During systemic inflammation, the liver becomes unresponsive to growth hormone (GH), resulting in decreased plasma insulin-like growth factor-I (IGF-I) with concomitant reductions in lean body mass. Transgenic mice that overexpress IL-6 also demonstrate impaired growth and decreased IGF-I. To determine whether IL-6 directly inhibits GH-inducible gene expression, CWSV-1 hepatocytes were incubated with IL-6 (10 ng/ml), then stimulated with recombinant human GH (500 ng/ml, 18 h). The increase in IGF-I and serine protease inhibitor 2.1 (Spi 2.1) mRNA in GH-treated cells was inhibited by treatment with IL-6 for 24 h. To investigate potential mechanisms, we examined the effects of IL-6 on GH receptor (GHR) expression and GH signaling via the JAK/signal transducer and activator of transcription (STAT) and MAP kinase pathways. Incubation of cells with IL-6 (10 ng/ml, 24 h) had no effect on GHR abundance or signaling proteins JAK2, STAT5b, and ERK1/2. Although GH transiently increased (2- to 5-fold) the tyrosine phosphorylation of GHR, JAK2, STAT5b, and ERK1/2, IL-6 did not alter these phosphorylation events. However, nuclear protein from IL-6-treated cells demonstrated reduced STAT5 DNA binding (by EMSA) at 15 min (-20%) and 60 min (-43%) after GH stimulation. To determine whether IL-6 inhibits GH-inducible promoter activity, CWSV-1 cells were transfected with Spi 2.1 or prolactin receptor promoter luciferase vectors, incubated with or without IL-6, then stimulated with GH. The induction of both Spi 2.1 (7.5-fold) and prolactin receptor (4-fold) promoter activity by GH was inhibited by IL-6. In summary, IL-6 mediates hepatic GH resistance by a time-dependent inhibition of GH-inducible promoter activity that is associated with reductions in STAT5 DNA binding.
Introduction For trauma triage, the US Army has developed a portable heart rate complexity (HRC) monitor, which estimates cardiac autonomic input and the activity of the hypothalamic-pituitary-adrenal (HPA) axis. We hypothesize that autonomic/HPA stress associated with predeployment training in U.S. Army Forward Surgical Teams will cause changes in HRC. Materials and Methods A prospective observational study was conducted in 80 soldiers and 10 civilians at the U.S. Army Trauma Training Detachment. Heart rate (HR, b/min), cardiac output (CO, L/min), HR variability (HRV, ms), and HRC (Sample Entropy, unitless), were measured using a portable non-invasive hemodynamic monitor during postural changes, a mass casualty (MASCAL) situational training exercise (STX) using live tissue, a mock trauma (MT) STX using moulaged humans, and/or physical exercise. Results Baseline HR, CO, HRV, and HRC averaged 72 ± 11b/min, 5.6 ± 1.2 L/min, 48 ± 24 ms, and 1.9 ± 0.5 (unitless), respectively. Supine to sitting to standing caused minimal changes. Before the MASCAL or MT, HR and CO both increased to ~125% baseline, whereas HRV and HRC both decreased to ~75% baseline. Those values all changed an additional ~5% during the MASCAL, but an additional 10 to 30% during the MT. With physical exercise, HR and CO increased to >200% baseline, while HRV and HRC both decreased to 40 to 60% baseline; these changes were comparable to those caused by the MT. All the changes were P < 0.05. Conclusions Various forms of HPA stress during Forward Surgical Team STXs can be objectively quantitated continuously in real time with a portable non-invasive monitor. Differences from resting baseline indicate stress anticipating an impending STX whereas differences between average and peak responses indicate the relative stress between STXs. Monitoring HRC could prove useful to field commanders to rapidly and objectively assess the readiness status of troops during STXs or repeated operational missions. In the future, health care systems and regulatory bodies will likely be held accountable for stress in their trainees and/or obliged to develop wellness options and standardize efforts to ameliorate burnout, so HRC metrics might have a role, as well.
TNF inhibits serine protease inhibitor 2.1 (Spi 2.1) and IGF-I gene expression by GH in CWSV-1 hepatocytes. The current study describes construction of a GH-inducible IGF-I promoter construct and investigates mechanisms by which TNF and nuclear factor-kappaB (NFkappaB) inhibit GH-inducible gene expression. CWSV-1 cells were transfected with GH-inducible Spi 2.1 or IGF-I promoter luciferase constructs, incubated with TNF signaling inhibitors (fumonisin B1 for sphingomyelinase and SP600125 for c-Jun N-terminal kinase), treated with or without TNF, and then stimulated with recombinant human GH. The 5- to 6-fold induction of Spi 2.1 and IGF-I promoter activity by GH was inhibited by TNF. Neither fumonisin B1 nor SP600125 prevented the inhibitory effects of TNF on GH-inducible promoter activity. Dominant-negative inhibitor-kappaBalpha (IkappaBalpha) expression vectors (IkappaBalphaS/A or IkappaBalphaTrunc), p65 and p50 expression vectors, and p65 deletion constructs were used to investigate the NFkappaB pathway. IkappaBalphaS/A and IkappaBalphaTrunc ameliorated the inhibitory effects of TNF on GH-inducible Spi 2.1 and IGF-I promoter activity. Cotransfection of CWSV-1 cells with expression vectors for p65 alone or p50 and p65 together inhibited GH-inducible Spi 2.1 and IGF-I promoter activity. Cotransfection with a C-terminal p65 deletion (1-450) enhanced GH-inducible promoter activity, whereas the N-terminal deletion (31-551) was inhibitory for IGF-I but not Spi 2.1. Cycloheximide did not antagonize the inhibitory effects of TNF on GH-inducible IGF-I expression. We conclude the inhibitory effects of TNF on GH-inducible promoter activity are mediated by NFkappaB, especially p65, by a mechanism that does not require protein synthesis.
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