Loss of RREB1 reduces adipogenesis and improves insulin sensitivity in mouse and human adipocytes

Abstract

There are multiple independent genetic signals at the Ras-responsive element binding protein 1 (RREB1) locus associated with type 2 diabetes risk, fasting glucose, ectopic fat, height, and bone mineral density. We have previously shown that loss of RREB1 in pancreatic beta cells reduces insulin content and impairs islet cell development and function. However, RREB1 is a widely expressed transcription factor and the metabolic impact of RREB1 loss in vivo remains unknown. Here, we show that male and female global heterozygous knockout (Rreb1 ^+/-) mice have reduced body length, weight, and fat mass on high-fat diet. Rreb1^+/- mice have sex- and diet-specific decreases in adipose tissue and adipocyte size; male mice on high-fat diet had larger gonadal adipocytes, while males on standard chow and females on high-fat diet had smaller, more insulin sensitive subcutaneous adipocytes. Mouse and human precursor cells lacking RREB1 have decreased adipogenic gene expression and activated transcription of genes associated with osteoblast differentiation, which was associated with Rreb1 ^+/- mice having increased bone mineral density in vivo. Finally, human carriers of RREB1 T2D protective alleles have smaller adipocytes, consistent with RREB1 loss-of-function reducing diabetes risk.

Keywords: Diabetes; RREB1; adipocyte; insulin sensitivity; transcription factor.

Fig. 1 ∣. Reduced length, body weight, and fat mass in Rreb1 heterozygous knockout mice.

a Length in mm of wildtype (grey) and Rreb1 heterozygous knockout (green) male mice at 29 weeks on HFD and LFD. n = 15-21. b Length in mm of wildtype (grey) and Rreb1 heterozygous knockout (green) male mice at 38 weeks on RM3 diet. n = 7-9. c Length in mm of wildtype (grey) and Rreb1 heterozygous knockout (purple) female mice at 29 weeks on HFD and LFD. n = 18-24. d Length in mm of wildtype (grey) and Rreb1 heterozygous knockout (purple) female mice at 38 weeks on RM3 diet. n = 9-11. e-g Biweekly measurements of (e) body weight in grams (g), (f) fat mass normalized to body weight (BW), and (g) lean mass normalized to body weight (BW) of wildtype (grey) and Rreb1 heterozygous knockout (green) male mice on HFD and LFD. n = 15-21. h-j Biweekly measurements of (h) body weight in grams (g), (i) fat mass normalized to body weight (BW), and (j) lean mass normalized to body weight (BW) of wildtype (grey) and Rreb1 heterozygous knockout (purple) female mice on HFD and LFD. n = 18-24. Data are presented as mean ± s.e.m. Statistical analyses were performed by (a-d) unpaired t test (d, with Welch correction) or (e-j) two-way ANOVA with Tukey’s multiple comparisons test. *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001.

Fig. 2 ∣. Rreb1 heterozygous knockout mice have differences in depot size of white adipose tissue.

a-d Comparisons of visceral gonadal (a, gWAT), mesenteric (b, mWAT), perirenal (c, pWAT), and subcutaneous inguinal (d, iWAT) white adipose tissue weight (% normalized to body weight) at 26 weeks in wildtype (grey) and Rreb1 heterozygous knockout (green) male mice on HFD and LFD. n = 15-21. e-h Comparisons of visceral gonadal (e, gWAT), mesenteric (f, mWAT), perirenal (g, pWAT), and subcutaneous inguinal (h, iWAT) white adipose tissue weight (% normalized to body weight) at 26 weeks in control (grey) and Rreb1 heterozygous knockout (purple) female mice on HFD and LFD. n = 18-24. i,j Plasma adiponectin (ng/mL) after an overnight fast at (i) 12 weeks and (j) 22 weeks for male wildtype (grey) and Rreb1 heterozygous knockout (green) mice on HFD and LFD. n = 15-22. k,l Plasma adiponectin (ng/mL) after an overnight fast at (k) 12 weeks and (l) 22 weeks for female wildtype (grey) and Rreb1 heterozygous knockout (purple) mice on HFD and LFD. n = 17-24. Data are presented as mean ± s.e.m. Statistical analyses were performed using a one-way ANOVA with Sidak’s multiple comparison test or (f,g,h,j) Brown-Forsythe and Welch ANOVA tests with Dunnett's T3 multiple comparisons test *p<0.05, **p<0.01, and ****p<0.0001.

