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. 2004 Feb;113(3):474-81.
doi: 10.1172/JCI18712.

Conditional disruption of IkappaB kinase 2 fails to prevent obesity-induced insulin resistance

Mathias Röhl  1 , Manolis Pasparakis, Stephanie Baudler, Julia Baumgartl, Dinesh Gautam, Marion Huth, Rossana De Lorenzi, Wilhelm Krone, Klaus Rajewsky, Jens C Brüning
Affiliations

Conditional disruption of IkappaB kinase 2 fails to prevent obesity-induced insulin resistance

Mathias Röhl et al. J Clin Invest. 2004 Feb.

Abstract

The inhibitor of NF-kappaB (IkappaB) kinases (IKK1[alpha] and IKK2[beta]), the catalytic subunits of the IKK complex, phosphorylate IkappaB proteins on serine residues, targeting them for degradation and thus activating the transcription factor NF-kappaB. More recently, IKK2 has been implicated in mediation of insulin resistance caused by obesity, lipid infusion, and TNF-alpha stimulation, since salicylate and aspirin, known inhibitors of IKK activity, can reverse insulin resistance in obese mouse models. To further genetically elucidate the role of IKK2 in obesity-mediated insulin resistance, we have conditionally inactivated the mouse IKK2 gene in adult myocytes by Cre-loxP-mediated recombination in vivo. We have investigated the development of obesity-induced insulin resistance in muscle-specific IKK2 knockout mice and mice exhibiting a 50% reduction of IKK2 expression in every tissue and have found that, after gold thioglucose treatment, wild-type and mutant mice developed obesity to a similar extent. Surprisingly, no difference in obesity-induced insulin resistance was detectable, either at a physiological or at a molecular level. Moreover, impaired glucose tolerance resulting from a high-fat diet occurred to the same degree in control and IKK2 mutant mice. These data argue against a substantial role for muscular IKK2 in mediating obesity-induced insulin resistance in these models in vivo.

