in body weight, serum lipid profiles, and liver function were monitored. The aorta and liver tissues were analyzed for gross pathology and histopathology. Eight-week administration of L. lactis-LZ8 with HFD ameliorated atherosclerosis by downregulating protein and gene expression associated with lipid metabolism and inflammation in the aortas. The rabbits receiving L. lactis-LZ8 exhibited a significant dose-dependent reduction in hepatic fat accumulation. RNA sequencing of the livers revealed that inflammatory genes in the L. lactis-LZ8 groups were downregulated compared to the HFD group. Disease ontology enrichment analysis indicated that these genes were involved in atherosclerosis. Gene set enrichment analysis plots revealed significant enrichment in the gene sets related to cholesterol homeostasis. CIBERSORT immune cell fraction analysis indicated significant infiltration by regulatory T cells, CD8+ T cells, activated dendritic cells, and natural killer cells in the L. lactis-LZ8 group. Our studies underscore LZ8’s role in precision nutrition, providing a potential solution to the current challenges in modifying atherosclerosis and NAFLD.

Untargeted Metabolomics Analysis Revealed Lipometabolic Disorders in Perirenal Adipose Tissue of Rabbits Subject to a High-Fat Diet.

Simply SummaryA high-fat diet is widely recognized as a significant modifiable risk for metabolic diseases. In this study, untargeted metabolomics, combined with liquid chromatography and high-resolution mass spectrometry, was used to evaluate perirenal adipose tissue metabolic changes. Our study revealed 206 differential metabolites. These metabolites were mainly associated with the biosynthesis of unsaturated fatty acids, the arachidonic acid metabolic pathway, the ovarian steroidogenesis pathway, and the platelet activation pathway. Our study revealed that a high-fat diet causes significant lipometabolic disorders; these metabolites may inhibit oxygen respiration by increasing adipocytes cells and density, cause mitochondrial and endoplasmic reticulum dysfunction, produce inflammation, and finally lead to insulin resistance, thereby increasing the risk of Type 2 diabetes, atherosclerosis, and other metabolic syndromes.A high-fat diet (HFD) is widely recognized as a significant modifiable risk for insulin resistance, inflammation, Type 2 diabetes, atherosclerosis and other metabolic diseases. However, the biological mechanism responsible for key metabolic disorders in the PAT of rabbits subject to HFD remains unclear. Here, untargeted metabolomics (LC-MS/MS) combined with liquid chromatography (LC) and high-resolution mass spectrometry (MS) were used to evaluate PAT metabolic changes. Histological observations showed that the adipocytes cells and density of PAT were significantly increased in HFD rabbits. Our study revealed 206 differential metabolites (21 up-regulated and 185 down-regulated); 47 differential metabolites (13 up-regulated and 34 down-regulated), comprising mainly phospholipids, fatty acids, steroid hormones and amino acids, were chosen as potential biomarkers to help explain metabolic disorders caused by HFD. These metabolites were mainly associated with the biosynthesis of unsaturated fatty acids, the arachidonic acid metabolic pathway, the ovarian steroidogenesis pathway, and the platelet activation pathway. Our study revealed that a HFD caused significant lipometabolic disorders. These metabolites may inhibit oxygen respiration by increasing the adipocytes cells and density, cause mitochondrial and endoplasmic reticulum dysfunction, produce inflammation, and finally lead to insulin resistance, thus increasing the risk of Type 2 diabetes, atherosclerosis, and other metabolic syndromes.

Growth, behavioural, serum biochemical and morphological changes in female rabbits fed high-fat diet.

This study aimed to determine whether high-fat diet (HFD) could cause growth, behavioural, biochemical and morphological changes in young female rabbits. Thirty-six female rabbits were randomly divided into two groups fed with either a high-fat diet (HFD) or a standard normal diet (SND) for 5weeks. Growth and behavioural changes were recorded during the 5-week feeding period. Tissue samples, including blood and adipose tissue, were obtained after slaughter. HFD rabbits weighed more by the end of the feeding period, had a higher percent body weight and adipose tissue weight change and had longer body and bust lengths than SND rabbits. HFD rabbits significantly reduced their feed intake and feeding frequency during the fourth and fifth weeks. HFD rabbits also showed lower frequency of drinking and resting and increased stereotypical behaviour. Besides, HFD rabbits showed significant physiological abnormalities. HFD rabbits had higher serum cholesterol (TC) and triglycerides (TG) levels than SND rabbits at the end of the feeding period, and higher free fatty acid (FFA) levels than rabbits in the SND group after the third week of feeding. Serum thyroxine (T4) increased significantly in week 2 and week 5 and triiodothyronine (T3) increased significantly in week four. However, there was no significant change in serum glucose (GLU) and insulin (INS) levels. Additionally, HFD reduced the area and diameter of perirenal and subcutaneous fat cells and increased their density. Our findings suggest that HFD rabbits had higher weight gains, accumulation of fat, and more behavioural changes than SND rabbits. Although high levels of fat in the diet had a low impact on hyperglycaemia, it could lead to hyperlipidemia and hyperthyroidism. Our results also suggest that sustained HFD may cause the proliferation of adipocytes in young female rabbits.

