Adipose expression of CREB3L3 modulates body weight during obesity

We found the hepatic transcription factor Cyclic-AMP Responsive Element Binding Protein 3-like-3 (CREB3L3) to be expressed in adipose tissue, and selectively downregulated in the more metabolically protective subcutaneous adipose tissue in obese mice and humans. We sought to elucidate the specific role of this factor in adipose biology. CREB3L3 fat-specific knockout mice were fed a high-fat diet to induce obesity and metabolic dysfunction. Additionally, we injected a flip-excision adeno-associated virus directly into the subcutaneous inguinal adipose tissue of Adiponectin-Cre mice to create a depot-specific overexpression model for further assessment. Fat-specific ablation of CREB3L3 enhanced weight gain and insulin resistance following high-fat feeding, as fat-specific knockout mice expended less energy and possessed more inflammatory adipose tissue. Conversely, inguinal fat CREB3L3 overexpression deterred diet-induced obesity and ameliorated metabolic dysfunction. Together, this study highlights the relevance of CREB3L3 in obese adipose tissue and demonstrates its role as a powerful body weight modulator.

. Adipose ablation of CREB3L3 enhances fat mass, but not body weight in mice fed a chow diet (a) Quantitative PCR for the expression of CREB3L3 message in mature adipocytes isolated from digested epididymal (eWAT) and inguinal (iWAT) white adipose tissue (n= 4-5 mice per group). CREB3L3 expression was normalized to TBP housekeeping gene. (b-c) Body weight and body composition NMR measurements of chow-fed control and fKO mice at 22 wks of age (n=7-9 mice per group). (d) Representative image of control and fKO mice following high-fat feeding. Data presented as mean +/-SEM. The difference in means was analyzed using Student's t-test where *P<0.05.
Figure S1   Figure S2. CREB3L3 fKO does not induce hepatic steatosis (a) Quantitative PCR for insulin-like growth factor binding protein 1 (Igfbp1) in control and fKO livers following high-fat feeding (n=5 mice per group). Igfbp1 expression was normalized to B-actin housekeeping gene. (b) Representative images of H&E-stained liver sections following high-fat feeding.
(d) Quantification of fasted plasma 3-hydroxybutyrate (3-HB) following high-fat feeding using colorimetric assay (n=7-8 mice per group). Data presented as mean +/-SEM. The difference in means was analyzed using Student's t-test where *P<0.05. Figure S2 a c Figure S3. CREB3L3 ablation promotes insulin resistance, crown-like structure formation, and lipogenesis in obese inguinal fat (a-b) Western blot measuring abundance of Akt and Akt phosphorylated at the S473 site in high fat-fed control and fKO iWAT following injection with PBS or insulin and (b) quantification of Western blot results, with abundance of S473 phosphorylation normalized to abundance of total Akt in iWAT (n=2 mice per PBS group; n=3-4 ins-stimulated mice per group).
(c) Representative images of H&E-stained iWAT sections following high-fat feeding and quantification of the number of crown-like structures per field of view. Arrows demarcate the presence of crown-like structures (3 images were taken per mouse. n=4-8 mice per group).
(d-e) Quantitative PCR for markers of (d) adipogenesis or (e) lipogenesis in control and fKO iWAT following high-fat feeding (n=5-6 mice per group). Expression was normalized to TBP housekeeping gene and presented as fold change over controls. Data presented as mean +/-SEM with sample sizes listed above. The difference in means was analyzed using Student's t-test where *P<0.05 and **P<0.01.   Figure S4. CREB3L3 ablation does not reduce expression of thermogenic markers in obese brown, epididymal, or inguinal fat, nor in cold-exposed inguinal fat (a) Quantitative PCR for markers of thermogenesis and adipocyte browning in brown adipose tissue from control and fKO mice following high-fat feeding (n=5 mice per group). Target gene expression was normalized to TBP and presented as fold change over controls.
(b) Quantification of plasma FGF-21 concentration following high-fat feeding using ELISA (n=9 mice per group). (c) Quantitative PCR for markers of thermogenesis and adipocyte browning in inguinal adipose tissue in lean control and fKO mice housed at 6 degrees for 7 days (n=3-4 mice per group).
(d-e) Quantitative PCR for markers of thermogenesis and adipocyte browning in (d) iWAT or (e) eWAT from control and fKO mice following high-fat feeding (n=5-6 mice per group).
(f-g) Quantitative PCR for markers of fatty acid oxidation in (f) eWAT or (g) iWAT from control and fKO mice following high-fat feeding (n=5-6 mice per group).
(h) Western blot measuring abundance of UCP1 in iWAT from control and fKO mice following high-fat feeding. Data presented as mean +/-SEM with sample sizes listed above. The difference in means was analyzed using Student's t-test where *P<0.05 and **P<0.01.  Figure S5. Ablation of CREB3L3 reduces expression of lipolytic markers in obese epidiymal, but not inguinal fat (a-b) Quantitative PCR for markers of lipolysis in (a) eWAT or (b) iWAT from control and fKO mice following high-fat feeding (n=5 mice per group). Target gene expression was normalized to TBP and presented as fold change over control. (c) Quantification of non-esterified fatty acids (NEFA) from the plasma of high-fat fed control and fKO mice 1h after injection with CL316,243 to induce adipocyte lipolysis (n=5-8 mice per group). Data presented as mean +/-SEM with sample sizes listed above. The difference in means was analyzed using Student's t-test where *P<0.05 and **P<0.01.

Figure S5
Hsl ATGL Adrb3 Lpl Plin1 Plin2  Figure S6. Overexpression of CREB3L3 in inguinal fat increases expression of inflammatory cytokines, but not adipogenic markers or UCP1 protein abundance (a) Quantitative PCR for the expression of CREB3L3 message in iWAT from ctrl and sOE mice following high-fat diet (n=6-8 mice per group). Target gene expression was normalized to TBP and presented as fold change over WT.
(b-c) Quantitative PCR for the expression of adipogenic or inflammatory markers in iWAT from ctrl and sOE mice following high-fat diet (n=6-8 mice per group). (d) Western blot measuring abundance of UCP1 in iWAT from ctrl and sOE mice following high-fat feeding. Data presented as mean +/-SEM with sample sizes listed above. The difference in means was analyzed using Student's t-test where *P<0.05.