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Figures

Figure 1
Figure 1
Efficient muscle-specific deletion of IKK2 in IKK2 mutant mice. (a) The upper panel shows a Western blot analysis of protein extracts prepared from control and conditionally IKK2-deficient mice (lanes 1–7) and from mouse embryonic fibroblasts of control (lane 8) and conventional IKK2-deficient mice (lane 9) probed with an anti-IKK2–specific antiserum. The genotypes of the mice are as follows: IKK2WT, lanes 1 and 5; IKK2Het, lanes 2 and 6; IKK2Mus, lane 3; IKK2Het/Mus, lanes 4 and 7. “NS” marks the position of a nonspecific band detected by the antiserum in skeletal muscle. The lower panel shows a Western blot analysis of the same blot shown in the upper panel, after stripping and reprobing with an anti-actin–specific antiserum. (b) Western blot analysis of IKK1 expression in skeletal muscle of different IKK2 mutant mice. The genotypes of the mice are as follows: IKK2WT, lanes 1 and 5; IKK2Het, lanes 2 and 6; IKK2Mus, lane 3; IKK2Het/Mus, lane 4. (c) Western Blot analysis of IKK2 expression in adipose tissue of different conditional IKK2 mutant mice. The genotypes of the mice are as follows: IKK2WT, lanes 1 and 5; IKK2Het, lanes 2 and 6; IKK2Mus, lane 3; IKK2Het/Mus, lane 4.
Figure 2
Figure 2
GTG treatment results in obesity. (a) Growth curves of lean male mice (open squares) as compared with GTG-treated male mice (filled squares). Data represent the mean ± SEM of at least 16 animals in each group. Weights are significantly different between lean and obese mice from week 8 on (P < 0.05). (b) The body weight of GTG-treated male mice, according to their IKK2 genotype. Data represent the mean ± SEM of at least six animals in each group. (c) Plasma leptin concentrations in lean and obese mice, given as mean ± SEM of at least 16 animals in each group (*P < 0.01). (d) Plasma leptin concentrations in GTG-treated male mice of the indicated genotype, given as mean ± SEM of at least 6 animals in each group, revealed no significant differences depending on IKK2 expression.
Figure 3
Figure 3
GTG treatment results in increased TNF-α expression in WAT, activation of TNF-α signaling in skeletal muscle, and decreased plasma adiponectin concentrations. (a) The upper panel shows a Northern blot analysis of RNA extracted from WAT of control and IKK2Het mice injected either with PBS or GTG. “TNF-α” marks the position of the murine TNF-α mRNA. The lower panel shows a photograph of the ethidium bromide–stained RNA gel prior to transfer onto a nylon membrane as a loading control. “18S” marks the position of the 18S RNA. (b) Protein extracts were prepared from skeletal muscle of lean and obese IKK2WT and IKK2Het/Mus mice and subjected to SDS-PAGE followed by Western transfer. After transfer onto nitrocellulose filters, blots were cut and the upper part was subjected to Western blot analysis with an antiserum against RasGap as a loading control, while the lower part was subjected to Western blot analysis with an antiserum that detects IκBα when phosphorylated at serine 32. The lower panel shows the densitometric quantification of IκBα phosphorylation as mean ± SEM of four to six animals in each group. (c) Plasma adiponectin concentrations in lean and obese mice, given as mean ± SEM of at least 16 animals in each group (*P < 0.01). (d) Plasma adiponectin concentrations in GTG-treated male mice of the indicated genotype, given as mean ± SEM of at least six animals in each group, revealed no significant differences depending on IKK2 expression.
Figure 4
Figure 4
Glucose metabolism in obese IKK2-deficient mice. (a) Random-fed blood glucose concentrations in male GTG-treated animals of the indicated genotype. Data represent the mean ± SEM of at least six animals in each group. Statistical analysis revealed no significant differences between the groups (unpaired Student’s t test). (b) Plasma insulin concentrations in lean and obese mice, given as mean ± SEM of at least 16 animals in each group (*P < 0.05). (c) Plasma insulin concentrations in male GTG-treated animals of the indicated genotype. Data represent the mean ± SEM of at least six animals in each group. Statistical analysis revealed no significant differences between the groups (unpaired Student’s t test). (d) The results of glucose-tolerance tests in male lean mice (filled squares), GTG-treated mice (open squares), and mice fed a high-fat diet (filled circles). Data represent the mean ± SEM of at least 12 animals in each group (*P < 0.05, **P < 0.01, unpaired Student’s t test). (e) The results from glucose-tolerance tests in male GTG-treated animals. Data represent the mean ± SEM of at least six animals in each group (IKK2WT mice, filled diamonds; IKK2Het mice, filled squares; IKK2Mus mice, open squares; IKK2Het/Mus mice, open diamonds). (f) The results from glucose-tolerance tests in male animals exposed to a high-fat diet. Data represent the mean ± SEM of at least six animals in each group (IKK2WT mice, filled diamonds; IKK2Het mice, filled squares; IKK2Mus mice, open squares; IKK2Het/Mus mice, open circles).
Figure 5
Figure 5
Insulin-stimulated IR activation in skeletal muscle of IKK2-deficient mice. (a) Protein extracts isolated from skeletal muscle of mice, which had been injected with 5 IU of regular insulin 5 minutes before harvesting, were immunoprecipitated with an anti–IR-specific antiserum. The upper panel shows Western blot analysis using an anti–phosphotyrosine-specific antibody (anti-PY). The lower panel shows the autoradiogram after reprobing of the same blot with an anti-IR antiserum. IB, immunoblot; IP, immunoprecipitation. (b) Shown is the densitometric quantification of insulin-stimulated tyrosine phosphorylation of the IR β subunit from multiple experiments as outlined in a. The insulin-stimulated IR tyrosine phosphorylation of obese control (IKK2WT) mice was arbitrarily set as 100%. The results represent the mean of two to five animals of the indicated genotype. Insulin-stimulated IR tyrosine phosphorylation was significantly decreased in obese versus lean animals (P < 0.05), but without significant differences between obese animals of the different IKK2 genotypes.
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