Transplantation of committed pre-adipocytes from brown adipose tissue improves whole-body glucose homeostasis

Abstract

Obesity and its co-morbidities including type 2 diabetes are increasing at epidemic rates in the U.S. and worldwide. Brown adipose tissue (BAT) is a potential therapeutic to combat obesity and type 2 diabetes. Increasing BAT mass by transplantation improves metabolic health in rodents, but its clinical translation remains a challenge. Here, we investigated if transplantation of 2-4 million differentiated brown pre-adipocytes from mouse BAT stromal fraction (SVF) or human pluripotent stem cells (hPSCs) could improve metabolic health. Transplantation of differentiated brown pre-adipocytes, termed "committed pre-adipocytes" from BAT SVF from mice or derived from hPSCs improves glucose homeostasis and insulin sensitivity in recipient mice under conditions of diet-induced obesity, and this improvement is mediated through the collaborative actions of the liver transcriptome, tissue AKT signaling, and FGF21. These data demonstrate that transplantation of a small number of brown adipocytes has significant long-term translational and therapeutic potential to improve glucose metabolism.

Keywords: Biological sciences; Endocrinology; Health sciences; Natural sciences; Physiology.

Graphical abstract

Figure 1

Transplantation of WT committed pre-adipocytes improves whole-body glucose metabolism of WT recipient mice (A–G) Body weight (A), fat mass (B), lean mass (C), glucose tolerance test (GTT) excursion curve (D), GTT area under curve (AUC) (E), insulin tolerance test (ITT) AUC (F), and plasma insulin levels after 12-h fast (ng/mL) (G), 12 weeks post-transplant (n = 5–46 per group; ˆp < 0.05, ˆˆp < 0.01, ˆˆˆp < 0.001, ˆˆˆˆp < 0.0001 vs. Chow; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001 vs. Sham-WT; ^$p < 0.05, ^$$p < 0.01, ^$$$p < 0.001, ^$$$$p < 0.0001 vs. SVF 1x; ^#p < 0.05, ^##p < 0.01 vs. SVF 2x; ^&p < 0.05, ^&p < 0.01 vs. iBAT 1x; two-tailed t-test). (H and I) Cold tolerance test in WT recipients with average rectal temperature [^oC] over 240 min of cold exposure [4°C] (H) and rectal temperature [^oC] 0, 30, 60, 120, 180, and 240 min after cold exposure [4°C] (I) (n = 5–7 per group). All data are represented as mean ± SEM.

Figure 2

Transplantation of WT committed pre-adipocytes increases pAKT/AKT signaling in tibialis anterior (TA) muscle, iBAT, and liver of WT recipient mice (A–C) Western blotting protein expression for pAKT/AKT and blots for pAKT and AKT in TA (A), iBAT (B), and liver (C) (n = 3–5 per group; insulin-stimulated conditions depicted using ‘i'; ∗p < 0.05, ∗∗p < 0.01 as described [∗ = insulin effect; ^# = group effect]; two-tailed t test). All data are represented as mean ± SEM.

Figure 3

Transplantation of WT committed pre-adipocytes alters pathways associated with glucose, lipid, insulin, and immune processing in the liver of WT recipient mice (A) Principal component analysis (PCA) plot of Sham-WT and iBAT 2x mice livers (n = 7–9 per group). (B) Volcano plot depicting differentially expressed genes in iBAT 2x livers versus Sham-WT livers (n = 6–8 per group; significance threshold: 1-fold-change, p < 0.05). (C and D) Tile plots highlighting significantly upregulated (C) or downregulated (D) Gene ontology (GO) biological process (BP) terms and the associated genes based on the over-representation analysis of iBAT 2x livers versus Sham-WT livers (n = 7–9 per group; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 vs. Sham-WT). (E) Gene expression analysis in liver of Sham-WT and iBAT 2x mice, 12 weeks post-transplant (n = 3 per group; ∗p < 0.05 vs. Sham-WT; two-tailed t test). (F) Western blotting protein expression for MUP1, MUP11, and MUP12 in liver of Sham-WT and iBAT 2x mice, 12 weeks post-transplant (n = 4–6 per group; ∗p < 0.05 vs. Sham-WT; two-tailed t test). All data are represented as mean ± SEM.

Figure 4

Transplantation of WT committed pre-adipocytes improves metabolic health in FGF21^−/− mice (A–C) Weekly body weight (A), fat mass (B), and lean mass (C), 12 weeks post-transplantation. (D) GTT AUC data at 4-, 8-, and 12-week post-transplant. (n = 4–31 per group; ∗p < 0.05. ∗∗p < 0.01 vs. Sham-WT; ^$$p < 0.01, ^$$$p < 0.001, ^$$$$p < 0.0001 vs. iBAT-2x; ^#p < 0.05 vs. Sham-KO; ^@p < 0.05 vs. 4 weeks post-transplant within group; two-tailed t test). (E) ITT AUC, 12 weeks post-transplant (n = 8–13 per group; ∗∗p < 0.01 vs. Sham-WT; ^$$$$p < 0.0001 vs. iBAT 2x; ^#p < 0.05 vs. Sham-KO; two-tailed t test). (F) Plasma insulin levels at 12 weeks post-transplantation after 12-h fast (ng/mL) (n = 3–5 per group; ∗∗p < 0.01 vs. Sham-KO; two-tailed t test). (G) Plasma FGF21 (ng/mL) after 12-h fast, 12 weeks post-transplant (n = 3–12 per group; ∗p < 0.05, ∗∗p < 0.01 vs. Sham-WT; two-tailed t test). (H and I) Cold tolerance test in FGF21^−/− recipients with average rectal temperature [^oC] over 240 min of cold exposure [4°C] (H) and rectal temperature [^oC] 0, 30, 60, 120, 180, and 240 min after cold exposure [4°C] (I) (n = 4–15 per group). All data are represented as mean ± SEM.

