Insulin modulates energy and substrate sensing and protein catabolism induced by chronic peritonitis in skeletal muscle of neonatal pigs

Abstract

Background: Acute infection promotes skeletal muscle wasting and insulin resistance, but the effect of insulin on energy and substrate sensing in skeletal muscle of chronically infected neonates has not been studied.

Methods: Eighteen 2-d-old pigs underwent cecal ligation and puncture (CLP) or sham surgery (CON) to induce a chronic infection for 5 d. On d 5, pancreatic-substrate clamps were performed to attain fasting or fed insulin levels but to maintain glucose and amino acids in the fasting range. Total fractional protein synthesis rates (K_s), translational control mechanisms, and energy sensing and degradation signal activation were measured in longissimus dorsi muscle.

Results: In fasting conditions, CLP reduced K_s and sirtuin 1 (SIRT1) and increased AMP-activated protein kinase α (AMPKα) activation and muscle RING-finger protein-1 (MuRF1). Insulin treatment increased K_s and mitochondrial protein synthesis, enhanced translation activation, and reduced SIRT1 in CON. In contrast, in CLP, insulin treatment increased K_s, protein kinase B (PKB) and Forkhead box O1 phosphorylation, antagonized AMPK activation, and decreased peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC-1α), MuRF1, and SIRT1.

Conclusion: Energy and substrate sensing in skeletal muscle by the PKB-AMPK-SIRT1-PGC-1α axis is impacted by chronic infection in neonatal pigs and can be modulated by insulin.

Figure 1

Schematic representation of protein synthesis, protein degradation and energy sensing signaling pathways in skeletal muscle and the effect of insulin (I) on relevant signaling after 5 d of cecal ligation and puncture (CLP)-induced peritonitis in neonatal pigs. CLP Insulin CLP + Insulin.

Figure 2

Body weight gain (a) and plasma concentrations of tumor necrosis a (TNF-α)(b), interleukin 8 (IL-8) (d), and 3-methylhistidine (3-MH)(c), after 5 d of CLP-induced peritonitis in neonatal pigs. Black bars represent sham animals while white bars represent CLP animals. Values are expressed as least square means ± SE. CON, control (n = 9); CLP, cecal ligation and puncture (n = 9).*P = 0.05.

Figure 3

Net whole body glucose (a) and amino acid (AA) disposal rates (b) during a 2-h pancreatic glucose-AA clamp after 5 d of peritonitis in neonatal pigs. CON, control, n = 4; CON + Insulin, n = 5; CLP, cecal ligation and puncture, n = 4; CLP + Insulin, n = 5. Values are means ± SE; n = 4–5 per group.* P=0.05,** P = 0.001.

Figure 4

Global (a) and mitochondrial (b) fractional protein synthesis rates during a 2-h pancreatic glucose-amino acid clamp after 5 d of peritonitis in skeletal muscle of neonatal pigs. CON, control, n = 4; CON + Insulin, n = 5; CLP, cecal ligation and puncture, n = 4; CLP + Insulin, n = 5.* P = 0.05,** P = 0.001.

Figure 5

Effect of insulin on the phosphorylation of protein kinase B (p-PKB) (a), tuberous sclerosis complex 2 (p-TSC2) (b), mammalian target of rapamycin (p-mTOR) (c), ribosomal protein S6 kinase-1 (p-S6K1) (d), and the abundance of eukaryotic initiation factor 4 complex (eIF4E-eIF4G) (e) during a 2-h pancreatic glucose–amino acid clamp after 5 d of peritonitis in skeletal muscle of neonatal pigs. CON, control, n = 4; CON + Insulin, n = 5; CLP,cecal ligation and puncture, n = 4; CLP + Insulin, n = 5. Values are means ± SE. * P = 0.05, ** P = 0.001.

Figure 6

Effect of insulin on the phosphorylation of forkhead box O1 (FOXO1) (a), FOXO3 (b), and FOXO4 (c), and the abundance of atrogin-1/Muscle Atrophy F-box (Atrogin-1) (d), muscle RINGfinger protein-1 (MuRF1) (e), and Pro-caspase 3 (f) during a 2-h pancreatic glucose-amino acid clamp after 5 d of peritonitis in skeletal muscle of neonatal pigs. CON, control, n = 4; CON + Insulin, n = 5; CLP, cecal ligation and puncture, n = 4; CLP + Insulin, n = 5. Values are means ± SE. * P = 0.05, ** P = 0.001.

Figure 7

Effect of insulin on the phosphorylation of AMP-activated protein kinase α (p-AMPKα) (a), and nuclear factor-κB (NF-κb) (b), protein abundance of sirtuin 1 (SIRT1) (c), and the mRNA abundance of peroxisome proliferator-activated receptor gamma coactivator1 a (PGC-1α) (d) during a 2-h pancreatic glucose–amino acid clamp after 5 d of peritonitis in skeletal muscle of neonatal pigs. CON, control, n = 4; CON + Insulin, n = 5; CLP, cecal ligation and puncture, n = 4; CLP + Insulin, n = 5. Values are means ± SE. * P = 0.05, ** P = 0.001.

Figure 8

Schematic representation of the pancreatic glucose–AA clamp after 5 d of cecal ligation and puncture (CLP) in neonatal pigs. AA, amino acid; Phe, phenylalanine.