Roles of Mitogen-Activating Protein Kinase Kinase Kinase Kinase-3 (MAP4K3) in Preterm Skeletal Muscle Satellite Cell Myogenesis and Mammalian Target of Rapamycin Complex 1 (mTORC1) Activation Regulation.

BACKGROUND Preterm skeletal muscle genesis is a paradigm for myogenesis. The role of mitogen-activating protein kinase kinase kinase kinase-3 (MAP4K3) in preterm skeletal muscle satellite cells myogenesis or its relationship to mammalian target of rapamycin complex 1 (mTORC1) activity have not been previously elaborated. MATERIAL AND METHODS Small interfering RNA (siRNA) interference technology was used to inhibit MAP4K3 expression. Leucine stimulation experiments were performed following MAP4K3-siRNA interference. The differentiation of primary preterm skeletal muscle satellite cells was observed after siRNA-MAP4K3 interference. Western blot analysis was used to determine the expression of MAP4K3, MyHC, MyoD, myogenin, p-mTOR, and p-S6K1. The immunofluorescence fusion index of MyHC and myogenin were detected. MAP4K3 effects on preterm rat satellite cells differentiation and its relationship to mTORC1 activity are reported. RESULTS MAP4K3 siRNA knockdown inhibited myotube formation and both MyoD and myogenin expression in primary preterm rat skeletal muscle satellite cells, but MAP4K3 siRNA had no effect on the activity of mTORC1. In primary preterm rat skeletal muscle satellite cells, MAP4K3 knockdown resulted in significantly weaker, but not entirely blunted, leucine-induced mTORC1 signaling. CONCLUSIONS MAP4K3 positively regulates preterm skeletal muscle satellite cell myogenesis, but may not regulate mTORC1 activity. MAP4K3 may play a role in mTORC1 full activation in response to leucine.

[Effects of feeding patterns after hospital discharge on increase rates of growth indices in preterm and low-birth-weight infants within 3 months after birth].

OBJECTIVE: To study the effects of post-discharge formula (PDF) for preterm infants, breast milk (BM) and term infant formula (TF) on increase rates of body weight, length and head circumference in preterm and low-birth-weight infants (PLBWIs) from discharge to 3 months after birth, and to provide a reference for the choice of feeding pattern for PLBWIs. METHODS: A total of 407 PLBWIs discharged from the newborn departments of ten hospitals in Guangzhou City and Foshan City in Guangdong Province, China were chosen for this study. According to feeding pattern, they were assigned to three groups: PDF-fed (n=258), BM-fed (n=58) and TF-fed (n=91). Their body weight, length and head circumference were measured at 3 months after birth, and the increase rates of growth indices relative to baseline values (at birth) were calculated and compared. RESULTS: At 3 months after birth, the PDF-fed group had significantly greater body weight, length and head circumference than the BM-fed and TF-fed groups (P<0.05). The increase rates of body weight and length were significantly higher in the PDF-fed group than in the BM-fed and TF-fed groups (P<0.05). CONCLUSIONS: Compared with those fed with BM and TF after discharge, the PDF-fed PLBWIs have higher increase rates of body weight and length and show greater body weight and length at 3 months after birth. However, further study is needed to investigate the long-term effects.

[The effect of birth weight and early growth on body fat composition and insulin sensitivity].