Fig. 3 ∣. Sex- and tissue-specific differences in adipocyte size of white adipose tissue in Rreb1 heterozygous knockout mice.

a-d Quantification of adipocyte area (μm²) within (a,b) visceral gonadal (gWAT, both sexes: Rreb1^+/+ n = 5, Rreb1^+/− n = 5 mice) and (c,d) subcutaneous inguinal (iWAT, both sexes: Rreb1^+/+ n = 6, Rreb1^+/− n = 6 mice) fat depots from (a,c) male and (b,d) female Rreb1^+/+ and Rreb1^+/− mice fed a HFD at 29 weeks of age. Dotted line denotes the interval where the dataset converges. e-h Quantification of adipocyte area (μm²) within (e,f) visceral gonadal (gWAT, both sexes: Rreb1^+/+ n = 3, Rreb1^+/− n = 6 mice) and (g,h) subcutaneous inguinal (iWAT, male: Rreb1^+/+ n = 6, Rreb1^+/− n = 5; female: Rreb1^+/+ n = 6, Rreb1^+/− n = 6 mice) fat depots from (e,g) male and (f,h) female Rreb1^+/+ and Rreb1^+/− mice fed a LFD at 29 weeks of age. Data are presented as mean adipocyte frequency within each 250 μm² sized bin ± s.e.m. Statistical analyses were performed on the area under the curve for adipocytes in (a) <4500 and >5750 μm² or (d) <4500 and >5250 μm² adipocyte size ranges by (a,d,e,f,h) unpaired t test or (b,c,g) Mann-Whitney two tailed test, depending on the normality of the data distribution. **p<0.01 and ****p<0.0001.

Fig. 4 ∣. Global Rreb1 heterozygous knockout mice have ectopic fat in the liver.

a,b,c Plasma (a) alkaline phosphatase (ALP; U/L), (b) alanine aminotransferase (ALT; U/L), and (c) aspartate aminotransferase (AST; U/L) levels in Rreb1^+/+ and Rreb1^+/− male mice at 29 weeks on HFD and LFD. n = 15-20. d,e,f Plasma (d) alkaline phosphatase (ALP; U/L), (e) alanine aminotransferase (ALT; U/L), and (f) aspartate aminotransferase (AST; U/L) levels in Rreb1^+/+ and Rreb1^+/− female mice at 29 weeks on HFD and LFD. n = 14-23. g,h Liver weight (adjusted for body weight, %) of Rreb1^+/+ and Rreb1^+/− on HFD and LFD at 29 weeks of age in (g) male (n = 15-21) and (h) female (n = 17-23) mice. Data are presented as mean ± s.e.m. Statistical analysis performed by (a,g) Brown-Forsythe and Welch ANOVA tests with Dunnett's T3 multiple comparisons test, (b,c,e,f) ordinary one-way ANOVA with Sidak’s multiple comparison tests, and (d) ANOVA Kruskal-Wallis test. (a,b,e,f) data were transformed Y=log(Y) before statistical analysis. * p<0.05, ** p<0.01, and **** p<0.0001.

Fig. 5 ∣. Reduced food intake and energy expenditure in Rreb1 heterozygous knockout mice.

a,b Comparisons of weekly food intake (g) measured from 6 to 24 weeks for Rreb1^+/+ and Rreb1^+/− (a) male and (b) female mice on HFD. (a) Rreb1^+/+ and Rreb1^+/− n = 11 cages, each containing 2 animals. (b) Rreb1^+/+ n = 9 and Rreb1^+/− n = 11 cages, each containing 2 animals. c,d Heat production of Rreb1^+/+ and Rreb1^+/− (c) male and (d) female mice over a 24-hour period. (c) n = 22, (d) n = 20 mice. e,f Comparisons of brown adipose tissue (BAT) weight of Rreb1^+/− (e) male and (f) female mice with wildtype littermate control mice at 38 weeks on RM3 diet. (e) n = 6-7 mice, (f) n = 8-9 mice. g,h Circulating leptin levels in Rreb1^+/− (g) male and (h) female mice with wildtype littermate control mice. (g) n = 6-8, (h) n = 6-11 mice. Data are presented as mean ± s.e.m. (a,b) Area under the curve was calculated for statistical analysis (males: 6-24 weeks and females: 6-23 weeks). Statistical analyses were performed by two-tailed unpaired t test or (c) a Mann Whitney test. *p<0.05, ***p<0.001.

Fig. 6 ∣. Male Rreb1^+/− mice have differences in insulin sensitivity and fasting insulin levels.

a,b IPIST on Rreb1^+/+ (grey) and Rreb1^+/− (green) male mice aged (a) 16 weeks and (b) 26 weeks on HFD and LFD n = 15-21. c,d IPIST on Rreb1^+/+ (grey) and Rreb1^+/− (purple) female mice aged (c) 16 and (d) 26 weeks of age on HFD and LFD. n = 19-24. e-h Plasma insulin after an overnight fast at (e,g) 12 and (f,h) 22 weeks for (e,f) male and (g,h) female Rreb1^+/+ and Rreb1^+/− mice on HFD and LFD. n = 15-24. Data are shown as mean ± s.e.m. Area of curve (AOC) was calculated and statistical analyses performed between genotypes using (a,b,c,d) one way ANOVA with Sidak’s multiple comparisons test or (e,f,g) Brown-Forsythe and Welch ANOVA tests with Dunnett's T3 multiple comparisons test. Where necessary to normalize data distribution before analysis, data was transformed by (e,h) Y=1/Y or (f,g) Y=log(Y). *p<0.05, **p<0.01.

