| BackgroundProtein-energy malnutrition (PEM) has recognized as a common problem among pediatric patients in developing countries, hospitalized (and especially elderly hospitalized) patients, and cancer patients.Protein-energy malnutrition can lead to poor response to treatment in patients after surgery, delayed wound healing, immune dysfunction, increased infectious and non-infectious complications, prolonged hospitalization and increased mortality and other adverse clinical outcomes.The study found that in patients with severe protein restriction (kwashiorkor) or protein-energy deprivation, growth retardation is associated with low serum insulin-like growth factor (IGF)-1 levels despite elevated serum growth hormone (GH) concentrations.Human growth hormone is synthesis and secretion from the hypothalamus gland of anterior pituitary cells, the molecular size of 22kDa protein peptide chain.GHRH can induce its synthesis and secretion, and by insulin-like growth factor-Ⅰ (IGF-Ⅰ) negative feedback inhibition of its synthesis and secretion.The release of GH is pulsatile with bursts occurring especially during sleep; however, the size and the number of bursts is influenced by age, gender, illnesses and nutritional status. GH regulates the production of IGF-Ⅰ, and together GH and IGF-Ⅰ stimulate skeletal growth. IGF-1 is a member of the insulin super-family. It was originally discovered as a mediator of GH action on somatic cell growth but it has also been shown to be an important regulator of cell metabolism, differentiation, and survival.IGF-1 is mainly synthesized in the liver. It is found in blood and other body fluids as a complex with specific high-affinity IGF-binding proteins (IGFBP-1 to IGFBP-6).The body IGF-1 content is affected by many factors, and nutritional status is a key regulator of circulating and tissue-bound IGF-1.Protein and energy content of the diet influence plasma IGF-1 concentrations and IGF-1 levels are reduced and increased the amount of urinary nitrogen loss during conditions of energy restriction such as short-term fasting and malnutrition. Furthermore, it was shown that IGF-1 promotes protein synthesis within the body.IGF-1 synthesis is a well-regulated process that depends on the availability of energy and amino acids. Leucine activates the phosphoinositide-3 kinase (PI3K)-Akt anabolic pathway, which in turn triggers a cascade of intracellular signals that culminate in the activation of protein translation. Our previous study found that leucine-enriched high-protein enteral nutrition could promote IGF-1 system synthesis in a rat model of trauma-hemorrhagic shock. Although, intravenous infusion or ingestion of leucine can acutely increase the protein synthesis rate, only few studies have tested whether long-term supplementation with leucine promotes significant changes in the GH-IGF-1 system during severe malnutrition.Therefore, the purpose of this study was to investigate if long-term dietary supplementation with different doses of leucine could regulate GH-IGF-1 system function and mTOR-related signal transduction in skeletal muscles in a rat model of severe malnutrition.ObjectiveFirst,The aim of this study was to establish a rat model of protein-energy malnutrition to simulate the clinical high catabolic state of in patients with malnutrition, and further to investigate the changes of GH-IGF-1 system function, as well as explore malnutrition lead to acquired growth hormone resistance mechanisms.Second, Our study aimed at investigating if long-term leucine supplementation could modulate GH-insulin-like growth factor (IGF)-1 system function and mTOR-related signal transduction in skeletal muscles in a rat model of severe malnutrition. It could provide a theoretical basis for clinicians dealing with malnourished patients with GH resistance.MethodsPart one:1.1 Male Sprague-Dawley rats were fed the AIN-93M diet.Animals were housed in individual cages in a climatized environment at 23±2℃ and a relative air humidity of 55±10% under a 12-h light/12-h dark cycle ad libitum for 2 weeks to let them adapt to the new environmental conditions and to determine their food intake. The rats’mean food intake was used to determine the amount of chow offered daily during the food restriction phase. Animals were randomized into 2 groups:the control group(CON,n=10)receiving a chow diet for4 weeks and ad libitum water, the malnourished group(M,n=10)subjected to 50% food restriction for 4 weeks and ad libitum water. After 4 weeks the rats were sacrificed under anesthesia by intraperitoneal collect tissue and blood specimens.1.2 Data are expressed as means±tandard deviation (SD). The two groups were compared using the t test.using the SPSS19.0 software. A p value of<0.05 was considered statistically significant.Part two:2.1 Male Sprague-Dawley rats were fed the AIN-93M diet.Animals were housed in individual cages in a climatized environment at 23±2℃ and a relative air humidity of 55±10% under a 12-h light/12-h dark cycle ad libitum for 2 weeks to let them adapt to the new environmental conditions and to determine their food intake. The rats’mean food intake was used to determine the amount of chow offered daily during the food restriction phase. Animals were randomized into 4 groups:the control group (CON,n=10)receiving a chow diet for 