Pair-fed Rats Research Articles

Development of ghrelin resistance in a cancer cachexia rat model using human gastric cancer-derived 85As2 cells and the palliative effects of the Kampo medicine rikkunshito on the model.

Cancer cachexia (CC) is a multifactorial disease characterized by decreased food intake and loss of body weight due to reduced musculature with or without loss of fat mass. Patients with gastric cancer have a high incidence of cachexia. We previously established a novel CC rat model induced by human gastric cancer-derived 85As2 cells in order to examine the pathophysiology of CC and identify potential therapeutics. In patients with CC, anorexia is often observed, despite elevation of ghrelin, suggesting that ghrelin resistance may develop in these patients. In this study, we aimed to clarify the occurrence of ghrelin resistance in CC rats accompanied by anorexia and we investigated whether rikkunshito (RKT), a traditional Japanese Kampo medicine that potentiates ghrelin signaling, ameliorated CC-related anorexia through alleviation of ghrelin resistance. 85As2-tumor-bearing rats developed severe CC symptoms, including anorexia and loss of body weight/musculature, with the latter symptoms being greater in cachectic rats than in non-tumor-bearing or pair-fed rats. CC rats showed poor responses to intraperitoneal injection of ghrelin. In CC rats, plasma ghrelin levels were elevated and hypothalamic anorexigenic peptide mRNA levels were decreased, whereas hypothalamic growth hormone secretagogue receptor (GHS-R) mRNA was not affected. In vitro, RKT directly enhanced ghrelin-induced GHS-R activation. RKT administrated orally for 7 days partly alleviated the poor response to ghrelin and ameliorated anorexia without affecting the elevation of plasma ghrelin levels in CC rats. The expression of hypothalamic orexigenic neuropeptide Y mRNA but not hypothalamic GHS-R mRNA was increased by RKT. Thus, the 85As2 cell-induced CC rat model developed ghrelin resistance, possibly contributing to anorexia and body weight loss. The mechanism through which RKT ameliorated anorexia in the CC rat model may involve alleviation of ghrelin resistance by enhancement of ghrelin signaling. These findings suggest that RKT may be a promising agent for the treatment of CC.

Capsaicin reduces Alzheimer-associated tau changes in the hippocampus of type 2 diabetes rats.

Type 2 diabetes (T2D) is a high-risk factor for Alzheimer’s disease (AD) due to impaired insulin signaling pathway in brain. Capsaicin is a specific transient receptor potential vanilloid 1 (TRPV1) agonist which was proved to ameliorate insulin resistance. In this study, we investigated whether dietary capsaicin could reduce the risk of AD in T2D. T2D rats were fed with capsaicin-containing high fat (HF) diet for 10 consecutive days (T2D+CAP). Pair-fed T2D rats (T2D+PF) fed with the HF-diet of average dose of T2D+CAP group were included to control for the effects of reduced food intake and body weight. Capsaicin-containing standard chow was also introduced to non-diabetic rats (NC+CAP). Blood glucose and insulin were monitored. The phosphorylation level of tau at individual sites, the activities of phosphatidylinositol 3 kinase/protein kinase B (PI3K/AKT) and glycogen synthase kinase-3β (GSK-3β) were analyzed by Western blots. The results revealed that the levels of phosphorylated tau protein at sites Ser199, Ser202 and Ser396 in hippocampus of T2D+CAP group were decreased significantly, but these phospho-sites in T2D+PF group didn’t show such improvements compared with T2D group. There were almost no changes in non-diabetic rats on capsaicin diet (NC+CAP) compared with the non-diabetic rats with normal chow (NC). Increased activity of PI3K/AKT and decreased activity of GSK-3β were detected in hippocampus of T2D+CAP group compared with T2D group, and these changes did not show in T2D+PF group either. These results demonstrated that dietary capsaicin appears to prevent the hyperphosphorylation of AD-associated tau protein by increasing the activity of PI3K/AKT and inhibiting GSK-3β in hippocampus of T2D rats, which supported that dietary capsaicin might have a potential use for the prevention of AD in T2D.

Long-Term Ethanol Exposure Decreases the Endotoxin-Induced Hepatic Acute Phase Response in Rats.

