LN Rats Research Articles

Effect of phenotype on thyroidal parameters and metabolic sequelae in Wistar fatty rats

To determine the effects of phenotype on parameters of peripheral thyroid hormone and actions, groups of adult male lean (LN) and obese (OB) Wistar Fatty Rats were fed a nutritionally adequate semisynthetic diet containing 54% carbohydrate, 16% mixed fats, 20% protein plus essential vitamins, minerals, and cellulose fiber for 8 weeks. Measures of weight gain (WG), feed efficiency ratio (FER), Resting (RMR) and norepinephrine (NE) stimulated VO2, serum triiodothyronine (T3), and in vitro T4-5’ deiodinase (5’DI) activity in liver (LVR), kidney (KID), gastrocnemius muscle (GNM) and interscapular brown adipose tissue (IBAT) were determined at baseline and after dithiothreitol (DTT) stimulation to optimize in vitro deiodination activity. The WG and FER of OB >> LN phenotype and FER increased over time in both phenotypes, with the greatest increase in the OB phenotype. The RMR of LN > OB, and NE-stimulation (200 µg/kg BW, sc) increased VO2 by 180% in LN but only ~30% in OB phenotype. Serum T3 of LN >> OB. Baseline 5’DI of OB > LN in LIV and KID and were of similar magnitude in both phenotypes in GNM and IBAT. After DTT stimulation, LIV increased ~200% in LN, ~30% in OB; In LN rats, KID +DTT increased ~200%, but only modest increases in OB phenotype, while in LN GNM, DTT resulted in 264% increase vs. +70% in OB rats. In IBAT, DTT resulted in ~ 30-fold increase in 5’DI in LN, and a 16-fold increase in 5’DI in the OB phenotype. These results indicate that parameters of thyroidal actions including circulating plasma levels of T3 and maximum capacity to generate T3 in peripheral tissues via 5’DI, although increased in the OB phenotype, were associated with decreases in RMR and NE-stimulated VO2. These observations occurred in association with an improved efficiency of FER and weight gain and dysregulation of intracellular T3 actions including parameters of T3 receptor affinity kinetics are likely to be among key contributors to the epigenetic expression of the OB+NIDDM phenotype in this strain and are consistent with a reduced affinity of T3- mediated cellular components of intermediary metabolism.

Diietary Fat Retention In Cortical Bone In Rats Fed High Fat Diet

Excess adiposity during childhood and adolescence are associated with poorer bone quality and increased risk of fracture. A high fat diet (HFD) may promote marrow adipogenesis at the expense of osteogenesis and may also promote excess lipid accumulation in osteoblasts and osteocytes. Exercise is prescribed for bone health primarily because of its mechanical loading attributes, but little is known about how obesity or exercise training status affects nutrient trafficking and retention in bone. PURPOSE: To determine the effects of obesity and exercise on fat retention in bone. METHODS: Female Wistar rats, 5 weeks in age (n=18), were fed a high fat diet (HFD) for 20 weeks and designated as obese (OB) or lean (LN) based on weight gain. OB and LN rats performed treadmill running (EX) (5 d/w, 1h/d, 15 m/min) or sedentary control (SED) for 10 weeks. In a 24-hour tracer study, rats were given ad libitum access to 14C oleate and 14C palmitate blended in food. Marrow was removed from hind limb bones and lipid was extracted from the remaining cortical bone. Data are mean ± SEM. RESULTS: Fat intake was not different between groups. Obesity status had no significant effect on fat retention in cortical bone. Lipid in hindlimb cortical bone was higher in EX compared to SED (EX: 6.66 ± 1.64 vs SED: 1.50 ± 1.47 mg, p<0.05). LNEX had the highest dietary fat retention in cortical bone lipid compared to other groups (LNEX: 0.69 ± 0.14, OBEX: 0.24 ± 0.14, LNSED: 0.27 ± 0.13, OBSED: 0.41 ± 0.13 mg, interaction p<0.05). The proportion of the meal retained, and the magnitude of lipid turnover did not differ between groups. Dietary fat retention in bone was positively (p<0.05) associated with fat intake, bone mineral density and bone mineral content (BMC; r=0.51-0.64). BMC in bone was associated (r=0.54, p<0.05) with total energy expenditure. CONCLUSION: Although cortical bone constitutes a minor fraction of overall dietary fat retention, we demonstrate that fat is trafficked to and retained in cortical bone in proportion to meal size and bone size. Future work is needed to test whether fat accumulation influences the ability to adapt to mechanical loading.

Characterization of liver changes in ZSF1 rats, an animal model of metabolic syndrome.

