CD44 Plays a Critical Role in Regulating Diet-Induced Adipose Inflammation, Hepatic Steatosis, and Insulin Resistance

<div><p>CD44 is a multifunctional membrane receptor implicated in the regulation of several biological processes, including inflammation. CD44 expression is elevated in liver and white adipose tissue (WAT) during obesity suggesting a possible regulatory role for CD44 in metabolic syndrome. To study this hypothesis, we examined the effect of the loss of CD44 expression on the development of various features of metabolic syndrome using CD44 null mice. Our study demonstrates that CD44-deficient mice (CD44KO) exhibit a significantly reduced susceptibility to the development of high fat-diet (HFD)-induced hepatic steatosis, WAT-associated inflammation, and insulin resistance. The decreased expression of genes involved in fatty acid synthesis and transport (Fasn and Cd36), <i>de novo</i> triglyceride synthesis (Mogat1), and triglyceride accumulation (Cidea, Cidec) appears in part responsible for the reduced hepatic lipid accumulation in CD44KO(HFD) mice. In addition, the expression of various inflammatory and cell matrix genes, including several chemokines and its receptors, osteopontin, and several matrix metalloproteinases and collagen genes was greatly diminished in CD44KO(HFD) liver consistent with reduced inflammation and fibrogenesis. In contrast, lipid accumulation was significantly increased in CD44KO(HFD) WAT, whereas inflammation as indicated by the reduced infiltration of macrophages and expression of macrophage marker genes, was significantly diminished in WAT of CD44KO(HFD) mice compared to WT(HFD) mice. CD44KO(HFD) mice remained considerably more insulin sensitive and glucose tolerant than WT(HFD) mice and exhibited lower blood insulin levels. Our study indicates that CD44 plays a critical role in regulating several aspects of metabolic syndrome and may provide a new therapeutic target in the management of insulin resistance.</p> </div

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.

<p>In liver, HFD induces the expression of many lipogenic genes, including Cd36, Fasn, Elovl, Apoa4, Mogat and Cidec and Cidea, which enhance the transport, synthesis, and accumulation of triglycerides. In addition, the expression of several inflammatory genes, such as Ccl2, Ccl7, Ccr2, and Opn, and fibrotic genes, including Col3a1, Mmp13, and Col1a1, are elevated and related to the observed increase in hepatic inflammation and the onset of hepatic fibrogenesis. In adipose tissue, HFD induces expression of several lipogenic genes and inflammatory genes (e.g., Il1rn, Mmp12, Ccr1, Cxcl14). The induction of these genes is in part responsible for the increased lipid accumulation and inflammation in WAT. Hepatosteatosis and WAT-associated inflammation subsequently promote the development of insulin resistance and glucose intolerance. The reduced expression of lipogenic, inflammatory, and fibrogenic genes in CD44-deficient mice is linked to their resistance to develop diet-induced hepatosteatosis. The inhibition of inflammatory genes and of the infiltration of macrophages and CD8⁺ lymphocytes in WAT of CD44KO mice results in reduced WAT-associated inflammation, while elevated expression of lipogenic genes is at least in part related to the observed increase in adiposity. The inhibition of hepatosteatosis and WAT-associated inflammation protects CD44-deficient mice against the development of insulin-resistance and glucose intolerance.</p

Hepatic steatosis was dramatically decreased in CD44KO(HFD).

<p>A–D) Representative H&E stained sections of liver were presented. Scale bar indicates 200 µm. E) Comparison of hepatic triglyceride and cholesterol levels. F) Comparison of gene expression in the livers of WT or CD44KO mice fed a normal diet (ND) or a high fat diet (HFD) (n = 5–6 mice per each group). Data present mean±SEM, *p<0.05, **p<0.01, ***p<0.001.</p

Comparison of total body mass and the relative tissue weight in CD44KO(HFD) and WT(HFD) mice.

<p>A) The expression of CD44 mRNA was analyzed in liver and WAT from 2 month-old WT (n = 6) or 7 month-old (n = 5) mice or mice fed a normal (ND) or HFD by QRT-PCR. B) Twelve week-old male mice (WT, n = 8; CD44KO, n = 8) were fed an HFD for 21 weeks and weight gain monitored. (C) Total body weight of mice fed an HFD for 21 weeks was plotted (WT, n = 8; CD44KO, n = 8). (D) Comparison of the relative weights of liver, WAT, kidneys, and BAT after 21 weeks on an HFD. Data present mean±SEM, *p<0.05, **p<0.01, ***p<0.001.</p

Adipose tissue inflammation was reduced in CD44KO(HFD) compared to WT(HFD) mice.

