Data_Sheet_1_The Dipeptide Pro-Gly Promotes IGF-1 Expression and Secretion in HepG2 and Female Mice via PepT1-JAK2/STAT5 Pathway.docx

<p>It has been shown that IGF-1 secretion is influenced by dietary protein or amino acid. However, whether the dipeptides elicit regulatory effects on IGF-1 secretion remains largely unclear. Thus, this study aimed to investigate the effects of the dipeptide Pro-Gly on IGF-1 expression and secretion in HepG2 cells and mice, and explore the underlying mechanisms. The in vitro results indicated that Pro-Gly, but not Pro plus Gly, promoted the expression and secretion of IGF-1 in HepG2. Meanwhile, the expression of the peptide transporter 1 (PepT1) was elevated by Pro-Gly, whereas knockdown of PepT1 with siRNA eliminated the increase of IGF-1 expression induced by Pro-Gly. In addition, Pro-Gly activated JAK2/STAT5 signaling pathway in a PepT1-dependent manner. Furthermore, Pro-Gly enhanced the interaction between JAK2 and STAT5, and the translocation of phospho-STAT5 to nuclei. Moreover, inhibition of JAK2/STAT5 blocked the promotive effect of Pro-Gly on IGF-1 expression and secretion. In agreement with the in vitro results, the in vivo findings demonstrated that Pro-Gly, but not Pro plus Gly, stimulated the expression and secretion of IGF-1 and activated JAK2/STAT5 signaling pathway in the liver of mice injected with Pro-Gly or Pro+Gly acutely or chronically. Besides, acute injection of JAK2/STAT5 inhibitor abolished the elevation of IGF-1 expression and secretion induced by Pro-Gly in mice. Collectively, these findings suggested that the dipeptide Pro-Gly promoted IGF-1 expression and secretion in HepG2 cells and mice by activating JAK2/STAT5 signaling pathway through PepT1. These data provided new insights to the regulation of IGF-1 expression and secretion by the dipeptides.</p

Dietary protein-induced hepatic IGF-1 secretion mediated by PPARγ activation

<div><p>Dietary protein or amino acid (AA) is a crucial nutritional factor to regulate hepatic insulin-like growth factor-1 (IGF-1) expression and secretion. However, the underlying intracellular mechanism by which dietary protein or AA induces IGF-1 expression remains unknown. We compared the IGF-1 gene expression and plasma IGF-1 level of pigs fed with normal crude protein (CP, 20%) and low-protein levels (LP, 14%). RNA sequencing (RNA-seq) was performed to detect transcript expression in the liver in response to dietary protein. The results showed that serum concentrations and mRNA levels of IGF-1 in the liver were higher in the CP group than in the LP group. RNA-seq analysis identified a total of 1319 differentially expressed transcripts (667 upregulated and 652 downregulated), among which the terms “oxidative phosphorylation”, “ribosome”, “gap junction”, “PPAR signaling pathway”, and “focal adhesion” were enriched. In addition, the porcine primary hepatocyte and HepG2 cell models also demonstrated that the mRNA and protein levels of IGF-1 and PPARγ increased with the increasing AA concentration in the culture. The PPARγ activator troglitazone increased IGF-1 gene expression and secretion in a dose dependent manner. Furthermore, inhibition of PPARγ effectively reversed the effects of the high AA concentration on the mRNA expression of IGF-1 and IGFBP-1 in HepG2 cells. Moreover, the protein levels of IGF-1 and PPARγ, as well as the phosphorylation of mTOR, significantly increased in HepG2 cells under high AA concentrations. mTOR phosphorylation can be decreased by the mTOR antagonist, rapamycin. The immunoprecipitation results also showed that high AA concentrations significantly increased the interaction of mTOR and PPARγ. In summary, PPARγ plays an important role in the regulation of IGF-1 secretion and gene expression in response to dietary protein.</p></div

Validation of microarray results by qPCR and hierarchical cluster analysis of differentially expressed genes.

<p>(A) Comparison of expression ratios (log 2, <i>y</i>-axis; genes, <i>x</i>-axis) measured by qPCR and microarray in the 19 selected genes. Ratios by microarray and qPCR were averaged for triplicates. (B) The levels of differentially expressed genes were calculated by log2 and compared between 20% crude protein diet (CP, <i>n</i> = 3) and 14% crude protein diet (LP, <i>n</i> = 3) groups. (C) Differentially expressed genes in PPAR signaling pathway. The red color denotes high expression, whereas the green color indicates low expression.</p

Statistics of transcript expression.

<p>Statistics of transcript expression.</p

Effects of dietary protein regulated the serum index and IGF expression in porcine liver.

<p>Serum IGF-1 (A), albumin (B), and urea nitrogen (C) levels were detected in 63 day-old piglets (<i>n</i> = 6) fed with 20% crude protein diet (CP) and 14% crude protein diet (LP) using commercial kits. Total RNA was harvested and analyzed by qPCR for IGF-1 (D) and IGFBP-1 (E) mRNA expression in liver tissue (<i>n</i> = 6). Data represent the mean ± SEM. * <i>P</i> < 0.05, ** <i>P</i> < 0.01 vs. LP.</p

Effects of AA on IGF-1 and IGFBP-1 mRNA expression were mediated by PPARγ.

<p>IGF-1 secretion (A) and IGF-1 mRNA expression (B) were measured after treatment with the PPARγ agonist troglitazone. Values with different letters were significantly different (<i>P</i> < 0.05, <i>n</i> = 6). (C and D) Cells treated with HepG2 in media with 1× or 4× physiological AA concentrations, which contained 10 μM of the PPARγ inhibitor GW9662. IGF-1 and IGFBP-1 mRNA expression were analyzed by qPCR. *<i>P</i> < 0.05 (<i>n</i> = 6). Results were expressed as mean ± SEM.</p

Major enrichment pathways recognized by DAVID.

<p>Major enrichment pathways recognized by DAVID.</p

mTOR was involved in the AA-induced activation of PPARγ.

<p>HepG2 cells were cultured in media with 1× and 4× physiological AA concentrations for 48 h. One fraction of the total protein was used to determine the total and phosphorylated levels of the mTOR (A and B), GAPDH, PPARγ (C), and IGF-1 (D) proteins by Western blot analysis. All results contained three replicates (<i>n</i> = 3). The other total protein extracts were analyzed by immunoprecipitation (IP) with anti-PPARγ (E) capture antibodies. Data were expressed as the mean ± SEM. Values with different letters were significantly different (<i>P</i> < 0.05).</p