In this study we show, for the first time, that loss of TAK1 protects mice against age- and HFD-induced metabolic syndrome. TAK1−/− mice remain lean and show reduced adiposity and hepatic steatosis during aging or when fed a HFD. Moreover, TAK1−/− mice are protected against the development of age- and diet-induced adipose tissue-associated inflammation, insulin resistance, and glucose intolerance. These observations indicate that the nuclear receptor TAK1 plays a critical role in the control of energy balance and lipid homeostasis.
Livers of TAK1−/−
mice showed a reduced lipid accumulation compared with their WT littermates. Hepatic triglyceride accumulation is controlled at several levels, including fatty acid uptake, synthesis and storage of triglycerides, fatty acid oxidation, and lipolysis. Gene expression profiling revealed a great number of differences in gene expression between livers from 1-year-old WT and TAK1−/−
mice, including genes that are critical in the regulation of lipid, fatty acid, carbohydrate, and xenobiotic metabolism, and gene transcription (). The expression of many of these genes has been reported to be elevated in hepatic steatosis (31
). One of these genes is CD36, which encodes a multifunctional protein implicated in angiogenesis, immunity, and in several metabolic disorders, such as obesity, hepatic steatosis, and insulin resistance (28
). In several cell types, including adipocytes and hepatocytes, CD36 facilitates long-chain fatty acid uptake. Thus, the reduced CD36 expression observed in TAK1−/−
liver may lead to diminished hepatic fatty acid uptake and, at least in part, be responsible for the resistance to hepatic steatosis.
Cidea and Cidec were also among the genes that were the most dramatically downregulated in TAK1−/−
mice. Cide proteins promote triglyceride accumulation within lipid droplets and regulate lipolysis, and their expression correlates positively with the development of obesity and hepatic steatosis (25
). Deficiency in Cidea or Cidec in mice resulted in increased energy expenditure and lipolysis, and yielded a lean phenotype in mice and resistance to diet-induced obesity (25
). Therefore, the repression of these genes in TAK1−/−
mice may also have contributed to the reduction in hepatic triglyceride levels and resistance to hepatic steatosis in TAK1−/−
mice. Although the expression of Cidea and Cidec, as well as CD36, was greatly repressed in the liver of TAK1−/−
mice, TAK1 did not appear to regulate the expression of these genes in WAT, suggesting a tissue-dependent regulation.
Mogat1, another gene that was dramatically downregulated in TAK1−/−
liver, is part of an alternative, less-studied pathway of triglyceride synthesis. The main pathway of triglyceride synthesis is catalyzed by glycerol-3-phosphate acyltransferase (GPAT), acyl-glycerol-3-phosphate acyltransferases (AGPATs), and diacylglycerol transferase (DGAT) in the final step of synthesis (37
). The expression of DGAT1 was not altered; however, the expression of GPAT1 and AGPAT6 was significantly reduced in TAK1−/−
liver. The latter is interesting because AGPAT6-deficiency has been reported to cause lipodystrophy and resistance to obesity (38
). Thus, the lower levels of Mogat1, GPAT1, and AGPAT6 expression may be part of the mechanism by which triglyceride synthesis and storage is reduced in TAK1−/−
liver. Thus, the regulation of several genes with functions related to fatty acid uptake (CD36
), triglyceride synthesis (Mogat1
), and storage (Cidea
) suggests that TAK1 affects several aspects of lipid accumulation. In contrast, no significant changes in fatty acid oxidation were observed.
In contrast to aged mice, 4- to 5-month-old mice fed with a normal diet did not show histologic signs of hepatic steatosis; however, the hepatic expression of Cidea, Cidec, Mogat1, CD36, and Retn was significantly lower in TAK1−/−
mice than WT littermates. Consistent with a previous study (19
), young TAK1 KO mice were also more glucose tolerant and insulin sensitive than WT mice (supplementary Fig. 4). These observations suggest that TAK1 affects changes in hepatic gene expression and insulin sensitivity at an early age.
