Amino acids change liver growth factors gene expression in malnourished rats

Background: Glutamine and proline are metabolized in the liver and may collaborate on its regeneration. Parenteral nutrition (PN) containing either glutamine or proline was given to partially hepatectomized rats. The total RNA content and growth factor gene expression in hepatic remnants was measured, to determine the effects of these amino acid supplementation on the expression of growth factors during liver regeneration. Methods: Wistar rats nourished (HN) and malnourished (HM) were hepatectomized and divided in two groups: 20 receiving PN enriched with Alanyl-Glutamine (HN-Gln and HM-Gln) and 20 PN enriched with proline+alanine (HN-Pro and HM-Pro). The control groups comprised 7 nourished (CN) and 7 malnourished (CM) rats that didn`t undergo surgery. Growth factor and thymidine kinase mRNA levels were measured by RT-PCR. Results: In nourished rats, total hepatic RNA levels were lower in the HN-Gln and HN-Pro groups (0.75 and 0.63 mu g/mg tissue, respectively) than in control group (1.67 mu g/mg tissue) (P<0.05). In malnourished rats, total hepatic RNA content was higher in the HM-Pro group than FIN-Pro, HM-Gln, and CM (3.18 vs. 0.63, 0.93 and 1.10 mu g/mg, respectively; P<0.05). Hepatocyte growth factor mRNA was more abundant in the HM-Gln group when compared to CM (031 vs. 0.23 arbitrary units) and also in HM-Pro in relation to HM-Gln, HN-Pro, and CM (0.46 vs. 033 and 0.23, respectively, P<0.05). Conclusions: Proline or glutamine supplementation in malnourished rats improves total RNA content in the remnant hepatic tissue. Amino acids administration increased HGF gene expression after partial hepatectomy in malnourished rats, with a greater effect of proline than glutamine.

Influence of preoperative supplementation of omega-3 fatty acid in the healing of colonic anastomoses in malnourished rats receiving paclitaxel

OBJECTIVE: To evaluate the effect of preoperative supplementation of omega-3 fatty acids on the healing of colonic anastomoses in malnourished rats receiving paclitaxel. METHODS: we studied 160 male Wistar rats, divided in two groups: one subjected to malnutrition by pair feeding (M) for four weeks, and another that received food ad libitum (W). In the fourth week, the groups were further divided into two subgroups that received omega-3 or olive oil by gavage. The animals were submitted to colonic transection and end-to-end anastomosis. After the operation, each of the four groups was divided into two subgroups that received intraperitoneal isovolumetric solutions of saline or paclitaxel. RESULTS: mortality was 26.8% higher in the group of animals that received paclitaxel (p = 0.003). The complete rupture strength was greater in well-nourished-oil Paclitaxel group (WOP) compared with the the malnourished-oil Paclitaxel one (MOP). The collagen maturation index was higher in well-nourished-oil saline group (WOS) in relation to the malnutrition-oil-saline group (MOS), lower in malnourished-oil-saline group (MOS) in relation to malnourished-ômega3-saline one (M3S) and lower in the well-nourished-omega3-saline group (W3S) compared with the malnourished-omega3-saline (M3S). The blood vessel count was higher in the malnourished-oil-saline group (MOS) than in the malnourished-oil-paclitaxel group (MOP) and lower in the malnourished-oil-saline group (MOS) in relation to the malnourished-omega3-paclitaxel group (M3P). CONCLUSION: supplementation with omega-3 fatty acids was associated with a significant increase in the production of mature collagen in malnourished animals, with a reversal of the harmful effects caused by malnutrition associated with the use of paclitaxel on the rupture strength, and with a stimulus to neoangiogenesis in the group receiving paclitaxel.

Response threshold to aversive stimuli in stimulated early protein-malnourished rats

Two animal models of pain were used to study the effects of short-term protein malnutrition and environmental stimulation on the response threshold to aversive stimuli. Eighty male Wistar rats were used. Half of the pups were submitted to malnutrition by feeding their mothers a 6% protein diet from 0 to 21 days of age while the mothers of the other half (controls) were well nourished, receiving 16% protein. From 22 to 70 days all rats were fed commercial lab chow. Half of the animals in the malnourished and control groups were maintained under stimulating conditions, including a 3-min daily handling from 0 to 70 days and an enriched living cage after weaning. The other half was reared in a standard living cage. At 70 days, independent groups of rats were exposed to the shock threshold or to the tail-flick test. The results showed lower body and brain weights in malnourished rats when compared with controls at weaning and testing. In the shock threshold test the malnourished animals were more sensitive to electric shock and environmental stimulation increased the shock threshold. No differences due to diet or environmental stimulation were found in the tail-flick procedure. These results demonstrate that protein malnutrition imposed only during the lactation period is efficient in inducing hyperreactivity to electric shock and that environmental stimulation attenuates the differences in shock threshold produced by protein malnutrition

Can calories from ethanol contribute to body weight preservation by malnourished rats?

