Mice Deficient in <i>Sfrp1</i> Exhibit Increased Adiposity, Dysregulated Glucose Metabolism, and Enhanced Macrophage Infiltration

<div><p>The molecular mechanisms involved in the development of obesity and related complications remain unclear. Wnt signaling plays an important role in preadipocyte differentiation and adipogenesis. The expression of a Wnt antagonist, secreted frizzled related protein 1 (SFRP1), is increased in response to initial weight gain, then levels are reduced under conditions of extreme obesity in both humans and animals. Here we report that loss of <i>Sfrp1</i> exacerbates weight gain, glucose homeostasis and inflammation in mice in response to diet induced obesity (DIO). Sfrp1^-/- mice fed a high fat diet (HFD) exhibited an increase in body mass accompanied by increases in body fat percentage, visceral white adipose tissue (WAT) mass, and adipocyte size. Moreover, <i>Sfrp1</i> deficiency increases the mRNA levels of key <i>de novo</i> lipid synthesis genes (<i>Fasn</i>, <i>Acaca</i>, <i>Acly</i>, <i>Elovl</i>, <i>Scd1</i>) and the transcription factors that regulate their expression (<i>Lxr-α, Srebp1, Chreb</i>, and <i>Nr1h3</i>) in WAT. Fasting glucose levels are elevated, glucose clearance is impaired, hepatic gluconeogenesis regulators are aberrantly upregulated (<i>G6pc</i> and <i>Pck1</i>), and glucose transporters are repressed (<i>Slc2a2</i> and <i>Slc2a4</i>) in Sfrp1^-/- mice fed a HFD. Additionally, we observed increased steatosis in the livers of Sfrp1^-/- mice. When there is an expansion of adipose tissue there is a sustained inflammatory response accompanied by adipokine dysregulation, which leads to chronic subclinical inflammation. Thus, we assessed the inflammatory state of different tissues and revealed that Sfrp1^-/- mice fed a HFD exhibited increased macrophage infiltration and expression of pro-inflammatory markers including <i>IL-6, Nmnat, Tgf-β2</i>, and <i>SerpinE1</i>. Our findings demonstrate that the expression of <i>Sfrp1</i> is a critical factor required for maintaining appropriate cellular signaling in response to the onset of obesity.</p> </div

Analysis of de novo lipid synthesis and the inflammatory signature in the gonadal fat pad.

<p>(A) Total RNA from the gonadal fat pad in each group (n=6) was utilized for real-time PCR analysis of <i>Fasn</i>, Acly, and <i>Acaca, Elov</i>16, and <i>Scd</i>1 gene expression. The results shown represent experiments performed in duplicate and normalized to the amplification of β-actin mRNA. Bars represent mean ± SEM of the difference in fold change compared with control ND fed mice. (B) Real-time PCR was carried out utilizing RNA from the gonadal fat pad of control mice and Sfrp1^-/- mice on a ND to measure the relative mRNA expression of the following transcription factors: <i>Srebp1, Chrebp-α</i>, <i>Chrebp</i>-β, and <i>Nr1h3</i>. The experiments were carried out as described above and bars represent mean ± SEM of the difference in fold change compared with control ND fed mice. (C) <i>Left </i><i>panel</i>, gonadal fat pad sections were subjected to immunohistochemical analysis, stained for F4/80 (brown chromogen), and representative images were captured at 100X and are displayed for mice in each treatment group (scale bar 200 μm). <i>Right </i><i>upper </i><i>panel</i>, gondal fat pad RNA was used for real-time PCR analysis of F4/80 mRNA, experiments were carried out as described, and bars represent mean ± SEM of the relative expression with respect to ND fed mice. <i>Right </i><i>lower </i><i>panel</i>, The total number of CLSs (see arrow inset) were counted in F4/80 stained gonadal fat pad sections (n=4/genotype) (D) Real-time PCR analysis of liver Il-6<i>, Tnf</i>-α, Il-1β, and <i>Nampt</i> gene expression was carried out as described above. Bars represent mean ± SEM of the difference in fold change compared with control ND fed mice. (*p<0.05, **p<0.01, ***p<0.001 significantly different from control mice fed a ND using Bonferroni’s <i>t</i> test after a two-way ANOVA; ^#p<0.05, ^##p<0.01, significantly different from respective ND fed mice using student’s <i>t</i> test.).</p

Macrophage infiltration and cytokine expression in the mammary gland.

