In this study, we examined the effect of CD44 deficiency on the development of diet-induced obesity and associated pathologies in mice. We demonstrated that CD44KO mice are protected against the development of diet-induced hepatic steatosis as indicated by the lower levels of hepatic triglycerides and reduced hepatotoxicity, inflammation, and fibrogenesis. In addition, our data showed that CD44KO mice are considerably less susceptible to the development of diet-induced adipose inflammation, insulin resistance, and glucose-intolerance.
The accumulation of triglycerides during hepatic steatosis is mediated by several mechanisms, including changes in lipid transport, de novo
synthesis, storage, fatty acid oxidation, and lipolysis. Our study demonstrated that the diet-induced increase in the expression of several genes implicated in the regulation of lipid metabolism was diminished considerably in the liver of CD44(HFD) mice. Of the genes analyzed, the expression of the cell death-inducing DFF45-like effector (CIDE) genes, Cidea and Cidec, was the most dramatically affected by CD44 deficiency (). Both Cidea and Cidec play a critical role in the regulation of lipid storage, lipid droplet formation, and lipolysis 
. The expression of CD36, which among other things facilitates the transport of fatty acids into the liver, as well as the expression of several genes involved in fatty acid biosynthesis, including fatty acid synthase (Fasn), and Elovl5 and -7, were also significantly reduced in CD44(HFD) liver compared to WT(HFD) liver. Moreover, the expression of Mogat1, which is part of an alternative pathway of de novo
triglyceride synthesis, was greatly diminished in CD44(HFD) liver, while the expression of Dgat1 and Dgat2, which are important in the classical de novo
triglyceride synthesis pathway, was not different between the two genotypes. In addition to their control of lipid metabolism, several of these lipogenic genes, including Cidea, Cidec, and Cd36, play a critical role in regulating the development of hepatic steatosis and insulin sensitivity 
. Mice deficient in Cidea or Cidec have been shown to exhibit reduced hepatic lipid accumulation and improved insulin sensitivity, while overexpression of Cidea in mouse liver promoted hepatic steatosis. Cd36 deficiency has been reported to protect mice against diet-induced hepatic steatosis and insulin resistance, while increased expression has the inverse effect 
. The reduced expression of Cidec, Cidea, Cd36, Mogat1, and Fasn, as well as Elovl5 and 7, may provide a mechanism for the diminished hepatic fatty acid uptake and triglyceride synthesis, and the decreased lipid accumulation and be at least in part responsible for the reduced hepatic steatosis and insulin resistance observed in CD44KO mice ().
Schematic view of the links between CD44 deficiency and the development of diet-induced hepatic steatosis, inflammation, and fibrogenesis, adipose-associated inflammation, and insulin resistance.
In addition to triglyceride accumulation, inflammation, hepatotoxicity, and fibrogenesis are important features in the progression of NAFLD 
. We provided evidence suggesting that hepatotoxicity was greatly reduced in CD44KO(HFD) mice compared to WT(HFD) mice as indicated by the significantly lower blood levels of the biomarkers, ALT and AST. We further observed that the liver of CD44KO(HFD) liver displayed less inflammation than WT(HFD) liver as indicated by gene expression profiling. The expression of a number of proinflammatory cytokines, including several chemokines (Ccl2, Ccl5, Ccl7) and their receptors (Ccr2, Ccr5), metalloproteinases, and Opn, was reduced in CD44KO liver. Several of these genes, including the Ccr2 and Ccr5 signaling pathways, have been implicated in the mobilization of monocytes 
. These observations are consistent with previous reports showing a link between Opn expression and progression of hepatic injury and inflammation and an association between the level of Ccl2 expression and the severity of hepatosteatosis 
. Ccl2 is produced by hepatocytes and hepatic macrophages and functions as a potent chemoattractive mediator for bone marrow-derived macrophages, while both Ccl2 and Opn can function as activators of M1 macrophages 
. A strong link has been established between the regulation of inflammation and fibrogenesis 
. The reduced hepatic expression of matrix metalloproteinases and several collagens in CD44KO(HFD) mice is consistent with the conclusion that these mice are protected against the onset of hepatosteatosis-associated fibrosis. The latter is consistent with recent findings on the role of CD44 in lung fibrosis 
. Thus, the observed decreased expression of various chemokines and their receptors as well as several cellular matrix genes in CD44KO(HFD) mice might be at least in part responsible for the reduced hepatic inflammation injury, and fibrogenesis, and consequently for its protective effect against hepatosteatosis ().
In most mouse obesity models, the development of hepatic steatosis and increased adiposity are often associated 
. Unexpectedly however, in contrast to the decrease in hepatic steatosis, lipid accumulation was enhanced in WAT CD44KO(HFD) mice, which appeared to be due to an increase in lipid storage. This may be in part related to the enhanced expression of Fasn, Elovl3 and -6, FABP1, and Cidec, a key factor regulating triglyceride storage and lipid droplet size in WAT 
rather than changes in lipolysis since the expression of the major lipases, Hasl and Atgl, was not significantly different between WAT of WT and CD44KO mice. As the expression of Pparγ was not different between genotype groups, increased lipid storage in CD44KO WAT may not be due to expansion or development of adipocytes (Figure S4)
. Differences in adiposity might involve altered energy intake and expenditure; however, no significant differences in food intake, oxygen consumption, CO2
production, and heat generation were observed between WT and CD44KO mice (Figure S6
). Thus, the effects on hepatic steatosis and adiposity appear to be related to an immune-mediated modulation of lipid metabolism, rather than changes in food intake or energy expenditure in CD44KO mice.