Our objective was to compare the use of calories from ethanol by well-nourished and malnourished rats in terms of body weight. Female Wistar rats weighing 170-180 g at the beginning of the study were used. The animals were divided into two groups (N = 12 each): group W received water ad libitum and group E an ethanol solution ad libitum as the only source of liquid throughout the experiment. The concentration of ethanol was increased weekly from 0 to 5, 10, 20 and 40% (v/v). In the well-nourished phase (A), all rats received food ad libitum (AW and AE). Ethanol treatment (AE) was then interrupted and water was offered to both groups. After 2 weeks both AW and AE rats were submitted to food restriction (50% of group AW food consumption), thus initiating the malnutrition phase (M). Liquid was offered as described before to the same W (MW) and E (ME) groups. The weight gain during the 1-week treatment of AE rats was similar to that of AW animals only when AE rats received the 5% (v/v) ethanol solution (9.16 vs 10.47 g). Weight loss was observed after exposure to 10% ethanol (P < 0.05) in spite of maintenance of caloric intake. Malnourished rats presented weight loss, which was attenuated by ethanol intake up to the 20% (v/v) solution and was related to an increased caloric offer. This effect was not observed with the 40% ethanol solution (-9.98 g). These data suggest that calories from ethanol were used to maintain body weight up to the concentration of 10% (v/v) (well-nourished) and 20% (v/v) (malnourished) and that ethanol has a toxic profile which depends on nutritional status.

The interaction of housing condition and acute immobilization stress on the elevated plus-maze behaviors of protein-malnourished rats

Protein malnutrition induces structural, neurochemical and functional alterations in the central nervous system, leading to behavioral alterations. In the present study, we used the elevated plus-maze (EPM) as a measure of anxiety to evaluate the interaction between acute immobilization and housing conditions on the behavior of malnourished rats. Pups (6 males and 2 females) were fed by Wistar lactating dams receiving a 6% (undernourished) or 16% (well-nourished) protein diet. After weaning, the animals continued to receive the same diets ad libitum until 49 days of age when they started to receive a regular lab chow diet. From weaning to the end of the tests on day 70, the animals were housed under two different conditions, i.e., individual or in groups of three. On the 69th day, half of the animals were submitted to immobilization for 2 h, while the other half were undisturbed, and both groups were tested 24 h later for 5 min in the EPM. Independent of other factors, protein malnutrition increased, while immobilization and social isolation per se decreased, EPM exploration. Analysis of the interaction of diet vs immobilization vs housing conditions showed that the increased EPM exploration presented by the malnourished group was reversed by acute immobilization in animals reared in groups but not in animals reared individually. The interaction between immobilization and housing conditions suggests that living for a long time in social isolation is sufficiently stressful to reduce the responses to another anxiogenic procedure (immobilization), while living in groups prompts the animals to react to acute stress. Thus, it is suggested that housing condition can modulate the effects of an anxiogenic procedure on behavioral responses of malnourished rats in the EPM.

Differential response to gepirone but not to chlordiazepoxide in malnourished rats subjected to learned helplessness

The learned helplessness (LH) paradigm is characterized by learning deficits resulting from inescapable events. The aims of the present study were to determine if protein-calorie malnutrition (PCM) alters learning deficits induced by LH and if the neurochemical changes induced by malnutrition alter the reactivity to treatment with GABA-ergic and serotonergic drugs during LH. Well-nourished (W) and PCM Wistar rats (61 days old) were exposed or not to inescapable shocks (IS) and treated with gepirone (GEP, 0.0-7.5 mg/kg, intraperitoneally, N = 128) or chlordiazepoxide (0.0-7.5 mg/kg, intraperitoneally, N = 128) 72 h later, 30 min before the test session (30 trials of escape learning). The results showed that rats exposed to IS had higher escape latency than non-exposed rats (12.6 ± 2.2 vs 4.4 ± 0.8 s) and that malnutrition increased learning impairment produced by LH. GEP increased the escape latency of W animals exposed or non-exposed to IS, but did not affect the response of PCM animals, while chlordiazepoxide reduced the escape deficit of both W and PCM rats. The data suggest that PCM animals were more sensitive to the impairment produced by LH and that PCM led to neurochemical changes in the serotonergic system, resulting in hyporeactivity to the anxiogenic effects of GEP in the LH paradigm.

