Dietary-induced obesity (DIO) is accompanied by a depression-like phenotype in mice

To determine whether the consumption of a fat-dense diet plays a causative role in the development of depression, we first examined depression-related behaviors among mice fed a HFD for 3 or 8 weeks (Fig. 1a), where 60% of caloric intake is derived from fat. Induction of depression-like behavior, as assessed by increased immobilization time during the tail suspension and forced swim tests, was observed after just 3 weeks and persisted at 8 weeks (Fig. 1b, c). Consumption of an HFD was also accompanied by the consumption of less sucrose solution than was observed for wild-type (WT) aged-matched control mice maintained on a normal diet (ND), a test related to anhedonia (Supplementary Fig. S1A), a characteristic feeling of depressed patients that describes their inability to experience pleasure by enjoyable activities.

Fig. 1: Dietary or genetically induced obesity is accompanied by a depression-like phenotype in mice. a Schematic of the experimental plan for dietary-induced obesity (DIO) and a series of behavioral tests (EPM elevated plus maze, FST forced swim test, HFD high-fat diet, ND normal diet, OF open field, SPT sucrose preference test, TST tail suspension test). b TST and c FST for aged-matched wild-type (WT) C57BL/6J mice maintained for a period of 3 weeks or 8 weeks on either ND or HFD (n = 10 per group, experiment repeated twice; *P < 0.05, **P < 0.01 by linear mixed model fit by restricted maximum likelihood (REML). d Schematic of the experimental plan for genetically induced obesity (GIO) and a series of behavioral tests. e TST and f FST for wild-type (WT) C57BL/6J and ob/ob mice maintained on a ND for a period of 12–16 weeks (n = 8–10 per group, experiment repeated twice; **P < 0.01, ****P < 0.0005 by unpaired Student's t-test) Full size image

As expected, mice fed an HFD gained substantially more weight than the control mice fed ND, even from the first week of the intervention (Supplementary Fig. S1B). Increased body weight did not correlate with increased immobilization during the tail suspension and forced swim tests after 3 weeks (Supplementary Fig. S1C), suggesting that the performance of the mice in these tests was not affected by their increased body weight. In agreement with that, the depression-like phenotype developed on mice fed an HFD was not accompanied by less locomotor or rearing activity during the open field test compared with mice on ND (Supplementary Fig. S2A).

These results suggest that consumption of an HFD can contribute to the development of depression-like behavior.

GIO is accompanied by a depression-like phenotype in mice

To determine whether GIO also results in the depression-like phenotype, we conducted the behavioral tests with the leptin-deficient mice (ob/ob), which develop obesity from the third week of age even when maintained on an ND (Fig. 1d). During both the tail suspension and forced swim tests, the immobilization time was greater in 8-week-old ob/ob mice than in WT aged-matched mice (Fig. 1e, f). As expected, even from the third week of life, ob/ob mice on an ND gained significantly more weight than WT mice on an ND (Supplementary Fig. S2B). Even though the DIO did not affect the locomotor activity of mice measured by the open field test, the ob/ob mice had less locomotor and rearing activity compared with their WT aged-matched control mice (Supplementary Fig. S2A).

These results suggest that like DIO, GIO promotes the development of a depressive-like phenotype in mice.

DIO alters gene expression profiles in the hypothalamus

Given the early onset of the depression-like phenotype in the group of mice fed an HFD, which did not correlate with body weight, we hypothesized that consumption of an HFD alters the molecular signaling pathways in the hypothalamus, which is a brain region with major role in the control of both obesity and depression36. We used genome-wide microarray analysis to determine the hypothalamic gene expression profile of WT mice fed an ND versus WT mice fed an HFD for a period of 4 or 8 weeks.

