A number of studies now suggest that obesity generates a state of chronic, systemic low-grade inflammation (Hotamisligil, 2006
; Shoelson et al., 2007
; Wellen and Hotamisligil, 2005
). Numerous genetic and pharmacological approaches indicate that the NFκB pathway may play a crucial role in this response, although the tissues involved, the precise cell types that are activated in those tissues, and the signaling processes remain uncertain. To investigate the role of this pathway in obesity, we directly examined NFκB activation in vivo
using HLL mice, a transgenic line bearing the NFκB-responsive promoter of HIV LTR fused to luciferase (Sadikot et al., 2001
). These studies revealed that HFD activates NFκB in fat and liver, with the most profound increase in adipocytes and ATMs. The signals that lead to the activation of NFκB during high fat feeding are unknown, as are the potential consequences of sustained NFκB activation in metabolic tissues. We report here that the protein kinase IKKε, which is induced by activation of the NFκB pathway, might play a major role as a mediator of this multifaceted process independent of any role in acute immune responses.
IKKε is a direct transcriptional target of the NFκB pathway, and is likely to amplify further inflammatory signals via phosphorylation of other transcription factors. We show here that IKKε gene and protein expression and enzyme activity are increased in liver, adipocytes and ATMs after high fat feeding, coincident with the activation of NFκB. IKKε KO mice are rendered resistant to the deleterious effects of HFD on insulin sensitivity, exhibiting normal glucose, pyruvate and insulin tolerance. Their circulating cholesterol levels are markedly reduced, and adiponectin is elevated while leptin is lowered. Interestingly, these mice do not gain as much weight as wild type mice on HFD, due to increased energy expenditure. Additionally, these mice do not develop hepatic steatosis, macrophage infiltration in adipose tissue, or elevations in pro-inflammatory gene expression in fat and liver expected with an inflammatory state.
These dramatic effects on glucose and lipid metabolism, energy expenditure and inflammation beg an important question, what is the primary effect of HFD-induced IKKε elevation, and where does it occur? Increased energy expenditure without changes in RQ indicates that there is no shift in fuel substrate preference in these animals. Although adiponectin levels were elevated in KO mice, transgenic mice overexpressing adiponectin at levels significantly higher than seen here do not phenocopy the metabolic profile of IKKε KO mice (Otabe et al., 2007
), suggesting that adiponectin alone is unlikely to account for the phenotype. However, there was an elevation in UCP-1 expression in WAT, and IKKε KO mice exhibited increased temperatures, indicating that increased energy expenditure may be a result of enhanced thermogenesis. The molecular mechanisms underlying this event are unknown, and could involve increased expression or activity of PPARγ (Kelly et al., 1998
; Laplante et al., 2003
), or alleviation of a direct inhibitory effect of IKKε on the UCP-1 promoter through phosphorylation of transcriptional regulator(s). One interesting possibility is that IKKε normally functions to repress an adaptive increase in expression of UCP-1 in WAT in response to HFD.
It is possible that both metabolic and inflammatory changes in the IKKε KO mice are secondary to lower body weight. Weight reduction would be expected to reduce macrophage infiltration in adipose tissue and improve glucose and insulin tolerance. However, this difference in energy expenditure was only seen in HFD mice, and weight-matched animals showed the same differences in rectal temperature, inflammation and hepatosteatosis (data not shown), suggesting that weight loss alone is unlikely to explain these traits. It is also possible that IKKε gene deletion in macrophages prevents the initial inflammatory response to HFD, subsequently leading to metabolic resistance to diet. Indeed, many studies have shown that deletion of inflammatory genes can restore global glucose and insulin tolerance. However, other targeted gene disruptions that block adipose tissue inflammation not prevent weight gain from HFD (Kanda et al., 2006
; Lesniewski et al., 2007
; Moller, 2000
; Weisberg et al., 2006
). While blockade of adipose tissue inflammation alone may not explain the entire phenotype, we cannot exclude the possibility that simultaneously reducing inflammation in liver and fat can produce these changes.
