Methionine supplementation spares body protein by regulating the expression of mTORC1 downstream factors in rats fed a soy protein diet with sufficient sulfur amino acids: a pilot study.

Dietary supplementation with methionine and threonine spares body protein in rats fed a low protein diet, but the effect is not observed for other essential amino acids. Although the requirement for sulfur amino acids is relatively high in rodents, the precise mechanisms underlying protein retention are not fully understood. The aim of this study was to explore whether the activation of mammalian target of rapamycin complex 1 (mTORC1) downstream factors in skeletal muscle by supplementation with threonine and/or methionine contributes to protein retention under sufficient cystine requirement. Male Sprague-Dawley rats were freely fed a 0% protein diet for 2 weeks. These experimental rats were then fed a restricted diet (14.5 g/day) containing 12% soy protein supplemented with both cystine and, methionine and threonine (MT), methionine (M), threonine (T), or neither (NA) (n = 8) for an additional 12 days. Two additional groups were freely fed a diet containing 0% protein or 20% casein as controls (n = 6). Body weight and gastrocnemius muscle weight were higher, and blood urea nitrogen and urinary nitrogen excretion were lower, in the M and MT groups than in the T and NA groups, respectively. p70 S6 kinase 1 abundance was higher, and eukaryotic translation initiation factor 4E-binding protein 1 abundance and mRNA levels were lower, in the skeletal muscles of the M and MT groups. These results suggest that methionine regulates mTORC1 downstream factors in skeletal muscle, leading to spare body protein in rats fed a low protein diet meeting cystine requirements.

Undigested low-methoxy pectin prevents diarrhea and induces colonic contraction during liquid-diet feeding in rats.

OBJECTIVES: Dietary fibers, such as pectins, are blended in liquid diets (LDs) to prevent diarrhea; however, which type of pectin is more effective, along with its mechanism of action, remains unclear. This study aimed to investigate the gelling characteristics, fermentability, fecal properties, and motility of the colon during the administration of LDs blended with pectins. METHODS: Male Sprague-Dawley rats were administered LDs containing high-methoxy pectin (HM), low-methoxy amidated pectin (LMA), low-methoxy pectin (LM), and very low-methoxy amidated pectin (VLMA) ad libitum. The amount of pectin in the feces was assessed by measuring galacturonic acid content. The contractile motility of the rats' descending colons was measured with a force transducer. RESULTS: HM was well fermented, but VLMA was significantly less fermented. LM and LMA displayed intermediate fermentability. An LD that contained LM and VLMA gelled with calcium ions in artificial gastric juice did not cause diarrhea, as opposed to other pectin types. Contractile motility was significantly lower and stools were looser when pectin or calcium was excluded from the LD. CONCLUSIONS: In the colon, LM or VLMA could form a water-holding gel with calcium ions to produce normal feces. The mechanical stimulation of the formed fecal mass might induce physiological colonic contractions.

Enteral feeding with low-methoxyl pectin accelerates colonic anastomosis healing in rats.

OBJECTIVE: Enteral feeding with pectin has proven beneficial for anastomosis healing in rats. The aim of this study was to investigate the effects of low-methoxyl pectin (LMP) or high-methoxyl pectin (HMP), on colonic anastomosis healing in rats. METHODS: Male Sprague-Dawley rats (age 7 wk) were fed liquid diets containing LMP, HMP, or no pectin (pectin-free [PF]) for 14 d (n = 10/group). The rats underwent colonic anastomosis surgery on day 7 and were sacrificed on day 14. Bursting pressure, breaking strength, and salt-soluble hydroxyproline at the anastomosis site were used as indices of anastomosis healing. Short-chain fatty acids (SCFAs) in the cecal contents were analyzed. RESULTS: Breaking strength was higher in the LMP group than in the other two groups (P < 0.001). The salt-soluble hydroxyproline content was higher in LMP group than in the PF group (P < 0.01). Bursting pressure did not differ among the three groups. The LMP group produced normal, formed stools, whereas watery stools were observed in HMP and PF groups throughout the experimental period. Cecal SCFAs were highest in LMP group. CONCLUSIONS: These results suggest that LMP promotes healing of colonic anastomosis more effectively than HMP, which may be explained by the mechanical stresses generated by the movement of normally formed stool though the colon.

Role of serine 249 of ezrin in the regulation of sodium-dependent phosphate transporter NaPi-IIa activity in renal proximal tubular cells.

Type IIa sodium-dependent phosphate transporter (NaPi-IIa) is responsible for renal phosphate reabsorption and maintenance of systemic phosphate homeostasis in mammals. Macromolecular complex formation of NaPi-IIa with sodium-proton exchanger related factor-1 (NHERF-1) and ezrin is important for apical membrane localization in the proximal tubular cells. Here, we investigated the interactions of the ezrin phosphomimetic mutation of serine to aspartic acid at 249 with NHERF-1 and the inhibition of apical membrane localization of NaPi-IIa. In vitro phosphorylation analysis revealed that serine 249 of human ezrin serves as a phosphorylation site for protein kinase A. The N-terminal half of ezrin had a dominant negative effect on the phosphate transport activity and inhibited the apical localization of NaPi-IIa in renal proximal tubular cells. We found that the phosphomimetic S249D mutant interfered with the inhibitory effects of the dominant negative mutant on the transport and localization of NaPi-IIa. The S249D mutant also inhibited the interaction with NHERF-1. Therefore, serine 249 of ezrin can play important roles in the regulation of the complex formation and membrane localization of NaPi-IIa.

Analysis of different complexes of type IIa sodium-dependent phosphate transporter in rat renal cortex using blue-native polyacrylamide gel electrophoresis.

Type IIa sodium-dependent phosphate transporter (NaPi-IIa) can be localized in the apical plasma membrane of renal proximal tubule to carry out a rate-limiting step of phosphate reabsorption. For the apical localization, NaPi-IIa is required to form a macromolecular complex with some adaptor proteins such as Na(+)/H(+) exchanger regulatory factor 1 (NHERF-1) and ezrin. However, the detail of macromolecular complex containing NaPi-IIa in the apical membrane of the renal proximal tubular cells has not been clarified. In this study, we identified at least four different complexes (220, 480, 920, 1,100 kDa) containing NaPi-IIa by using blue-native polyacrylamide gel electrophoresis. Interestingly, LC-MS/MS analysis and immunoprecipitation analysis reveal that megalin is a component of larger complexes (920 and 1,100 kDa). In addition, NaPi-IIa can be heterogeneously co-localized with ezrin and megalin on the apical membrane of renal proximal tubuler cells by fluorescence microscopy analysis. These results suggest that NaPi-IIa can form some different complexes on the apical plasma membrane of renal proximal tubular cells.