White adipocytes in subcutaneous fat depots require KLF15 for maintenance in preclinical models

Abstract

Healthy adipose tissue is essential for normal physiology. There are 2 broad types of adipose tissue depots: brown adipose tissue (BAT), which contains adipocytes poised to burn energy through thermogenesis, and white adipose tissue (WAT), which contains adipocytes that store lipids. However, within those types of adipose, adipocytes possess depot and cell-specific properties that have important implications. For example, the subcutaneous and visceral WAT confers divergent risk for metabolic disease. Further, within a depot, different adipocytes can have distinct properties; subcutaneous WAT can contain adipocytes with either white or brown-like (beige) adipocyte properties. However, the pathways that regulate and maintain this cell and depot-specificity are incompletely understood. Here, we found that the transcription factor KLF15 is required for maintaining white adipocyte properties selectively within the subcutaneous WAT. We revealed that deletion of Klf15 is sufficient to induce beige adipocyte properties and that KLF15's direct regulation of Adrb1 is a critical molecular mechanism for this process. We uncovered that this activity is cell autonomous but has systemic implications in mouse models and is conserved in primary human adipose cells. Our results elucidate a pathway for depot-specific maintenance of white adipocyte properties that could enable the development of therapies for obesity and associated diseases.

Keywords: Adipose tissue; Cell biology; Endocrinology.

Figure 1. Acute modulation of Klf15 expression in white adipocytes induces a beige fat expression profile.

(A) RT-qPCR quantifying the expression levels of Klf15 in adipocytes isolated from distinct fat pads. 1-way ANOVA, n = 3. (B) RT-qPCR quantifying the expression levels of Klf15 in adipocytes after isoproterenol (Iso) treatment for 4 hours. Student’s t test, n = 6. (C) RT-qPCR quantifying the expression levels of Klf15 in iWAT isolated from mice after they were injected with CL316243 (CL) (1 mg/kg/day) for 7 days. Student’s t test, n = 4. (D) RT-qPCR quantifying the relative expression levels of Adrb1, Adrb2, and Adrb3 in iWAT, gWAT, and BAT with iWAT set to 1 for each litter. 1-way ANOVA, n = 5. (E) Light phase microscopy images of adipocytes from differentiated SVF harvested from the iWAT of Klf15-fl/fl mice and infected with adenovirus expressing Cre (Ad-Cre) or adenovirus control (Ad-control). Scale bar: 25 μm. (F) RT-qPCR quantifying the expression levels of thermogenic genes and panadipocyte marker Adipoq. Student’s t test, n = 4. (G) RT-qPCR quantifying the expression levels. Student’s t tests followed by Holm-Šidák correction, n = 7. (H) Immunoblots detecting and quantifying the relative levels of β1AR following acute Klf15 deletion in adipocytes. Student’s t test, n = 3. (I) Immunoblots detecting and quantifying the relative levels of phosphorylation of p38 MAPK following acute Klf15 deletion in adipocytes. Student’s t test, n = 3. (J) RT-qPCR quantifying the change in Ucp1 expression levels with SB202190 pretreatment. Student’s t test, n = 3–4. (K) RT-qPCR quantifying the expression levels of Ucp1 following acute Klf15 deletion in adipocytes treated with Isoproterenol. 2-way ANOVA, n = 3. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 2. Adipocyte-specific Klf15 KO promotes beige adipocyte formation in iWAT.

(A) RT-qPCR quantifying the expression levels of Klf15 in iWAT, gWAT, BAT, and liver from WT and Adipo-Klf15 mice. Student’s t tests followed by Holm-Šidák correction, n = 3. (B) RT-qPCR quantifying the expression levels of Klf15 in the SVF and adipocyte fraction of iWAT from WT and Adipo-Klf15 mice. Student’s t test followed by Holm-Šidák correction, n = 3. (C) Representative images of in situ iWAT in WT and Adipo-Klf15 mice. Scale bar: 5 mm. (D) Quantification of iWAT mass as a percent of body weight in WT and Adipo-Klf15 littermates. Ratio paired t test, n = 5. (E) RT-qPCR quantifying the expression levels of thermogenic genes in iWAT of WT and Adipo-Klf15 littermates. Student’s t test, n = 5. (F–H) RT-qPCR quantifying the expression levels of adrenergic receptors in iWAT, gWAT, and BAT from WT and Adipo-Klf15 mice. Student’s t test followed by Holm-Šidák correction, n = 5. (I) RT-qPCR quantifying the expression levels of Adrb1 versus Adrb3 in iWAT in littermates of WT and Adipo-Klf15 mice. 1-tailed ratio paired t test, n = 4. (J) Immunoblots detecting the levels of β1AR protein in iWAT from WT and Adipo-Klf15 mice compared with the levels of β-actin controls. (K) Quantifying the relative protein level of β1AR. Student’s t test, n = 4. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3. Enhanced beiging occurs in the iWAT of Prx1-Klf15–cKO mice.

