Pro-insulin C-peptide Enhances pH Sensitivity of the Anabolic Amino Acid Transporter SNAT2 in L6 Rat Skeletal Muscle Cells

Abstract

Muscle wasting is a major problem that accompanies chronic (CKD) especially in CKD patients with diabetes mellitus. By decreasing output of muscle-derived anti-inflammatory cytokines, muscle wasting could increase risk of life-threatening chronic inflammation. A major factor in CKD which drives muscle wasting is acidosis which inhibits the pH-sensitive anabolic amino acid transporter SNAT2. It has been reported that Pro-insulin C-peptide has a potent stimulatory effect on SNAT2 in rat L6 myoblasts. C-peptide acts in L6 myoblasts by stimulating anabolic amino acid transporter SNAT2, potentially reversing the inhibitory effect of acidosis which is thought to be important in muscle wasting in acidotic CKD patients. The pH sensitivity of SNAT2 occurs through the pH sensor protein IRR. The aim is to confirm the previously reported stimulation by C-Pep of SNAT2 in L6 cells. To measure the effect of C-Pep on the pH sensitivity of SNAT2 and to determine by Q-PCR whether pH sensor IRR is expressed in L6 and whether its expression responds to C-Pep or Insulin. Transport experiments were performed in L6 myoblast Cells under air in Hepes-buffered Saline adjusted to pH. SNAT2 was measured from the uptake of (14C-MeAIB) into intact L6-Myoblasts in 5 min incubations at room temperature and expressed relative to total cell protein. To show the gene expression of IRR in response to C-Pep stimulation with insulin used as positive control. RT-Q-PCR was performed with rat IRR & rat primers Cyclophilin (house-keeping gene). Relative gene expression was calculated by the Delta delta Ct method. Neither C-Pep nor Insulin significantly increased transport at a normal extracellular pH of 7.4. However, the cells showed the normal stimulation of transport when the pH of the medium was increased. This pH sensitivity increased after pre-incubated for 1h with 0.03nM C-Pep. This stimulation was also seen with Insulin and Vanadate. Gene expression of IRR was detectable in the cells and was stimulated by C-Pep and Insulin but its sensitivity to C-Pep is probably too weak to explain the effect of C-Pep on SNAT2. C-Pep failed to stimulate SNAT2 activity in rat L6 myoblasts. However a low dose of C-Pep increased the pH sensitivity of SNAT2. Insulin exerted a similar effect but required higher doses.