The non-alcoholic fatty liver disease is the hepatic counterpart of the metabolic syndrome. ZSF1 rats are a metabolic syndrome animal model in which liver changes have not been described yet. The characterization of liver histological and innate immunity changes in ZSF1 rats. Five groups of rats were included (n = 7 each group): healthy Wistar-Kyoto control rats (Ctrl), hypertensive ZSF1 lean (Ln), ZSF1 obese rats with a normal diet (Ob), ZSF1 obese rates with a high-fat diet (Ob-HFD), and ZSF1 obese rats with low-intensity exercise training (Ob-Ex). The animals were sacrificed at 20 weeks of age, their livers were collected for: a) measurements of the area of steatosis, fibrosis and inflammation (histomorphological analysis); and b) innate immunity (toll-like receptor [TLR] 2, TLR4, peroxisome proliferator-activated receptor γ [PPARγ], toll interacting protein [TOLLIP]) and inflammatory marker (tumor necrosis factor-alpha [TNFα], interleukin 1 [IL-1]) expression analysis by real-time PCR. Ob, Ob-HFD and Ob-Ex were significantly heavier than Ln and Ctrl animals. Ob, Ob-HFD and Ob-Ex animals had impaired glucose tolerance and insulin resistance. ZSF1 Ob, Ob-HFD and Ob-Ex presented a higher degree of steatosis (3,5x; p < 0.05) than Ctrl or ZSF1 Ln rats. Steatohepatitis and fibrosis were not observed in any of the groups. No differences in expression were observed between Ctrl, Ln and Ob animals (except for the significantly higher expression of TOLLIP observed in the Ob vs Ln comparison). Ob-HFD and Ob-Ex rats showed increased expression of PPARγ and TOLLIP as compared to other groups. However, both groups also showed increased expression of TLR2 and TLR4. Nevertheless, this did not translate into a differential expression of TNFα or IL-1 in any of the groups. The ZSF1 model is associated with liver steatosis but not with steatohepatitis or a significantly increased expression of innate immunity or inflammation markers.

Effect of chronic intake of liquid nutrition on stomach and duodenum morphology

Changes in the quantity and/or quality of food intake have been shown to be associated with morphological and functional alterations of the gastrointestinal system. To examine this, we investigated the effect of chronic liquid nutrition intake (Fresubin) on stomach and duodenum morphology in Wistar rats fed liquid nutrition during different developmental periods. We used four groups of rats: a) control group (CON) fed pelleted chow for 130days; b) liquid nutrition group (LN) fed liquid nutrition for 130days; c) liquid nutrition juvenile group (LNJ) fed liquid nutrition for 70days and then pelleted food for 60days; d) liquid nutrition adult group (LNA) fed pelleted chow for 70days and then liquid nutrition for 60days. We found that LN and LNA rats showed a significant reduction of empty stomach mass compared to CON animals, while stomach and duodenal longitudinal muscle layer thickness did not differ between groups. Villus height was increased only in LNA animals, while villus width was increased in both LN and LNA rats. Crypt depth was reduced in LNJ. However, liquid nutrition intake did not affect villus height/crypt depth ratio, nor number of goblet cells. We found that chronic intake of liquid nutrition affects some morphological parameters of the stomach and duodenum but these changes were not homogenous between experimental groups. Interestingly, transition from liquid nutrition to solid food reversed the alterations of stomach weight as well as villus width induced by intake of liquid nutrition in LNA rats. Our data indicate that morphological and functional changes in the gastrointestinal system induced by qualitative and quantitative changes in food intake are at least partially reversible. Therefore, specific diets may be used potentially as adjuvant treatment for modulating the progression of gastrointestinal diseases by affecting stomach and small intestine morphology.

Sugar Consumption Produces Effects Similar to Early Life Stress Exposure on Hippocampal Markers of Neurogenesis and Stress Response.