Reduced pancreatic β-cell mass is associated with decreased FoxO1 and Erk1/2 protein phosphorylation in low-protein malnourished rats

A low-protein diet leads to functional and structural pancreatic islet alterations, including islet hypotrophy. Insulin-signaling pathways are involved in several adaptive responses by pancreatic islets. We determined the levels of some insulin-signaling proteins related to pancreatic islet function and growth in malnourished rats. Adult male Wistar rats (N = 20 per group) were fed a 17% protein (normal-protein diet; NP) or 6% protein (low-protein diet; LP), for 8 weeks. At the end of this period, blood glucose and serum insulin and albumin levels were measured. The morphometric parameters of the endocrine pancreas and the content of some proteins in islet lysates were determined. The β-cell mass was significantly reduced (≅65%) in normoglycemic but hypoinsulinemic LP rats compared to NP rats. Associated with these alterations, a significant 30% reduction in insulin receptor substrate-1 and a 70% increase in insulin receptor substrate-2 protein content were observed in LP islets compared to NP islets. The phosphorylated serine-threonine protein kinase (pAkt)/Akt protein ratio was similar in LP and NP islets. The phosphorylated forkhead-O1 (pFoxO1)/FoxO1 protein ratio was decreased by 43% in LP islets compared to NP islets (P < 0.05). Finally, the ratio of phosphorylated-extracellular signal-related kinase 1/2 (pErk1/2) to total Erk1/2 protein levels was decreased by 71% in LP islets compared to NP islets (P < 0.05). Therefore, the reduced β-cell mass observed in LP rats is associated with the reduction of phosphorylation in mitogenic-related signals, FoxO1 and Erk proteins. The cause/effect basis of this association remains to be determined.

Augmentation of insulin secretion by leucine supplementation in malnourished rats: possible involvement of the phosphatidylinositol 3-phosphate kinase/mammalian target protein of rapamycin pathway

A regimen of low-protein diet induces a reduction of pancreatic islet function that is associated with development of metabolic disorders including diabetes and obesity afterward. In the present study, the influence of leucine supplementation on metabolic parameters, insulin secretion to glucose and to amino acids, as well as the levels of proteins that participate in the phosphatidylinositol 3-phosphate kinase (PI3K) pathway was investigated in malnourished rats. Four groups were fed with different diets for 12 weeks: a normal protein diet (17%) without (NP) or with leucine supplementation (NPL) or a low (6%)-protein diet without (LP) or with leucine supplementation (LPL). Leucine was given in the drinking water during the last 4 weeks. As indicated by the intraperitoneal glucose tolerance test, LPL rats exhibited increased glucose tolerance as compared with NPL group. Both NPL and LPL rats had higher circulating insulin levels than controls. The LPL rats also showed increased insulin secretion by pancreatic islets in response to glucose or arginine compared with those observed in islets from LP animals. Glucose oxidation was significantly reduced in NPL, LP, and LPL isolated islets as compared with NP; but no alteration was observed for leucine and glutamate oxidation among the 4 groups. Western blotting analysis demonstrated increased PI3K and mammalian target protein of rapamycin protein contents in LPL compared with LP islets. A significant increase in insulin-induced insulin receptor substrate I associated PI3K activation was also observed in LPL compared with LP islets. These findings indicate that leucine supplementation can augment islet function in malnourished rats and that activation of the PI3K/maminalian target protein of rapamycin pathway may play a role in this process. (C) 2010 Elsevier Inc. All rights reserved.