Modulation of the Inflammatory Process by Hypercholesterolemia in Osteoarthritis.

Objective: Several studies have linked metabolic syndrome to the development of osteoarthritis (OA) through hypercholesterolemia, one of its components. However, epidemiological studies showed contradictory results, and it is not clear how hypercholesterolemia itself, or oxidized LDL (oxLDL)—a pathological molecule potentially involved in this relationship—could be affecting OA. The objectives of this study were to investigate the effect of hypercholesterolemia induced by high-fat diet (HFD) in cartilage from OA rabbits, and how oxLDL affect human chondrocyte inflammatory and catabolic responses.Design: New Zealand rabbits were fed with HFD for 18 weeks. On week 6, OA was surgically induced. At the end of the study, cartilage damage and IL-1β, IL-6, MCP-1, MMP-13, and COX-2 expression in articular cartilage were evaluated. In addition, cultured human OA articular chondrocytes were treated with oxLDL at concentrations equivalent to those expected in synovial fluid from HFD rabbits, in the presence of IL-1β and TNFα. The effect of oxLDL on cell viability, nitric oxide production and catabolic and pro-inflammatory gene expression was evaluated.Results: HFD intake did not modify cartilage structure or pro-inflammatory and catabolic gene expression and protein presence, both in healthy and OA animals. OxLDL did not affect human chondrocyte viability, ADAMTS5 and liver X receptor (LXR) α gene expression, but decreased the induction of IL-1β, IL-6, MCP-1, MMP-13, iNOS, and COX-2 gene expression and MMP-13 and COX-2 protein presence, evoked by cytokines.Conclusions: Our data suggest that cholesterol intake per se may not be deleterious for articular cartilage. Instead, cholesterol de novo synthesis and altered cholesterol metabolism could be involved in the associations observed in human disease.

Testosterone improves muscle fiber asset and exercise performance in a metabolic syndrome model.

Lifestyle modifications, including physical exercise (PhyEx), are well-known treatments for metabolic syndrome (MetS), a cluster of metabolic and cardiovascular risk factors often associated to hypogonadism. Given the trophic role of testosterone on skeletal muscle (SkM), this study was aimed at evaluating the effects of testosterone treatment on SkM metabolism and exercise performance in male rabbits with high-fat diet (HFD)-induced MetS. HFD rabbits, treated or not with testosterone (30 mg/kg/week) for 12 weeks, were compared to regular diet animals (RD). A subset of each group was exercise-trained for 12 weeks. HFD increased type-II (fast, glycolytic) and decreased type-I (slow, oxidative) muscle fibers compared to RD as evaluated by RT-PCR and histochemistry. Testosterone reverted these effects, also inducing the expression of mitochondrial respiration enzymes and normalizing HFD-induced mitochondrial cristae reduction. Moreover, testosterone significantly increased the expression of myogenic/differentiation markers and genes related to glucidic/lipid metabolism. At the end of the PhyEx protocol, when compared to RD, HFD rabbits showed a significant reduction of running distance and running time, while testosterone counteracted this effect, also decreasing lactate production. In the trained groups, muscle histology showed a significant reduction of oxidative fibers in HFD compared to RD and the positive effect of testosterone in maintaining oxidative metabolism, as also demonstrated by analyzing mitochondrial ultrastructure, succinate dehydrogenase activity and ATP production. Our results indicate that testosterone could be useful to promote oxidative muscle metabolism altered by MetS, thus improving exercise performance. Conversely, testosterone administration to otherwise eugonadal rabbits (RD) only increased muscle fiber diameter but not endurance performance.