OBJECTIVE: To investigate the effect of birth weight and early growth on body fat composition and insulin sensitivity. METHODS: The birth and growth data of 258 children of 6 to 7 years old in Guangzhou were collected from Jun.2009 to Feb. 2010. Physical and laboratory examination were preformed, which included body weight, body height and body fat composition index (body mass index (BMI), percentage of body fat (PBF), waist circumference to height ratio (WtHR), etc). Fasting blood glucose and insulin were measured. The homeostasis model assessment model for insulin resistance index (HOMA-IR) was calculated. According to birth weight, the children were divided into three groups from light to heavy: BW-I, BW-II, BW-III group. Then according to change in weight SDS between 0 and 36 months, the children were divided into three groups: changers up (CU), non-changers (NC), changers down (CD) group. The effect of birth weight and early growth on body fat composition and insulin sensitivity were analyzed. RESULTS: Change in weight SDS between 0 and 36 months was higher in BW-I group (1.06 ± 1.29) than in the BW-II group (-0.19 ± 0.94) and BW-III group (-0.10 ± 1.20) (all P values < 0.01). Birth weight of the CU group ((2.90 ± 0.47) kg) was lower than that of the NC group ((3.22 ± 0.34) kg) and the CD group ((3.57 ± 0.37) kg) (all P values < 0.01). The body fat composition index of BMI, PBF and WtHR were higher in the BW-III group ((16.35 ± 2.13) kg/m(2), (17.03 ± 5.88)%, (0.479 ± 0.033)) than in the BW-I group ((15.46 ± 2.06) kg/m(2), (14.06 ± 5.25)%, (0.459 ± 0.032)) and BW-II group ((15.47 ± 1.58) kg/m(2), (14.09 ± 5.01)%, (0.460 ± 0.025)) (P < 0.01), while there was no significant difference between the BW-I group and the BW-II group (P > 0.05). The body fat composition index of BMI, PBF and WtHR were higher in the CU group ((16.44 ± 2.20) kg/m(2), (16.51 ± 5.78)%, (0.473 ± 0.034)) than in the NC group ((15.62 ± 1.74) kg/m(2), (14.49 ± 5.30)%, (0.463 ± 0.030)) and the CD group ((15.26 ± 1.85) kg/m(2), (14.24 ± 5.54)%, (0.462 ± 0.031)) (all P values < 0.05). In the CU group, BMI, PBF and WtHR were higher in the BW-III-CU group ((18.76 ± 2.56) kg/m(2), (22.19 ± 8.28)%, (0.512 ± 0.029)) than in the BW-I-CU group ((16.04 ± 2.14) kg/m(2), (15.54 ± 5.28)%, (0.467 ± 0.034)) and BW-II-CU group ((16.70 ± 1.36) kg/m(2), (17.12 ± 4.44)%, (0.474 ± 0.017)) (all P values < 0.05), while there was no significant difference between the BW-I-CU group and the BW-II-CU group (P > 0.05). HOMA-IR was higher in the CU group (1.27 ± 0.44) than in the NC group (1.08 ± 0.31) and the CD group (1.00 ± 0.36) (all P values < 0.01). In the CU group, HOMA-IR was higher in the BW-III-CU group (1.69 ± 0.48) than in the BW-I-CU group (1.21 ± 0.41) and the BW-II-CU group (1.27 ± 0.44) (all P values < 0.01), while there was no significant difference between the BW-I-CU and BW-II-CU group (P > 0.05). CONCLUSION: According to birth weight tertile, both lower birth weight individuals with more weight change-up growth postnatal early and higher birth weight individuals had greater body fat composition in childhood. They were high-risk people of insulin resistance.

Effect of early nutrition on intestine development of intrauterine growth retardation in rats and its correlation to leptin.