Long-term excessive alcohol intake predisposes to infectious diseases. The hepatic acute-phase response is a component of the innate immune system and is part of the first line of defense against invading pathogens, which may be compromised by alcohol. We aimed to investigate whether an induced acute-phase response is impaired in long-term ethanol (EtOH)-fed rats. For 6weeks, rats were either fed a Lieber-DeCarli EtOH-containing (36% as calories) liquid diet adlibitum or calorically pair-fed. Then, the rats were injected intraperitoneally with a low dose of lipopolysaccharide (LPS) (0.5mg/kg) to induce an acute-phase response. Two hours after LPS, we measured the plasma concentrations of an array of inflammatory cytokines. Twenty-four hours after LPS, we measured the hepatic mRNA expression and serum concentrations of prominent rat acute-phase proteins. EtOH-fed rats showed either no liver histopathological changes or varying degrees of steatosis. EtOH feeding decreased the spontaneous liver mRNA expression of the prevailing acute-phase protein alpha-2-macroglobulin (α2M) by 30% (p<0.01). LPS immediately increased plasma tumor necrosis factor-alpha and interleukin-6 more than 100-fold in both feeding groups (p<0.001, all) and approximately twice as much in the EtOH-fed rats (p<0.05 and p=0.08, respectively). LPS also induced a variable but marked amplification of (α2M), haptoglobin, alpha-1-acid glycoprotein, and lipocalin-2 liver mRNA expression levels and serum concentrations in both feeding groups (p≤0.01 to 0.001). However, the LPS-induced increases in serum (α2M) and haptoglobin were less pronounced in the EtOH-fed rats, averaging approximately 60% of the concentrations in the pair-fed rats (p<0.01 and p<0.001, respectively). Long-term EtOH exposure in rats reduces the spontaneous hepatic mRNA expression of (α2M) and markedly impairs the hepatic acute-phase response to endotoxin, despite higher pro-inflammatory cytokine release. The same phenomenon may contribute to the increased susceptibility to infections observed in humans with long-term excessive alcohol intake.

Alterations to mitochondrial fatty-acid use in skeletal muscle after chronic exposure to hypoxia depend on metabolic phenotype.

We investigated the effects of chronic hypoxia on the maximal use of and sensitivity of mitochondria to different substrates in rat slow-oxidative (soleus, SOL) and fast-glycolytic (extensor digitorum longus, EDL) muscles. We studied mitochondrial respiration in situ in permeabilized myofibers, using pyruvate, octanoate, palmitoyl-carnitine (PC), or palmitoyl-coenzyme A (PCoA). The hypophagia induced by hypoxia may also alter metabolism. Therefore, we used a group of pair-fed rats (reproducing the same caloric restriction, as observed in hypoxic animals), in addition to the normoxic control fed ad libitum. The resting respiratory exchange ratio decreased after 21 days of exposure to hypobaric hypoxia (simulated elevation of 5,500 m). The respiration supported by pyruvate and octanoate were unaffected. In contrast, the maximal oxidative respiratory rate for PCoA, the transport of which depends on carnitine palmitoyltransferase 1 (CPT-1), decreased in the rapid-glycolytic EDL and increased in the slow-oxidative SOL, although hypoxia improved affinity for this substrate in both muscle types. PC and PCoA were oxidized similarly in normoxic EDL, whereas chronic hypoxia limited transport at the CPT-1 step in this muscle. The effects of hypoxia were mediated by caloric restriction in the SOL and by hypoxia itself in the EDL. We conclude that improvements in mitochondrial affinity for PCoA, a physiological long-chain fatty acid, would facilitate fatty-acid use at rest after chronic hypoxia independently of quantitative alterations of mitochondria. Conversely, decreasing the maximal oxidation of PCoA in fast-glycolytic muscles would limit fatty-acid use during exercise.NEW & NOTEWORTHY Affinity for low concentrations of long-chain fatty acids (LCFA) in mitochondria skeletal muscles increases after chronic hypoxia. Combined with a lower respiratory exchange ratio, this suggests facility for fatty acid utilization at rest. This fuel preference is related to caloric restriction in oxidative muscle and to hypoxia in glycolytic one. In contrast, maximal oxidation for LCFA is decreased by chronic hypoxia in glycolytic muscle and can explain glucose dependence at exercise.

Effect of pectin feeding on obesity development and duodenal alkaline phosphatase activity in Sprague-Dawley rats fed with high-fat/high-energy diet.