PS-04-010 Effects of physical exercise on metabolic syndrome-associated hypogonadotropic hypogonadism and erectile dysfunction

Metabolic syndrome (MetS) clusters cardiovascular and metabolic risk factors, along with hypogonadism (HG) and erectile dysfunction. Lifestyle modifications, including physical exercise (PhyEx), are well-known treatments for this condition. We now report studies on the effect of PhyEx on hypothalamus-pituitary-testis axis and penile relaxation. In this study, we used a non-genomic animal MetS model, previously established in rabbits fed a high fat diet (HFD). Rabbits fed a regular diet (RD) were used as controls. A subset of both groups was trained on a treadmill. In vitro contractility studies were performed to evaluate responsiveness to acetylcholine and electrical field stimulation in corpora cavernosa (CC) strips. Tissue specimens of CC, testis and hypothalamus were obtained at sacrifice and studied by immunohistochemistry and RT-qPCR. HFD rabbits showed typical MetS features including HG (reduced testosterone, T, and LH) and impairment of erectile function. PhyEx in HFD rabbits completely restored plasma T and LH and the penile alterations. At testicular and hypothalamic levels, an HFD-induced inflammatory status was accompanied by reduced T synthesis and GnRH immunopositivity, respectively. In the testis, PhyEx normalized HFD-related macrophage infiltration and pro-inflammatory markers expression, while increasing the expression of steroidogenic enzymes and T synthesis. In the hypothalamus, PhyEx normalized HFD-induced gene expression changes related to inflammation and glucose metabolism, restored GnRH expression, particularly doubling mRNA levels, and regulated expression of molecules related to GnRH release (kisspeptin, dynorphin). Concerning MetS components, PhyEx significantly reduced circulating cholesterol and visceral fat. In multivariate analyses, cholesterol levels resulted the main determinant for MetS-related alterations in penile, testicular and hypothalamic districts.

PO-01-049 Testosterone counteracts metabolic syndrome-related changes in skeletal muscle fiber metabolism and improves exercise performance in the rabbit

Lifestyle modifications, including physical exercise (PhyEx) are well-known treatments for metabolic syndrome (MetS), a cluster of metabolic and cardiovascular risk factors often associated to hypogonadism. Given the trophic role of testosterone (T) on skeletal muscle (SkM) mass, this study was aimed at evaluating the effects of T treatment on SkM metabolism and exercise performance in male rabbits with high fat diet (HFD)-induced MetS. HFD rabbits, treated or not with T (HFD+T; 30 mg/kg/week) for 12 weeks were compared to regular diet animals (RD). A subset of each group were exercise-trained on a treadmill for 12 weeks. SkM samples were collected for subsequent analyses. HFD determined an increased gene expression of type-II (fast, glycolytic) and a significant decrease of type-I (slow, oxidative) muscle fiber markers, as compared to RD group. T reverted these effects, also inducing the expression of mitochondrial respiration chain enzymes and normalizing HFD-induced mitochondrial cristae reduction. Fiber typing by PAS-staining in SkM cross-sections confirmed a shift from type-I to type-II fibers in HFD, which was normalized by T. Moreover, T significantly increased the expression of both myogenic and muscle metabolism (insulin-dependent signaling, lipid turnover) markers. Preliminary Results in rabbit SkM satellite cells confirmed the positive effect of T on myogenesis. At the end of the PhyEx protocol, when compared to RD, HFD rabbits showed a significant reduction of both running distance and running time, while T counteracted this effect, also decreasing the lactate production. Muscle histology evidenced a further reduction of type-I fibers in HFD compared to RD and the positive effect of T in maintaining oxidative metabolism, as also demonstrated by mitochondrial ultrastructure analysis.

Comparative hypolipidemic efficacy of homeopathic mother tincture Allium Sativa Q, Curcuma Longa Q and statin in normal and cholesterol fed rabbits