AIM: To investigate the intestine and body development of intrauterine growth retardation (IUGR) rats under early different protein diet and to analyze the correlation between leptin and intestine and body development. METHODS: An IUGR rat model was established by food restriction of pregnant female rats. Fifty-six neonatal IUGR rats and 24 neonatal normal rats were randomly divided into normal control group (C group), IUGR model group (SC group), low protein diet IUGR group (SL group), and high protein diet IUGR group (SH group). Eight rats were killed per group at wk 0, 4, and 12. Serum leptin, body weight (BW), body length (BL), intestinal weight (IW), intestinal length (IL), and intestinal disaccharidase (including lactase, maltase, and saccharase) were detected. RESULTS: BW (4.50+/-0.41 g), BL (5.96+/-0.40 cm), IW (0.05+/-0.01 g), and IL (15.9+/-2.8 cm) in neonatal IUGR rats were much lower than those in C group (6.01+/-0.55 g, 6.26+/-0.44 cm, 0.10+/-0.02 g, 21.8+/-2.7 cm, P<0.05), while intestinal lactase and maltase activities were higher than those in C group. SH group showed the fastest catch up growth and their BW, BL, IW, and IL reached the C group level at wk 4. SC group showed relatively slower catch up growth than SH group, and their BW, BL, IW did not reach the C group level at wk 4. SL group did not show intestine and body catch up growth. Intestinal maltase (344+/-33 micromol/(min.g)) and saccharase activities (138+/-32 micromol/(min.g)) in SL group were both markedly lower than those in C group (751+/-102, 258+/-27 micromol/(min.g), P<0.05). There were no significant differences in lactase activities at wk 4 and disaccharidase activities at wk 12 among all groups (P>0.05). The leptin level in SL group (0.58+/-0.12 ng/mL) was the highest in all groups, and much lower in SH group (0.21+/-0.03 ng/mL) than that in any other IUGR groups at wk 4 (P<0.05). Leptin was negatively related to BW (r = -0.556, P = 0.001), IW (r = -0.692, P = 0.001) and IL (r = -0.738, P = 0.000) at wk 4, while no correlation was found at wk 12. CONCLUSION: High protein diet is a reasonable early nutritional mode to IUGR rats in promoting intestine and body catch up growth.

Treponemal antibody in CSF and cellular immunity in peripheral blood of syphilitic patients with persisting positive rapid plasma regain.

The ratio of patients with RPR constant positive more than 2 years despite receiving standard syphilis treatment has been reported to be 11.54%~31.3%. The current interpretations on this phenomenon are cellular immune function restrained and the existence of neurosyphilis or asymptomatic neurosyphilis. We conducted this study to detect the treponemal antibody in cerebrospinal fluid (CSF) and lymphocyte subsets in peripheral blood of syphilis patients with persisting RPR positive more than 2 years without neurologic signs, and then explore their relationship. In this study, Treponemal antibody in CSF of 46 syphilitic with HIV negative were measured by syphilis serum test and compared with that of 5 neurosyphilis. Lymphocyte subsets were measured by flow cytometry (FCM) and compared with that of 30 healthy controls. We observed that treponemal antibody in CSF was detected not only in 12 cases (25.21%) of 46 treated patients, but also in 5 neurosyphilis. The ratio of lymphocyte subsets revealed that CD3+, CD4+ T cells and natural killer (NK) cells showed no significant differences between the patient and healthy controls (P>0.05), while CD8+ T cells in patients were significant higher than that in healthy controls (P<0.001). Lymphocyte subsets showed no significant differences between the patients with treponemal antibody positive and negative in CSF (P>0.05). In conclusion, the treponemal antibody in CSF of treated patients suggests that part of them were asymptomatic neurosyphilis and with cellular immunodifeciency. And there is no significant relationship between asymptomatic neurosyphilis and cellular immunodeficiency in peripheral blood.

Leucine Promotes Proliferation and Differentiation of Primary Preterm Rat Satellite Cells in Part through mTORC1 Signaling Pathway.