Adverse early life experience is a known risk factor for psychiatric disorders. It is also known that stress influences food preference. We were interested in exploring whether the choice of diet following early life stress exerts long-lasting molecular changes in the brain, particularly the hippocampus, a region critically involved in stress regulation and behavioral outcomes. Here, we examined the impact of early life stress induced by limited nesting material (LN) and chronic sucrose availability post-weaning on an array of hippocampal genes related to plasticity, neurogenesis, stress and inflammatory responses and mitochondrial biogenesis. To examine mechanisms underlying the impact of LN and sugar intake on hippocampal gene expression, we investigated the role of DNA methylation. As females are more likely to experience adverse life events, we studied female Sprague-Dawley rats. After mating LN was imposed from days 2 to 9 postpartum. From 3 to 15 weeks of age, female Control and LN siblings had unlimited to access to either chow and water, or chow, water and 25% sucrose solution. LN markedly reduced glucocorticoid receptor (GR) and neurogenic differentiation 1 (Neurod1) mRNA, markers involved in stress and hippocampal plasticity respectively, by more than 40%, with a similar effect of sugar intake in control rats. However, no further impact was observed in LN rats consuming sugar. Hippocampal Akt3 mRNA expression was similarly affected by LN and sucrose consumption. Interestingly, DNA methylation across 4 CpG sites of the GR and Neurod1 promoters was similar in LN and control rats. In summary, early life stress and post-weaning sugar intake produced long-term effects on hippocampal GR and Neurod1 expression. Moreover we found no evidence of altered promoter DNA methylation. We demonstrate for the first time that chronic sucrose consumption alone produces similar detrimental effects on the expression of hippocampal genes as LN exposure.

Early Life Stress Induced by Limited Nesting Material Produces Metabolic Resilience in Response to a High-Fat and High-Sugar Diet in Male Rats.

Environmental conditions experienced in early life can profoundly influence long-term metabolic health, but the additive impact of poor nutrition is poorly understood. Here, we tested the hypothesis that early life stress (ELS) induced by limited nesting material (LN) combined with high-fat and high-sugar diet (HFHS) post-weaning would worsen diet-related metabolic risk. Sprague-Dawley male rats were exposed to LN, postnatal days 2–9, and at weaning (3 weeks), siblings were given unlimited access to chow or HFHS resulting in (Con-Chow, Con-HFHS, LN-Chow, and LN-HFHS, n = 11–15/group). Glucose and insulin tolerance were tested and rats were killed at 13 weeks. LN rats weighed less at weaning but were not different to control at 13 weeks; HFHS diet led to similar increases in body weight. LN-chow rats had improved glucose and insulin tolerance relative to Con-Chow, whereas LN-HFHS improved insulin sensitivity versus Con-HFHS, associated with increased peroxisome proliferator-activated receptor gamma co-activator-1-alpha (Pgc-1α) mRNA in muscle. No effect of LN on plasma or liver triglycerides was observed, and hepatic gluconeogenic regulatory genes were unaltered. In summary, this study demonstrates that ELS induced by LN conferred some metabolic protection against insulin and/or glucose intolerance in a diet-dependent manner during adulthood.

Exaggerated activity of HPA axis in obese rats fed normocaloric liquid nutrition.

Experimental and clinical studies have shown alterations in activity of systems responsible for neuroendocrine stress response in obese individuals. Therefore we investigated the effect of palatable normocaloric liquid nutrition (Fresubin) on alterations in activity of the hypothalamic-pituitary-adrenal (HPA) axis in male Wistar rats of different developmental stages. Control rats (CON) received standard pellet chow all the time from weaning (21st day of age) to 150 days. Fresubin was administered throughout the experiment (LN), only in juvenility (from 21st to 90th day of age; LNJ) or only in adulthood (from 90th to 150th day of age; LNA). Body weight and energy intake were periodically monitored. Adrenal gland and fat tissue weight and plasma corticosterone levels (CORT) was determined after sacrification. Fresubin intake induced obesity in LN and LNA rats. In LN and LNA rats were observed elevated serum CORT levels, but only in LN rats with significant twofold increase compared to LNJ rats. However, the weight of adrenal glands did not differ between LN, LNJ and LNA experimental groups. Based on our results, we suggest, that obesity induced by Fresubin in LN and LNA rats is accompanied by increased HPA activity represented by elevated plasma CORT levels in these rats.

Abstract 18610: Mitochondrial Transfer Restores Endothelial Dependent Vasodilation

