Obesity Resistance and Enhanced Insulin Sensitivity in <i>Ahnak</i>^-/- Mice Fed a High Fat Diet Are Related to Impaired Adipogenesis and Increased Energy Expenditure

<div><p>Objective</p><p>Recent evidence has suggested that AHNAK expression is altered in obesity, although its role in adipose tissue development remains unclear. The objective of this study was to determine the molecular mechanism by which <i>Ahnak</i> influences adipogenesis and glucose homeostasis.</p><p>Design</p><p>We investigated the <i>in vitro</i> role of AHNAK in adipogenesis using adipose-derived mesenchymal stem cells (ADSCs) and C3H10T1/2 cells. AHNAK-KO male mice were fed a high-fat diet (HFD; 60% calories from fat) and examined for glucose and insulin tolerances, for body fat compositions, and by hyperinsulinemic-euglycemic clamping. Energy expenditures were assessed using metabolic cages and by measuring the expression levels of genes involved in thermogenesis in white or brown adipose tissues.</p><p>Results</p><p>Adipogenesis in ADSCs was impaired in AHNAK-KO mice. The loss of AHNAK led to decreased BMP4/SMAD1 signaling, resulting in the downregulation of key regulators of adipocyte differentiation (<i>P<</i>0.05). AHNAK directly interacted with SMAD1 on the <i>Ppar</i>γ<i>2</i> promoter. Concomitantly, HFD-fed AHNAK-KO mice displayed reduced hepatosteatosis and improved metabolic profiles, including improved glucose tolerance (<i>P</i><0.001), enhanced insulin sensitivity (<i>P</i><0.001), and increased energy expenditure (<i>P<</i>0.05), without undergoing alterations in food intake and physical activity.</p><p>Conclusion</p><p>AHNAK plays a crucial role in body fat accumulation by regulating adipose tissue development via interaction with the SMAD1 protein and can be involved in metabolic homeostasis.</p></div

Putative model for the role of <i>Ahnak</i> in Smad1-dependent induction of PPARg2 by BMP signaling.

<p>Putative model for the role of <i>Ahnak</i> in Smad1-dependent induction of PPARg2 by BMP signaling.</p

<i>Ahnak</i>-KO mice display increased energy expenditure.

<p>(<b>A</b>) Food intake (WT: <i>n</i> = 6, KO: <i>n</i> = 5). (<b>B</b>) Serum leptin concentrations (n = 4). (<b>C</b>) Measurement of locomotor activity (WT: <i>n</i> = 6, KO: <i>n</i> = 5). (<b>D</b>) Whole-body oxygen consumption (VO₂) over the course of 24 h (WT: <i>n</i> = 6, KO: <i>n</i> = 5). (<b>E</b>) Average value of heat generation (WT: <i>n</i> = 6, KO: <i>n</i> = 5). (<b>F</b>) The respiratory exchange rate was calculated as CO₂ production/O₂ consumption (WT: <i>n</i> = 6, KO: <i>n</i> = 5). (<b>G</b>) Relative mRNA expression of genes involved in energy dissipation and in brown adipose-specific genes in WAT, as measured by qPCR (<i>n</i> = 6). Values were normalized to <i>36B4</i> expression. The data shown are the mean±SEM; *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001.</p

<i>Ahnak</i>-KO mice displayed reduced adiposity.

<p>(<b>A</b>) AHNAK expression of obese human subjects. (<b>B</b>) Changes in body weights during 12 weeks of feeding on RC or an HFD; <i>n</i> = 3 for WT and KO mice on regular chow diet (RC), <i>n</i> = 4 for WT and KO mice on HFD. (<b>C</b>) Body-composition changes in mice after 4 weeks on an HFD; WT: <i>n</i> = 6, KO: <i>n</i> = 5. (<b>D</b>) Representative pictures of fat pads from mice fed an HFD. (<b>E</b>) H&E staining (left) of eWAT and distribution of adipocyte sizes (right); <i>n</i> = 3 Scale bar, 200μm. (<b>F</b>) Relative mRNA levels of the indicated genes in WATs were measured by qPCR. Values were normalized to <i>36B4</i>. WT: <i>n</i> = 5, KO: <i>n</i> = 8 (<b>F</b>). The data shown are the mean±SEM. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001 between WT and KO mice. eWAT, epididymal white adipose tissue.</p

AHNAK plays a role in adipogenesis <i>in vitro</i>.

<p>(<b>A</b>) C3H10T1/2 cells were transfected with control siRNA and <i>Ahnak</i> siRNA. (<b>B</b>) C3H10T1/2 cells underwent adipocyte differentiation, as determined by oil red O staining. (<b>C</b>) Expression levels of adipogenic genes were measured by qPCR. All genes were normalized to <i>36B4</i>. (<b>D</b>) Representative pictures showing differentiated ADSCs stained with oil red O at day 8 after the induction of differentiation. (<b>E</b>) Immunoblot analysis of adipogenic proteins from differentiated cells. GAPDH was used as a loading control.</p

Metabolic parameters.

<p>(<b>A</b>) Serum triglyceride. (<b>B</b>) FFA. (<b>C</b>) Total cholesterol. (<b>D</b>) LDL-cholesterol in WT and KO mice on an HFD. Serum levels were measured after a 16-h fasting period; <i>n</i> = 9/group (a-d). (<b>E</b>) Glucose tolerance after an 8-week HFD; <i>n</i> = 9 for WT and n = 6 for KO mice. (<b>F</b>) Insulin tolerance after an 8-week HFD; <i>n</i> = 6 for WT and n = 6 for KO mice. (<b>G, H</b>) Insulin signaling pathway in WAT (<b>G</b>) and liver (<b>H</b>). The data shown are the mean±SEM. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001 between WT and KO mice.</p