Adipose PTEN acts as a downstream mediator of a brain-fat axis in environmental enrichment

Abstract

Background/objectives: Environmental enrichment (EE) is a physiological model to investigate brain-fat interactions. We previously discovered that EE activates the hypothalamic-sympathoneural adipocyte (HSA) axis via induction of brain-derived neurotrophic factor (BDNF), thus leading to sympathetic stimulation of white adipose tissue (WAT) and an anti-obesity phenotype. Here, we investigate whether PTEN acts as a downstream mediator of the HSA axis in the EE.

Methods: Mice were housed in EE for 4- and 16-week periods to determine how EE regulates adipose PTEN. Hypothalamic injections of adeno-associated viral (AAV) vectors expressing BDNF and a dominant negative form of its receptor were performed to assess the role of the HSA axis in adipose PTEN upregulation. A β-blocker, propranolol, and a denervation agent, 6-hydroydopamine, were administered to assess sympathetic signaling in the observed EE-PTEN phenotype. To determine whether inducing PTEN is sufficient to reproduce certain EE adipose remodeling, we overexpressed PTEN in WAT using an AAV vector. To determine whether adipose PTEN is necessary for the EE-mediated reduction in adipocyte size, we injected a rAAV vector expressing Cre recombinase to the WAT of adult PTEN^flox mice and placed the mice in EE.

Results: EE upregulated adipose PTEN expression, which was associated with suppression of AKT and ERK phosphorylation, increased hormone-sensitive lipase (HSL) phosphorylation, and reduced adiposity. PTEN regulation was found to be controlled by the HSA axis-with the hypothalamic BDNF acting as the upstream mediator-and dependent on sympathetic innervation. AAV-mediated adipose PTEN overexpression recapitulated EE-mediated adipose changes including suppression of AKT and ERK phosphorylation, increased HSL phosphorylation, and reduced adipose mass, whereas PTEN knockdown blocked the EE-induced reduction of adipocyte size.

Conclusions: These data suggest that adipose PTEN responds to environmental stimuli and serves as downstream mediator of WAT remodeling in the EE paradigm, resulting in decreased adipose mass and decreased adipocyte size.

Keywords: AAV; PTEN; adipose tissue; environmental enrichment; lipolysis; sympathetic nervous system.

Fig. 1

4-week EE induces PTEN in WAT. (A) Gene expression profile of the gWAT after 4 weeks in SE or EE housing (n ​= ​5 per group). (B) Relative whole-body fat mass after 4 weeks of housing (n ​= ​9 SE and n ​= ​8 ​EE). (C) Relative tissue weight at sacrifice (n ​= ​5 per group). (D) Western blots of gWAT lysates (n ​= ​5 per group). (E) Protein quantification of gWAT lysates (n ​= ​5 per group). Data are means ​± ​SEM. ​+ ​P ​< ​0.10, ∗P ​< ​0.05, ∗∗P ​< ​0.01, ∗∗∗P ​< ​0.001.

Fig. 2

16-week EE induces PTEN in WAT. (A) Glucose tolerance test at 14 weeks post housing (n ​= ​10 per group). (B) Area under the curve from (A) (n ​= ​10 per group). (C) Adiposity by echoMRI at 15 weeks post housing (n ​= ​10 per group). (D) Relative tissue weight at sacrifice (n ​= ​10 per group). (E) Western blots of gWAT lysates (n ​= ​5 per group). (F) Protein quantification of gWAT lysates (n ​= ​5 per group). Data are means ​± ​SEM. ​+ ​P ​< ​0.10, ∗P ​< ​0.05, ∗∗P ​< ​0.01, ∗∗∗P ​< ​0.001.

Fig. 3

HSA axis regulatesPtenexpression in WAT. (A) Gene expression profile of the gWAT in hypothalamic BDNF overexpression experiment (n ​= ​5 per group). (B) Gene expression profile of the gWAT in rAAV-TrkB.T1 hypothalamically-injected mice (n ​= ​5 per group). (C) Gene expression profile of the gWAT in BDNF ^± mice (n ​= ​4 per group). (D) Gene expression profile of the gWAT in adrenalectomized mice after 5 weeks in SE or EE housing (n ​= ​5 per group). (E) Gene expression profile of the gWAT in mice treated with oral propranolol 5 weeks in SE or EE housing (n ​= ​5 per group). (F) Pten expression within rWAT in SE or EE mice treated with 6OHDA denervation agent (n ​= ​5 per group). Data are means ​± ​SEM. ​+ ​P ​< ​0.10, ∗P ​< ​0.05, ∗∗P ​< ​0.01, ∗∗∗P ​< ​0.001.

Fig. 4

PTEN overexpression leads to reduced mass of targeted fat depot and adipocyte size. (A) Gene expression profile of gWAT at 4 weeks post injection of Rec2-Empty or Rec2-PTEN (n ​= ​5 per group). (B) Glucose tolerance test at 8 weeks post AAV injection (n ​= ​5 per group). (C) Relative tissue weight at sacrifice (n ​= ​10 per group). (D) Representative picture of gWAT depots. (E) Western blots of PTEN, p-AKT, and p-ERK 44/42 (T202/Y204) from gWAT lysates (n ​= ​5 per group). (F) Quantification of western blots in (E) (n ​= ​5 per group). (G) Western blot of p-HSL from gWAT lysates (n ​= ​4 Rec2-Empty and n ​= ​5 Rec-PTEN). (H) Quantification of western blots in (G). (I) Representative H&E images of gWAT. (J) Adipocyte area distribution curve with measures of interquartile range (n ​= ​4 animals per group; 6 images analyzed per animal). (K) Mean adipocyte area (mean area of all adipocytes analyzed per animal; n ​= ​4 animals per group). (L) Measure of the median adipocyte area (median area of all adipocytes analyzed per animal; n ​= ​4 animals per group). Unless otherwise noted, data are means ​± ​SEM. ​+ ​P ​< ​0.10, ∗P ​< ​0.05, ∗∗P ​< ​0.01, ∗∗∗P ​< ​0.001.

Fig. 5

EE PTEN knockdown experiment design and representative images of iWAT. (A) Schematic for the experiment design. (B) Representative H&E images of iWAT.

Fig. 6

PTEN knockdown in WAT blocks EE-induced reduction in adipocyte size. (A) Adipocyte area distribution curve (n ​= ​6 animals per group; 6 images analyzed per animal). (B) Adipocyte area distribution curve with measures of interquartile range (n ​= ​6 animals per group; 6 images analyzed per animal). (C) Mean adipocyte area (mean area of all adipocytes analyzed per animal; n ​= ​6 animals per group). (D) Measure of the median adipocyte area (median area of all adipocytes analyzed per animal; n ​= ​6 animals per group). Unless otherwise noted, data are means ​± ​SEM. ​+ ​P ​< ​0.10, ∗P ​< ​0.05, ∗∗P ​< ​0.01, ∗∗∗P ​< ​0.001.