Energy and lipid homeostasis is under the control of a complex network of transcription factors and coregulators (32
). Deficiencies in many of these factors have been associated with resistance to diet-induced obesity. For example, mice deficient in the nuclear receptors COUP-TFII and ERRα, or the coregulator RIP140 exhibit a lean phenotype; however, the expression of these genes was unaltered in TAK1−/−
liver. Because TAK1 itself functions as a transcription factor, one might expect that some of the differentially expressed genes be regulated directly by TAK1. Indeed, a recent report showed that TAK1 regulates CD36
transcription in macrophages by binding to TAK1 response elements in the CD36
gene promoter (14
), suggesting that CD36
is a direct TAK1 target gene. CD36
is also a known target of several other nuclear receptors, including PPARγ, LXR, and PXR (42
). Although the expression of PXR and LXR was unchanged, the expression of PPARγ was reduced by 50% in liver of TAK1−/−
mice. Therefore, hepatic CD36 expression might be regulated by TAK1 directly as well as indirectly through modulation of PPARγ expression (D
). The coregulators RIP140 and PGC-1α, and the receptor PPARγ have also been implicated in the regulation of Cidec (29
). TAK1 might cooperate with these transcriptional modulators to regulate the expression of these genes. Moreover, the downregulation of the transcription factor Srebf1, which promotes triglyceride synthesis (44
), may contribute to the reduced lipid accumulation in TAK1−/−
Our data also demonstrated that the expression of several lipogenic genes was dramatically decreased in TAK1−/− primary hepatocytes compared with WT hepatocytes. Restoration of TAK1 expression in TAK1−/− hepatocytes by Ad-TAK1 induced the expression of Mogat1, Cidea, and Cidec, whereas empty virus or expression of an inactive form of TAK1 had little effect on their expression level. Moreover, downregulation of TAK1 in Hepa1–6 cells by TAK1 siRNAs suppressed Cidec, whereas stable expression of TAK1-induced Cidec expression. These data indicate that these changes in gene regulation by TAK1 are hepatocyte cell autonomous and not a response to changes in other tissues. Whether these TAK1-responsive genes are direct targets of TAK1 transcriptional regulation needs further study.
Recent studies have provided evidence indicating that TAK1 functions as a ligand-dependent transcription factor. Certain fatty acids, including γ-linoleic acid and γ-linolenic acid, as well as several eicosanoids, have been shown to activate TAK1-mediated transcription, suggesting that TAK1 might function as a fatty acid sensor (13
). Consistent with this hypothesis, we speculate that during aging or when fed a HFD, elevated levels of fatty acids may result in increased activation of TAK1 and enhanced expression of TAK1-responsive genes, such as CD36, that promote fatty acid uptake and triglyceride accumulation, and subsequent obesity (D
). Hence, one could speculate that TAK1-selective antagonists would inhibit the expression of these genes and might be useful for the management of metabolic syndrome.
In addition to hepatic steatosis, adiposity is greatly reduced in aged TAK1−/−
(HFD) mice compared with WT mice. The adipocytes in TAK1−/−
mice were significantly smaller than in WT mice, suggesting reduced storage of triglycerides. Obesity is well known to be associated with chronic, low-grade inflammation, and there is considerable evidence that inflammation, insulin resistance, and aberrant lipid metabolism are interlinked in metabolic syndrome (3
). Hypertrophy of adipose tissues and infiltration of inflammatory cells have been recognized as important early events in the development of obesity-linked pathologies. The molecular process of the recruitment and function of macrophage infiltration is not fully understood; however, the release of various cytokines by adipose tissue is likely part of the recruitment of various immune cells (6
). In contrast to WT mice, TAK1−/−
mice are protected against the development of age- and diet-induced adipose tissue-associated inflammation, as indicated by reduced infiltration of macrophages and T lymphocytes. Crown-like structures were rarely observed in WAT of TAK1−/−
mice and the macrophage markers, F4/80 and Mac-2, were expressed at significantly lower levels. In addition, the expression of several proinflammatory genes, including Saa3
, and Tlr8
, were also reduced in adipose tissues of TAK1−/−
mice. T lymphocytes have also been implicated in the development of obesity-associated complications (6
effector T cells have been reported to exhibit an essential role in the initiation and maintenance of adipose tissue inflammation, including macrophage recruitment, during obesity. The observed reduction in the number of CD8+
cells in SVF might be linked to the diminished infiltration of macrophages and inflammatory response in TAK1−/−
mice. Moreover, the reduced WAT inflammation in TAK1−/−
mice may in part be responsible for the preservation of the insulin sensitivity and glucose tolerance observed in TAK1−/−
mice. In this regard, the repression of Il1rn expression in TAK1−/−
WAT is particularly interesting because upregulation of this gene has been reported to be associated with obesity whereas Il1rn KO mice have been shown to be resistant to obesity (45
). Therefore, repression of this gene may contribute to the resistance to obesity observed in TAK1 KO mice.
Finally, two important factors in energy balance are food intake and energy expenditure. Although their relative food intake was slightly higher than their WT littermates, TAK1−/− mice exhibited a lean phenotype compared with WT mice. Furthermore, TAK1−/− mice showed a significant increase in energy expenditure as indicated by increased oxygen consumption and CO2 production rates. The increase in energy expenditure by TAK1−/− mice is consistent with the elevated expression of UCP1 in BAT. UCP1 diverts energy derived from mitochondrial electron transport chain and generation of ATP into heat production. Thus, the elevated energy expenditure observed in TAK1−/−(HFD) mice may at least in part be responsible for the reduced weight gain and resistance to hepatic steatosis and insulin insensitivity.
In summary, in this study we show for the first time that TAK1−/− mice are protected against age- and HFD-induced obesity, hepatic steatosis, adipose tissue-associated inflammation, and insulin resistance. As a ligand-dependent nuclear receptor, TAK1 might provide a novel therapeutic target in the management and prevention of obesity and related pathologies, such as diabetes.