Signaling through the mammalian target of rapamycin (mTOR) in response to leucine modulates many cellular and developmental processes. However, in the context of satellite cell proliferation and differentiation, the role of leucine and mTORC1 is less known. This study investigates the role of leucine in the process of proliferation and differentiation of primary preterm rat satellite cells, and the relationship with mammalian target of rapamycin complex 1 (mTORC1) activation. Dissociation of primary satellite cells occurred with type I collagenase and trypsin, and purification, via different speed adherence methods. Satellite cells with positive expression of Desmin were treated with leucine and rapamycin. We observed that leucine promoted proliferation and differentiation of primary satellite cells and increased the phosphorylation of mTOR. Rapamycin inhibited proliferation and differentiation, as well as decreased the phosphorylation level of mTOR. Furthermore, leucine increased the expression of MyoD and myogenin while the protein level of MyoD decreased due to rapamycin. However, myogenin expressed no affect by rapamycin. In conclusion, leucine may up-regulate the activation of mTORC1 to promote proliferation and differentiation of primary preterm rat satellite cells. We have shown that leucine promoted the differentiation of myotubes in part through the mTORC1-MyoD signal pathway.

Effects of early nutrition intervention on IGF1, IGFBP3, intestinal development, and catch-up growth of intrauterine growth retardation rats.

OBJECTIVE: To investigate the effects of early nutritional intervention on the serum insulin-like growth factor-1 (IGF1), insulin-like growth factor binding protein 3 (IGFBP3), intestinal development, and catch-up growth of intrauterine growth retardation (IUGR) rats by giving the IUGR new born rats different protein level diet. METHODS: IUGR rat model was built by starvation of pregnant female rats. Twenty-four IUGR pups and 8 normal pups were divided randomly into 4 groups: normal control group (C group); IUGR control group (S group), IUGR low-protein diet group (SL group), and IUGR high-protein diet group (SH group). Detected the serum IGF1, IGFBP3, body weight, body length, intestinal weight length, intestinal villi height (VH), crypt depth (CD), villi absorbing area (VSA), mucous thickness (MT), and disaccharidase at the 4th week. RESULTS: (1) The SH group showed the fastest catch-up growth, serum IGF1, IGFBP3, VH, and VSA were significantly higher than those of normal control group and IUGR control group. The intestinal weight and length, and the activities of lactase and saccharase of the SH group also reached the normal control group level. (2) The SL group kept on small size, the serum IGF1, IGFBP3, and most of intestinal histological indexes were all significantly lower than other groups. (3) IGF1, IGFBP3 were positively correlated to intestinal VH, VSA, saccharase, body weight and length. CONCLUSIONS: The serum IGF1 was a sensitive index to the catch-up growth. The early nutritional intervention of high-protein diet after birth is helpful for the catch-up growth of IUGR through promoting the intestinal development and the absorption of nutrition.

[The effects of pregnancy malnutrition on the development of insulin resistance in rat offspring].

OBJECTIVE: To investigate the effects of pregnancy malnutrition on the occurrence of insulin resistance (IR) in rat offspring during adult stage and to find out the relationship between TNF-alpha and IR; and to find out a reasonable early nutritional intervention measure for the prevention of IR, through giving different diets to offspring. METHODS: An IUGR model was built by maternal nutrition restriction. 80 newborn IUGR female pups were randomly divided into 4 groups, the mother rats were given the following diet respectively for 3 weeks after delivery, pups were fed by mother milk: (1) The IUGR (intrauterine growth retardation) rat model was used and the animals were divided into: IUGR control group (group S/N) fed with normal diet, (2) IUGR high-caloric diet group (group A), (3) IUGR high-protein and high-caloric diet group (group B) and (4) IUGR high-protein isocaloric diet group (group C). Each group had 20 pups and another 20 normal female pups were fed with normal diet as the normal control group (group C/N). All pups were weaned at the 4th week of age and fed with normal diet till the end of the experiment. At the 12th week (adulthood) and 48th week (senility) of life, body weight and length, the fasting blood glucose, insulin concentration, TNF-alpha of adipose tissue and body weight were measured. Body mass index (BMI), ISI (insulin sensitive index), IRI (insulin resistant index) and HBCI (beta cell insulin excretion index) and their correlation to TNF-alpha were calculated. RESULTS: At 12th week and 48th week of life, the insulin sensitivity of IUGR model group was significantly lower than group C/N, although there was no significant difference of body weight between these two groups. TNF-alpha was negatively correlated with ISI, positively correlated with IRI and no relation to HBCI. Group A and B was fatter and developed more severe IR. There were no significant differences in ISI, IRI, HBCI and TNF-alpha between group C and group C/N. CONCLUSIONS: IUGR offspring of pregnancy malnutrition mother rats showed IR at the age of 12th week. TNF-alpha was closely related to the occurrence of IR in IUGR pups. IUGR pups fed with high caloric diet or high protein and caloric diet at the early postnatal period amplified the metabolic abnormality. The high protein isocaloric diet is effective early nutritional intervention measure for the prevention of occurrence of IR at adulthood.