(A) RT-qPCR quantifying the expression levels of Klf15 in iWAT, gWAT, BAT, and liver from WT and Prx1-Klf15 mice. Student’s t tests followed by Holm-Šidák correction, n = 3. (B) RT-qPCR quantifying the expression levels of Klf15 in the SVF and adipocyte fractions of iWAT from WT and Prx1-Klf15 mice. Student’s t tests followed by Holm-Šidák correction, n = 3. (C) Quantification of iWAT mass as a percent of body weight in WT and Prx1-Klf15 mice. Student’s t test, n = 9. (D) Representative images of in situ iWAT from female WT and Prx1-Klf15 mice. Scale bar: 5 mm. (E) Representative images of H&E stained histological sections of iWAT from female WT and Prx1-Klf15 mice. Bottom images are magnified from the indicated square in the top images. Scale bar: 100 μm. (F–H) RT-qPCR quantifying the expression levels of (F) panadipocyte markers, n = 4 (G) white adipocyte markers, n = 3–8, and (H) thermogenic genes in iWAT, n = 6–11. Student’s t test followed by Holm-Šidák correction (I) RT-qPCR quantifying the expression levels of adrenergic receptors in iWAT from WT and Prx1-Klf15 mice. Student’s t test followed by Holm-Šidák correction, n = 4. (J) RT-qPCR quantifying the expression levels of Adrb1 versus Adrb3 in iWAT from littermates of WT and Prx1-Klf15 mice. 1-tailed ratio paired t test, n = 4. (K) Immunoblot detecting UCP1 and GAPDH protein levels in the iWAT of WT and Prx1-Klf15 mice. (L) Time course quantification of OCRs of iWAT isolated from WT and Prx1-Klf15 littermates after exposure to Xamoterol using a Seahorse Bioanalyzer. 2-way ANOVA, n = 5. (M and N) Whole-body heat generation (kcal) of mice undergoing cold exposure. (M) 2-way ANOVA, (N) ANCOVA, n = 5. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4. KLF15 regulates the expression of Adrb1 in a pathway conserved in humans.

(A and B) RT-qPCR quantifying the expression levels of Adrb1 and Ucp1 in adipocytes with or without Xamoterol treatment. Student’s t test, n = 5. (C) Quantifying of unstimulated intracellular cAMP levels in adipocytes with deletion of Klf15. Student’s t test, n = 5. (D) Conservation of a canonical KLF15 binding site sequence (red) identified in the mouse Adrb1 gene. (E) Quantification of dual-luciferase assays on adipocytes transfected with the Adrb1 promoter-driven firefly luciferase reporter construct and pCMV-Klf15 or control plasmid and normalized by Renilla bioluminescence, facilitated by cotransfected pRL-TK plasmid. Student’s t test, n = 3. (F) RT-qPCR quantifying the amount of immunoprecipitated DNA containing the putative KLF15 binding site located in Adrb1 using a KLF15 antibody in iWAT adipocytes from WT and Prx1-Klf15–cKO mice. Student’s t test, n = 3. (G) RT-qPCR quantifying the amount of immunoprecipitated DNA containing the putative KLF15 binding site located in the Adrb1 using the FLAG compared to IgG antibody in iWAT adipocytes isolated from Klf15^3xFLAG mice. Student’s t test, n = 3. (H) Image of PCR amplicons in an agarose gel of the putative KLF15 binding site (Target region) compared with amplification of the control region from the ChIP of adipocytes from WT and Prx1-Klf15–cKO mice with the KLF15 antibody. (I) RT-qPCR quantifying the expression levels of Ucp1 in iWAT from mice injected with saline, Denopamine (10 μg/g/day), Xamoterol (8 ng/g/day), or Dobutamine (10 μg/g/day) for 7 days. 1-way ANOVA, n = 4–5. (J) Light phase microscopy images of human adipocytes. Scale bar: 25 μm. (K) RT-qPCR quantifying the expression levels of hKLF15, hADRB1, and hUCP1 in human adipocytes infected by Ad-shCtrl or Ad-shKLF15. Student’s t test followed by Holm-Šidák correction, n = 4. (L) OCRs and (M) respiratory profile in Ad-shKLF15 and Ad-shCtrl infected human adipocytes quantified using a Seahorse Bioanalyzer, 2-way ANOVA n = 8. (N) Time-course of OCRs of human adipocytes after exposure to Xamoterol. 2-way ANOVA, n = 8–9. *P < 0.05, **P < 0.01, ***P < 0.001.