Purpose The objective of this study was to evaluate whether pectin feeding would affect the small intestinal function and whether these changes would lead to obesity prevention in rats fed with high-fat diet. Three groups of weaned male rats (ad lib. fed; rats fed with diet containing 15% w/w of citrus pectin; restrictedly pair-fed rats) were fed with either a standard diet (9.5% fat) or a high-fat diet (30% fat) for 10 days. Results Our results revealed that pectin feeding led to significant decreases in body weight, energy intake and fat pad weight in rats fed with the standard as well as high-fat diet. Moreover, compared to the restrictedly pair-fed rats, in both groups of rats fed with the diet containing pectin, significant decrease in duodenal alkaline phosphatase (AP) activity was observed in histochemically stained cryostat sections. In contrast, despite their lower energy intake, restrictedly pair-fed rats showed similar fat pad deposition accompanied by unchanged values of AP activity in comparison to the controls. Conclusions Our findings indicate that daily pectin consumption could be beneficial in suppressing body weight gain and reducing probability of obesity risk in rats fed with a high-fat diet.

Myeloid Mixed Lineage Kinase 3 Contributes to Chronic Ethanol-Induced Inflammation and Hepatocyte Injury in Mice.

Proinflammatory activity of hepatic macrophages plays a key role during progression of alcoholic liver disease (ALD). Since mixed lineage kinase 3 (MLK3)-dependent phosphorylation of JNK is involved in the activation of macrophages, we tested the hypothesis that myeloid MLK3 contributes to chronic ethanol-induced inflammatory responses in liver, leading to hepatocyte injury and cell death. Primary cultures of Kupffer cells, as well in vivo chronic ethanol feeding, were used to interrogate the role of MLK3 in the progression of liver injury. Phosphorylation of MLK3 was increased in primary cultures of Kupffer cells isolated from ethanol-fed rats compared to cells from pair-fed rats. Kupffer cells from ethanol-fed rats were more sensitive to LPS-stimulated cytokine production; this sensitization was normalized by pharmacological inhibition of MLK3. Chronic ethanol feeding to mice increased MLK3 phosphorylation robustly in F4/80(+) Kupffer cells, as well as in isolated nonparenchymal cells. MLK3(-/-) mice were protected from chronic ethanol-induced phosphorylation of MLK3 and JNK, as well as multiple indicators of liver injury, including increased ALT/AST, inflammatory cytokines, and induction of RIP3. However, ethanol-induced steatosis and hepatocyte apoptosis were not affected by MLK3. Finally, chimeric mice lacking MLK3 only in myeloid cells were also protected from chronic ethanol-induced phosphorylation of JNK, expression of inflammatory cytokines, and increased ALT/AST. MLK3 expression in myeloid cells contributes to phosphorylation of JNK, increased cytokine production, and hepatocyte injury in response to chronic ethanol. Our data suggest that myeloid MLK3 could be targeted for developing potential therapeutic strategies to suppress liver injury in ALD patients.

Enhanced activity of hormone sensitive lipase (HSL) in mesenteric but not epididymal fat correlates with higher production of epinephrine in mesenteric adipocytes in rat model of cachectic rheumatoid arthritis

Cachectic rheumatoid arthritis, the less frequent form of the disease, is associated with loss of fat mass and often more severe course of the disease. Its experimental model represents rat adjuvant arthritis (AA) characterized by edema, lack of appetite, sharp body weight and fat loss. As individual fat depots display functional differences, here we studied lipolytic activity and sensitivity to lipolytic stimuli of nodeless epididymal fat (eWAT) and perinodal mesenteric fat (mWAT) depots at the peak of AA. We also examined changes in catecholamine and cytokine levels involved in lipolysis in plasma and/or isolated adipocytes from both WATs to identify the contribution of local, adipocyte-based processes and/or systemic events to adiposity loss in cachectic rheumatoid arthritis. AA was induced to male Lewis rats by complete Freund’s adjuvant. Groups of ad libitum-fed and pair-fed controls were used to distinguish the effects of food restriction from inflammation-induced cachexia. Adipose triglyceride lipase (ATGL), hormone-sensitive lipase (HSL) and its phosphorylated form (pHSL) were analyzed by western blot. CRP and catecholamine levels in plasma or adipocyte lysates were determined using ELISA kits. Cytokine-induced neutrophil chemoattractant-1 (CINC-1/CXCL1), monocyte chemoattractant protein-1 (MCP-1/CCL2), IL-1β, IL-6, IL-10 and leptin in adipocyte lysate were analyzed by quantitative protein microarray. Plasma glycerol and FFA were measured spectrophotometrically. AA rats developed severe cachexia, with lower adiposity in mWAT compared to normal and pair-fed controls, whereas in eWAT the adiposity was similarly reduced in AA and pair-fed groups. ATGL levels in both WATs were not affected by AA or pair feeding. AA upregulated levels of HSL, pHSL and pHSL/HSL ratio in mWAT, whereas none of these parameters has changed in eWAT of AA rats or in either WATs of pair-fed rats. In AA rats plasma glycerol was elevated, whereas FFA concentration was reduced. Plasma norepinephrine and epinephrine were increased in AA compared with both groups of controls. In eWAT adipocytes, AA but not pair feeding, upregulated norepinephrine levels. In mWAT adipocytes, AA rats showed higher epinephrine levels than pair-fed controls. Leptin levels in both WATs were depleted in AA animals in accordance with body weight loss. None of the measured cytokines in eWAT and mWAT was enhanced. Our results demonstrate augmented lipolytic activity in mWAT and not eWAT during cachectic arthritis. The adipocyte-derived cytokines do not seem to contribute to activated lipolysis. We first demonstrated enhanced presence of norepinephrine in perinodal adipocytes that may contribute to the regulation of local lipolytic activity by auto/paracrine fashion and thus provide independent fuel supply to activated lymph nodes.