Although, lipid lowering drugs regulate the blood serum lipids, they have several reported side effects. Therefore, a substance that is less toxic and yet effective would be beneficial. Here, we compared the antihypercholesterolemic effects of combinations of homeopathic mother tincture (HMT) Allium sativa Q (garlic) and Curcuma longa Q (turmeric), with standard lipid lowering drug statin. For experimentation, a total of 20 rabbits were recruited and divided into four groups with five rabbits (n = 5). Normal saline (NS) group served as control; second group served as high fat diet (HFD) group, third group received Allium sativa Q (garlic) and Curcuma longa Q (turmeric) and HFD (T and G group) while the fourth group received statin and HFD (statin group). At the end of the 8th week, blood was collected to analyze the comparative efficacy of Allium sativa Q, Curcuma longa Q and statin on total cholesterol (TC), low density lipoproteins (LDL), high density lipoproteins (HDL) and triglyceride (TG). Various parameters measured (mg/dl) in HFD rabbits were, TC (291.76±2.2), LDL (244.20±8.3) and triglycerides (243.79±4.3) increased significantly and HDL (32.19±3.3) was reduced to an alarming level as compared to the NS group after eight weeks. TC, LDL and triglycerides level reduced significantly in Tand G group while the HDL level increased significantly as compared to HFD. In statin group, TC (148.37±1.2), LDL (76.32 ±3.4) and TG (104.33 ±8.9) decreased while HDL increased (p LESS than 0.05) as compared to the HFD after eight weeks. It was therefore concluded that that homeopathic medicine Allium sativa Q and Curcuma longa Q is equally good like statin and can safely be used to regulate the circulating serum lipid profile

Physical activity counteracts metabolic syndrome-induced hypogonadotropic hypogonadism and erectile dysfunction in the rabbit.

Metabolic syndrome (MetS) clusters cardiovascular and metabolic risk factors along with hypogonadism and erectile dysfunction. Lifestyle modifications including physical exercise (PhyEx) are well-known treatments for this condition. In this study, we analyzed the effect of PhyEx on hypothalamic-pituitary-testis axis and erectile function by use of an animal MetS model, previously established in rabbits fed a high-fat diet (HFD). Rabbits fed a regular diet (RD) were used as controls. A subset of both groups was trained on a treadmill. HFD rabbits showed typical MetS features, including HG (reduced T and LH) and impairment of erectile function. PhyEx in HFD rabbits completely restored plasma T and LH and the penile alterations. At testicular and hypothalamic levels, an HFD-induced inflammatory status was accompanied by reduced T synthesis and gonadotropin-releasing hormone (GnRH) immunopositivity, respectively. In the testis, PhyEx normalized HFD-related macrophage infiltration and increased the expression of steroidogenic enzymes and T synthesis. In the hypothalamus, PhyEx normalized HFD-induced gene expression changes related to inflammation and glucose metabolism, restored GnRH expression, particularly doubling mRNA levels, and regulated expression of molecules related to GnRH release (kisspeptin, dynorphin). Concerning MetS components, PhyEx significantly reduced circulating cholesterol and visceral fat. In multivariate analyses, cholesterol levels resulted as the main factor associated with MetS-related alterations in penile, testicular, and hypothalamic districts. In conclusion, our results show that PhyEx may rescue erectile function, exert anti-inflammatory effects on hypothalamus and testis, and increase LH levels and T production, thus supporting a primary role for lifestyle modification to combat MetS-associated hypogonadism and erectile dysfunction.

High fat diet-induced metabolically obese and normal weight rabbit model shows early vascular dysfunction: mechanisms involved.

Obesity contributes significantly to the development and evolution of cardiovascular disease (CVD) which is believed to be mediated by oxidative stress, inflammation and endothelial dysfunction. However, the vascular health of metabolically obese and normal weight (MONW) individuals is not completely comprehended. The purpose of our study was to evaluate vascular function on the basis of a high fat diet (HFD)-MONW rabbit model. Twenty four male rabbits were randomly assigned to receive either a regular diet (CD, n = 12) or a high-fat diet (18% extra fat on the regular diet, HFD, n = 12) for 6 weeks. Body weight, TBARS and gluthathione serum levels were similar between the groups; fasting glucose, triglycerides, C reactive protein (CRP), visceral adipose tissue (VAT), triglyceride-glucose index (TyG index) were higher in the HFD group. Compared to CD, the HFD rabbits had glucose intolerance and lower HDL-cholesterol and plasma nitrites levels. Thoracic aortic rings from HFD rabbits exhibited: (a) a reduced acetylcholine-induced vasorelaxation; (b) a greater contractile response to norepinephrine and KCl; (c) an improved angiotensin II-sensibility. The HFD-effect on acetylcholine-response was reversed by the cyclooxygenase-2 (COX-2) inhibitor (NS398) and the cyclooxygenase-1 inhibitor (SC560), and the HFD-effect on angiotensin II was reversed by NS398 and the TP receptor blocker (SQ29538). Immunohistochemistry and western blot studies showed COX-2 expression only in arteries from HFD rabbits. Our study shows a positive pro-inflammatory status of HFD-induced MONW characterized by raised COX-2 expression, increase of the CRP levels, reduction of NO release and oxidative stress-controlled conditions in an early stage of metabolic alterations characteristic of metabolic syndrome. Endothelial dysfunction and increased vascular reactivity in MONW individuals may be biomarkers of early vascular injury. Therefore, the metabolic changes induced by HFD even in normal weight individuals may be associated to functional alterations of blood vessels.