Although endothelial dysfunction is a hallmark of vascular disease, the mechanisms underlying this impairment are unknown. Recently, impaired mitochondrial function was observed in vascular pathologies with endothelial dysfunction; yet a causal link between mitochondrial and endothelial function is not established. We sought to establish this link by demonstrating that restoration of mitochondrial function rescues endothelium-dependent dilation in a model of endothelial dysfunction (metabolic syndrome). Initially, we evaluated activities of mitochondrial complexes I-IV in aortas), and endothelial-dependent dilation (isolated, perfused mesenteric small arteries [MA’s], 289±32 µm, id) in Zucker Obese Fatty (ZOF) and Zucker Lean (LN) rats. Activity of complex I, II, III, or IV (per mg/mitochondrial protein) was lower (56%, 65%, 72% or 59% reduction, respectively) in ZOF than LN (P&lt;0.05). Dilation to the endothelium-independent agonist, nitroprusside (NP) was equivalent in LN and ZOF rats suggesting normal smooth muscle function. Dilation to vasoactive intestinal peptide (VIP, endothelium-dependent agonist) in LN was completely blocked by L-NAME (10 -4 M) (Figure 1). Dilation to VIP was markedly compromised in ZOF (Figure 1). To acutely restore mitochondrial function, mitochondria from livers of lean rats were isolated, labeled with Mitotracker, administered into the perfusion circuit, and “transferred” into the endothelium of MA’s of ZOF via electroporation (confirmed by Mitotracker fluorescence). Thirty minutes after electroporation, dilation of mitochondria-treated vessels (ZOF+Mito) was restored to near normal levels (Figure 1, ZOF+Mito vs LN) and significantly greater than untreated ZOF. Dilation to NP was unaffected by transfer (not shown). These results suggest that mitochondria play a key role in endothelial function, and that treatment regimens for vascular disease should consider mitochondrial directed therapies.

Abstract 892: The Importance of Mitochondrial Oxidative Stress and Integrity of Mitochondrial DNA in Coronary Arteriogenesis

The prediabetic metabolic syndrome (pdMS) leads to many pathological cardiac and vascular phenotypes including impaired arteriogenesis. Mitochondrial (mt) oxidative stress (mtOxStress) is postulated to contribute to the development of such phenotypes, but whether reduction of mtOxStress, or imposition of a maneuver to mimic its effects, would restore or inhibit coronary arteriogenesis (CA), respectively, has not been determined. We tested the hypothesis that in the pdMS, mtOxStress plays a key role in inhibition of coronary arteriogenesis. Lean (LN, n = 6) and obese (OB, n = 6) Zucker obese fatty rats (model of pdMS) were instrumented with a pneumatic snare around the left anterior descending artery (LAD), and subjected to a protocol of repetitive ischemia (RI: 24, 40 sec occlusions/day for 10 days) to stimulate growth. CA was determined from the ratio of flows (radioactive microspheres) measured during LAD occlusion to collateral-dependent and normal zones (CZ/NZ). Prior to RI, CZ/NZ in all groups was less than 0.2 indicating that the native collateral conductance could supply less than 20% of the normally occurring flow during LAD occlusion. After RI, CZ/NZ was 0.82Â ± 0.09 in LN, which was markedly higher (p &lt; 0.05) than that in the OB (0.21Â ± 0.08) indicating impaired CA in the OB rats. To reduce mtOxStress, OB animals (n = 5) were given mito-Quinone (mt-directed free radical scavenger) in drinking water (750 mg/L); this treatment restored CA (CZ/NZ = 0.79Â ± 0.03) comparable to that in LN. Because one manifestation of mt-OxStress is mt-DNA fragmentation, which results in impaired expression of key mt-proteins and compromised mt-function, we determined if administration of a recombinant protein engineered with a TAT sequence to enable intracellular delivery, a mt-localization sequence, and exonuclease ExoIII (mt-tat-ExoIII) to fragment mtDNA would impair coronary arteriogenesis in LN (n = 5). Mt-tat-ExoIII (iv, 0.2 ug/g, every other day) in LN rats impaired CA in response to RI (CZ/NZ = 0.32Â ± 0.09, p &lt; 0.05 vs. LN controls). We conclude that mitochondrial oxidative stress, and its effects on mtDNA integrity, are pivotal in the abrogation of CA in the pdMS.

Alleviation of hepatic steatosis accompanied by modulation of plasma and liver TNF-α levels by Trigonella foenum graecum (fenugreek) seeds in Zucker obese (fa/fa) rats