Improvement in glycemia after glucose or insulin overload in leptin-infused rats is associated with insulin-related activation of hepatic glucose metabolism.

BackgroundInsulin regulates glucose homeostasis through direct effects on the liver, among other organs, with leptin modulating insulin’s hepatic actions. Since central leptin may modify insulin signaling in the liver, we hypothesized that leptin infusion activates hepatic glycogen synthesis following peripheral administration of a bolus of glucose or insulin, thus regulating glycemia.FindingsOral glucose and intraperitoneal insulin tolerance tests were performed in control, intracerebroventricular leptin-treated and pair-fed rats during 14 days. An improvement in glycemia and an increase in hepatic free glucose and glycogen concentrations after glucose or insulin overload were observed in leptin-treated rats. In order to analyze whether the liver was involved in these changes, we studied activation of insulin signaling by Western blotting and multiplex bead immunoassay after leptin infusion. Our studies revealed an increase in phosphorylation of insulin receptor substrate-1 and Akt in leptin-treated rats. Examination of parameters related to glucose uptake and metabolism in the liver revealed an augment in glucose transporter 2 and a decrease in phosphoenolpyruvate carboxylase protein levels in this group.ConclusionsThese results indicate that central leptin increases hepatic insulin signaling, associated with increased glycogen concentrations after glucose or insulin overload, leading to an improvement in glycemia.

Hyperammonemia results in reduced muscle function independent of muscle mass.

The mechanism of the nearly universal decreased muscle strength in cirrhosis is not known. We evaluated whether hyperammonemia in cirrhosis causes contractile dysfunction independent of reduced skeletal muscle mass. Maximum grip strength and muscle fatigue response were determined in cirrhotic patients and controls. Blood and muscle ammonia concentrations and grip strength normalized to lean body mass were measured in the portacaval anastomosis (PCA) and sham-operated pair-fed control rats (n = 5 each). Ex vivo contractile studies in the soleus muscle from a separate group of Sprague-Dawley rats (n = 7) were performed. Skeletal muscle force of contraction, rate of force development, and rate of relaxation were measured. Muscles were also subjected to a series of pulse trains at a range of stimulation frequencies from 20 to 110 Hz. Cirrhotic patients had lower maximum grip strength and greater muscle fatigue than control subjects. PCA rats had a 52.7 ± 13% lower normalized grip strength compared with control rats, and grip strength correlated with the blood and muscle ammonia concentrations (r(2) = 0.82). In ex vivo muscle preparations following a single pulse, the maximal force, rate of force development, and rate of relaxation were 12.1 ± 3.5 g vs. 6.2 ± 2.1 g; 398.2 ± 100.4 g/s vs. 163.8 ± 97.4 g/s; -101.2 ± 22.2 g/s vs. -33.6 ± 22.3 g/s in ammonia-treated compared with control muscle preparation, respectively (P < 0.001 for all comparisons). Tetanic force, rate of force development, and rate of relaxation were depressed across a range of stimulation from 20 to 110 Hz. These data provide the first direct evidence that hyperammonemia impairs skeletal muscle strength and increased muscle fatigue and identifies a potential therapeutic target in cirrhotic patients.