OP.8B.05] TRANSGENERATIONAL INHERITANCE OF OBESITY AND HYPERTENSION

Objective: Maternal obesity increases the risk of developing hypertension in the offspring. Circulating leptin is closely linked to the control of sympathetic nerve activity (SNA) and blood pressure (BP) in the central nerve system. The ventromedial hypothalamus (VMH) is a key center of energy homeostasis, hemodynamic and sympathetic tone to renal vasculature. Exposure to overnutrition during early development changes the activity of the neurons and the receptors in the CNS, amplifying sympathetic output which leads to hypertension. We assessed the effects of maternal high fat diet (HFD) feeding during pregnancy on cardiovascular variables and sympathetic nerve activity (SNA) with changes in leptin and insulin signaling pathways in adulthood. Design and method: Breeder rabbits were fed a HFD (13%; HFD) or a control diet (4%; CD) during pregnancy and lactation. Offspring received CD after weaning. All rabbits had a VMH cannula and a renal nerve recording electrode. Mean arterial pressure (MAP), heart rate (HR) and renal sympathetic nerve activity (RSNA) were measured at baseline and after receiving increasing doses of αMSH (α-Melanocortin stimulating hormone, 0.3, 1nmol), SHU9119 (melanocortin receptor antagonist, 0.02, 0.04nmol), leptin receptor antagonist (5, 10 μg) or insulin receptor antagonist (0.01, 0.05U). Hypothalamic expression of leptin receptor (LepR) and Phosphoinositide 3-kinase (PI3K) was examined using real-time PCR. Results: Offspring from HFD fed rabbits (mHFD) showed higher MAP (+11%) and RSNA (+42%) than controls in adulthood (P < 0.05). αMSH into the VMH increased MAP, HR and RSNA (+6.6 ± 1mmHg, +23.4 ± 3.45 b/min and +3.66 ± 0.7nu) and SHU9119 reduced MAP and RSNA (−5.6 ± 0.8mmHg, −1.3 ± 0.13 nu) in mHFD rabbits. Leptin receptor antagonist normalized hypertension (−5.8 ± 0.8mmHg) in mHFD rabbits (P < 0.05). Insulin receptor antagonist administration into the VMH showed no changes in MAP, HR and RSNA in both mHFD and mCD rabbits. Offspring from maternal HFD rabbits exhibited an increase in LepR and PI3K expression in the hypothalamus compared to the control group (+47% and +74%). Conclusions: Exposure to over-nutrition during early development leads to permanently altered leptin and MC signaling pathways, due to programming of the key receptors and/or by neuronal plasticity, leading to sympathoexcitation and hypertension in adulthood.

Anti-atherogenic and anti-lipidemic effects of vanadium in high fat diet rabbits – An histological and biochemical study

Anti-atherogenic and anti-lipidemic effects of vanadium in high fat diet rabbits – An histological and biochemical study

Origin of Aberrant Blood Pressure and Sympathetic Regulation in Diet-Induced Obesity.

High fat diet (HFD)-induced hypertension in rabbits is neurogenic and caused by the central action of leptin, which is thought to be dependent on activation of α-melanocortin-stimulating hormone (α-MSH) and neuropeptide Y-positive neurons projecting to the dorsomedial hypothalamus (DMH) and ventromedial hypothalamus (VMH). However, leptin may act directly in these nuclei. Here, we assessed the contribution of leptin, α-MSH, and neuropeptide Y signaling in the DMH and VMH to diet-induced hypertension. Male New Zealand white rabbits were instrumented with a cannula for drug injections into the DMH or VMH and a renal sympathetic nerve activity (RSNA) electrode. After 3 weeks of an HFD (13.3% fat; n=19), rabbits exhibited higher RSNA, mean arterial pressure (MAP), and heart rate compared with control diet-fed animals (4.2% fat; n=15). Intra-VMH injections of a leptin receptor antagonist or SHU9119, a melanocortin 3/4 receptor antagonist, decreased MAP, heart rate, and RSNA compared with vehicle in HFD rabbits (P<0.05) but not in control diet-fed animals. By contrast, α-MSH or neuropeptide Y injected into the VMH had no effect on MAP but produced sympathoexcitation in HFD rabbits (P<0.05) but not in control diet-fed rabbits. The effects of the leptin antagonist, α-MSH, or neuropeptide Y injections into the DMH on MAP or RSNA of HFD rabbits were not different from those after vehicle injection. α-MSH into the DMH of control diet-fed animals did increase MAP, heart rate, and RSNA. We conclude that the VMH is the likely origin of leptin-mediated sympathoexcitation and α-MSH hypersensitivity that contribute to obesity-related hypertension.