Oral supplements of fenugreek (Trigonella foenum graecum) seeds (Fen) have been shown to treat glucose and lipid homeostasis in several metabolic disorders; however, its ability to alleviate obesity-associated pathologies is not known. The main objective of this study was to evaluate the effect of Fen in Zucker obese rats (Ob), an animal model of obesity and related disorders, such as dyslipidemia and hepatic steatosis. Female Zucker (6-week-old) Ob and lean (Ln) rats were randomly grouped (n = 8 rats/group) to receive either basal or 5% Fen-supplemented AIN-93G diets for 8 weeks, and then were euthanized. Histopathology and biochemical parameters in the liver together with plasma biochemistry were assessed. Obese rats had significantly higher (P < 0.05) body and liver weight, as well as plasma insulin, lactate, cholesterol, triglyceride and tumor necrosis factor (TNF)-alpha compared to their Ln counterparts. Fen significantly reduced (P < 0.05) the liver-weight of Ob rats in comparison to Ob rats fed basal diet (Control), while no effect was observed in the Ln rats. Fen treatment resulted in a lower P-value (P < 0.05). In addition, Ob rats on Fen-supplemented diets had fasting plama cholesterol and TNF-alpha levels, and significantly higher (P < 0.05) triglycerides in comparison to the control rats. Hepatic triglyceride level was significantly lower (P < 0.05) in Ob rats fed Fen supplemented diets in comparison to control. The levels of bound and soluble liver TNF-alpha (26 and 17 kDa, respectively) and TNF receptor-II (TNFR-II, 75 kDa) proteins were significantly lower (P < 0.05) in Ob than in Ln rats. Fen significantly lowered (P < 0.05) both the soluble and bound forms of TNF-alpha protein while significantly elevating (P < 0.05) TNFR-II in the livers of Ob rats compared to Ob Controls. These results demonstrate unequivocally that in a short-term preclinical evaluation, dietary Fen supplementation reduced the triglyceride accumulation in the liver, a hallmark feature of hepatic steatosis without affecting the plasma insulin or glucose levels in Zucker obese rats and suggest that TNF-alpha may play an important role in this process.

Obese state leads to elevated levels of TGF-β and COX isoforms in platelets of Zucker rats

Platelets are rich sources of growth factors and enzymes that are implicated in a number of diseases including obesity, atherosclerosis, heart disease, syndrome X, liver and kidney diseases and certain types of cancers. In this research we investigated, if platelets in Zucker obese rats differ from their lean counterparts with respect to the levels of TGF-beta and COX isoforms, implicated in the pathogenesis of chronic diseases. In addition, we investigated if energy intake of the animals affects the platelet physiology. Platelets were isolated from obese and lean rats bearing preneoplastic lesions in their colon. Prior to platelet isolation these rats were fed either ad libitum (Ob or Ln) or energy restricted (Ob-ER or Ln-ER) diets for 8 weeks (n = 8/group). The levels of TGF-beta1/-beta2 and COX-1/-2 proteins in platelets were analyzed by Western blot. The platelets of the Ob rats had significantly higher levels of TGF-beta1, COX-1/-2 (p < 0.001) than did the platelets of the Ln rats and were not affected by moderate energy restriction. There were no significant differences in the protein expression of platelet TGF-beta2 among any of the groups. These results demonstrate that cytokines and candidates playing a role in the pathogenesis of chronic diseases, such as TGF-beta1 and COX-1/-2, are over-expressed in platelets of Zucker obese rats by comparison to their lean counterparts. These findings also demonstrate that the genotype of the animals exerts a significant effect on the biochemical composition of the platelets and could contribute to the pathogenesis of colon cancer and other metabolic abnormalities associated with obesity.

Offspring myocardium alteration from dams submitted to nitric oxide synthesis inhibition during pregnancy

Abstract Background: Nitric oxide synthesis (NOS) inhibition during pregnancy alters offspring cardiac structure. Methods: Thirty female Wistar rats were assigned to two groups: control (15 rats) and LN (15 rats). LN rats received N ω-nitro- l-arginine methyl ester ( l-NAME) during 5 weeks (2 weeks before matching and the weeks of pregnancy). Offspring were separated and sacrificed in three age groups ( n=5): 2, 15 and 30 postnatal days. Left ventricular myocardium was analyzed with light microscopy and stereology. Results: BP increased significantly in LN matrices. No significant difference in neonate C-R length, body mass (BM) nor heart mass (HM) occurred in control and LN groups with 2 days of life. HM was greater in LN group than in control group from 15 days of life; BM was greater in LN group than in control group only with 30 days of life. HM/BM ratio was greater in LN neonates than in control neonates in all age groups. Vasculature total length (L[VE]) increased from days 2 to 15 and from days 15 to 30 and total number of cardiomyocyte nuclei profiles ( N[CM]) also increased from days 2 to 30 in both LN and control neonates. Conclusion: The offspring main cardiac structural alteration due to prenatal NOS inhibition is compatible with neonatal cardiac hypertrophy with intramyocardial vasculature enhancement. Furthermore, the prenatal NOS inhibition apparently stimulates the cardiomyocyte proliferation that can be observed until the end of lactation, which is when the number of cardiomyocytes nuclei profiles seems to become stable.

Hypersensitivity of the adrenal cortex to trophic and secretory effects of angiotensin II in Lyon genetically-hypertensive rats.