Preliminary report: Leucine supplementation enhances glutamate dehydrogenase expression and restores glucose-induced insulin secretion in protein-malnourished rats

Low-protein diet impairs insulin secretion in response to nutrients and may induce several metabolic disorders including diabetes, obesity, and cardiovascular disease. In the present study, the influence of leucine supplementation on glutamate dehydrogenase (GDH) expression and glucose-induced insulin secretion (GIIS) was investigated in malnourished rats. Four groups were fed with different diets for 12 weeks: a normal-protein diet (17%) without or with leucine supplementation or a low (6%)-protein diet without (LP) or with leucine supplementation (LPL). Leucine (1.5%) was supplied in the drinking water. Western blotting analysis revealed reduced GIN! expression in LP, whereas LPL displayed improved GDH expression, similar to control. The GHS and leucinc-induced insulin release were also enhanced in LPL compared with LP and similar to those observed in rats fed a normal-protein diet without leucine supplementation. In addition, GDH allosteric activators produced an increased insulin secretion in LPL. These findings indicate that leucine supplementation was able to increase GDH expression leading to Cl IS restoration, probably by improved leucine metabolic pathways. (C) 2010 Elsevier Inc. All rights reserved.

Reduced pancreatic β-cell mass is associated with decreased FoxO1 and Erk1/2 protein phosphorylation in low-protein malnourished rats

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

The gastroprotective effect of the essential oil of Croton cajucara is different in normal rats than in malnourished rats

It has been shown previously that malnourished rats are resistant to acute gastric lesions but not to subchronic gastric ulceration. It also has been demonstrated that the essential oil obtained from the bark of Croton cajucara (Sacaca) has antiulcer properties. In the present study, the ability of this essential oil to prevent the formation of gastric ulcers in rats fed a diet with 17 % protein (normoproteic rats) or 6 % protein (malnourished rats) was investigated. At a dose of 100 mg/kg body weight, orally, the essential oil significantly reduced the gastric injury caused by indomethacin (25 % after 2 h and 70 % after 4 h) only in normoproteic rats. In the pylorus ligature model, the essential oil increased the pH and gastric volume, but decreased the total acid concentration in both groups when compared to the respective control group. The essential oil significantly increased prostaglandin E2 production in glandular cells by 50 % compared to the controls in both groups of rats. In addition, the amount of gastric mucus was two-fold higher in malnourished rats than in normoproteic rats. The present results show that the enhanced protective effect of essential oil in malnourished rats involved an increase in prostaglandin E2 production and mucus secretion, which are both factors that protect the gastric mucosa against damage. In agreement with this, malnourished rats always had a lower number of acute gastric ulcers.

The antiulcer effect of Croton cajucara Benth in normoproteic and malnourished rats

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Tensile strength and histological changes of abdominal aorta of malnourished rats

The resistance of the abdominal aorta of rats after 6, 7 and 8 weeks of malnutrition, compared with control animals, was evaluated by longitudinal tensiometry. Weakness of this vessel in malnourished rats was demonstrated; microscopic examination of the aorta stained by Masson, Calleja and hematoxylin-eosin methods showed a decrease in amorphous ground substance and an increase in the width of elastic laminae. There was no visible alteration either in the endothelial lining layer or in the smooth muscle fibers. Such alterations of the aorta are, to the authors' knowledge, the first reported modifications in the peripheral vasculature after malnutrition.

Reduced insulin secretion in response to nutrients in islets from malnourished young rats is associated with a diminished calcium uptake

Changes in Ca-45 uptake and insulin secretion in response to glucose, leucine, and arginine were measured in isolated islets derived from 4-week-old rats born of mothers maintained with normal protein (NP, 17%) or low protein (LP, 6%) diet during pregnancy and lactation. Glucose provoked a dose-dependent stimulation of insulin secretion in both groups of islets, with basal (2.8 mmol/L glucose) and maximal release (27.7 mmol/L glucose) significantly reduced in LP compared with NP islets. In the LP group the concentration-response curve to glucose was shifted to the right compared with the NP group, with the half-maximal response occurring at 16.9 and 13.3 mmol/L glucose, respectively. In LP islets, glucose-induced first and second phases of insulin secretions were drastically reduced. In addition, insulin response to individual amino acids, or in association with glucose, was also significantly reduced in the LP group compared with NP islets. Finally, in LP islets the Ca-45 uptake after 5 minutes or 90 minutes of incubation (which reflect mainly the entry and retention, respectively, of Ca2+), was lower than in NP islets. These data indicate that in malnourished rats both initial and sustained phases of insulin secretion in response to glucose were reduced. This poor secretory response to nutrients seems to be the consequence of an altered Ca2+ handling by malnourished islet cells. (J. Nutr. Biochem. 10:37-43, 1999) (C) Elsevier B.V. 1999. All rights reserved.