Antihyperlipidemic activity of adenosine triphosphate in rabbits fed a high-fat diet and hyperlipidemic patients

Context Recently, adenosine triphosphate (ATP) was occasionally found to decrease the triglyceride (TG) levels in several hyperlipidemic patients in our clinical practice.Objective The study investigates the anti-hyperlipidemic effects of ATP in a high-fat fed rabbit model and hyperlipidemic patients.Materials and methods Twenty-four rabbits were randomly divided into three groups of eight animals each as follows: normal diet, high-fat diet and high-fat diet + ATP group. ATP supplementation (40 mg/day) was started at the 20th day and lasted for 10 days. Serum concentrations of total cholesterol (TC), TG, LDL-C, HDL-C were measured on the 20th day and 30th day. Heart, liver and aorta were subjected histopathological examination. Twenty outpatients diagnosed primary hyperlipidemia took ATP at a dose of 60 mg twice a day for 1 week.Results Feeding rabbits with a high-fat diet resulted in a significant elevation of lipid parameters including TC, TG, LDL-C, VLDL-C compared to the normal diet group (p < 0.01). ATP treatment significantly decreased serum TG level (p < 0.01), whilst other parameters remained statistically unaltered. Meanwhile, ATP significantly reduced the thickness of fat layer in cardiac epicardium (p < 0.05) and pathological gradation of ballooning degeneration in hepatocytes (p < 0.05). After taking ATP for 1 week, hyperlipidemia patients exhibited a significant decrease of TG (p < 0.01), but other lipid parameters had no significant change.Discussion and conclusion The study indicates that ATP selectively decreases serum TG levels in high-fat diet rabbits and hyperlipidemic patients. Therefore, ATP supplementation may provide an effective approach to control TG level.

Central proopiomelanocortin but not neuropeptide Y mediates sympathoexcitation and hypertension in fat fed conscious rabbits.

High-fat diet (HFD)-induced hypertension in rabbits is neurogenic because of the central sympathoexcitatory actions of leptin. Hypothalamic melanocortin and neuropeptide Y (NPY) neurons are recognized as the major signalling pathways through which leptin exerts its central effects. In this study, we assessed the effects of specific antagonists and agonists to melanocortin and NPY receptors on HFD-induced sympathoexcitation and hypertension. Rabbits were instrumented with intracerebroventricular cannula, renal sympathetic nerve activity (RSNA) electrode, and blood pressure telemetry transmitter. After 3 weeks HFD (13.5% fat, n = 12) conscious rabbits had higher RSNA (+3.8 nu, P = 0.02), blood pressure (+8.6 mmHg, P < 0.001) and heart rate (+15 b/min, P = 0.01), and brain-derived neurotrophic factor levels in the hypothalamus compared with rabbits fed a control diet (4.2% fat, n = 11). Intracerebroventricular administration of the melanocortin receptor antagonist SHU9119 reduced RSNA (-2.7 nu) and blood pressure (-8.5 mmHg) in HFD but not control rabbits, thus reversing 100% of the hypertension and 70% of the sympathoexcitation induced by a HFD. By contrast, blocking central NPY Y1 receptors with BVD10 increased RSNA only in HFD rabbits. Intracerebroventricular α-melanocortin stimulating hormone increased RSNA and heart rate (P < 0.001) in HFD rabbits but had no effect in control rabbits. These findings suggest that obesity-induced hypertension and increased RSNA are dependent on the balance between greater activation of melanocortin signalling through melanocortin receptors and lesser activation of NPY sympathoinhibitory signalling. The amplification of the sympathoexcitatory effects of α-melanocortin stimulating hormone also indicates that the underlying mechanism is related to facilitation of leptin-melanocortin signalling, possibly involving chronic activation of brain-derived neurotrophic factor.