A total of 68 genes exhibited altered expression patterns in the hypothalamus of mice fed an HFD for 8 weeks compared with mice fed an ND, with false discovery rate (FDR) < 0.05 (Fig. 2a). Moreover, the most highly significant upregulated and downregulated genes affected by the consumption of a HFD are shown (Fig. 2a). The PKA signaling was the most affected pathway upon the consumption of HFD for 8 weeks (P = 0.0000398) (Fig. 2b, c). Genes regulating the PKA signaling pathway were significantly decreased after 8 weeks on an HFD (Table 1). Other pathways were also suppressed, including the GPCR signaling nodes and GABA receptor signaling pathways, which are involved in neuronal functions (Fig. 2b, c). Gene ontology (GO) enrichment analysis revealed that the hypothalamic adenylate cyclase pathway, a major contributor in the regulation of PKA signaling, were also affected by consumption of an HFD (Fig. 2d). Analysis of hypothalamic samples from mice fed either ND or HFD uncovered a decrease in the total phosphorylation levels of PKA substrates in samples from HFD-fed animals (Fig. 2e) and decreased phosphorylation at serine 133 of CREB, a key downstream target of PKA (Fig. 2f).

Fig. 2: Dietary-induced obesity alters gene expression profiles in the hypothalamus. a (Left) Global gene expression analysis of hypothalamic samples taken from wild-type (WT) C57BL/6J mice maintained on either a normal diet (ND) or a high-fat diet (HFD) for a period of 4 or 8 weeks. Colors represent values as log 10 after normalization by z-score. Low expression level is shown in green and high expression level in red. Each column represents a different mouse and in each condition there are three different mice. (Right) The most upregulated and downregulated genes affected by HFD compared with ND. b Heat map of the key genes altered by HFD and are involved in the three main signaling pathways affected by the consumption of an HFD. Colors represent values as log 10 after normalization by z-score. c Microarray analysis by IPA Ingenuity Pathway Analysis software of the canonical pathways affected by HFD compared with ND. Grayscale represents P-values of the statistics by the IPA Ingenuity pathway analysis. d Gene ontology (GO) enrichment pathways that were affected by the consumption of an HFD compared with ND. Grayscale represents the P-values statistics of each pathway by GO. e (Left) Western blot analysis detecting the phosphorylated status of protein kinase A (PKA) substrates of whole-hypothalamic homogenates from WT C57BL/6J mice fed either ND or HFD for 3 weeks. pPKA refers to the sum of all phosphorylated substrates of PKA. GAPDH was used as a loading control. (Right) Quantification of the total phosphorylated status of PKA substrates between whole-hypothalamic homogenates from mice fed either ND or HFD. Each line represents a different mouse. (n = 4 mice per group, ****P < 0.0001 by two-tail unpaired Student's t-test). f (left) Western blot analysis of total phosphorylated cAMP response element-binding protein (p-Creb) and cAMP response element-binding protein (CREB) protein levels in whole-hypothalamic homogenates of mice fed either ND or HFD for 3 weeks. (Right) Quantification of the total phosphorylated levels of Creb between whole-hypothalamic homogenates from mice fed either ND or HFD for 3 weeks. Each line represents a different individual mouse. (n = 3 mice per group, **P < 0.01 by two-tail unpaired Student's t-test). All data in the figure are represented as mean ± SEM Full size image

Table 1 Changes in expression of genes regulating the PKA signaling pathway following HFD Full size table

These results suggest that the consumption of a HFD regulates the PKA signaling pathway in the hypothalamus and might be responsible for the development of the obesity-induced depression-like phenotype in mice.

HFD increases expression levels and activity of PDE4A5 in the hypothalamus

Next, we sought to investigate whether DIO alters the activity of PDE4 enzymes in the hypothalamus. There was a slight trend, but one that did not reach statistical significance, for increased total PDE4 activity in mice fed an HFD for 3 weeks vs. mice fed an ND (Supplementary Fig. S3A).

A variety of different PDE4 isoforms are expressed in the brain, so we decided to perform real-time PCR analysis to investigate whether DIO or GIO in mice can alter the mRNA levels of particular PDE4 isoforms in the hypothalamus. Levels of PDE4B mRNA in the hypothalamus were undetectable, whereas no statistically significant difference was found for PDE4D transcripts among mice fed ND, mice fed HFD or ob/ob mice (Supplementary Fig. S3B). In contrast to this, the total levels of the PDE4A isoforms were somewhat increased, in response to DIO and GIO, although such changes did not attain statistical significance (Supplementary Fig. S3C). However, when we analyzed transcript levels for each of the different PDE4A isoforms encoded by the PDE4A gene, then we found that transcripts for the PDE4A5 isoform were specifically upregulated in response to both DIO and GIO (Fig. 3a). Furthermore, PDE4A5 protein levels were increased in the hypothalamus after 3 weeks on the HFD (Fig. 3b), as was the level of PKA-mediated phosphorylation of the PDE4A5 population that was located within the membrane fraction (Fig. 3c). PKA phosphorylation of PDE4 long isoforms, such as PDE4A5, has been shown to elicit their activation, which serves as a critical negative feedback loop by engendering increased cAMP degradation37.

