The somatotropic axis is required for a number of biological processes, including growth, metabolism, and aging. Due to its central effects on growth and metabolism and with respect to its positive effects on muscle mass, regulation of the GH/IGF-system during endurance exercise is of particular interest. In order to study the control of gene expression and adaptation related to physical performance, we used a non-inbred mouse model, phenotype-selected for high running performance (DUhTP). Gene expression of the GH/IGF-system and related signaling cascades were studied in the pituitary gland and muscle of sedentary males of marathon and unselected control mice. In addition, the effects of three weeks of endurance exercise were assessed in both genetic groups. In pituitary glands from DUhTP mice, reduced expression of Pou1f1 (p = 0.002) was accompanied by non-significant reductions of Gh mRNA (p = 0.066). In addition, mRNA expression of Ghsr and Sstr2 were significantly reduced in the pituitary glands from DUhTP mice (p ≤ 0.05). Central downregulation of Pou1f1 expression was accompanied by reduced serum concentrations of IGF1 and coordinated downregulation of multiple GH/IGF-signaling compounds in muscle (e.g., Ghr, Igf1, Igf1r, Igf2r, Irs1, Irs2, Akt3, Gskb, Pik3ca/b/a2, Pten, Rictor, Rptor, Tsc1, Mtor; p ≤ 0.05). In response to exercise, the expression of Igfbp3, Igfbp 4, and Igfbp 6 and Stc2 mRNA was increased in the muscle of DUhTP mice (p ≤ 0.05). Training-induced specific activation of AKT, S6K, and p38 MAPK was found in muscles from control mice but not in DUhTP mice (p ≤ 0.05), indicating a lack of mTORC1 and mTORC2 activation in marathon mice in response to physical exercise. While hormone-dependent mTORC1 and mTORC2 pathways in marathon mice were repressed, robust increases of Ragulator complex compounds (p ≤ 0.001) and elevated sirtuin 2 to 6 mRNA expression were observed in the DUhTP marathon mouse model (p ≤ 0.05). Activation of AMPK was not observed under the experimental conditions of the present study. Our results describe coordinated downregulation of the somatotropic pathway in long-term selected marathon mice (DUhTP), possibly via the pituitary gland and muscle interaction. Our results, for the first time, demonstrate that GH/IGF effects are repressed in a context of superior running performance in mice.

Forced expression of insulin-like growth factor binding proteins (IGFBPs) in transgenic mice has clearly revealed inhibitory effects on somatic growth. However, by this approach, it cannot be solved if or how IGFBPs rule insulin-like growth factor (IGF)-dependent growth under normal conditions. In order to address this question, we have used growth-selected mouse models (obese and lean) and studied IGF-1 and IGFBPs in serum with respect to longitudinal growth activity in males and females compared with unselected controls. In mice of both genders, body weights were recorded and daily weight gains were calculated. Between 2 and 54 weeks of age, serum IGF-1 was determined by ELISA and intact IGFBP-2, -3 and -4 were quantified by Western ligand blotting. The molar ratio of IGF-1 to the sum of IGFBP-2 to -4 was calculated for all groups and plotted against the daily weight gain curve. Growth-selected mice are characterized by higher daily weight gains and extended periods of elevated growth activity if compared to matched unselected controls. Therefore, adult mice from the obese and lean groups can achieve more than twofold increased body weight in both genders (p < 0.001). Between 2 and 11 weeks of age, in obese and lean mice of both genders, serum IGF-1 concentrations are increased more prominently if compared to unselected controls (p < 0.001). Instead, substantial decreases of IGFBPs, particularly of IGFBP-2, are observed in males and females of all groups at the age of 2 to 4 weeks (p < 0.001). Due to the strong increase of IGF-1 but not of IGFBPs between two and four weeks of age, the ratio of IGF-1 to IGFBP-2 to -4 in serum significantly increased in all groups and genders (p < 0.05). Notably, the IGF-1 to IGFBP ratio was higher in male and female obese mice if compared to unselected controls (p < 0.05).

The bioactivity of the IGF system is not a function of isolated hormone concentrations in a given biological matrix. Instead, the biological activities of IGFs are regulated by IGFBPs, IGFBP proteases, and inhibitors of IGFBP proteases. Therefore, assays based on IGF-related bioactivity may describe functions of the complete IGF system in a given biological matrix. Of particular interest are the IGF system effects on the AKT/mTOR pathway, as a dominant system for controlling growth, metabolism, and aging. In order to improve the sensitivity of IGF-dependent bioactivity, we made use of the known short-term and enhancing effects of IGFBP2 on the intracellular PI3K pathway. As a specific readout of this pathway, and further as a marker of the mTOR pathway, we assessed the phosphorylation of AKT-Ser473. Preincubation using IGFBP2 enhanced IGF1-dependent AKT-Ser473 phosphorylation in our experimental system. The assay’s specificity was demonstrated by inhibition of IGF1 receptors outside or inside the cell, using antiserum or small molecule inhibitors, which reduced AKT phosphorylation in response to exogenous IGF1 (p < 0.05). The maximal response of AKT phosphorylation was recorded 15 to 60 min after the addition of IGF1 to cell monolayers (p < 0.001). In our cellular system, insulin induced AKT phosphorylation only at supra-physiological concentrations (µM). Using this novel assay, we identified the differential biological activity of the IGF system in AKT-Ser473 phosphorylation in serum (mouse, naked mole rat, and human), in cerebrospinal fluid (human), and in colostrum or mature milk samples (dairy cow). We have developed a sensitive and robust bioassay to assess the IGF-related activation of the AKT/mTOR pathway. The assay works efficiently and does not require expensive cell culture systems. By using capillary immuno-electrophoresis, the readout of IGF-related bioactivity is substantially accelerated, requiring a minimum of hands-on time. Importantly, the assay system is useful for studying IGF-related activity in the AKT/mTOR pathway in a broad range of biological matrices.

In farmed animals, carcass weight represents an important economic trait. Since we had demonstrated that IGFBP-2 represents a potent inhibitor of muscle accretion in inbred mice, we wanted to quantify the inhibitory effects of IGFBP-2 under conditions of elevated protein mass in growth selected non-inbred mice (DU6P). Therefore, we crossed male DU6P mice with female IGFBP-2 transgenic mice. Male IGFBP-2 transgenic offspring (DU6P/IGFBP-2) were characterized by more than 20% reductions of carcass mass compared to male non-transgenic littermates. The carcass mass in males was also significantly lower (p < 0.001) than in transgenic female DU6P/IGFBP-2 mice, which showed a reduction of less than 10% (p < 0.05) compared to non-transgenic female DU6P/IGFBP-2 mice. Although transgene expression was elevated in the muscle of both sexes (p < 0.001), serum levels were normal in female, but significantly reduced in male transgenic DU6P/IGFBP-2 mice (p < 0.001). In this group, also IGFBP-3 and IGFBP-4 were significantly reduced in the circulation (p < 0.01). Particularly in male transgenic mice, we were able to identify proteolytic activity against recombinant IGFBP-2 included in diluted serum. IGFBP-proteolysis in males correlated with massive reductions of IGF-1 in serum samples and the presence of elevated levels of IGFBP-2 fragments. From our data, we conclude that elevated tissue expression of IGFBP-2 is an essential effector of muscle accretion and may block more than 20% of carcass mass. However, in the circulation, intact IGFBP-2 contained no reliable biomarker content. Notably, for the estimation of breeding values in meat-producing animal species, monitoring of IGFBP-2 expression in muscle appears to be supported by the present study in a model system.