Preventing Gut Leakiness and Endotoxemia Contributes to the Protective Effect of Zinc on Alcohol-Induced Steatohepatitis in Rats1–3

Background: Zinc deficiency has been well documented in alcoholic liver disease.Objective: This study was undertaken to determine whether dietary zinc supplementation provides beneficial effects in treating alcohol-induced gut leakiness and endotoxemia.Methods: Male Sprague Dawley rats were divided into 3 groups and pair-fed (PF) Lieber-DeCarli liquid diet for 8 wk: 1) control (PF); 2) alcohol-fed (AF; 5.00–5.42% wt:vol ethanol); and 3) AF with zinc supplementation (AF/Zn) at 220 ppm zinc sulfate heptahydrate. The PF and AF/Zn groups were pair-fed with the AF group. Hepatic inflammation and endotoxin signaling were determined by immunofluorescence and quantitative polymerase chain reaction (qPCR). Alterations in intestinal tight junctions and aldehyde dehydrogenases were assessed by qPCR and Western blot analysis.Results: The AF rats had greater macrophage activation and cytokine production (P < 0.05) in the liver compared with the PF rats, whereas the AF/Zn rats showed no significant differences (P > 0.05). Plasma endotoxin concentrations of the AF rats were 136% greater than those of the PF rats, whereas the AF/Zn rats did not differ from the PF rats. Ileal permeability was 255% greater in the AF rats and 19% greater in the AF/Zn rats than in the PF rats. The AF group had reduced intestinal claudin-1, occludin, and zona occludens-1 (ZO-1) expression, and the AF/Zn group had upregulated claudin-1 and ZO-1 expression (P < 0.05) compared with the PF group. The intestinal epithelial expression and activity of aldehyde dehydrogenases were elevated (P < 0.05) in the AF/Zn rats compared with those of the AF rats. Furthermore, the ileal expression and function of hepatocyte nuclear factor 4α, which was impaired in the AF group, was significantly elevated in the AF/Zn group compared with the PF group.Conclusions: The results demonstrate that attenuating hepatic endotoxin signaling by preserving the intestinal barrier contributes to the protective effect of zinc on alcohol-induced steatohepatitis in rats.

Pectin supplementation in rats mitigates age-related impairment in insulin and leptin sensitivity independently of reducing food intake.

This study investigates whether pectin supplementation in adult rats can ameliorate age-associated disturbances in peripheral insulin and leptin actions. Seven-month-old male Wistar rats were divided into three groups: control (rats fed ad libitum a standard-diet), pectin (rats fed ad libitum a standard-diet supplemented with 10% pectin), and pair-fed (rats pair-fed to the pectin group). They were sacrificed after 1 month. Pectin and pair-fed rats showed lower body weight gain and food intake than controls and underwent a decrease in leptin levels and an increase in adiponectin levels. Pectin-treated animals, but not pair-fed ones, showed lower body-fat content and HOMA-IR index after dietary intervention. Compared to controls, pectin-treated rats showed a decline in the expression of genes related to energy uptake (WAT) and lipogenesis (WAT and liver), and increased expression levels of lipolysis- and fatty-acid oxidation-related genes (liver). Some of the changes were not evidenced in the pair-fed group. These effects appear to be associated with improved leptin signaling. Ten percent pectin supplementation for 1 month in adult rats decreases body-fat content and ameliorates age-related insulin and leptin resistance more intensely than what could be attributed to the decrease in energy intake, overall contributing to better metabolic health.

Ginsenoside Rb1 increases insulin sensitivity by activating AMP-activated protein kinase in male rats.

Although ginseng has been reported to ameliorate hyperglycemia in animal models and clinical studies, the molecular mechanisms are largely unknown. We previously reported that chronic treatment with ginsenoside Rb1 (Rb1), a major component of ginseng, significantly reduced fasting glucose and improved glucose tolerance in high-fat diet (HFD)-induced obese rats. These effects were greater than those observed in pair-fed rats, suggesting a direct effect of Rb1 on glucose homeostasis, and this possibility was confirmed in the present study. In lean rats fed standard rodent chow, 5-day treatment with Rb1 significantly improved glucose tolerance and enhanced insulin sensitivity. Notably, those effects were not accompanied by reduced food intake or changed body weight. To elucidate the underlying molecular mechanisms, rats fed a HFD for 4 weeks were treated with Rb1 for 5 days. Subsequently, euglycemic-hyperinsulinemic clamp studies found that compared to vehicle, Rb1, while not changing food intake or body weight, significantly increased glucose infusion rate required to maintain euglycemia. Consistent with this, insulin-induced inhibition of hepatic gluconeogenesis was significantly enhanced and hepatic phosphoenolpyruvate carboxykinase and glucose-6-phosphatase gene expression was suppressed. Additionally, glucose uptake was significantly increased in skeletal muscle. While proximal insulin signaling was not changed after Rb1 treatment, increased phosphorylation of TBC1D4, a downstream target of AMPK signaling, appears to be a key part of the mechanism for Rb1-stimulated glucose uptake in skeletal muscle. These findings indicate that Rb1 has multiple effects on glucose homeostasis, and provide strong rationale for further evaluation of its potential therapeutic role.