<p>A) Macrophages in crown-like structures (CLS) were identified by immunohistochemical staining with an F4/80 antibody as indicated by arrows. Scale bar indicates 250 µm. B) The number of CLS was decreased in CD44KO(HFD) compared to WT(HFD) mice. F4/80 positive cells in at least 4 randomly selected fields in sections from 4 different mice were counted. C) Comparison of the expression of macrophage markers, F4/80 and Mac-2, in WAT of WT or CD44KO mice fed a normal diet (ND) or a high fat diet (HFD) (n = 5–6 mice per each group). D) SVF cells from WAT of WT(HFD) mice (n = 6) and CD44KO(HFD) mice (n = 8) were examined by FACS analysis with F4/80, CD11b, CD11c, and CD206 antibodies. The percentage and ratio of pro-inflammatory macrophage (M1: F4/80⁺CD11C⁺CD206⁻) and anti-inflammatory macrophage (M2: F4/80⁺CD11C⁻CD206⁺) were determined. Expression of several macrophage markers (E) and inflammatory markers (F) was analyzed in WAT of WT or CD44KO mice fed a normal diet (ND) or a high fat diet (HFD) (n = 5–6 mice per each group).</p

CD44KO mice are protected against HFD-induced insulin resistance and glucose intolerance.

<p>A) Glucose tolerance test (GTT) and insulin tolerance test (ITT) were analyzed in WT and CD44KO mice fed an HFD for 6 weeks, 12 weeks, or 21 weeks (n = 6–8 mice for each group). Blood glucose levels were analyzed every 20 min for up to 2 hrs after glucose or insulin injection, for GTT or ITT respectively. B) Blood insulin levels were analyzed in WT and CD44KO mice fed a HFD for 21 weeks (WT, n = 7; CD44KO, n = 8). Data represent mean±SEM. *p<0.05, **p<0.01, ***p<0.001.</p

The CD3⁺ lymphocyte population and level of activated p38 MAPK in WAT of CD44KO(HFD) is reduced compared to WT (HFD) mice.

<p>The percentages of different CD4/CD8 cell populations were determined by FACS analysis. The percentage of CD8 single-positive T lymphocytes (CD4⁻CD8⁺) was decreased in WAT of CD44KO(HFD) compared to WT (HFD) mice. (n = 5–6 mice per each group). Data represent mean±SEM. *p<0.05, ***p<0.001. (B) Protein lysates were extracted from WAT of WT(HFD) and CD44KO(HFD) mice, and Western blot analysis was performed using phosphorylated or total p38 MAPK antibodies and phosphorylated JNK antibody.</p

Total body fat was increased in CD44KO(HFD) compared to WT(HFD) mice.

<p>A) Total body fat mass from mice fed a HFD for 21 weeks was analyzed by Piximus densitometry. B) Representative H&E stained sections of WAT were presented. Scale bar indicates 200 µm. C) Comparison of the cell size of WAT adipocytes from WT(HFD) and CD44KO(HFD). Cell diameters in sections of WAT from 4 mice in each group were measured and the percentages of the different cell sizes calculated and plotted. D) Increased Cidec and Fasn expression in WAT of CD44KO(HFD) compared to WT(HFD) mice (n = 5–6 mice per each group). E) Comparison of gene expression in WAT of WT or CD44KO mice fed a normal diet (ND) or a high fat diet (HFD) (n = 5–6 mice per each group).</p

Activity of hepatic injury associated enzymes was decreased in CD44KO(HFD) compared to WT(HFD) mice.

<p>Levels of serum lipid (A–D), and alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activity (E–F) were analyzed in WT and CD44KO mice fed a normal diet (ND) or a high fat diet (HFD) (n = 6–8 mice for each group). Data present mean±SEM, **p<0.01, ***p<0.001.</p

Perturbation of microRNAs in Rat Heart during Chronic Doxorubicin Treatment

<div><p>Anti-cancer therapy based on anthracyclines (DNA intercalating Topoisomerase II inhibitors) is limited by adverse effects of these compounds on the cardiovascular system, ultimately causing heart failure. Despite extensive investigations into the effects of doxorubicin on the cardiovascular system, the molecular mechanisms of toxicity remain largely unknown. MicroRNAs are endogenously transcribed non-coding 22 nucleotide long RNAs that regulate gene expression by decreasing mRNA stability and translation and play key roles in cardiac physiology and pathologies. Increasing doses of doxorubicin, but not etoposide (a Topoisomerase II inhibitor devoid of cardiovascular toxicity), specifically induced the up-regulation of miR-208b, miR-216b, miR-215, miR-34c and miR-367 in rat hearts. Furthermore, the lowest dosing regime (1 mg/kg/week for 2 weeks) led to a detectable increase of miR-216b in the absence of histopathological findings or alteration of classical cardiac stress biomarkers. <em>In silico</em> microRNA target predictions suggested that a number of doxorubicin-responsive microRNAs may regulate mRNAs involved in cardiac tissue remodeling. In particular miR-34c was able to mediate the DOX-induced changes of Sipa1 mRNA (a mitogen-induced Rap/Ran GTPase activating protein) at the post-transcriptional level and in a seed sequence dependent manner. Our results show that integrated heart tissue microRNA and mRNA profiling can provide valuable early genomic biomarkers of drug-induced cardiac injury as well as novel mechanistic insight into the underlying molecular pathways.</p> </div