Figure 5

Transplantation of WT committed pre-adipocytes attenuates AKT phosphorylation in skeletal muscle and liver of FGF21^−/− recipients (A–C) Western blotting protein expression for pAKT/AKT and blots for pAKT and AKT in TA (A), iBAT (B), and liver (C) (n = 3 per group; insulin-stimulated conditions depicted using ‘i'; ∗p < 0.05, ∗∗p < 0.01 as described [∗ = insulin effect; ^# = group effect]; two-tailed t test). (D) Gene expression analysis in liver of Sham-KO and WT-to-KO mice, 12 weeks post-transplant (n = 3 per group; ∗p < 0.05, ∗∗p < 0.01 vs. Sham-KO; two-tailed t test). (E) Western blotting protein expression for MUP1, MUP11, and MUP12 in liver of Sham-KO and WT-to-KO mice, 12 weeks post-transplant (n = 4–6 per group; ∗p < 0.05 vs. Sham-KO; two-tailed t test). All data are represented as mean ± SEM.

Figure 6

Transplantation of committed pre-adipocytes from BAT SVF of FGF21^−/− mice improves insulin tolerance in WT, but not FGF21^−/− recipients (A–C) Weekly body weight (A), fat mass (B), and lean mass (C), 12 weeks post-transplant (n = 5–24 per group). (D and E) GTT AUC data at 4-, 8-, and 12-week post-transplant (n = 4–46 per group; ^@p < 0.05, ^@@p < 0.01, ^@@@p < 0.001 vs. 4 weeks post-transplant within group; two-tailed t test). (F) ITT AUC, 12 weeks post-transplant (n = 5–16 per; ∗p < 0.05 vs. Sham-WT; ^###<p0.001 vs. Sham-KO; ^$$p < 0.05 vs. KO-to-KO; two-tailed t test). (G) Plasma insulin levels at 12 weeks post-transplantation after 12-h fast (ng/mL) (n = 3–14 per group; ∗p < 0.05 vs. Sham-WT, two-tailed t test). (H) Plasma FGF21 (ng/mL) after 12-h fast, 12 weeks post-transplant (n = 2–12 per group; ∗p < 0.05, ∗∗p < 0.01 vs. Sham-WT; ^##p < 0.01 vs. KO-to-WT; two-tailed t test). (I and J) Cold tolerance test in WT recipients with average rectal temperature [^oC] over 240 min of cold exposure [4°C] (I) and rectal temperature (^oC) 0, 30, 60, 120, 180, and 240 min after cold exposure [4°C] (J) (n = 5–16 per group). (K and L) Cold tolerance test in FGF21^−/− recipients with average rectal temperature [^oC] over 240 min of cold exposure [4°C] (K) and rectal temperature [^oC] 0, 30, 60, 120, 180, and 240 min after cold exposure [4°C] (L) (n = 6–8 per group). All data are represented as mean ± SEM.

Figure 7

Transplantation of committed pre-adipocytes from FGF21^−/− donors increases AKT signaling in TA, iBAT, and liver of WT recipient mice but not FGF21^−/− recipient mice (A–C) Western blotting protein expression for pAKT/AKT and blots for pAKT and AKT in WT recipients in TA (A), iBAT (B), and liver (C). (D–F) Western blotting protein expression for pAKT/AKT and blots for pAKT and AKT in FGF21^−/− recipients in TA (D), iBAT (E), and liver (F) (n = 3 per group; insulin-stimulated conditions depicted using ‘i'; ∗p < 0.05, ∗∗p < 0.01 as described [∗ = insulin effect; ^# = group effect]; two-tailed t test). All data are represented as mean ± SEM.

Figure 8

Differentiated human brown adipocytes (hBAs) from AgeX-NP88 cell line improve glucose and insulin tolerance in NSG mice (A–C) Weekly body weight (A), fat mass (B), and lean mass (C), 16-week post-transplant. (n = 9–10 per group). (D) GTT AUC data at 16-week post-transplant (n = 9–10 per group; ∗∗p < 0.01 vs. Sham 16-week post-transplant; two-tailed t test). (E) ITT AUC data at 16-week post-transplant (n = 9–10 per group). (F) Plasma insulin levels at 20 weeks post-transplantation after 12-h fast (ng/mL) (n = 6–9 per group; ∗p < 0.05 vs. Sham, two-tailed t test). All data are represented as mean ± SEM.