Single‐pedicled Fasciocutaneous Flap Survival In Aged Rat Model of Chronic Alcoholism

Impact of chronic ethanol consumption on signaling events underlying composite pedicled flap survival was examined in isocaloric pair-fed Sprague-Dawley rats that consumed Lieber-DeCarli '82 liquid 1000 kcal/L diet consisting of 15.1% protein, 35.9% fat and either 49% carbohydrates (control group) or 13.5% carbohydrates and 35.5% ethanol (alcoholic group). Six pairs of rats that were fed such diet for at least one year underwent surgery, where a 3:8 width to length ratio pedicled fasciocutaneous flap based on the inferior epigastric artery was raised in each rat and rotated into a defect created in the ventral surface. Tissue from the contralateral recipient site was snap-frozen to serve as baseline (day 0) for comparison with the flap harvested on 9th post-operative day. The endogenous expression of VEGF-A, FGF-2, IL-6, IL-10, TNFα, TIMP-1, MMP-9, Hsp47, NF-κB, cleaved Caspase-9, β-Actin, GAPDH and activating phosphorylation of Akt, STAT3, PLC-γ, eNOS, Src, FAK, p38 MAPK and ERK1/2 proteins were measured by Western Blotting in homogenates of proximal, middle and distal segments of the flaps to reflect the pro-angiogenic, inflammatory, anti-apoptotic, stress, motogenic and mitogenic cellular responses. The middle and distal segments were less prone to necrosis in control than in alcohol-fed animals that, in turn, expressed significantly lower levels of activated Akt, STAT3 and p-ERK. This data implies that chronic alcohol consumption leads to flap failure by impairing multiple signaling pathways whose cross-talk is essential for cell survival and expedient wound healing.

Scopoletin 보충이 만성 알코올을 급여한 흰쥐의 인슐린저항성 및 항산화방어계에 미치는 영향

This study investigated the effects of scopoletin (6-methoxy-7-hydroxycoumarin) supplementation on insulin resistance and the antioxidant defense system in chronic alcohol-fed rats. Rats were fed a Lieber-Decarli liquid diet containing 5% ethanol with or without two doses of scopoletin (0.01 and 0.05 g/L) for 8 weeks. Pair-fed rats received an isocaloric carbohydrate liquid diet. Chronic alcohol did not affect fasting serum glucose levels, although it induced glucose intolerance and hyperinsulinemia compared with the pair-fed group and led to insulin resistance. Both doses of scopoletin similarly improved glucose intolerance, serum insulin level, and insulin resistance. Scopoletin supplementation significantly activated phosphatidyl inositol 3-kinase, which was inhibited by chronic alcohol. Two doses of scopoletin up-regulated hepatic mRNA expression and activity of glucokinase as well as down-regulated mRNA expression and activity of glucose-6-phosphatase compared with the alcohol control group. Both doses of scopo- letin significantly reduced cytochrome P450 2E1 activity and elevated aldehyde dehydrogenase 2 activity, resulting in a lower serum acetaldehyde level compared with the alcohol control group. Chronic alcohol suppressed hepatic mRNA expression and activities of antioxidant enzymes such as superoxide dismutase, catalase, and glutathione perox- idase; however, they were reversed by scopoletin supplementation, which reduced hydrogen peroxide and lipid peroxide levels in the liver. These results indicate that dietary scopoletin attenuated chronic alcohol-induced insulin resistance and activated the antioxidant defense system through regulation of hepatic gene expression in glucose and antioxidant metabolism.

Dietary intake of high-dose biotin inhibits spermatogenesis in young rats.