IKKε KO mice also exhibited gene expression changes that are consistent with improved insulin sensitivity. In adipose tissue, PPARγ mRNA was increased, along with changes in genes (adiponectin, CAP, CD36 and TNFα) sensitive to PPARγ activation. Moreover, there was a significant reduction in adipocyte size, along with an increase in cell number that is expected with PPARγ activation and increased adipogenesis (Okuno et al., 1998
; Tsuchida et al., 2005
). At the same time, overexpression of wild type IKKε produced a substantial reduction in insulin-stimulated glucose uptake in 3T3-L1 adipocytes, indicating that the IKKε-dependent insulin resistance in adipocytes might be at least partially cell autonomous.
IKKε KO also improved glucose and lipid metabolism in the liver. In addition to the almost complete prevention of steatosis, there were large reductions in circulating cholesterol. Transcriptional profiling of IKKε KO mice revealed reduced levels of PDK4, PPARγ, FABP4 and CD36 mRNA, with increases in PPARα, lipin1 and glucokinase, all of which are consistent with enhanced insulin sensitivity and decreased lipid synthesis and export. These genes may be regulated by transcription factors that are direct targets of IKKε. In this regard, overexpression of IKKε in hepatoma cells increased expression of selected gluconeogenic, lipogenic and proinflammatory genes, indicating that at least some of these effects were direct and cell autonomous in the liver.
We are currently investigating the kinase substrates of IKKε in tissues that respond to HFD. We hypothesize that some of these targets may be transcriptional activators or repressors that regulate metabolic genes. A number of the genes that changed in IKKε KO mice in both liver and fat are transcriptionally regulated by interferon regulatory factor (IRF) family members in 3T3-L1 adipocytes (Eguchi et al., 2008
), although the mechanism(s) by which IRF3 acts as a repressor remains unknown. Additionally, IKKε can catalyze STAT1 phosphorylation on serine 708 in response to interferon β treatment in vitro
(Tenoever et al., 2007
). Phosphorylation of S708 favors the formation of STAT1/STAT2 heterodimers for assembly of the ISGF3 (interferon-stimulated gene factor 3) complex. This complex binds to interferon-stimulated response elements in the promoters of interferon-stimulated genes, some of which, including IFI205, IFIT2, and IFIT3, were identified in the microarray analyses, and thus might lie downstream of this pathway.
The increased expression of IKKε in liver, adipocytes and ATMs, coupled with the complex nature of the IKKε KO phenotype, suggest that IKKε is likely to exert its effects through all three tissues in a manner that involves crosstalk among these cells. Although IKKε is unlikely to play a role in the initial inflammatory response to high fat feeding, it may be required for a sustained inflammatory state. Although it is important to note that IKKε KO mice are susceptible to lethal viral infections (Tenoever et al., 2007
), the specificity of the apparent actions of IKKε, the nature of the enzyme, and the profound resistance of KO mice to high fat diet, make it an especially appealing drug target for the treatment of metabolic disease.
Immunohistochemistry and confocal microscopy
IHC and confocal microscopy were performed as described (Lumeng et al., 2007a
; Lumeng et al., 2008
). Adipocyte cross-sectional area from caveolin stained adipose tissue images (150–200 adipocytes/mouse, 3 mice/genotype) was calculated using CellProfiler image analysis software. Adipocyte number was calculated from adipocyte diameters using established formulas (Hirsch and Batchelor, 1976
In vitro IKKε kinase assay in Cos cells
Cos cells were transfected with plasmid encoding FLAG-IKKε for 24 h and treated with LPS (1μg/ml) for 30min before harvested. FLAG-IKKε in whole cell lysates was immunoprecipitated and subjected to an in vitro kinase assay using MBP as substrate. Following fractionation of samples by SDS-PAGE, the [r-32P]-ATP-labeled MBP on the lower half of the gel was quantified by autoradiography; the upper half of gel was blotted for the immunoprecipitated kinase using anti-FLAG antibody.