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, 9 (1), 14779

Adipocyte PU.1 Knockout Promotes Insulin Sensitivity in HFD-fed Obese Mice

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Adipocyte PU.1 Knockout Promotes Insulin Sensitivity in HFD-fed Obese Mice

Denise E Lackey et al. Sci Rep.

Abstract

Insulin resistance is a key feature of obesity and type 2 diabetes. PU.1 is a master transcription factor predominantly expressed in macrophages but after HFD feeding PU.1 expression is also significantly increased in adipocytes. We generated adipocyte specific PU.1 knockout mice using adiponectin cre to investigate the role of PU.1 in adipocyte biology, insulin and glucose homeostasis. In HFD-fed obese mice systemic glucose tolerance and insulin sensitivity were improved in PU.1 AKO mice and clamp studies indicated improvements in both adipose and liver insulin sensitivity. At the level of adipose tissue, macrophage infiltration and inflammation was decreased and glucose uptake was increased in PU.1 AKO mice compared with controls. While PU.1 deletion in adipocytes did not affect the gene expression of PPARg itself, we observed increased expression of PPARg target genes in eWAT from HFD fed PU.1 AKO mice compared with controls. Furthermore, we observed decreased phosphorylation at serine 273 in PU.1 AKO mice compared with fl/fl controls, indicating that PPARg is more active when PU.1 expression is reduced in adipocytes. Therefore, in obesity the increased expression of PU.1 in adipocytes modifies the adipocyte PPARg cistrome resulting in impaired glucose tolerance and insulin sensitivity.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Pu1 expression. (A) Relative Pu1 expression in various cell types from normal chow (NC) and high fat diet (HFD)-fed mice. (B) Relative Pu1 gene expression in 3T3-L1 adipocytes harvested 7 d post-differentiation, treated with or without TNFa for 48 h. (C) Relative Pu1 expression in various cell types/tissues in fl/fl and PU.1 AKO mice after 14 wk HFD, normalized to adipocyte fl/fl expression. (D) Quantification of western blot detection of PU.1 in eWAT from fl/fl and PU.1 AKO mice, relative to HSP90 expression (see Supplemental Fig. 1C). Values are fold induction of gene expression normalized to the housekeeping gene Rplp0 and expressed as mean ± SEM, n = 5 per group, *p < 0.05 comparing two groups using a t-test.
Figure 2
Figure 2
Improved glucose tolerance and insulin sensitivity in PU.1 AKO mice. (A) Glucose tolerance test (GTT) (B) Area under curve (AUC) of GTT. (C) Fasting insulin after 10 wks of HFD, and (D) insulin tolerance test after 11 wks of HFD in fl/fl and PU.1 AKO. (E) Body weight, (F) glucose infusion rate (GIR), (G) basal and clamp hepatic glucose production (HGP), (H) percent HGP suppression, (I) percent free fatty acid (FFA) suppression, (J) basal glucose disposal rate (GDR), and (K) insulin stimulated (IS)-GDR during hyperinsulinemic-euglycemic clamp after 12 wks HFD. Insulin-stimulated AKT phosphorylation in (L) liver, (M) eWAT, and (N) skeletal muscle 14 wk after HFD. (O) Representative H&E staining of paraffin embedded liver sections. (P) Triglyceride (TG) concentration in livers from HFD-fed fl/fl and PU.1 AKO mice. Expression of genes associated with pro-inflammation (Q) and anti-inflammation (R) in livers from HFD-fed fl/fl and PU.1 AKO mice. Values are expressed as mean ± SEM, n = 7–10 per group, *p < 0.05 by t-test.
Figure 3
Figure 3
PU.1 negatively affects adipocyte metabolism. (A) Relative mRNA expression of adipogenic-associated genes in eWAT from fl/fl and PU.1 AKO mice after 14 wk HFD feeding and (B). Adipocyte diameter was quantified and sorted by bins with Lowess curve fitting in both f/f mice (B) and PU.I AKO mice (C). (D) Epididymal adipose weight. Glucose uptake, as a percent of basal 3H-2-deoxy-D-glucose uptake, in (E). Cultured primary adipocytes from eWAT from HFD-fed fl/fl and PU.1 AKO mice. Plasma concentrations of (F). leptin, (G). Total PAI-1, (H). Adiponectin, (I). Resistin, (J). Free fatty acids (FFA), and (K). Triglycerides (TG) in fl/fl and PU.1 AKO mice after 14 wk HFD feeding. Values are expressed as mean ± SEM, *p < 0.05 by t-test. All groups are significantly different from each other for each gene analyzed in B by 2-way ANOVA followed by Tukey’s post-hoc test. N = 10–14 per group in (A) and (FK), n = 6–8 per group in (BE).
Figure 4
Figure 4
PU.1 affects eWAT inflammation in obesity. (A) Paraffin embedded eWAT sections were stained with the macrophage marker F4/80 by immunohistochemistry. (B) Flow cytometry gating strategy for analysis of F4/80+ ‘M1-like’ (F4/80+ CD11b+ CD11c+) and ‘M2-like (F4/80+ CD11b+ CD11c+) stromal vascular fraction from fl/fl and PU.1 AKO eWAT after 14 wk HFD feeding and (C). “M1-like” and “M2-like” macrophages. (D) LTB4 local concentration in eWAT in fl/fl and PU.1 AKO HFD fed mice. (E) PKH26 fluorescently-labeled total macrophage, (F). M1-like macrophage, and (G). Ratio of ‘M1-like:M2-like’ macrophages tracking in eWAT from fl/fl and PU.1 AKO recipient mice after 14 wk HFD feeding. (H) Chemotaxis of WT intraperitoneal-macrophages (IP-Mac) toward conditioned medium from fl/fl or PU.1 AKO cultured primary eWAT adipocytes from HFD mice. Analysis of Inflammation-associated gene expression from (I). Adipocytes or (J). Stromal vascular cells from eWAT from HFD fed fl/fl or PU.1 AKO mice. Values are expressed as mean ± SEM, *p < 0.05 by t-test (DG), or ANOVA followed by Tukey’s post-hoc test. N = 4–8 per group in (AC), 10–14 per group in (D,HJ) and n = 6–8 per group in (EG).
Figure 5
Figure 5
Adipocyte PU.1 expression negatively affects PPARg activity. PPARg target gene expression in (A). fl/fl or PU.1 AKO HFD eWAT. (B) Western blot of PPARg phosphorylation (Ser273),pERK and pCDK5 in eWAT from HFD-fed fl/fl and PU.1 AKO mice. (C) Quantification of western blot. Values are expressed as mean ± SEM, n = 5–10 per group, *p < 0.05 by t-test in (A,C).

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