To characterize a new function of the water-soluble vitamin, biotin, in reproduction and early growth in mammals, the effects of high dietary doses of biotin on early spermatogenesis were biochemically and histologically investigated in male rats. Weaned rats were fed a CE-2 (control) diet containing 0.00004% biotin, or a control diet supplemented with 0.01%, 0.1%, or 1.0% biotin. Pair-fed rats were fed a control diet that was equal in calories to the amount ingested by the 1.0% biotin group, because food intake was decreased in the 1.0% biotin group. Food intake and body weight gain were lower in the 1.0% biotin group than in the control group. The kidney, brain and testis weights were significantly lower in the 1.0% biotin group than in the pair-fed group after 6 weeks of feeding. The accumulation of biotin in the liver and testis increased in a dose-dependent manner. In the 1.0% biotin group, the number of mature sperm was markedly lower, that of sperm with morphologically abnormal heads, mainly consisting of round heads, had increased. In addition, the development of seminiferous tubules was inhibited, and few spermatogonia and no spermatocytes were histologically observed. These results demonstrated that the long-term intake of high-dose biotin inhibited spermatogenesis in young male rats.

Pathophysiological Changes by Short-term Food Restriction in Obese Diabetic Rats

Aim: Reduced calorie intake by food restriction leads to extension of life span by exerting beneficial effects on metabolism. Zucker diabetic fatty (ZDF) rat is a type 2 diabetic model with obesity, showing the incidence of diabetes after 7 weeks of age. The present study investigated effects of 2-week food restriction on the pancreas in ZDF rats. Methods: ZDF rats were pair-fed with Zucker lean (ZL) rats from 7 to 9 weeks of age (The amount of food in ZDF rats was calculated based on the daily food consumption in ZL rats). Body weight and biochemical parameters such as serum glucose, triglyceride, total cholesterol, non-esterified fatty acid and insulin levels, were evaluated. After necropsy, islet size and insulin content in pancreas were measured. Original Research Article Miyajima et al.; BJMMR, 9(6): 1-7, 2015; Article no.BJMMR.18712 2 Results: The ZDF rats showed increased blood glucose, insulin and lipid levels at 7 weeks as compared with those in ZL rats. After food restriction for 2 weeks, the blood glucose, insulin, and triglyceride levels in pair-fed ZDF rats were decreased. The islet size in pair-fed ZDF rats decreased as compared with that in ad lib-fed ZDF rats, but the insulin content in pancreas of pairfed ZDF rats increased remarkably. Conclusion: ZDF rats showed improvement of pancreatic disorders by food restriction. Caloric restriction applied at an insulin resistant pre-diabetic stage is an effective means for preventing the pancreatic disorder in diabetes with obesity.

Effects on Fat Distribution by Food Restriction in Zucker Diabetic Fatty Rats

Aim: Zucker diabetic fatty (ZDF) rat is a type 2 diabetic model with hyperphagia, obesity, and the overt fat storage. The present study investigated effects of food restriction for 15 weeks on the fat distribution in ZDF rats. Methods: ZDF rats were pair-fed with Zucker Lean (ZL) rats from 9 to 24 weeks of age. Body weight and blood chemistry parameters, such as glucose, insulin, triglyceride, total cholesterol and non-esterified fatty acid, were measured every two weeks. The visceral and subcutaneous fat weights were measured at 24 weeks of age by computed tomography (CT) analysis, and the total fat weight and the ratio of visceral fat weight to subcutaneous fat weight (V/S ratio) were determined. Results: The ZDF rats showed obesity, hyperglycemia, hyperinsulinemia, and hyperlipidemia as compared with the ZL rats. Pair-fed ZDF rats showed a temporary decrease in body weight and a suppression of hyperglycemia, but the blood insulin and lipid levels increased. Total fat weight was about 2.4 times higher in the ZDF rats than the ZL rats. The total fat weight in Pair-fed ZDF rats Short Research Article Ohta et al.; BJMMR, 7(8): 699-704, 2015; Article no.BJMMR.2015.378 700 was increased by 56%, but the V/S ratio was decreased by 38% at 24 weeks of age. Conclusion: The change of fat distribution by dietary restriction may be related to the improvement of glucose and lipid metabolic disorders in diabetes mellitus with obesity.