Fig. 3: PDE4A is involved in the depression-like phenotype induced by obesity and HFD increases expression levels and activity of phosphodiesterases 4A5 in the hypothalamus. a Real-time PCR analysis of PDE4A5 mRNA in the hypothalamus of (left) WT C57BL/6J mice fed either ND or HFD (1 and 3 weeks) (n = 4 per condition; *P < 0.05, by one-way ANOVA with Dunnett’s multiple comparison test) and (right) between WT C57BL/6J mice and ob/ob mice (n = 4 per condition; **P < 0.01 by two-tail unpaired Student's t-test). b (Left) Western blot analysis of Pde4a5 expression in WT C57BL/6J mice fed either ND or HFD. (n = 4 mice per condition, **P < 0.01 by two-tail unpaired Student's t-test). Representative blot (left) and (right) its quantification. c Western blot analysis of phosphorylated-Pde4a5 (p-Pde4a5) expression in WT C57BL/6J mice fed either ND or HFD. (n = 4 mice per condition, ***P < 0.001 by two-tail unpaired Student's t-test). Representative blot (left) and its quantification (right). Gapdh was used as the loading control for all western blots shown in this figure. All data in the figure are represented as mean ± SEM. d Tail suspension and e forced swim tests on PDE4A+/+ and PDE4A−/− mice fed either normal diet (ND) or high-fat diet (HFD) (n = 8–10 mice per condition; ****P < 0.0001 by two-way ANOVA with Sidak’s multiple comparison test) and ob/ob vs ob/ob::PDE4A−/− mice fed ND (n = 7–10 mice per condition; **P < 0.01, *P < 0.05 by two-tail unpaired Student's t-test) DIO: diet-induced obesity. f PDE4 activity in the membrane fraction of hypothalamus collected from PDE4A+/+ and PDE4A−/− mice fed either ND or HFD (n = 5–6 mice per group; **P < 0.01 by two-way ANOVA). All data in the figure are represented as mean ± SEM Full size image

Such results of ours revealed that both DIO and GIO lead to the specific upregulation of the PDE4A5 isoform in the hypothalamus. Furthermore, the level of protein expression and the PKA phosphorylation-mediated activation status of PDE4A5 were both increased in the hypothalamus of HFD-fed mice.

PDE4A is involved in the depression-like phenotype induced by obesity

Given the potential central role of PDE4A, we assessed whether mice lacking PDE4A (PDE4A−/−) were protected from the depression-like behavior induced by obesity. Genetic ablation of PDE4A in vivo prevented both DIO and GIO depression, as shown by the tail suspension and forced swim tests (Fig. 3d, e). PDE4A−/− and their WT litter mate controls (PDE4A+/+) showed similar increases in body weight when maintained on ND or HFD (Supplementary Fig. S3D, S3E). The ob/ob and the double knockout PDE4A−/−:ob/ob showed similar body weight gains when fed an ND (Supplementary Fig. S3E). These results suggest that loss of PDE4A protects mice from obesity-associated depression phenotype, despite similar weight gains in response to an HFD.

To elucidate further any subcellular regulation of PDE4, due to the consumption of HFD, PDE4 activity assays were performed on both cytosolic and membrane fractions from hypothalamus. PDE4 activity was greater in the membrane fraction of mice fed an HFD for 3 weeks than on an ND (Fig. 3f). This increase was abolished in the PDE4A−/− mice (Fig. 3f), suggesting that membrane-associated PDE4A, namely PDE4A5, is the functionally relevant PDE4A species whose activity is upregulated in the hypothalamus after the consumption of a HFD for 3 weeks. No difference was detected for PDE4 activity in the cytosolic fraction of either WT or PDE4A−/− mice maintained on ND or HFD (Supplementary Fig. S3F).

