Voluntary physical activity counteracts the proliferative tumor growth microenvironment created by adipose tissue via high-fat diet feeding in female rats

Abstract

The adipokine secretion profile created from adipose tissue may represent the molecular mechanism behind the obesity-breast cancer association. Two adipokines, adiponectin (ADIPO), and leptin (LEP), are altered with obesity and exert antagonistic effects on breast cancer proliferation. We set out to determine whether the adipose-dependent tumor promoting growth environment created by a high-fat diet (HFD) in female Sprague-Dawley rats is altered compared to established responses in male rats and whether voluntary physical activity (PA) ameliorates any HFD-dependent effects. We found that conditioned media (CM) created from the adipose tissue of female HFD-fed rats increased the proliferation of MCF7 cells compared to those cells grown in CM prepared from lean adipose tissue. HFD-CM inhibited AMPK and activated Akt signaling, decreased p27 phosphorylation at T198, reduced total p27 and AdiporR1 protein levels and promoted cell-cycle entry. PA reversed the proliferative effects of HFD-CM on MCF7 cells by preventing the effects of HFD on AMPK, Akt, p27 and AdipoR1, ultimately resulting in cell-cycle withdrawal. Overexpressing AdipoR1 abolished the proliferative effects of the HFD-CM on MCF7 cells and enhanced the anti-proliferative effects PA on the HFD-CM Thus, PA represents a means to prevent deleterious obesity-related alterations in tumor growth environment which are brought about by changes in adipokine secretion profile from adipose tissue in the presence of estrogen. Furthermore, although adipose produces hundreds of adipokines, the ADIPO:LEP ratio may serve to indicate the contribution of adipose in creating a tumor growth microenvironment.

Keywords: Adipokines; breast cancer; estrogen; obesity; physical activity.

Figure 1

High‐fat diet increases total visceral fat and is ameliorated with PA. Body mass changes over the 6‐week protocol (A). Body weight adjusted total visceral fat mass in CD (open bar), HFD (black bar), HFD + LPA (light grey bar), HFD + HPA (dark gray bar) and CD + LPA (hatched bar) animals (B). Body weight adjusted muscle mass of the gastrocnemius (C), soleus (D) and tibialis anterior (E) muscles. Physical activity alters cytochrome C oxidase enzyme activity in the gastrocnemius muscles of CD, HFD, HFD + LPA, HFD + HPA and CD + LPA animals (F). *In A indicates different from HFD and HFD + LPA animals, **Indicates different from all other groups (P < 0.05). Different letters (B–F) indicate groups that are significantly different from each other (P < 0.05, n = 6/group). HFD, high‐fat diet; CD, chow diet; HFD + LPA, high‐fat diet + low physical activity; HFD + HPA, high‐fat diet + high physical activity; CD + LPA, chow diet + low physical activity.

Figure 2

ADIPO is decreased, LEP and E2 increased in serum of HFD animals and reversed with HPA. Circulating serum ADIPO (open bar; μg/mL), LEP (closed bar; ng/mL) and E2 (gray bar; ng/mL) concentrations at time of killing (A). Circulating serum ADIPO:LEP ratio at time of killing in CD (open bar), HFD (black bar), HFD + LPA (light gray bar), HFD + HPA (dark gray bar) and CD + LPA (hatched bar) animals at time of killing (B). Plotting of ADIPO:LEP ratio in CM versus daily km run (C). Dotted line indicates divider between HPA and LPA groups. Western blots showing levels of Akt and β‐actin in adipose tissues from the indicated groups (D). *In A indicates different from HFD (P < 0.05). Different letters (B) indicate groups that are significantly different from each other (P < 0.05, n = 6/group). HFD, high‐fat diet; CD, chow diet; HFD + LPA, high‐fat diet + low physical activity; HFD + HPA, high‐fat diet + high physical activity; CD + LPA, chow diet + low physical activity; ADIPO, adiponectin; LEP, leptin.

Figure 3

Physical activity abolishes the effect of an HFD on adipose‐dependent tumor growth microenvironment in MockT MCF7 cells. Representative western blots for selected proteins showing the effects of treatment with CM prepared from CD (open bar), HFD (black bar), HFD + LPA (light gray bar), HFD + HPA (dark gray bar) and CD + LPA (hatched bar) animals on MockT MCF7 cells (A). Graphical representations of multiple experiments showing the effects of CM on pAMPK^{T 172} (B), pAKT^{T 308} (C), p27 (D), p27^T198 (E) and AdipoR1 (F) protein levels. β‐actin was used as a loading control. Different letters indicate groups that are significantly different from each other (P < 0.05, n = 6/group). HFD, high‐fat diet; CD, chow diet; HFD + LPA, high‐fat diet + low physical activity; HFD + HPA, high‐fat diet + high physical activity; CD + LPA, chow diet + low physical activity.

Figure 4

Overexpression of AdipoR1 can counteract the effects of HFD and accentuates the effects of PA. Representative western blots for selected proteins showing the effects of treatment with CM prepared from CD (open bar), HFD (black bar), HFD + LPA (light gray bar), HFD + HPA (dark gray bar) and CD + LPA (hatched bar) animals on AdipoR1 transfected (AdipoR1‐T) MCF7 cells (A). Graphical representations of multiple experiments showing the effects of CM on pAMPK^{T 172} (B), pAKT^{T 308} (C), p27 (D), p27^T198 (E) and AdipoR1 (F) protein levels. β‐actin was used as a loading control. Different letters indicate groups that are significantly different from each other (P < 0.05, n = 6/group). HFD, high‐fat diet; CD, chow diet; HFD + LPA, high‐fat diet + low physical activity; HFD + HPA, high‐fat diet + high physical activity; CD + LPA, chow diet + low physical activity.

Figure 5

Adipose‐dependent growth environment causes cell‐cycle changes in CM experiments. Typical cell‐cycle profiles in MockT MCF7 cells (A) and stably transfected AdipoR1 overexpressing MCF7 cells treated with HFD‐CM (B). Graphical representation of multiple cell‐cycle profile experiments observing effects of diet on CM effects in CD (open bar), HFD (black bar) animals in MockT MCF7 cells (C) and in AdipoR1‐T MCF7 cells (D). Graphical representation of multiple cell‐cycle profiles showing the effects of exercise and diet CD + LPA (hatched bar), HFD + HPA (dark gray bar) and HFD + LPA (light gray bar) on MockT MCF7 cells (E) and MCF7 cells stably overexpressing AdipoR1 (F). *Indicate groups that are significantly different from HFD‐treated cells (P < 0.05, n = 6/group). HFD, high‐fat diet; CD, chow diet; HFD + LPA, high‐fat diet + low physical activity; HFD + HPA, high‐fat diet + high physical activity; CD + LPA, chow diet + low physical activity.