Tadalafil reduces visceral adipose tissue accumulation by promoting preadipocytes differentiation towards a metabolically healthy phenotype: Studies in rabbits

Development of metabolically healthy adipocytes within dysfunctional adipose tissue may represent an attractive way to counteract metabolic syndrome (MetS). In an experimental animal model of high fat diet (HFD)-induced MetS, in vivo, long- and short-term tadalafil treatments were able to reduce visceral adipose tissue (VAT) accumulation and hypertriglyceridemia, and to induce the expression in VAT of the brown fat-specific marker, uncoupling protein 1 (UCP1). VAT preadipocytes (PAD), isolated from the tadalafil-treated HFD rabbits, showed: i) a multilocular morphology; ii) an increased expression of brown fat-specific genes (such as UCP1 and CIDEA); iii) improved mitochondrial structure and dynamic and reduced superoxide production; iv) improved insulin sensitivity. Similar effects were obtained after in vitro tadalafil treatment in HFD rPAD. In conclusion, tadalafil counteracted HFD-associated VAT alterations, by restoring insulin-sensitivity and prompting preadipocytes differentiation towards a metabolically healthy phenotype.

PP.12.13

Objective: High fat diet (HFD) induced hypertension in rabbits is neurogenic and due to the central action of leptin. This action is dependent on secondary neuronal activation in alpha-melanocortin stimulating hormone (alpha-MSH) and neuropeptide Y (NPY) positive cells. Neurons in the ventromedial hypothalamus (VMH) and Dorsomedial hypothalamus (DMH) are innervated by both neuronal populations and transduce leptin signaling from the Arcuate to other hypothalamic and hindbrain nuclei. The VMH and DMH are also capable of responding to leptin signals directly, independent of NPY or alpha-MSH neurons. In the present study we assessed the contribution of leptin, alpha-MSH and NPY neurons in the VMH and DMH on development of diet-induced neurogenic hypertension. Design and method: Male New Zealand White rabbits were instrumented with a VMH or DMH cannula and a renal sympathetic nerve electrode. Blood pressure was measured by means of an intra-arterial catheter. Following 3 weeks of a HFD (13.5 % fat, n = 10) conscious rabbits had higher renal sympathetic nerve activity (RSNA), blood pressure and heart rate compared with control diet-fed animals (3.5 % fat, n = 10). Results: Rabbits exhibited higher blood pressure, heart rate and renal sympathetic nerve activity when fed a high fat diet compared to controls (n = 6–10). Alpha-melanocyte-stimulating hormone injection into the ventromedial hypothalamus increased blood pressure and renal sympathetic nerve activity (P < 0.05) in high fat diet rabbits. By contrast, no changes were observed in blood pressure or renal sympathetic nerve activity following alpha-melanocyte-stimulating hormone injections into the dorsomedial hypothalamus. Leptin antagonist injection into ventromedial and dorsomedial hypothalamus decreased blood pressure (P < 0.05) when given to high fat diet rabbits. Neuropeptide Y injection had no effect on blood pressure or renal sympathetic nerve activity in either nuclei. Conclusions: We conclude that the VMH is the likely origin of leptin-mediated sympathoexcitation, alpha-MSH hypersensitivity and altered central responsiveness to NPY.

Estrogen mediates metabolic syndrome-induced erectile dysfunction: a study in the rabbit.

Estrogen receptor (ER) α is critical in mediating the harmful effects of hyperestrogenism in fetal or neonatal life on the developing penis. In contrast, little is known on the impact of an excess of estrogens on penile function in adulthood. To investigate the effect of estrogens on metabolic syndrome (MetS)-associated erectile dysfunction (ED). We employed a recently established animal model of high fat diet (HFD)-induced MetS. Subgroups of MetS rabbits were dosed with either testosterone (T) or tamoxifen. We evaluated penile responsiveness to acetylcholine (Ach) as well as the expression of genes related to penile smooth muscle relaxation and contractility. Associations between MetS-induced penile alterations and sex steroids were investigated in an animal model of HFD-induced MetS. To understand the role of either androgen deficiency or estrogen excess on ED, we treated subgroups of MetS rabbits with either T or tamoxifen, a classical ER antagonist. Feeding an HFD-induced MetS was associated to elevated estradiol (E2) and low T levels. E2, but not T, was independently and negatively associated with genes able to affect penile erection. Smooth muscle-related markers decreased as a function of E2 and were positively associated with all the variables investigated. Increasing concentrations of circulating E2 were negatively associated with Ach-induced relaxation. In HFD rabbits, in vivo T dosing significantly improved MetS and completely normalized circulating E2. Conversely, in vivo tamoxifen dosing reduced visceral adiposity and partially restored T level. Ach-induced relaxation was severely impaired by HFD and significantly restored, up to the control level, by both tamoxifen and T dosing. In rabbit smooth muscle cells cultures 17β-E2 (1 nM) significantly reduced the expression of α-smooth muscle actin, transgelin, and phosphodiesterase type 5. The effects of 17β-E2 were completely reverted by tamoxifen (100 nM). This study demonstrates, for the first time, that HFD-induced ED is more associated with a high E2, rather than to a low T, milieu. HFD-induced ED is partially restored by in vivo treatment not only with T but also with the nonsteroidal ER antagonist, tamoxifen.

