Peripheral tumor necrosis factor α regulation of adipose tissue metabolism and adipokine gene expression in neonatal pigs

Abstract

The neonatal pig is susceptible to stress and infection, conditions which favor tumor necrosis factor α (TNFα) secretion. This study examined whether TNFα can alter metabolic activity and cytokine gene expression within neonatal pig adipose tissue. Cell cultures were prepared from neonatal subcutaneous adipose tissue using standard procedures. Cultures (5 experiments) were incubated with medium containing (14)C-glucose for 4 h to measure glucose conversion to lipid in the presence of combinations of TNFα (10 ng), insulin (10 nM) and an anti-pig TNFα antibody (5 μg). Basal lipogenesis was not affected by TNFα treatment (P > 0.05). However, insulin stimulated lipogenesis was reduced by TNFα (P < 0.02). For gene expression studies, cultures were incubated with 0, 2.5, 5.0 or 10 ng TNFα for 2, 4 or 24 h (n = 4 experiments). Interleukin 6 and TNFα gene expression were acutely (2-4 h) stimulated by exogenous TNFα treatment (P < 0.05), as analyzed by real-time PCR. Adiponectin mRNA abundance was reduced (P < 0.001) while monocyte chemotactic gene expression was increased by TNFα treatment at all time points (P < 0.001). Chronic treatment (24 h) was required to increase monocyte multiplication inhibitory factor or suppress lipoprotein lipase gene expression (P < 0.02). These data suggest conditions which increase serum TNFα, like sepsis, could suppress lipid accumulation within adipose tissue at a time of critical need in the neonate and induce a variety of adipose derived cytokines which may function to alter adipose physiology.

Regulation of cytokine gene expression by orosomucoid in neonatal swine adipose tissue

“…Since the observed reduction was relatively small, some caution must be used in the interpretation. Previous research has demonstrated that TNFα can interfere with insulin mediated glucose metabolism in neonatal porcine adipose tissue [ 17 ], thereby creating an insulin resistant state that is characteristic of adipose tissue inflammation in obesity, diabetes and metabolic syndrome [ 31 ]. The present data suggest that ORM1 does not contribute to adipose tissue inflammation in the pig at the low concentrations tested by stimulating TNFα expression or secretion, despite these same ORM1 concentrations (1 and 10 μg/mL medium) reducing glucose utilization by the tissue in vitro [ 14 ].…”

“…TNFα can regulate the expression of a number of cytokines within neonatal porcine adipose tissue, including stimulating the expression of the pro-inflammatory cytokines IL6, MIF and MCP [ 17 ]. A reduction in IL6 mRNA level by ORM1 has been previously reported for murine 3T3-L1 adipocytes [ 30 ].…”

“…Euthanasia was performed in accordance with the ethical standards of the institutional Animal Care and Use Committee and were approved by the committee. Primary cultures containing pig preadipocytes and SV cells were prepared by conventional methods previously published [ 17 ]. Cells were seeded on six well tissue culture plates at a density of 1.0 × 10 4 cells/cm 2 in DMEM/F12 containing 10 % FBS (vol/vol), 10,000 U/L penicillin sodium, 100 mg streptomycin sulfate/L, 250 μg amphotericin B (plating medium).…”

“…All primer sets were designed to span an intron as previously described and utilized for real-time PCR (Table 1 ), [ 13 , 17 , 23 ]. The specific genes of interest were selected based upon their reported roles in the inflammatory response within adipose tissue [ 12 ].…”

“…Thermal cycling and data acquisition were performed with a Bio-Rad iCycler iQ system (Bio-Rad Laboratories Inc., Hercules, CA). Reverse transcription (RT) and real time PCR analysis were performed in a two tube assay as previously described [ 13 , 17 ]. Reverse transcription was done using a SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen, Carlsbad, CA) according to the manufacturer’s directions.…”