The amygdala is involved in depression with many neuronal circuits within the hypothalamus; however, this brain region showed no statistical difference in PDE4 activity levels between the ND and HFD in either WT or PDE4A−/− mice (Supplementary Fig. S4A, S4B). Other brain areas involved in the depression-related behaviors, such as the cortex, hippocampus, and cerebellum, also showed no differences in PDE4 activity differences among mice fed either an ND or HFD (Supplementary Fig. S4C-E), further suggesting that the hypothalamus is a key locus affected in obesity-induced depression that leads to the upregulation of PDE4 activity.

The behavior phenotype induced by the GIO and DIO was not due to the development of any motor or anxiety deficits (Supplementary Fig. S5A, S5B). Measurement of motor anxiety (open field test) revealed no major differences between WT and PDE4A−/− fed either ND or HFD or between the ob/ob and PDE4A−/−:ob/ob mice (Fig. S5A). Use of the elevated plus maze test for the anxiety phenotype showed no difference between WT and PDE4A−/− mice or between the ob/ob and PDE4A−/−:ob/ob (Supplementary Fig. S5B).

The loss of PDE4A gene products prevented both the DIO and GIO in mice. Moreover, from all the brain regions that have been shown to be involved in the neurocircuitry of depression hypothalamus is the specific brain region with increased PDE4 activity due to the development of DIO. Furthermore, we found that the membrane compartmentalization of cAMP hydrolyzing PDE4A activity is critical for the cAMP/PKA signaling pathway in the depression-like phenotype.

Saturated fats accumulate specifically in the hypothalamus of mice fed the HFD and they can regulate PKA signaling in a neuronal cell line

Next, we hypothesized that dietary fatty acids might play pivotal roles as molecular transducers of cell signaling in the hypothalamus to regulate mood disorders such as depression. Increased accumulation of FFAs in the hypothalamus was found among mice fed an HFD for either 4 or 8 weeks on HFD compared with mice fed an ND (Fig. 4a, Supplementary S6A). One of the fatty acids with the highest upregulation in the hypothalamic samples of mice fed an HFD compared with mice maintained on the ND was the palmitic acid (Fig. 4a, Supplementary S6A). By contrast, fatty acid profile analysis of the cortex revealed no differences between the two dietary groups (Fig. 4a), suggesting that the hypothalamus was a specific brain region with FFAs accumulation after HFD.

Fig. 4: Saturated fats accumulate specifically in the hypothalamus of mice fed the HFD and they can regulate PKA signaling in a neuronal cell line. a Fatty acid profile (heatmaps) for hypothalamic (left) and cortical (right) samples collected from wild-type (WT) C57BL/6J mice fed either a normal diet (ND) or a high-fat diet (HFD) for a period of 4 or 8 weeks. Heatmaps were divided in two groups of high or low representation of fatty acids in the brain. Colors represent values as log 10 after normalization by z-score. Low expression level is shown in green and high expression level in red. (n = 3–4 mice per condition analyzed by two-way ANOVA with Bonferroni post-hoc test). b Mouse neuroblastoma cell line (N2a) transfected with rat PDE4A5-wt and PKA-R1 Förster (or fluorescence) resonance energy transfer (FRET) and treated with forskolin alone, c 100 μM palmitic acid before forskolin stimulation, d 100 μM oleic acid before forskolin stimulation, or e 100 μM myristic acid before forskolin stimulation. At the end of each experiment, cells were treated with the generic PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX) to test for their responsiveness. f Quantification of the PKA activation by resonance energy transfer in N2a cells pretreated with 100 μM of palmitic, oleic, or myristic acids. (n = 6–8 individual N2a cells per condition, ***P < 0.001 by one-way ANOVA with Dunnett’s multiple comparison test). g Measurement of cAMP in N2a cells treated with forskolin alone or in combination with palmitic acid (n = 3 independent replicas per condition; *P < 0.05, by unpaired two-tail Student's t-test. Two independent experiments). All data in the figure are represented as mean ± SEM Full size image