In Lyon hypertensive (LH) rats, a model of low-renin genetic hypertension, we investigated adrenal sensitivity to angiotensin II in terms of angiotensin II receptor (AT1 and AT2 receptors) regulation, morphological changes, and aldosterone and corticosterone secretion. Twelve-week-old LH rats, compared with normotensive LN and LL rats, were either untreated or treated for 4 weeks with AT1 receptor antagonist irbesartan (50 mg/kg/d), angiotensin-converting enzyme inhibitor perindopril (3 mg/kg/d), or perindopril (3 mg/kg/d) plus angiotensin II infusion (200 ng/kg/min). At 16 weeks, untreated LH rats had high systolic blood pressure (P<0.05), low aldosterone (P<0.05), and increased corticosterone (P<0.05) plasma levels. AT1-receptor binding density in the zona glomerulosa was similar in the three strains. In LH rats, angiotensin II infusion increased the relative adrenal weight from 10.5+/-0.3 to 16.7+/-0.7 mg/100g (P<0.05), whereas this change was very modest in normotensive rats. Zona glomerulosa enlarged and plasma aldosterone increased after angiotensin II infusion in the 3 strains, but more markedly in LH versus normotensive rats (2.4- versus 1.3- and 1.6-fold, respectively; 20- versus 10-fold in normotensive rats, P<0.05). Surprisingly, after angiotensin II infusion, despite the absence of angiotensin II receptors in the three strains, the zona fasciculata-reticularis enlarged 1.5-fold and plasma corticosterone increased 1.7-fold only in LH rats (P<0.05), suggesting an indirect control of this compartment by angiotensin II. The hypertrophy and hypersecretory activity of both zona glomerulosa and zona fasciculata-reticularis in LH rats in response to angiotensin II point to the adrenal cortex as a pivotal tissue in the pathophysiology of hypertension in LH rats.

ETA receptor-mediated Ca2+ signaling in thin descending limbs of Henle's loop: Impairment in genetic hypertension

Endothelins (ET) have diuretic and natriuretic actions via ETB receptors that are found in most renal tubular segments, although the thin limbs have not been studied. Data also suggest that dysfunction of the renal ET system may be important in the pathogenesis of hypertension. The present study was aimed at determining the presence and nature of ET receptors in the thin limbs of Henle's loop and their ability to activate a Ca2+-dependent signaling pathway, as well as whether ET-induced Ca2+ signals are altered in hypertension. Reverse transcription-polymerase chain reaction (RT-PCR) and Fura 2 fluoreselected strains of Lyon rats with low-normal (LL), normal (LN), and high (LH) blood pressure. In SD rats, ET induced Ca2+ signals in DTL of long-looped nephrons, but not in DTL of short loops, or in ascending thin limbs. Ca2+ increases were abolished by BQ123, an antagonist of the ETA receptor, but not by BQ788, an antagonist of the ETB subtype. Endothelin-3 and sarafotoxin 6c, two ETB receptor agonists, were both inactive. RT-PCR showed the presence of both ETA and ETB receptor mRNA. Ca2+ signals measured scence measurements of [Ca2+]i were made to characterize ET receptors in descending thin limbs (DTL) of Sprague-Dawley rats, spontaneously hypertensive (SH) rats, and control Wistar-Kyoto (WKY) rats, and the three in DTL of WKY LL and LN rats were similar to those in Sprague-Dawley rats, but were significantly diminished (LH) or abolished (SH) in hypertensive rats. A functional ETA receptor activating a Ca2+-dependent pathway is expressed in DTL. This ETA-induced calcium signaling is impaired in two strains of genetically hypertensive rats.

Oxidative stress in fatty livers of obese Zucker rats: Rapid amelioration and improved tolerance to warm ischemia with tocopherol

Fatty livers in humans and rats are less tolerant of ischemia, endotoxin, and alcohol. We hypothesized that fatty livers of obese (Ob) Zucker rats are oxidatively stressed and oxidative stress could be relieved by antioxidant treatment, leading to improved tolerance to ischemia. Total glutathione (GSH), tocopherol (TOC), ascorbic acid (AA), catalase (CAT), superoxide dismutase (SOD), and selenium–glutathione peroxidase (Se-GPx) were measured in the livers of Ob and lean (Ln) Zucker rats before and after treatment with high-dose TOC and ascorbate. Also, survival in treated Ob rats following a lethal 90 minutes of partial in vivo warm ischemia was examined. Fatty livers of Ob rats contained significantly less GSH, TOC, and CAT, in comparison with livers of Ln rats. Immunoblotting showed significantly decreased CAT protein without changes in mRNA in fatty livers. There were no significant differences in AA, SOD, and Se-GPx between the 2 groups. Pretreatment with TOC and ascorbate over 48 hours completely corrected the decreases in GSH, TOC, and CAT. Most importantly, TOC with or without ascorbate pretreatment significantly improved survival in Ob rats following ischemia in a dose-dependent manner. In conclusion, TOC administration corrected the oxidative stress in fatty livers of Ob Zucker rats and improved survival following lethal ischemia. Additional studies are needed to determine the efficacy of TOC—a relatively inexpensive agent—in treating patients with fatty livers in a variety of clinical conditions, possibly including liver transplantation. (HEPATOLOGY 2001;34:13-18.)

