Connexin43 in mesenchymal lineage cells regulates body adiposity and energy metabolism in mice

Figures

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  Figure 1.. Gja1 ablation in mesenchymal lineage cells leads to metabolic benefits in regular chow died (RCD) fed male mice on standard diet. (A) Body weight, and (B) DXA-determined percent body fat and (C) lean mass in 7-month-old wild type (WT, blue) and cKOTW2 mice (red). (D) White adipose tissue (WAT) weight normalized to body weight (BW) in inguinal (iWAT) and gonadal (gWAT) depots. (E) Weight of suprascapular brown adipose tissue (BAT) depots normalized to BW. (F) Hematoxylin-eosin-stained histologic sections of BAT and iWAT from wild type and cKOTW2 mice (bar=100 µm). Each image is representative of 6 wild type and 9 cKOTW2 mice. (G) Intraperitoneal glucose tolerance test: blood glucose before and after an intraperitoneal load of 1.5g/kg D-glucose. (H) Areas under the curve (AUC) calculated between 0 and 120 minutes for animals included in panel G. Data are presented as boxplots representing the interquartile range with median (inside bar). (A-E, H) Groups were compared using two-tailed Mann-Whitney U-test. (G) P-values represent the effect of genotype, time and their interaction by two-way ANOVA (genotype, F=3.082, p=0.103; time: F=97.14, p<0.001; time x genotype: F=3.032, p=0.016).

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  Figure 2.. Cx43 is up-regulated by high fat diet (HFD) in brown adipose tissue (BAT), and down-regulated during adipogenic differentiation. (A) Expression of connexin mRNA by qPCR in inguinal white adipose tissue (iWAT and BAT) in 5-month-old male mice fed either a regular chow diet (RCD) or HFD for 12 weeks. Data are presented as boxplots representing the interquartile range with median (inside bar). Groups were compared using two-tailed Mann-Whitney U-test. (B) Western blot of whole cell lysates of confluent, undifferentiated cultures of IngWAT pre-adipocytic cells, cells isolated from the stromal-vascular fraction (SVF) of WAT, and the osteogenic cell line, MC3T3-E1, as control. (C) Western blot of whole cell lysates and (D) qRT-PCR analysis of mRNA of IngWAT cells before and during adipogenic differentiation (median and range, n=6; p<0.001 vs. time 0 at all time points). (E) Immunohistochemisty stain (brown color) for Cx43 in WAT and BAT isolated from WT mice kept of either RCD or HFD and in cKOTW2 mice. Scale bar: 500 µm. Each image is representative of three mice per condition.

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  Figure 3.. Gja1 ablation in mesenchymal lineage cells is partially protective of high fat diet-induced obesity, hyperglycemia, and reduced glucose tolerance in male mice. (A) Body weight of 2-month-old wild type (WT, blue) and cKOTW2 (red) male mice during 8 weeks on HFD feeding. Data are shown as median and interquartile range; P-values represent the effect of genotype, time and their interaction by repeated measures two-way ANOVA (genotype, F=53.53, p<0.001; time: F=288.3, p<0.001; time x genotype: F=42.08, p<0.001). (B) Percent body fat and (C) lean mass by DXA after HFD in the two genotype groups. (D) Intraperitoneal glucose tolerance test: blood glucose before and after an intraperitoneal load of 1.5g/kg D-glucose (mean ± 95% CI; two-way ANOVA: genotype, F=8.202, p=0.013; time: F=70.87, p<0.001; time x genotype: F=5.972, p<0.001). (E) Areas under the curve (AUC) calculated between 0 and 120 minutes for animals included in panel D. (F) Intraperitoneal insulin tolerance test: blood glucose before and after an intraperitoneal injection of 0.75U/kg insulin (mean ± 95% CI; two-way ANOVA: genotype, F=4.131, p=0.063; time: F=6.634,p<0.001; time x genotype: F=1.347,p>0.10 ). (G) Areas under…

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  Figure 4.. Gja1 ablation in mesenchymal lineage cells protects high fat diet-induced expansion of fat depots, adipocyte hypertrophy, hyperinsulinemia, and hyperlipidemia in male mice. (A) Representative morphology of inguinal white adipose tissue (iWAT) in 2-month-old WT and cKOTW2 male mice after 12 weeks on a HFD. (B) Percent iWAT and gonadal WAT (gWAT) relative to body weight in the two genotypes after HFD (boxplots represent interquartile range with median); P values were determined by two-sided Mann-Whitney U-test. (C) Representative H&E-stained histological sections of iWAT and (D) liver of WT (n=7) and cKOTW2 (n=8) mice after 12 weeks on HFD. Scale bar: 50 µm. Serum levels of (E) insulin, (F) C-peptide, (G) Igf-1, (H) glucagon, (I) triglyceride, (J) cholesterol, and (K) fatty acid measured in WT and cKOTW2 mice after 12 weeks on HFD. Group data are in boxplots representing the interquartile range with median (inside bar), and P values were determined by two-sided Mann-Whitney U-test.