Fig. 7 ∣. Improved insulin-stimulated glucose uptake in male Rreb1^+/− mice.

a,b Basal (a) and insulin-stimulated (b) glucose uptake ((CPM)/mg of wet tissue weight) in all white adipose tissues (WAT), inguinal (iWAT), gonadal (gWAT), perirenal (pWAT), brown adipose tissue (BAT), liver and quadricep in male wildtype (grey) and Rreb1^+/− (green) mice. c,d Basal (c) and insulin-stimulated (d) glucose uptake ((CPM)/mg of wet tissue weight) in all white adipose tissues (WAT), inguinal (iWAT), gonadal (gWAT), perirenal (pWAT), brown adipose tissue (BAT), liver and quadricep in female wildtype (grey) and Rreb1^+/− (purple) mice. Data are shown as mean ± s.e.m. Statistical analyses were performed between genotypes using an unpaired t-test. n = 4-7. *p<0.05.

Fig. 8 ∣. Loss of Rreb1 decreases adipocyte formation and expression of pro-adipogenic genes.

a Quantification of adipocyte formation using Oil Red O staining (absorbance at 500 nm) in stromal vascular fraction (SVF) cells from wildtype (Rreb1^+/+; grey) and knockout (Rreb1^+/−; green) mice. n = 5-6. b Quantification of BrdU+ S-phase cells during in vitro differentiation of SVF cells from Rreb1^+/+ (grey) and Rreb1^+/− (green) male mice. n = 3. c RREB1 expression 48 hours following transient transfection of siRNAs against RREB1 (siRREB1) or non-targeting control (siNT) siRNAs in SGBS cells. n = 4. d Gene expression analysis of CEBPA, PPARG, and ADIPOQ at day 5 of in vitro differentiation of SGBS cells to adipocytes. SGBS were treated with siNT and siRREB1 48 hours before differentiation. n = 4. e Glucose uptake of SGBS cells at day 12 of in vitro differentiation. SGBS were treated with siNT and siRREB1 48 hours before differentiation. n = 3. f Gene enrichment analysis of RREB1 knockdown SGBS cells at day 12. The number of differentially expressed genes (count) in a subset of gene ontologies relating to mesenchymal stem cell differentiation (blue), SMAD pathway (purple), and lipids (red). g RREB1 transcript expression normalized to PPIA in human induced pluripotent stem cell (hiPSC), mesoderm, mesenchymal progenitor cells (MPC), and adipocytes. h Flow cytometry analysis of CD105 and CD73 co-expression in wildtype (RREB1^WT/WT; grey) and RREB1 knockout (RREB1^KO/KO; blue) hiPSC-derived mesenchymal progenitor cells (MPC). i,j hiPSC wildtype (RREB1^WT/WT; grey) and RREB1 knockout (RREB1^KO/KO; blue) cells were differentiated to adipocyte and osteoblast lineages. The expression of adipocyte genes (i) PPARG and (j) CEBPA were measured by qPCR and normalized to PPIA. Data are presented as mean ± s.e.m. Statistical analyses were performed by unpaired t test or one-way ANOVA. *p<0.05, **p<0.01, ***p<0.001.

Fig. 9 ∣. Increased bone mineral density in Rreb1 heterozygous knockout mice.

a,b Bone mineral density (BMD; g/cm²) on HFD and LFD of (a) male (n = 14-20) and (b) female (n = 17-24) mice at 29 weeks. c,d,e,f MicroCT analysis of male and female mice on HFD for (c) trabecular thickness (Tb.Th), (d) trabecular separation (Tb.Sp), (e) bone volume/tissue volume (BV/TV), and (f) trabecular number (Tb.N). n = 5-16. Data are presented as mean ± s.e.m. Statistical analyses were performed by one-way ANOVA with Šídák's multiple comparisons test. *p<0.05 and **p<0.01.

Fig. 10 ∣. Changes in human adipocyte area in RREB1 rs112492319 variant carriers.

a,b Adipocyte area of (a) subcutaneous and (b) visceral fat from human donors carrying rs112492319 variants. Left graph: all individuals (pooled and matched); middle graph: males; right graph: females. Data are presented as mean ± s.e.m. Statistical analyses were performed by unpaired t-test. Individual p values are labelled in each graph.

Fig. 11 ∣. RREB1 loss-of-function protects against type 2 diabetes.

Overview of significant differences in measured phenotypes in the global Rreb1 heterozygous knockout male and female mice on HFD, LFD, and RM3 diet. Created with BioRender.com.