<p><i>Left </i><i>panel</i>, 3^rd & 4^th inguinal mammary gland sections were subjected to immunohistochemical analysis, stained for F4/80 (brown chromogen), and representative images were captured at 100X are displayed for mice in each treatment group (scale bar 200 μm). <i>Right </i><i>upper </i><i>panel</i>, total RNA was harvested from 5^th inguinal mammary glands and employed for real-time PCR analysis of F4/80 mRNA, experiments were carried out as described, and bars represent mean ± SEM of the relative expression with respect to ND fed mice. <i>Right </i><i>lower </i><i>panel</i>, The total number of CLSs (see arrow inset) were counted in F4/80 stained mammary gland sections (n=4/treatment group) (D) Real-time PCR analysis of liver Il-6<i>, Tnf</i>-α, Il-1β, <i>Nampt, Tgf</i>-β2, and <i>Serpine</i>1 gene expression was carried out as described above. Bars represent mean ± SEM of the difference in fold change compared with control ND fed mice. (*p<0.05, ***p<0.001 significantly different from control mice fed a ND using Bonferroni’s <i>t</i> test after a two-way ANOVA; ^#p<0.05, ^##p<0.01, significantly different from respective ND fed mice using student’s <i>t</i> test.).</p

Aberrant glucose homeostasis and insulin secretion observed in Sfrp1^-/- mice.

<p>(A) Glucose tolerance test (GTT) was performed at three separate time points during the study (2 weeks, 6 weeks, and 12 weeks). After a 16-18 h fast, mice were injected with 2g/kg BW glucose and blood glucose levels were monitored for 2 h. (B) Blood glucose levels were measured prior to GTT initiation. (C) Plasma insulin levels were measured before (0 min) and after (15 min) glucose injection. (D) For real-time PCR analysis of <i>G6pc</i>, <i>Pck</i>1, and <i>Slca2</i> gene expression, total RNA was isolated from liver of mice in each treatment group (n=6). The results shown represent experiments performed in duplicate and normalized to the amplification of β-actin mRNA. Bars represent mean ± SEM of the relative expression with respect to ND fed mice. (E) <i>Left </i><i>panel</i>, For real-time PCR analysis of <i>Slca4</i> gene expression, total RNA was isolated from the gonadal fad pad from mice in each treatment group (n=6). Real-time PCR analysis was carried out as described above. <i>Right </i><i>panel</i>, gonadal fat pad sections were subjected to immunohistochemical analysis and stained for <i>Slc2a4</i> (brown chromogen).Representative images were captured at 100X and are displayed for mice in each treatment group (scale bar 200 μm). (F) Muscle sections were stained for <i>Slc2a4</i> and 200X images were captured as described above (scale bar 100 μm). (*p<0.05,**p<0.01, ***p<0.001 significantly different from control mice fed a ND using Bonferroni’s <i>t</i> test after a two-way ANOVA; ^#p<0.05, ^##p<0.01, ^###p<0.001 significantly different from respective ND fed mice using student’s <i>t</i> test.) .</p

Hepatic steatosis and inflammation exhibited by Sfrp1^-/- mice.

<p>(A) Steatosis is distinguished by Oil Red O staining of lipids. Images were captured at 400X and represent control mice and Sfrp1^-/- mice on a HFD (scale bar 50 μm). (B) H&E staining of livers and captured as described above. (C) Total RNA from the livers in each group (n=6) was utilized for real-time PCR analysis of <i>Fasn</i>, Acly, and <i>Acaca, Elov</i>16, and <i>Scd</i>1 gene expression. The results shown represent experiments performed in duplicate and normalized to the amplification of β-actin mRNA. Bars represent mean ± SEM of the relative expression with respect to ND fed mice. (D) Real-time PCR analysis of liver Il-6<i>, Tnf</i>-α, Il-1β, and <i>Nampt</i> gene expression was carried out as described above. Bars represent mean ± SEM of the difference in fold change compared with control ND fed mice. (*p<0.05, **p<0.01, ***p<0.001 significantly different from control mice fed a ND using Bonferroni’s <i>t</i> test after a two-way ANOVA; ^#p<0.05, ^##p<0.01, ^###p<0.001 significantly different from respective ND fed mice using student’s <i>t</i> test.).</p

Effects of high-fat diet-induced obesity on weight gain and adiposity.

<p>(A) <i>Left </i><i>panel</i>, The body weight of control and Sfrp1^-/- mice put on a ND and HFD was monitored every 3 days after being put on the diet and changes are displayed graphically as percent initial body weight. <i>Right </i><i>panel</i>, Final body weight after 12 weeks on the diet. (B) Body composition was analyzed by NMR-MRI at the start of the diet (0 weeks), mid-study (6 weeks), and at the end of the study (12 weeks) and fat mass was normalized to body weight and expressed as percent body fat. (C) Average weight of upper mammary glands (3^rd & 4^th inguinal), lower mammary gland (5^th inguinal), gonadal fat pad, renal fat pad, mesenteric fat pad, scapular fat pad, and brown adipose tissue harvested from mice fed a HFD for 12 weeks. (D) <i>Upper </i><i>panel</i>, Representative images of H&E staining of gonadal fad pads dissected from mice fed a HFD for 12 weeks. <i>Lower </i><i>panel</i>, Quantification of gonadal adipocyte size. (*p<0.05,**p<0.01, ***p<0.001 significantly different from control mice fed a ND using Bonferroni’s <i>t</i> test after a two-way ANOVA; ^#p<0.05, ^##p<0.01, ^###p<0.001 significantly different from control mice fed a HFD using student’s <i>t</i> test.) .</p