High-protein diets alters body composition and improves insulin resistance in a rat model of low birth weight.

OBJECTIVE: We aimed to investigate the effects of early high-protein supplementation on low birth weight (LBW)-associated adult metabolic disturbances. MATERIALS AND METHODS: This study involved 32 LBW rat pups that were fed a normal protein (20% of energy intake) diet or high-protein (30% of energy intake) diet on their first 4 weeks of life. Sixteen rat pups with normal birth weight (NBW) fed the normal-protein diet were included as control. Biochemical measurements were performed at 4 and 12 weeks of age. RESULTS: Low birth weight offspring showed significantly (P < 0.05) increased fat mass percentage and adipocyte size and decreased lean mass percentage and muscle fiber size relative to NBW offspring. These LBW-related changes in body composition were corrected by high-protein diet intervention. At 12 weeks of age, the fasting insulin level (7.14 ± 0.83 vs 9.27 ± 0.67 mU/L) and homeostasis model of insulin resistance (1.71 ± 0.35 vs 2.30 ± 0.44) were significantly lower in high protein-fed LBW offspring than in normal protein-fed LBW offspring. Low birth weight rat pups showed a significant (P < 0.05) reduction in serum adiponectin concentrations, glucose transporter 4 mRNA abundance, and phosphorylation levels of AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) relative to NBW controls. These LBW-associated alterations in gene expression were reversed by early high-protein treatment. CONCLUSIONS: Early postnatal high-protein intake alters the body composition and improves insulin resistance in adults with LBW, which is associated with activation of the AMPK and mTOR pathways.

[Expression of tumor necrosis factor alpha mRNA in adipose cell of intrauterine growth retarded rats and its relation to insulin resistance].

OBJECTIVE: To investigate the association between the expression of turnor necrosis factor alpha (TNF-alpha) mRNA in fat tissue of intrauterine growth retarded (IUGR) rats and insulin resistance, and the long-term effects of early different nutritional diet. METHODS: The IUGR rat model was established by food restriction of pregnant rats. A total of 32 newborn IUGR rats were randomly divided into 4 groups: IUGR model (S/N) group, IUGR high caloric diet (A) group, IUGR high caloric and high protein diet (B) group, IUGR high protein diet (C) group. Only the mother rats were given those different diets individually, and all IUGR newborn pups were lactated for 3 weeks. From the beginning of the 4(th) week, all IUGR pups were weaned and fed with normal diet till the end of the experiment. Eight normal birth weight newborn rats were used as the control group fed with the normal diet. Weight, perirenal fat weight, fasting glucose and insulin concentration and quantified TNF-alpha mRNA expression in adipose cell were measured at the 48(th) week. The insulin sensitive index (ISI) and the relation index between TNF-alpha mRNA and fat weight, fat weight/body weight (fw/bw) ratio and ISI were calculated. RESULTS: ISI of IUGR model group, IUGR A and B groups was lower than normal control group, while perirenal fat weight, fw/bw and the expression of TNF-alpha mRNA in adipose cells were all significantly higher (P < 0.05 or 0.01). There were no significant differences in these indexes between IUGR C group and normal control groups (P > 0.05). A positive correlation was found between TNF-alpha mRNA and fat weight and fw/bw (r(1) = 0.755, r(2) = 0.782, P = 0.000). Significant inverse associations between ISI and TNF-alpha mRNA (r = -0.556, P = 0.000) and fw/bw (r = -0.513, P = 0.02) were also found. CONCLUSION: The occurrence of insulin resistance in IUGR rats is possibly associated with central obesity and accumulation of the abdominal fat and adipose cell over-expression of TNF-alpha. The adipose TNF-alpha may be an important pathogenic factor of insulin resistance of IUGR. High protein diet is a reasonable nutritional intervention. Because it promotes the skeleton muscle catch-up growth but not fat catch-up growth, it can avoid the occurrence of central obesity and insulin resistance in IUGR rats.