6 weeks and ad libitum water, the M-CON group (subjected to 50% food restriction for 4 weeks, followed by 2 weeks of receiving a chow diet,n=10), the MC-L group (subjected to 50% food restriction for 4 weeks, followed by 2 weeks of receiving a chow diet supplemented with low-dose leucine [0.675g leucine/kg of body weight] by oral gavage,n=10), and the MC-H group (subjected to 50% food restriction for 4 weeks, followed by 2 weeks of receiving a chow diet supplemented with high-dose leucine [1.35g leucine/kg of body weight] by oral gavage,n=10). After 6 weeks the rats were sacrificed under anesthesia by intraperitoneal collect tissue and blood specimens.2.2 Data are expressed as means±standard deviation (SD). Statistical evaluation among groups was performed by one-way ANOVAfollowed by the Fisher’s least significant difference post-hoc analysis, using the SPSS 19.0 software.In the case of heterogeneous variances, we used the Dunnet’s T3test. A p value of <0.05 was considered statistically significant.ResultsPart one:1.Serum GH, IGF-1, IGFBP-1, and IGFBP-3 concentrationsCompared with CON group, the serum GH and IGFBP-1 levels were significantly increased in M group(p<0.01).Compared with CON group, the serum IGF-1 and IGFBP-3 levels were significantly decreased in M group(p<0.01).2.Western Blot ResultsCompared with CON group, the skeletal muscle tissue phosphorylated of Akt, mTOR and S6K1 levels were significantly decreased in M group(p<0.01).Compared with CON group,GHR levels in hepatic tissue was significantly reduced in M group(p<0.01).3. Hepatic expression of GHR, IGF-1, IGFBP-1, and IGFBP-3 mRNACompared with the control group, the malnourished group rat liver tissue GHR, IGF-1 and IGFBP-3 mRNA relative expression levels were significantly reduced(p <0.01).4. Changes in body weight of ratsGradually increased the weight of rats in the control group during the experiment, and gradually reduced the body weight of rats malnourished group After 4 weeks, the M animals lost 40% of their body weight(p<0.01).5. Changes in rat skeletal muscle wet weightCompared with CON group, the gastrocnemius, soleus and extensor digitorum longus wet weight were significantly decreased in M group (p<0.01).Part TWO:1.Serum GH, IGF-1, IGFBP-1, and IGFBP-3 concentrationsAt the study end point (after re-feeding),no statistical differences in GH levels were noted between the M-CON, M-L, M-H, and CON groups (p>0.05).Serum IGF-1 levels were significantly lower in the M-CON groups compared to the CON group (p< 0.01).Moreover, no differences in IGF-1 levels were observed between the M-L, M-H, and CON groups(p>0.05).Serum IGFBP-1 levels in the CON group were markedly lower than in the other roups at the study end point. The M-CON group showed higher IGFBP-1 concentrations than the M-L and M-H groups (p<0.01)Following re-feeding, we detected significantly reduced IGFBP-3 levels in all groups, when compared with the CON group(p<0.01).However,IGFBP-3 levels in the M-L and M-H groups were higher than those in the M-CON group at the end of the study(p<0.01).2. Western Blot Resultsphosphorylated of Akt, mTOR and S6K1 and the in hepatic tissue GHR levels in the M-CON group was greatly suppressed in comparison to the CON, M-L, and M-H groups (p<0.01, p< 0.01, and p<0.01, respectively).And there was no statistically significant differenceamong other groups(p> 0.05).3. Hepatic expression of GHR, IGF-1, IGFBP-1, and IGFBP-3 mRNAThe relative expression of GHR mRNA was significantly lower in M-CON rats than in CON,, M-L, and M-H rats (p< 0.01, p< 0.01, and p< 0.01,respectively). And there was no statistically significant differenceamong other groups(p> 0.05).The relative expression of IGF-1 mRNA was lower in the M-CON and M-H groups than the CON group (p< 0.01)IGFBP-1 mRNA levels were significantly higher in the M-CON than the CON group(p< 0.01).However, levels in the M-CON group were significantly higher than in M-H groups (p<0.01).After re-feeding, the relative expression of IGFBP-3 mRNA in the M-CON group was lower than that in all other groups. We observed significant differences in IGFBP-3mRNA levels among the M-L, M-H, and CONgroups, but no difference was found between the M-L and M-H groups.4. Changes in body weight of ratsBody weight was low in all M-CON,M-L,and M-H group rats throughout the study, but did increase during the re-feeding period. Re-feeding was beneficial for achieving weight gain; however, no significant differences between the groups were observed.5. Changes in rat skeletal muscle wet weightThe wet weights of the gastrocnemius, soleus, and extensor digitorum longus muscles in the M-L and M-H groups were significantly higher than those in the M-CON group at the study end point (i.e., after re-feeding) (p< 0.01). However, no significant differences were observed between the M-L, M-H, and CON groups.Conclusions1.Malnutrition lead to acquired growth hormone resistance mechanisms is varied.2.Long-term supplementation with leucine might promote skeletal muscle protein synthesis by regulating downstream Akt-mTOR anabolic signaling transduction.3.Our data are the first to demonstrate that long-term supplementation with leucine improved acquired growth hormone resistance in rats with protein-energy malnutrition. |
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