In contrast to adiposity, WAT-associated inflammation was significantly reduced in CD44(HFD) mice. This was indicated by gene expression profile analysis showing that a number of inflammatory genes, including Il17a, Il33, Il1rn, Ccrl1, and Ccl6, were significantly reduced in WAT of CD44KO(HFD) mice (Table
). In addition, the number of macrophages associated with WAT of CD44KO(HFD) mice was significantly lower compared to WT(HFD) WAT as indicated by the presence of considerably fewer crown-like structures and significantly lower levels of expression of the macrophage markers, F4/80, Cd11c, and Mac-2. Furthermore, the expression of the cytokine receptor Ccr5, which plays a critical role in the recruitment and activation of WAT macrophages 
, was significantly lower in CD44KO(HFD) WAT than WT(HFD) WAT. Moreover, the ratio of anti-inflammatory M2 macrophages over pro-inflammatory M1 macrophages was significantly higher in CD44KO(HFD) WAT (). Together, these observations support the conclusion that chemotaxis as well as the activation of pro-inflammatory macrophages is significantly reduced in CD44KO(HFD) WAT. In addition to the reduction in macrophages, the number of cytotoxic CD8+
T lymphocytes, which play an essential role in the initiation and propagation of adipose inflammation 
, and the expression Cd3 and Cd8 antigens was significantly lower in WAT of CD44KO(HFD) mice suggesting a role for CD44 in regulating the migration and recruitment of these cells as well. It is well recognized that obesity is associated with low-grade systemic inflammation and that WAT-associated inflammation plays a key role in obesity-linked pathologies, including insulin resistance 
. Thus, the reduced inflammation observed in WAT of CD44KO(HFD) mice might be a part of the mechanism that protects these mice against insulin resistance and glucose-intolerance ().
Adipokines produced by adipocytes play a critical role in regulating inflammation, lipid metabolism as well as insulin resistance 
. Adiponectin plays a protective role against inflammation by reducing expression of proinflammatory cytokines through inhibition of NF-κB signaling pathway 
and regulate fatty acid oxidation in liver though activation of PPARα and adenosine monophosphate-activated protein kinase (AMPK). However, the expression of adiponectin in WAT as well as the circulating level of adiponectin were not changed between genotypes (Figure S4
). This suggests that the protection against adipose-associated inflammation and hepatosteatosis in CD44KO(HFD) mice is not causally related to differences in adiponectin levels. Our observation that the level of expression of PPARα and genes involved in fatty acid oxidation was not significantly different between WT and in CD44KO liver is consistent with our data that adiponectin levels are unchanged between WT and CD44KO mice.
While this manuscript was in preparation, an expression-based genome-wide association (eGWAS) study linked CD44
to type 2 diabetes in humans and reported that CD44-deficiency ameliorates insulin resistance in mice and humans 
. Our study showing that CD44KO mice are protected against the development of diet-induced adipose inflammation and insulin resistance, is consistent with that report. However, our study provides additional insights into the crucial role of CD44 in diet-induced hepatic steatosis, inflammation, and fibrogenesis, and WAT- and liver-associated inflammation and suggests that migration and activation of inflammatory cells may be critical elements affected by the absence of CD44 ().
The precise mechanism by which this multi-functional protein regulates diet-induced inflammation and insulin resistance needs further study. CD44 might regulate the expression of certain lipogenic and inflammatory genes by a direct mechanism involving the interaction of its intracellular domain with respective promoter regions as recently reported for MMP-9
. It is intriguing that the pro-inflammatory cytokine Opn, which interacts with CD44, has also been reported to play a role in cell migration, macrophage activation, and inflammation in obesity 
. Like CD44KO mice, Opn-deficient mice are protected against the development of HFD-induced hepatic steatosis, WAT-associated inflammation, and insulin resistance, while lipid storage in WAT is increased 
. Although Opn acts through several receptor mechanisms, one might hypothesize that the decreased susceptibility observed in both knockout mouse models might be due in part to disruption of the Opn-CD44 signaling pathway. This hypothesis is consistent with recent reports showing that Opn mediates obesity-induced migration and infiltration of macrophages in WAT 
and this modulation appears to involve the CD44 pathway 
. Therefore, one might hypothesize that the reduced hepatic steatosis and WAT-associated inflammation in CD44KO mice might be due in part to the inability of Opn to activate the CD44 pathway. It is interesting to note that obesity in humans and mice is associated with increased expression of Opn in both liver and WAT 
. Our study shows that the expression of Opn was greatly repressed in liver of CD44KO(HFD) mice, but not in WAT. Thus, suppression of Opn expression in liver may provide an additional mechanism for the reduced susceptibility CD44KO(HFD) mice to hepatic steatosis and insulin resistance.
In summary, our study demonstrates that mice deficient in CD44 are considerably resistant to diet-induced hepatic steatosis, fibrogenesis, and inflammation, adipose-associated infiltration of M1 macrophages, glucose intolerance, and insulin resistance (). These observations suggest that CD44 provides a critical link between metabolic changes and the development of inflammation and insulin resistance. Because CD44 functions as a receptor, it may provide a convenient therapeutic target in the management of lipid dysregulation in diet-induced liver disease and type 2 diabetes.