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. 2024 Mar;35(1):13-30.
doi: 10.1007/s00335-023-10022-3. Epub 2023 Oct 26.

Adipose tissue depot specific expression and regulation of fibrosis-related genes and proteins in experimental obesity

Kristina Eisinger  1 , Philipp Girke  2 , Christa Buechler  3 , Sabrina Krautbauer  1
Affiliations

Adipose tissue depot specific expression and regulation of fibrosis-related genes and proteins in experimental obesity

Kristina Eisinger et al. Mamm Genome. 2024 Mar.

Abstract

Transforming growth factor beta (Tgfb) is a well-studied pro-fibrotic cytokine, which upregulates cellular communication network factor 2 (Ccn2), collagen, and actin alpha 2, smooth muscle (Acta2) expression. Obesity induces adipose tissue fibrosis, which contributes to metabolic diseases. This work aimed to analyze the expression of Tgfb, Ccn2, collagen1a1 (Col1a1), Acta2 and BMP and activin membrane-bound inhibitor (Bambi), which is a negative regulator of Tgfb signaling, in different adipose tissue depots of mice fed a standard chow, mice fed a high fat diet (HFD) and ob/ob mice. Principally, these genes were low expressed in brown adipose tissues and this difference was less evident for the ob/ob mice. Ccn2 and Bambi protein as well as mRNA expression, and collagen1a1 mRNA were not induced in the adipose tissues upon HFD feeding whereas Tgfb and Acta2 mRNA increased in the white fat depots. Immunoblot analysis showed that Acta2 protein was higher in subcutaneous and perirenal fat of these mice. In the ob/ob mice, Ccn2 mRNA and Ccn2 protein were upregulated in the fat depots. Here, Tgfb, Acta2 and Col1a1 mRNA levels and serum Tgfb protein were increased. Acta2 protein was, however, not higher in subcutaneous and perirenal fat of these mice. Col6a1 mRNA was shown before to be higher in obese fat tissues. Current analysis proved the Col6a1 protein was induced in subcutaneous fat of HFD fed mice. Notably, Col6a1 was reduced in perirenal fat of ob/ob mice in comparison to the respective controls. 3T3-L1 cells express Ccn2 and Bambi protein, whose levels were not changed by fatty acids, leptin, lipopolysaccharide, tumor necrosis factor and interleukin-6. All of these factors led to higher Tgfb in 3T3-L1 adipocyte media but did not increase its mRNA levels. Free fatty acids induced necrosis whereas apoptosis did not occur in any of the in vitro incubations excluding cell death as a main reason for higher Tgfb in cell media. In summary, Tgfb mRNA is consistently induced in white fat tissues in obesity but this is not paralleled by a clear increase of its target genes. Moreover, discrepancies between mRNA and protein expression of Acta2 were observed. Adipocytes seemingly do not contribute to higher Tgfb mRNA levels in obesity. These cells release more Tgfb protein when challenged with obesity-related metabolites connecting metabolic dysfunction and fibrosis.

