Acute vs chronic exposure to high fat diet leads to distinct regulation of PKA

Abstract

The cAMP-dependent protein kinase (PKA) is an essential regulator of lipid and glucose metabolism that plays a critical role in energy homeostasis. The impact of diet on PKA signaling has not been defined, although perturbations in individual PKA subunits are associated with changes in adiposity, physical activity and energy intake in mice and humans. We hypothesized that a high fat diet (HFD) would elicit peripheral and central alterations in the PKA system that would differ depending on length of exposure to HFD; these differences could protect against or promote diet-induced obesity (DIO). 12-week-old C57Bl/6J mice were randomly assigned to a regular diet or HFD and weighed weekly throughout the feeding studies (4 days, 14 weeks; respectively), and during killing. PKA activity and subunit expression were measured in liver, gonadal adipose tissue (AT) and brain. Acute HFD-feeding suppressed basal hepatic PKA activity. In contrast, hepatic and hypothalamic PKA activities were significantly increased after chronic HFD-feeding. Changes in AT were more subtle, and overall, altered PKA regulation in response to chronic HFD exposure was more profound in female mice. The suppression of hepatic PKA activity after 4 day HFD-feeding was indicative of a protective peripheral effect against obesity in the context of overnutrition. In response to chronic HFD-feeding, and with the development of DIO, dysregulated hepatic and hypothalamic PKA signaling was a signature of obesity that is likely to promote further metabolic dysfunction in mice.

Keywords: PKA; adipose tissue; diet-induced obesity; hypothalamus; liver.

Figure 1

Hepatic PKA enzymatic activity is differentially regulated by acute and chronic HFD intake and overall dysregulation of PKA system is observed after chronic HFD intake in mice. (a) Basal and cAMP-stimulated (5 μM cAMP) PKA activities after 4d CD- or HFD-feeding, n=6–8/group. (b) Representative western blot and quantification of pCREB in liver nuclear fraction after 4d CD- or HFD-feeding; ratio of pCREB to CREB was first normalized to the loading control, H3; membrane image was cut and rejoined to remove the central lane with molecular weight ladder. (c) Hepatic mRNA expression of PKA subunits after 4d CD or HFD exposure, n=6–8/group. (d) Representative western blot and quantification of PKA catalytic subunits α and β in liver after 4d CD- or HFD-feeding, n=6–8/group. (e) Basal and cAMP-stimulated (5 μM cAMP) PKA activities after 14 wk CD- HFD-feeding (n=6–8/group). (f) Representative western blot and quantification of pCREB in liver nuclear fraction after 14 week HFD-feeding; membrane image was cut and rejoined to remove a single poorly resolved lane. (g) mRNA expression of PKA subunits in liver from mice after 14 week CD- or HFD-feeding, n=6–8/group. (h) Representative western blot and quantification of PKA catalytic subunits α and β in liver after 14 wk CD- or HFD-feeding, n=6/group. All values are mean ±SEM; *, p < 0.05. Western blot and PKA activity experiments were repeated two to three times each. Abbreviations: pCREB, phospho-cAMP response element binding protein (S133); CREB, cAMP response element binding protein; H3, histone 3 protein; Cα, PKA catalytic subunit α protein; Cβ, PKA catalytic subunit β protein.

Figure 2

Phosphorylation and expression of downstream PKA targets in liver are impacted by altered PKA activity resulting from acute and chronic HFD feeding in mice. (a) Representative western blots and quantification of PEPCK in liver of mice after 4d or 14 wk access to CD or HFD. (b) Representative western blots and quantification of pACC and ACC in liver of mice after 4d or 14 wk access to CD or HFD; membrane image was cut and rejoined to remove a single poorly resolved lane. (c) Representative western blots and quantification of pAMPK (T-172) in liver of mice after 4d CD or HFD-feeding. All values are mean ±SEM;*, p < 0.05. Western blot experiments were each repeated three or more times from a pool of 6–8 samples/group.

Figure 3

Hypothalamic PKA activity is increased in response to chronic HFD and unchanged after acute HFD access in mice. (a) Basal and cAMP-stimulated (5 μM cAMP) PKA activities after acute 4d CD- or HFD-feeding, n=5–7/group. (b) Representative western blots and quantification of PKA Cα and Cβ in hypothalamus of mice after 4d CD- or HFD-feeding. (c) Basal and cAMP-stimulated (5 μM cAMP) PKA activities after acute 4d CD- or HFD-feeding, n=5–7/group. (d) Representative western blots and quantification of PKA Cα and Cβ in hypothalamus of mice after 14 wk CD- or HFD-feeding. Experiments repeated in two cohorts of mice. All values are mean ±SEM; *, p < 0.05.

Figure 4

Phosphorylation of CREB (S133) varies in Arc, VMH and DMH in fasted vs. fed states and is dysregulated after chronic HFD-feeding in mice. (a) Representative immunofluorescent staining for pCREB (S133) (Alexa fluor 594), counterstained with DAPI in hypothalamus of 16h-fasted and non-fasted mice that were chronically exposed to either HFD or CD; scale bars = 200 μm. (b) Representative immunofluorescent staining for pCREB (S133) (Alexa fluor 594) in DMH; scale bars = 100 μm. (c) Representative immunofluorescent staining for pCREB in Arc (S133) (Alexa fluor 594); scale bars = 100 μm. Immunofluorescence experiments were repeated in 3 cohorts of mice totaling 4–6 mice per treatment group. Abbreviations: Arc: arcuate nucleus, VMH: ventromedial hypothalamus, DMH: dorsomedial hypothalamus.

Figure 5

Chronic HFD-feeding impacts PKA expression in gonadal AT while PKA signaling is only modestly altered. (a) Basal and cAMP-stimulated (5 μM cAMP) PKA activities after acute 4d CD- or HFD-feeding, n=6–8/group. (b) Representative western blots and quantification of PKA Cα and RIIβ in gonadal AT of mice after 4d CD- or HFD-feeding, n=6–8/group. (c) Basal and cAMP-stimulated (5 μM cAMP) PKA activities after chronic 14wk CD- or HFD-feeding and ratio of basal to cAMP-stimulated PKA activities, n=6–8/group. (d) Representative western blots and quantification of PKA Cα and RIIβ in AT of mice after 14wk CD- or HFD-feeding, n=6–8/group. (e) mRNA expression of PKA subunits in AT after 14 week HFD-feeding (n=8/group). All values are mean ±SEM; *, p < 0.05. PKA enzymatic activity assays were repeated twice and western blots repeated three or more times.