We subcloned cDNAs encoding Flag-tagged, constitutively active24-26
, human IKK-β (IKBKB) S177E,S181E or hemagglutinin-tagged human IκBα (NFKBIA) S32A,S36A superrepressor31
into a truncated β-globin expression vector40
having a 3.0-kb fragment of the mouse albumin (Alb1
) promoter upstream from exon 1 (). DNA was excised from the cloning vector, purified and microinjected into mouse oocytes, which were introduced into pseudopregnant females. We genotyped transgenic offspring by Southern analysis and PCR. At least two independent lines were obtained for both LIKK and LISR mice and each had the same phenotype. The mice were housed in groups of four per cage, maintained under alternating 12-h light and dark cycles and had free access to food and water. Animal experiments were conducted in accordance with the US National Institutes of Health guidelines under protocols approved by the Institutional Animal Care and Use Committee of the Joslin Diabetes Center.
We isolated IKK complexes from mouse liver lysates using anti-IKK-γ antibodies (PharMingen) coupled to protein A-Sepharose, and conducted in vitro
kinase assays at 30 °C by adding [γ-32
P]ATP and 2 μg of GST-IκBα (1-54)12
. We determined NK-κB activity using electrophoretic mobility shift assay and enzyme-linked immunosorbent assay–based DNA binding methods41,42
, blood glucose using a Glucometer Elite (Bayer), insulin and leptin (Crystal Chem Inc.) and nonesterified fatty acids (Dako), and triglycerides in 50-mg liver samples43
(Sigma) with the indicated kits. We determined plasma concentrations of cytokines, including IL-1β, IL-6 and TNF-α, using a mouse Luminex kit (Linco). The clinical laboratory at Children's Hospital in Boston measured serum salicylate, lactic acid dehydrogenase and alanine aminotransferase levels.
We performed the clamps in conscious mice following an overnight fast44
, maintaining blood glucose concentrations (5.5 mM) during the 5-h, steady-state human insulin infusions (4 mU/kg/min) by infusing 20% glucose at variable rates. We initiated [3-3
H]glucose and 2-deoxy-D-[1-14
C]glucose (NEN) infusions during the final 1 h of the clamps to determine whole-animal glucose fluxes and tissue-specific glucose uptake, respectively. Liver, fat and muscle samples were rapidly removed and flash frozen in liquid nitrogen.
Western blotting. We homogenized animal tissues for 1.0 min at 4 °C in lysis buffer (30 mM HEPES, 150 mM NaCl, 1.0 mM phenylmethylsulfonyl fluoride, 3.0 μM aprotinin, 10 μM leupeptin, 5.0 μM pepstatin A, 25 mM benzamidine, 2 mM sodium vanadate, 5.0 mM glycerol phosphate, 100 mM NaF, 1.0 mM ammonium molybdate, 30 mM tetrasodium pyrophosphate, 5.0 mM EGTA, 10% glycerol and 1% Triton X-100, pH 7.4). Proteins were immunoprecipitated with appropriate antibodies, separated by SDS-PAGE, transferred to nitrocellulose membranes and identified by immunoblotting. Primary antibodies used included those directed against Flag (Strategene), hemagglutinin (Cell Signaling), phosphotyrosine (4G10, Upstate Biotechnology); phosphorylated Stat-3 and Stat-3 (Cell Signaling), IKK-β, IκBα, GSK3β, phosphorylated GSK3β, IRS-1, IRS-2 and insulin receptor α (Santa Cruz). Secondary, HRP-conjugated antibodies were specific for rabbit or mouse (Pierce).
Quantitative real-time RT-PCR. We extracted total RNA from frozen, pulverized mouse tissues using TRIzol (Invitrogen) and synthesized cDNA using oligo (dT) primers with the Advantage RT-for-PCR kit (BD Biosciences). Primers spanned intronic regions to generate 300–400-bp PCR products. We quantified PCR amplifications using SYBR Green PCR Master Mix (Applied Biosystems) and normalized results against TATA box binding protein (Tbp) and glyceraldehydes-3-phosphate dehydrogenase (Gapd) gene expression.
IL-6 neutralization and salicylate therapy. We administered antibody to IL-6 or isotype-matched nonimmune rat IgG (BD Biosciences) by tail vein injection (50 μg every other day for 10 d). We conducted glucose tolerance tests and other measures of insulin resistance 2 d after the last injection. Sodium salicylate (6 mg/ml) was dissolved in the drinking water (pH 7.0) and incorporated into the chow (0.5 mg/g). Mice were allowed free access to food and water.
Immunohistochemistry and morphometric analyses.
We treated fixed sections of liver (6 μm) with goat antibody to mouse IL-6 (R&D) and rat antibody to mouse Cd68 (PharMingen), followed by FITC- and rhodamine-conjugated secondary antibodies (Jackson). Naive goat and rat IgG were used as negative controls. We took sections (6 μm) from paraffin-embedded pancreas (Bouins, Sigma) as full footprints to include head, body and tail of the organ. These were immunostained with guinea pig antibody to insulin and rabbit antibody to glucagon (Linco), followed by FITC- and Texas Red–conjugated secondary antibodies (Jackson). We calculated beta-cell mass according to the method of Weibel45
using NIH Image 1.60 analysis software.
Statistical analyses. Data are presented as mean ± s.e.m. Student t-tests were used for statistical analysis. P < 0.05 was considered significant.