Effects of prepartum 2,4-thiazolidinedione on insulin sensitivity, plasma concentrations of tumor necrosis factor-α and leptin, and adipose tissue gene expression.

Administration of peroxisome proliferator-activated receptor gamma (PPARγ) ligands, thiazolidinediones (TZD), to prepartum dairy cattle has been shown to improve dry matter intake and decrease circulating nonesterified fatty acids (NEFA) around the time of calving. The objective of this work was to elucidate mechanisms of TZD action in transition dairy cattle by investigating changes in plasma leptin, tumor necrosis factor-α (TNFα), the revised quantitative insulin sensitivity check index (RQUICKI), and adipose tissue gene expression of leptin, PPARγ, lipoprotein lipase (LPL), and fatty acid synthase (FAS). Multiparous Holstein cows (n=40) were administered 0, 2.0, or 4.0 mg of TZD/kg of body weight (BW) by intrajugular infusion once daily from 21 d before expected parturition until parturition. Plasma samples collected daily from 22 d before expected parturition through 21 d postpartum were analyzed for glucose, NEFA, and insulin. Plasma samples collected on d -14, -3, -1, 1, 3, 7, 14, and 49 relative to parturition were also analyzed for leptin and TNFα. Adipose tissue was collected on d 7 before expected parturition from a subset of cows, and gene expression was examined via quantitative real-time PCR. A tendency for a treatment by time effect on plasma leptin prepartum was observed such that values were similar on d -14 but cows receiving 2.0 mg/kg of BW of TZD tended to have lower circulating leptin as calving approached. Postpartum leptin tended to be increased linearly (2.3, 2.4, and 2.5±0.1 ng/mL for 0, 2.0, and 4.0 mg/kg treatments, respectively) in cows that received TZD prepartum. Plasma TNFα increased linearly (2.6, 3.7, and 4.0±0.1 pg/mL) in response to TZD treatment and decreased through the first week postpartum. Calculation of RQUICKI 1/[log(glucose)+log(insulin)+log(NEFA)] suggested altered insulin sensitivity in cows administered TZD that may depend on day relative to calving. Administration of TZD increased adipose tissue expression of PPARγ mRNA (11.0, 13.3, and 12.8±1.9). Administration of TZD had a quadratic effect on gene expression of leptin (16.2, 10.7, and 17.4±1.6) and no effect on LPL expression, and expression of FAS was lower for TZD-treated cows than for controls (8.2, 4.2, and 6.1±1.8, respectively). Results imply altered expression and plasma concentrations of leptin, increased plasma TNFα concentrations, and increased expression of PPARγ in adipose tissue as potential mechanisms for the effects of TZD administration on transition dairy cattle.

Technical note: validation of an ELISA for measurement of tumor necrosis factor alpha in bovine plasma.

Tumor necrosis factor α (TNFα) is an inflammatory cytokine that is involved in immune function and is proposed to play a role in metabolic disorders. Although some bovine-specific methods have been published recently, assays used for determining plasma TNFα concentration in bovine disease models often do not offer acceptable precision for measurement of basal concentrations in healthy animals. The objective of this work was to develop an effective, low-cost sandwich ELISA procedure with improved sensitivity. A protocol developed for use with cell culture supernatant was modified for use with bovine plasma and serum by optimizing antibody concentrations, incubation times and temperatures, and standard diluents. The coating antibody concentration was decreased from 10 to 6.8 µg/mL, whereas the detection antibody concentration remained 2.5 µg/mL. Sample incubation was increased from 1h at room temperature to an overnight incubation at 4°C, which increased the sensitivity of the assay. Multiple matrices were tested for dilution of standards and were assessed by determining recovery of bovine TNFα spiked into bovine serum and plasma. Recoveries were acceptable in both bovine serum and plasma (71-103%) when quantified with standards diluted in human serum or phosphate-buffered saline. The modified bovine TNFα ELISA offers a detection range of 2 to 250 pg/mL. This detection limit is at least an order of magnitude lower than previously reported, and will allow for greater precision in determining basal TNFα concentrations in bovine plasma. The improved sensitivity of this ELISA will be critical to assessing current hypotheses concerning the metabolic effects of moderately elevated TNFα concentrations.