COMMENT Physiological Hyperinsulinemia Stimulates p70 Phosphorylation in Human Skeletal Muscle*

Using tracer methods, insulin stimulates muscle protein synthesis in vitro, an effect not seen in vivo with physiological insulin concentrations in adult animals or humans. To examine the action of physiological hyperinsulinemia on protein synthesis using a tracer-independent method in vivo and identify possible explanations for this discrepancy, we measured the phosphorylation of ribosomal protein S6 kinase (P70) and eIF4E-binding protein (eIF4E-BP1), two key proteins that regulate messenger ribonucleic acid translation and protein synthesis. Postabsorptive healthy adults received either a 2-h insulin infusion (1 mU/minzkg; euglycemic insulin clamp; n 5 6) or a 2-h saline infusion (n 5 5). Vastus lateralis muscle was biopsied at baseline and at the end of the infusion period. Phosphorylation of P70 and eIF4E-BP1 was quantified on Western blots after SDSPAGE. Physiological increments in plasma insulin (42 6 13 to 366 6 36 pmol/L; P 5 0.0002) significantly increased p70 (P , 0.01), but did not affect eIF4E-BP1 phosphorylation in muscle. Plasma insulin declined slightly during saline infusion (P 5 0.04), and there was no change in the phosphorylation of either p70 or eIF4E-BP1. These findings indicate an important role of physiological hyperinsulinemia in the regulation of p70 in human muscle. This finding is consistent with a potential role for insulin in regulating the synthesis of that subset of proteins involved in ribosomal function. The failure to enhance the phosphorylation of eIF4E-BP1 may in part explain the lack of a stimulatory effect of physiological hyperinsulinemia on bulk protein synthesis in skeletal muscle in vivo. (J Clin Endocrinol Metab 85: 4900–4904, 2000) A INTERESTING and unexplained dichotomy arises from studies examining insulin’s actions on muscle protein synthesis in vitro and in vivo. Abundant in vitro data clearly indicate that insulin strongly stimulates bulk cellular protein synthesis in a variety of cells and perfused organs (1–3) and that insulin acts primarily to enhance messenger ribonucleic acid (mRNA) translation (4, 5). However, using steady state tracer infusion methods (6–11), the floodingbolus tracer technique (12, 13), and more recently even measurements of aminoacyl transfer RNA labeling (14, 15), investigators have almost without exception (16) reported that insulin at physiological concentrations does not stimulate either whole body or muscle protein synthesis in vivo. This contrasts with the prominent synthetic effect seen in vivo when a balanced mixture of amino acids (6, 11), GH (17), insulin-like growth factor (11, 18), or pharmacological doses of insulin (19) are infused. Recent data suggest that insulin stimulates mRNA translation in vitro at least in part by enhancing the activity of the translation initiation step that begins with eIF4E forming the preinitiation complex (20, 21). Insulin increases eIF4E availability by phosphorylating the 4E-binding protein (eIF4E-BP1), which then dissociates from 4E, thereby increasing the availability of the latter to form the eIF4F cap binding complex of mRNA. In addition to eIF4E-BP1, p70 is phosphorylated in a variety of tissues in response to insulin (22). Phosphorylation of p70 and subsequently ribosomal protein S6 regulates the synthesis of a selected subset of proteins that includes ribosomal proteins, translation initiation and elongation factors, and other proteins with oligopyrimidine sequences at the transcriptional start site (23, 24). Whether it has a major role in the translation of the bulk of cellular proteins is not clear. In the current study we examined whether physiological hyperinsulinemia in vivo affects the phosphorylation state of these two proteins in an effort to use a nontracer based method to identify how physiological hyperinsulinemia regulates protein synthesis in vivo in human muscle. Subjects and Methods