The role of ventral midline thalamus in cholinergic-based recovery in the amnestic rat

The thalamus is a critical node for several pathways involved in learning and memory. Damage to the thalamus by trauma, disease or malnourishment can impact the effectiveness of the prefrontal cortex (PFC) and hippocampus (HPC) and lead to a profound amnesia state. Using the pyrithiamine-induced thiamine deficiency (PTD) rat model of human Wernicke–Korsakoff syndrome, we tested the hypothesis that co-infusion of the acetylcholinesterase inhibitor physostigmine across the PFC and HPC would recover spatial alternation performance in PTD rats. When cholinergic tone was increased by dual injections across the PFC–HPC, spontaneous alternation performance in PTD rats was recovered. In addition, we tested a second hypothesis that two ventral midline thalamic nuclei, the rhomboid nucleus and nucleus reuniens (Rh–Re), form a critical node needed for the recovery of function observed when cholinergic tone was increased across the PFC and HPC. By using the GABAA agonist muscimol to temporarily deactivate the Rh–Re the recovery of alternation behavior obtained in the PTD model by cholinergic stimulation across the PFC–HPC was blocked. In control pair-fed (PF) rats, inactivation of the Rh–Re impaired spontaneous alternation. However, when inactivation of the Rh–Re co-occurred with physostigmine infusions across the PFC–HPC, PF rats had normal performance. These results further demonstrate that the Rh–Re is critical in facilitating interactions between the HPC and PFC, but other redundant pathways also exist.

Acute Acidosis Attenuates Leucine Stimulated Signal Transduction and Protein Synthesis in Rat Skeletal Muscle

Background: Critical illnesses are often complicated by acute metabolic acidosis, which if persistent, adversely affects outcome. Among the harmful effects that it might cause are impaired utilization of nutrients, increased proteolysis and depressed protein synthesis, leading to muscle wasting. As the amino acid leucine stimulates protein synthesis by activating mTOR signaling, we explored whether in acidosis, impaired leucine-stimulated signaling might be a contributor to the depressed protein synthesis. Methods: Male pair-fed rats were gavaged with NH₄Cl (acidosis) or NaCl (control) for 2 days and then gavaged once with leucine and sacrificed 45 min later. Extensor digitorum longus muscles were isolated, incubated with or without leucine and protein synthesis measured. The anterior tibial muscle signaling was analysed by Western immunobloting. Results: Despite pair-feeding, acidotic rats lost body and muscle weight vs. controls. Moreover, leucine-induced protein synthesis in isolated muscle from acidotic rats was impaired. In-vivo, 45 min after an oral leucine load, anterior tibial muscle mTOR and 4E-BP1 phosphorylation increased significantly and comparably in control and acidotic rats. In contrast, leucine-stimulated phosphorylation of S6K1, a regulator of translation initiation and protein synthesis, was attenuated to approximately 56% of the control value (p < 0.05). Conclusion: This study reveals that an acute metabolic acidosis impairs leucine-stimulated protein synthesis and activation of signaling downstream of mTOR at the level of S6K1. We propose that this S6K1abnormality may account in part, for the resistance to leucine-stimulated muscle protein synthesis, and may thereby contribute to the impaired nutrient utilization and ultimately the muscle wasting that develops in acidosis.

Dietary umbelliferone attenuates alcohol-induced fatty liver via regulation of PPARα and SREBP-1c in rats

This study investigated the effects of umbelliferone (UF) on alcoholic fatty liver and its underlying mechanism. Rats were fed a Lieber–DeCarli liquid diet with 36% of calories as alcohol with or without UF (0.05 g/L) for 8 weeks. Pair-fed rats received an isocaloric carbohydrate liquid diet. UF significantly reduced the severity of alcohol-induced body weight loss, hepatic lipid accumulation and droplet formation, and dyslipidemia. UF decreased plasma AST, ALT, and γGTP activity. UF significantly reduced hepatic cytochrome P450 2E1 activities and increased alcohol dehydrogenase and aldehyde dehydrogenase 2 activities compared to the alcohol control group, which resulted in a lower plasma acetaldehyde level in the rats that received UF. Chronic alcohol exposure inhibited hepatic AMPK activation compared to the pair-fed rats, which was reversed by UF supplementation. UF also significantly suppressed the lipogenic gene expression (SREBP-1c, SREBP-2, FAS, CIDEA, and PPARγ) and elevated the fatty acid oxidation gene expression (PPARα, Acsl1, CPT, Acox, and Acaa1a) compared to the alcohol control group, which could lead to inhibition of FAS activity and stimulation of CPT and fatty acid β-oxidation activities in the liver of chronic alcohol-fed rats. These results indicated that UF attenuated alcoholic steatosis through down-regulation of SREBP-1c-mediated lipogenesis and up-regulation of PPARα-mediated fatty acid oxidation. Therefore, UF may provide a promising natural therapeutic strategy against alcoholic fatty liver.