Angiotensin II Increases Enos Expression in the Rat Myocardium Independent of Pressor Effect.

P5 There is accumulating evidence that nitric oxide antagonizes angiotensin II (AII) effects at multiple regulatory levels of the cardiovascular system. In the present study, we first determined the expression of endothelial (eNOS) and neuronal (nNOS) isoforms of nitric oxide synthase (eNOS) in the ventricles of two kidney Goldblatt(2K1C, 2 months) and L-NAME treated (LN, 2 months)hypertensive rats with Western blot. Systolic arterial pressures were 130±3, 165±4, 160±5 mmHg in control (n=7) and 2K1C (n=6)and LN treated (n=6) rats, respectively. nNOS was barely detected in ventricles of normotensive and 2K1C and LN hypertensive rats. In contrast, eNOS was detected in ventricles of all groups of rats and was increased by 100±8% and by 88±12%in 1K1C and LN rats. Experiments performed with blocking peptide (100:1; blocking peptide to antibody proportion) indicated that the ∼130 kDa bands identified in membranes blotted with anti-eNOS antibody was specific for eNOS. Another group of rats was continuously infused with AII (D1= 10, n=5; D2= 40, n=4 ng/kg/min) for 6 days and arterial pressure measured daily (1 h, continuous, sampled at 100 Hz). Arterial pressure did not change significantly during infusion of D1 and increased by 22±3 mmHg during D2 infusion. Both, D1 and D2 increased eNOS expression by ∼95% while, again, no change was observed in nNOS expression. These data indicate that 1) eNOS expression is up-regulated in 2K1C and LN hypertensive rats; 2) AII infusion enhances the eNOS expression by a level similar to that observed in 1K1C and LN hypertensive rats; and 3) The influence of AII on eNOS expression is independent of its pressor effect. In conclusion,our data suggest that AII increases the expression of eNOS, independent of the its hemodynamic effect and could be responsible for the increase in eNOS expression in 1K1C hypertensive rats. In addition, this modulation is specific for eNOS since nNOS is present only in small amounts in the rat myocardium and is not modulated by AII. The mechanism responsible for the increase in eNOS expression in LN rats is not clear, but could be related to a modulation mediated by a product derived from eNOS action on its substrate.

Angiotensin II Increases Enos Expression in the Rat Myocardium Independent of Pressor Effect.

P5 There is accumulating evidence that nitric oxide antagonizes angiotensin II (AII) effects at multiple regulatory levels of the cardiovascular system. In the present study, we first determined the expression of endothelial (eNOS) and neuronal (nNOS) isoforms of nitric oxide synthase (eNOS) in the ventricles of two kidney Goldblatt(2K1C, 2 months) and L-NAME treated (LN, 2 months)hypertensive rats with Western blot. Systolic arterial pressures were 130±3, 165±4, 160±5 mmHg in control (n=7) and 2K1C (n=6)and LN treated (n=6) rats, respectively. nNOS was barely detected in ventricles of normotensive and 2K1C and LN hypertensive rats. In contrast, eNOS was detected in ventricles of all groups of rats and was increased by 100±8% and by 88±12%in 1K1C and LN rats. Experiments performed with blocking peptide (100:1; blocking peptide to antibody proportion) indicated that the ∼130 kDa bands identified in membranes blotted with anti-eNOS antibody was specific for eNOS. Another group of rats was continuously infused with AII (D1= 10, n=5; D2= 40, n=4 ng/kg/min) for 6 days and arterial pressure measured daily (1 h, continuous, sampled at 100 Hz). Arterial pressure did not change significantly during infusion of D1 and increased by 22±3 mmHg during D2 infusion. Both, D1 and D2 increased eNOS expression by ∼95% while, again, no change was observed in nNOS expression. These data indicate that 1) eNOS expression is up-regulated in 2K1C and LN hypertensive rats; 2) AII infusion enhances the eNOS expression by a level similar to that observed in 1K1C and LN hypertensive rats; and 3) The influence of AII on eNOS expression is independent of its pressor effect. In conclusion,our data suggest that AII increases the expression of eNOS, independent of the its hemodynamic effect and could be responsible for the increase in eNOS expression in 1K1C hypertensive rats. In addition, this modulation is specific for eNOS since nNOS is present only in small amounts in the rat myocardium and is not modulated by AII. The mechanism responsible for the increase in eNOS expression in LN rats is not clear, but could be related to a modulation mediated by a product derived from eNOS action on its substrate.