[Effect of different early nutritional interventions on catch-up growth of rats with intrauterine growth retardation].

OBJECTIVE: About 20 - 50% individuals with intrauterine growth retardation (IUGR) could not achieve catch-up growth and remain small in size till adulthood. There are few reports on the relation between intestinal development and body catch-up growth of IUGR. Studies showed that early "nutritional programming" would results in long-term effects on the body growth and organic function, and gastrointestinal development is closely related to the body development as well. The authors aimed to study the effect of early nutritional interventions on serum IGF1, IGFBP3, intestinal development and catch-up growth of pups with IUGR by using diets with different protein and caloric levels during the first four weeks of life. METHODS: An IUGR rat model was established by maternal nutrition restriction during pregnancy. Thirty-two IUGR female pups were divided randomly into 4 groups (8 pups in each group) and eight normal female pups as control. The groups and interventions were (1) Normal control group (C group); (2) IUGR control group (S group), (3) IUGR low-protein diet group (SL group); (4) IUGR high-protein diet group (SH group); (5) IUGR high-caloric group (SA group). The serum IGF1, IGFBP3, body weight, body length, and intestinal weight, length, intestinal villi height (VH), crypt depth (CD), villi absorbing area (VSA), mucous thickness (MT) were measured at the 4(th) week of life. RESULTS: (1) At the 4(th) week, the serum IGF1 (724.0 +/- 153.5 ng/ml), IGFBP3 (9.69 +/- 3.13 ng/ml), and VH (416.9 +/- 46.3 microm), VSA (115.9 +/- 24.0 x 10(3) microm(2)), MT (583.9 +/- 68.5 microm) in the SH group were significantly higher than those of normal control group (539.4 +/- 198.4 ng/ml, 4.77 +/- 2.98 ng/ml and 322.1 +/- 25.8 microm, 85.8 +/- 17.8 x 10(3) microm(2), 480.0 +/- 61.5 microm) and IUGR control group (P < 0.05). The intestinal weight (1.91 +/- 0.16 g) and length (80.67 +/- 9.47 cm) in the SH group was not significantly different from the normal control group (2.24 +/- 0.22 g and 74.77 +/- 9.06 cm, P > 0.05). The SH group showed the fastest catch-up growth. Their body weights (40.14 +/- 11.03 g) at the 3(rd) week and body lengths (23.61 +/- 0.49 cm) at the 4(th) week of life reached the normal ranges of the control group (44.65 +/- 5.36 g and 23.10 +/- 1.42 cm, P > 0.05). (2) The serum IGF1 (346.7 +/- 85.3 ng/ml), IGFBP3 (1.4 +/- 0.21 ng/ml), body weight (21.41 +/- 3.54 g) and body length (15.96 +/- 1.29 cm) and the most of intestinal indexes in the SL group were markedly lower than other groups at the 4(th) week of life (P < 0.05). CONCLUSION: The serum IGF1 was a sensitive marker to reflect the catch-up growth and nutritional status, and IGF1 was positively correlated with the intestinal development and body growth. When given different nutritional interventions during the first four weeks of life, high protein diet is more helpful for the IUGR catch-up growth by promoting the intestinal development and the absorption of nutrition.