To test whether dietary fatty acids have a direct role in regulating PKA signaling in a neuronal cell line, we used a Förster (or fluorescence) resonance energy transfer (FRET)-based biosensor, based on the structure of PKA, to gauge dynamic cAMP signaling38. This probe enables quantitative, real-time detection of rapid changes in cytosolic PKA activity after cell treatment. Treatment of a neuronal cell line, which had been co-transfected to express both PDE4A5 and the PKA-R1 FRET sensor, with forskolin, an adenylyl cyclase activator, led to a marked increase in cellular PKA activity (Fig. 4b, f). However, pretreatment of such cells with palmitic acid abolished the forskolin-induced PKA activation (Fig. 4c, f). This effect was specific to palmitic acid as neither oleic acid nor myristic acid had any effect on forskolin-induced PKA activation in such cells (Fig. 4d–f). In accordance with this, pretreatment of the cells with palmitic acid abolished the forskolin-induced increase in cAMP levels in such cells (Fig. 4g).

These data indicate a differential effect of fatty acids on intracellular PKA activity. Namely, consumption of an HFD leads to an efflux of dietary fatty acids specifically in the hypothalamus and the entrance of dietary palmitic acid in the brain suppress the PKA pathway. Moreover, different fatty acids have differential effects on the PKA signaling cascade in a neuronal cell line.

The increased accumulation of the dietary fatty acid palmitic correlates with the upregulation of the FFAR1 and modulates the association of this receptor with PDE4A5

We next sought to investigate whether the expression of the different FFA receptors is altered in the hypothalamus of mice upon DIO or GIO. Real-time PCR analysis revealed a statistically significant upregulation of FFAR1 in the hypothalamus in response to an HFD and in the ob/ob mouse (Fig. 5a). Hypothalamic gene expression of FFAR3, a receptor that belongs in the same family as FFAR1, was not affected after the consumption of the HFD (Supplementary Fig. S6B); however, the FFAR4 receptor was increased in the hypothalamus of the ob/ob mouse (Supplementary Fig. S6C).

Fig. 5: The increased accumulation of the dietary fatty acid palmitic correlates with the upregulation of the FFAR1 and modulates the interaction of this receptor with Pde4a5. a Real-time PCR analysis of FFAR1 mRNA in the hypothalamus of wild-type (WT) C57BL/6J mice fed a normal diet (ND), WT C57BL/6J mice fed a high-fat diet (HFD) for 1 week and 3 weeks, and ob/ob mice (n = 4–9 mice per condition. Experiment was repeated three times, *P < 0.05, by one-way ANOVA with Bonferroni multiple comparison test). Data are represented as mean ± SEM. b Co-immunoprecipitation of Pde4a5 with Ffar1 in a human embryonic kidney cell line (HEK293) treated with 500 μM of palmitic acid at various time points. Pde4a5 was tagged with VSV (Vesicular stomatitis virus). β-Arrestin-2 (Arb2) was tagged with Flag. VSV tag and Arb2 levels normalized to Flag tag were quantified by densitometry (Δ represents fold changes). Similar results from two independent experiments were obtained and a representative immunoblot is shown. c Membrane fractionation of N2a cells treated with 500 μM of palmitic acid at various time points. Ffar1 was used to identify the membrane fraction. Pde4a5 and Arb2 levels normalized to total levels and quantified by densitometry (Δ represents fold changes). Similar results from two independent experiments were obtained and a representative immunoblot is shown Full size image

Next, we tested whether the PDE4A5 isoform interacts with Ffar1. In vitro co-immunoprecipitation assays from lysates derived from a human embryonic kidney cell line (HEK293) exhibited a time-dependent interaction between Ffar1 and PDE4A5 after treatment with palmitic acid (Fig. 5b). Palmitic acid treatment increased the translocation of the PDE4A5 protein, as well as β-arrestin-2, to the membrane fraction of the N2a cells in a time-dependent manner (Fig. 5c). Given that PDE4A5 is known to bind to β-arrestin-239, these data are consistent with the translocation of a PDE4A5–β-arrestin-2 complex to the membrane (Fig. 5b, c). Oleic acid treatment did not induce the translocation of PDE4A5 to the membrane, highlighting the specificity of the fatty acid receptor–ligand signaling cascade (Supplementary Fig. S6D).

These data suggest that HFD specifically increases FFAR1 gene expression in the mouse hypothalamus, which in turn can lead to a potential association of Ffar1 and PDE4A5. The translocation of PDE4A5 to the membrane fraction will lead to a re-programming of the pattern of compartmentalization of the cAMP signaling pathway in these cells.