Metabolic syndrome-associated sperm alterations in an experimental rabbit model: Relation with metabolic profile, testis and epididymis gene expression and effect of tamoxifen treatment

The influence of metabolic syndrome (MetS) on sperm quality and function is debated. Using a well-established high fat diet (HFD) rabbit model resembling human MetS, including development of hypogonadism, we demonstrate that HFD decreased sperm motility, morphology and acrosome reaction in response to progesterone and increased sperm cholesterol content. All the above parameters were associated with most MetS features, its severity and plasma testosterone (T) at univariate analysis. After T adjustment, sperm morphology and motility retained a significant association, respectively, with mean arterial pressure and circulating cholesterol levels. MetS modified the expression of inflammatory and tissue remodelling genes in the testis and of aquaporins in the epididymis. In a multivariate analysis, sperm morphology resulted associated with testis expression of fibronectin and collagen type 1 genes, whereas motility with epididymis aquaporin 1 gene. Administration of tamoxifen, used in the treatment of idiopathic male infertility, to HFD rabbits partially restored motility, but further decreased morphology and increased spontaneous acrosome reaction, without restoring responsiveness to progesterone. Overall our results indicate that development of MetS produces detrimental effects on sperm quality and functionality by inducing metabolic disorders leading to alterations in testis and epididymis functions and evidence a role of hypertension as a new determinant of abnormal sperm morphology, in line with a previous human study from our group.

Metformin in vitro and in vivo increases adenosine signaling in rabbit corpora cavernosa.

In subjects with erectile dysfunction responding poorly to sildenafil, metformin was reported to improve erections. The aim of this study is to investigate metformin's mechanism of action on erectile function, particularly focusing on adenosine (ADO) and nitric oxide (NO) signaling in an animal model of high-fat diet (HFD)-induced metabolic syndrome. In vitro contractility studies of penile strips. Penile expression of genes related to ADO or NO signaling was also evaluated. In vitro contractility studies were used to investigate the effect of in vivo and ex vivo metformin administration on ADO- or acetylcholine (Ach)-induced relaxation of penile strips from HFD as compared with animals fed a regular diet (RD). Expression of ADO receptor type 3 (A3 R), ADO deaminase (ADA), AMP deaminase type 1 (AMPD1), and 2 (AMPD2) was decreased in HFD as compared with RD. Accordingly, in HFD the ADO relaxant effect was potentiated as compared with RD (P < 0.02). In vivo metformin treatment in both RD and HFD significantly increased the ADO relaxing effect (P < 0.0001 and P < 0.01, respectively, vs. relative untreated groups) although to a different extent. In fact, the half-maximal inhibitory concentration (IC50 )/IC50 ratio in RD increased fourfold vs. HFD (RD IC50 ratio = 13.75 ± 2.96; HFD IC50 ratio = 2.85 ± 0.52). In corpora cavernosa (CC) from HFD, in vivo metformin (i) normalized A3 R, ADA, and AMPD1; (ii) further decreased AMPD2; (iii) increased dimethylarginine dimethylamino-hydrolase; and (iv) partially restored impaired Ach-induced relaxation. Ex vivo metformin time and dose dependently increased the relaxant effect of ADO in RD. The potentiating effect of metformin on ADO-induced relaxation was significantly reduced by preincubation with NO synthase inhibitor N(ω) -Nitro-L-arginine methyl ester hydrochloride (L-NAME). Interestingly, in vivo testosterone supplementation in HFD rabbits (i) increased penile expression of endothelial NO synthase and AMPD2 and (ii) restored metformin's potentiating effect on ADO-induced relaxation up to RD level. Metformin in vivo and ex vivo increases ADO signaling in CC, most probably interfering with NO formation and ADO breakdown.