Keywords: Actin alpha 2 smooth muscle; Bambi; Brown fat; Collagen; Lipopolysaccharide; White fat.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1
Fig. 1
Expression of Ccn2/Ccn2, Tgfb, Acta2/Acta2, Bambi/Bambi, Col1a1 and Col6a1 in subcutaneous (SC), epididymal (EPI), perirenal (REN) and brown adipose tissue (BAT) from 21-week-old mice fed a standard diet. Expression of a Ccn2 b Tgfb c Acta2 d Bambi and e Col1a1 in fat tissues. Identical letters in the figures indicate significantly different expression between these two tissues. p < 0.05 (one letter), p < 0.01 (two letters), p < 0.001 (three letters) (n = 7 per group). f Immunoblot of Ccn2 and Acta2 in these tissues. g Immunoblot of Bambi and Col6a1 in these tissues. h Quantification of Ccn2, Acta2, Bambi and Col6a1 in the fat tissues of 3 mice. *p < 0.05, **p < 0.01. i Sirius Red stained fat tissues. j % Picrosirius red stained area of subcutaneous, epididymal and perirenal fat. Because of the brownish color of BAT, this tissue was not suitable for ImageJ analysis
Fig. 2
Fig. 2
Expression of fibrosis-related genes and proteins in fat tissue of mice fed a standard diet (SD) or a high fat diet (HFD). a Ccn2 mRNA in the different fat depots of mice fed a SD or HFD. b Bambi mRNA in these tissues. c Ccn2 and BAMBI protein in the different white fat depots of two mice fed a SD and two mice fed a HFD. Coomassie (Coom) stained membrane is shown as loading control. d Tgfb, e Acta2 and f Col1a1 mRNA in the different fat depots of mice fed a SD or HFD. g Acta2 and Col6a1 protein in subcutaneous and perirenal fat of two mice fed a SD and two mice fed a HFD. h Tgfb protein in the serum of these mice *p < 0.05, **p < 0.01, ***p < 0.001 for comparison of gene expression between SD and HFD fed mice (n = 7 per group for mRNA expression analysis, n = 6 per group for immunoblot analysis and n = 5 per group for data shown in (h)
Fig. 3
Fig. 3
Expression of Ccn2/Ccn2 and Bambi/Bambi in fat tissue of wild type (WT) and ob/ob (OB) mice. a Ccn2 mRNA in the different fat depots. b Quantification of Ccn2 protein in the different fat depots. c Immunoblot of Ccn2 and Bambi protein in the different fat depots of two WT and 2 OB mice. Coomassie (Coom) stained membrane is shown as loading control. d Bambi mRNA in the different fat depots. e Quantification of Bambi protein in the different fat depots of mice. *p < 0.05, **p < 0.01 for comparison of gene expression between ob/ob and WT mice (n = 5 per group)
Fig. 4
Fig. 4
Serum Tgfb and expression of Tgfb, Acta2/Acta2, Col1a1 and Col6a1 in fat tissue of wild type (WT) and ob/ob (OB) mice. a Tgfb mRNA in the different fat depots. b Serum Tgfb of WT and ob/ob mice. c Acta2 mRNA in the different fat depots. d Col1a1 mRNA in the different fat depots. e Acta2 and Col6a1 protein in subcutaneous and perirenal fat of two WT and two ob/ob mice. f Quantification of Acta2 and Col6a1 protein in subcutaneous and perirenal fat. (n = 5 per group). *p < 0.05, **p < 0.01, ***p < 0.001 for comparison of gene/protein expression between ob/ob and WT mice
Fig. 5
Fig. 5
Effect of palmitic acid (PA), oleic acid (OA), leptin (Lpt) and lipopolysaccharide (LPS) on Ccn2, Bambi and Tgfb expression and on Ccn2 protein levels of 3T3-L1 cells. a Expression of these genes in 3T3-L1 adipocytes which were incubated with 100 µM fatty acids for 24 h after differentiation to mature cells (n = 3). b Expression of these genes in 3T3-L1 adipocytes, which were differentiated in the presence of 100 µM fatty acids (n = 3). c Immunoblot of Ccn2 and Bambi in the cells described in (b). d Expression of these genes in 3T3-L1 adipocytes, which were incubated with 100 ng/ml leptin for 24 h after differentiation to mature cells. e Expression of these genes in 3T3-L1 adipocytes, which were differentiated in the presence of 100 ng/ml leptin (n = 3). f Expression of these genes in 3T3-L1 adipocytes, which were differentiated in the presence of 10 ng/ml LPS (n = 6). g Immunoblot of Ccn2 in 3T3-L1 cells differentiated in the presence of 100 ng/ml leptin. h Immunoblot of Ccn2 in 3T3-L1 adipocytes, which were incubated with 100 ng/ml leptin for 24 h after differentiation to mature cells. i Immunoblot of Ccn2 in 3T3-L1 cells differentiated in the presence of 10 ng/ml LPS. Arbitrary units, au
Fig. 6
Fig. 6
Effect of palmitic acid (PA), oleic acid (OA), leptin (Lpt), lipopolysaccharide (LPS), TNF and IL-6 on Tgfb, Ccn2 and Bambi protein levels of 3T3-L1 cells. a Tgfb in cell media of 3T3-L1 adipocytes, which were incubated with fatty acids for 24 h after differentiation to mature cells (n = 4) or were differentiated in the presence of 100 µM fatty acids (n = 6). b Tgfb in cell media of 3T3-L1 adipocytes, which were incubated with LPS for 24 h after differentiation to mature cells (n = 6) or were differentiated in the presence of 10 ng/ml LPS (n = 6). c Tgfb in cell media of 3T3-L1 adipocytes, which were incubated with leptin for 24 h after differentiation to mature cells (n = 4) or were differentiated in the presence of 100 ng/ml leptin (n = 3). d Tgfb in cell media of 3T3-L1 adipocytes, which were differentiated in the presence of TNF, IL-6 or both (n = 3). e Immunoblot of Ccn2 and Bambi in 3T3-L1 adipocytes, which were differentiated in the presence of 10 pg/ml TNF. f Immunoblot of Ccn2 and Bambi in 3T3-L1 adipocytes, which were differentiated in the presence of 200 pg/ml IL-6. g Immunoblot of Ccn2 and Bambi in 3T3-L1 adipocytes, which were differentiated in the presence of 10 pg/ml TNF and 200 pg/ml IL-6. Cyclophilin A (Cyclo) was used as loading control
Fig. 7
Fig. 7
Cell death markers in 3T3-L1 cells treated with palmitic acid (PA), oleic acid (OA), leptin (Lpt), lipopolysaccharide (LPS), TNF and IL-6. a Cyclophilin A (Cyclo) in media of cells differentiated in the presence of PA. b Cyclophilin A (Cyclo) in media of cells differentiated in the presence of OA. c Cyclophilin A (Cyclo) in media of mature cells incubated with LPS for 24 h. d Cyclophilin A (Cyclo) in media of mature cells incubated with leptin for 24 h. e Cyclophilin A in cell media of 3T3-L1 adipocytes, which were differentiated in the presence of TNF and IL-6. f Quantification of cyclophilin A in cell media (n = 3). *p < 0.05, **p < 0.01. g PARP in cells differentiated in the presence of PA or OA. h PARP in cells differentiated in the presence of LPS. i PARP in cells incubated with leptin for 24 h. j PARP in 3T3-L1 adipocytes, which were differentiated in the presence of TNF and IL-6. k Ratio of uncleaved to cleaved PARP (n = 3). l Tgfb in 3T3-L1 adipocytes, which were differentiated in the presence of TNF and IL-6
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