Cyclooxygenases 1 and 2 and thromboxane synthase in kidneys of Lyon hypertensive rats

Compared with their two normotensive (LN and LL) controls, genetically hypertensive rats of the Lyon strain (LH) exhibit increased renal vascular resistance and a blunted pressure natriuresis function as well as an increased urinary excretion of vasoconstrictor prostanoids. The aim of this study was to assess in the kidneys of these animals the synthesis of vasoconstrictor or sodium-retaining prostanoids. The relative abundance of the mRNAs of cyclooxygenases (COX) 1 and 2 and of thromboxane A 2 synthase (TXS), was measured by reverse-transcription polymerase chain reaction (RT-PCR) in renal cortex and medulla dissected in groups of male LH, LN, and LL rats either in baseline conditions or after 1 week of salt loading (1.5% NaCl in the drinking water). In basal conditions, at 3 and 11 weeks of age COX1 was expressed in the kidneys of all rats more markedly in medulla than in cortex. COX2 was poorly expressed in the whole kidney. TXS expression was usually too low to be quantified. No difference could be observed among LH, LN, and LL rats. After salt loading, the expression of COX1 was enhanced in the medulla and that of COX2 reduced in the cortex. LH rats differed from controls by a significantly more marked increase in medullary COX1 expression. The present work excludes any primary generalized increase in the renal expression of the genes that control the synthesis of vasoconstrictor prostanoids in LH rats, but suggests that medullary COX1 is upregulated by salt in these animals.

Nitric oxide and preglomerular vascular lesions in lyon spontaneously hypertensive rats.

Accumulation of Sudan black-stainable (SB+) lipids is a hallmark of the focal inflammato-proliferative lesions that develop along preglomerular vessels in N G-nitro-L-arginine methyl ester (L-NAME) and angiotensin II hypertensive rats. We extended our findings to genetically hypertensive Lyon (LH) rats aged 14 and 30 weeks and to age-matched normotensive (LN) rats. Vessels were isolated by HCl maceration. Despite high systolic blood pressure (SBP), hypercholesterolaemia, albuminuria and increased interlobular and afferent arteriolar media thickness, SB+ lesions were rarely found in LH rats, regardless of age. To probe nitric oxide as a potential source of vascular protection, 14-week-old LN and LH rats received L-NAME for 10 days (20 mg kg-1 day-1, per os), which increased SBP to 174 +/- 5 and to >200 mmHg, respectively. It induced formation of focal SB+ lesions less frequently in LN than LH rats, in which they affected 39 +/- 7, 44 +/- 5 and 15 +/- 5% of arcuate arterial branches, interlobular arteries and afferent arterioles, respectively. Immunoreactive endothelin-1 was found to accumulate at the level of SB+ lesions. Co-administered with L-NAME, hydralazine (15 mg kg-1 day-1, per os) limited SBP rise to approximately 10 mmHg in both LN and LH rats. As a result, SB+ lesions were rare in LN rats, but were frequent in LH rats. In conclusion, preglomerular SB+ lesions are spontaneously lacking in LH rats. Endogenous nitric oxide production provides protection against vascular barotrauma. Endothelin-1 likely plays an autocrine/paracrine role in vascular lesion formation.

Renal effects of cP450 arachidonate metabolites in the Lyon hypertensive rat.

This study is aimed at evaluating the influence of the cytochrome P450 arachidonate metabolites on the renal alterations exhibited by the Lyon hypertensive (LH) rat. To that purpose, kidneys were isolated from LH rats and their normotensive (LN) controls and single-pass perfused at different pressure levels before (control conditions) and after cytochrome P450 inhibition by 7-ethoxyresorufin (7-ER, 1 microM). In control conditions, LH kidneys differed from LN ones by an increased preglomerular resistance and a blunted pressure natriuresis. 7-ER, which did not affect the function of LN kidneys, decreased the vascular resistance of LH kidneys, increased their glomerular filtration rate but had no effect on their pressure natriuresis. These results indicate that the renal cytochrome P450 arachidonate metabolism differs between LN and LH rats and is presumably involved in the functional alterations exhibited by LH kidneys.