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  Figure 5.. Gja1 ablation in mesenchymal lineage cells upregulates adipogenic genes and promotes glucose uptake without altering lipolysis in white adipose tissue (WAT). (A) Expression of mRNA by RT-qPCR of adipogenic genes, (B) glucose transporters, (C) 2-NBDG uptake, (D) lipolysis-associated genes, (E) glycerol release relative to tissue weight, in the presence or absence of 10 µM isoproterenol, and (F ) Gja1 mRNA expression in inguinal white adipose tissue of 2-month-old WT (blue) and cKOTW2 male mice (red) after 12 weeks on HFD. Boxplots represent interquartile range with median (inside bar); P values were determined by two-sided Mann-Whitney U-test.

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  Figure 6.. Gja1 ablation in mesenchymal lineage cells increases locomotor activity, food consumption, and energy expenditure. Two-month-old cKOTW2 (red, n=6) and wild type (WT: blue; n=8) male mice were placed in metabolic cages after being fed a high fat diet for 12 weeks, and continuously monitored for 48 hours. (A, B) Food consumption, (C, D) energy expenditures, (E, F) respiratory exchange rate (VCO2/O2), and (G. H) locomotor activity. Data are presented as both hourly averages (A, C, E, G), analyzed using general linear models or ANOVA (detailed results in Table S2; p values are given for genotype effect), and daily averages over the 2-day experiment for each time period (B, D, F, H), with groups compared using one-way ANOVA.

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  Figure 7.. Gja1 ablation in mesenchymal lineage cells protects from obesity-induced BAT whitening and increases thermogenesis and lipolysis in diet-induced obese mice. (A) Morphology of suprascapular brown adipose tissue (BAT) depots, (B) percent of BAT weight relative to body weight, and (C) representative H&E-stained histological sections of BAT of WT and cKOTW2 mice fed a HFD for 12 weeks. Scale bar: 100 µm. Each image is representative of 7 WT and 8 cKOTW2 mice. After a 12-week high fat diet (HFD), WT or cKOTW2 mice were exposed to cold temperature (4°C) for 6 hours. (D) Core body temperature during the acute cold challenge. Data are shown as average ± 95% CI; P-values represent the effect of genotype, time and their interaction by mixed effects analysis (genotype, F=9.371, p=0.009; time: F=38.74, p<0.001; time x genotype: F=0.6624, p=0.680). Expression of (E) brown adipose tissue (BAT) identity genes, and Gja1 mRNA, and (F) lipolysis-associated genes in suprascapular BAT depots of 2-month-old WT (blue) and cKOTW2 mice (red) after 12 weeks on HFD. (G) Glycerol release relative to tissue weight, in the presence or absence of 10 µM isoproterenol. (H) Expression of β-oxidation and (I)…

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  Figure 8.. Gja1 ablation in adipocytic cells does not affect diet-induced obesity and worsens glucose tolerance in mice. (A) Body weight of 2-month-old wild type (WT: blue) and cKOAdipoq male mice (red) during 8 weeks on HFD feeding. Data are shown as median ± interquartile range; P-values represent the effect of genotype, time and their interaction by mixed-effects analysis (genotype, F=0.1452, p=0.710; time: F=51.53, p=0.001; time x genotype: F=0.3383, p=0.9337). (B) Percent body fat by DXA after HFD in the two genotype groups. Percent of (C) inguinal and gonadal white adipose tissue (iWAT, gWAT) and (D) brown adipose tissue depot in the two genotypes after HFD. (E, F) Intraperitoneal glucose tolerance test: blood glucose before and after an intraperitoneal load of 1.5 g/kg glucose. Data points represent the mean ± 95% CI; P-values represent the effect of genotype, time and their interaction by two-way ANOVA (genotype, F=2.603, p=0.135; time: F=6.922, p=0.023; time x genotype: F=0.9072, p=0.483). (G, H ) Intraperitoneal insulin tolerance test: blood glucose before and after an intraperitoneal injection of 0.75U/kg insulin. Data points represent the mean ± 95% CI; P-values represent the…

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  Figure 9.. Schematic representation of the effect of Gja1 ablation on the metabolic response to a high fat diet. (Left column) In normal mice, high dietary calorie intake alters energy metabolism resulting in excess energy storage in fat depots and other organs leading to obesity, hyperinsulinemia, high serum lipids and glucose intolerance. In WAT depots (bottom row), fat accumulation occurs primarily by adipocyte hypertrophy; in BAT (top row), it leads to ‘whitening” as cells become engulfed by lipid droplets. (Right column) Genetic ablation of Gja1 in the mesenchymal lineage (cKOTw2) results in smaller WAT depots and increased glucose uptake and utilization under high calorie intake. At the organism level (middle row), Cx43-deficient mice are more active and more cold tolerant, burn more energy and utilize more glucose than control littermates under high calorie intake. We propose that the increased energy consumption for physical activity and thermogenesis reduces fat accumulation, WAT hypertrophy, and BAT whitening, resulting in less severe obesity, partially preserved glucose tolerance, and better circulating lipid profile than control littermates.