Elevated levels of adenosine in the DIO mice

Adenosine is a natural ligand for adenosine receptors. To examine whether there is any change of adenosine in the DIO animals, we fed male C57 BL/6 mice with a regular chow or high-fat diet (HFD). After 24 weeks of dietary treatment, we analysed the plasma adenosine level by using a sensitive fluorometric Elisa. The results showed that HFD feeding led to significantly elevated plasma adenosine level (Fig. 1a), and the plasma adenosine levels were well correlated with the body weights (Fig. 1b), suggesting adenosine metabolism was abnormal in the DIO mice. In addition, we measured the adenosine level in the cerebrospinal fluid (CSF) of 24 weeks HFD-fed mice. The CSF adenosine levels in these mice were evidently higher than the controls (Fig. 1c), and were also correlated with body weights (Fig. 1d). Moreover, when the hypothalamic adenosine was examined, we found that its contents in DIO mice were significantly elevated compared to chow-fed controls (Fig. 1e). Hypothalamic adenosine contents were also correlated to the body weights (Fig. 1f). To examine whether the change of brain adenosine occurs before the animal’s body weight is significantly increased, we measured the CSF levels in chow- or 2 weeks HFD-fed mice, at which time the animals’ body weights did not significantly differ (Chow, 21.50±0.88 g; HFD, 22.41±0.79 g. P=0.45, two-tailed Student’s t-test.). We found that the mean adenosine level was moderately, but significantly increased in HFD-fed animals (Supplementary Fig. 1a). Moreover, to exclude the effect of diet, we analysed the CSF adenosine levels in chow-fed ob/ob and wild-type mice. The result demonstrated that CSF level of adenosine was also elevated in the ob/ob mice (Supplementary Fig. 1b), indicating that alteration of brain adenosine is related to obesity, but not diet. Together, these results suggest that hypothalamic adenosine signalling might be involved in dietary obesity.

Figure 1: Aberration of the adenosine receptor signalling pathway in the hypothalamus of DIO mouse. (a) Plasma adenosine levels of chow- or 24 weeks HFD-fed mice. n=6 (Chow), 7 (HFD). (b) Correlation of plasma adenosine level with body weight, r, Pearson’s r; P, P value. (c) Adenosine levels in the CSF of chow- or 24 weeks HFD-fed mice. n=7. (d) Correlation of CSF adenosine level with body weight. (e) Hypothalamic adenosine contents of chow- or 24 weeks HFD-fed mice. n=7 (Chow), 6 (HFD). (f) Correlation of hypothalamic adenosine content with body weight. (g) Effect of i.c.v. administered adenosine on food intake. Ctrl, control. n=12 (Ctrl), 9 (0.1), 7 (0.5), 15 (1.0). (h) qRT-PCR analysis of the hypothalamic expression levels of adenosine receptors in chow- or HFD-fed mice. n=7 (Chow), 8 (HFD). (i) Western blot analysis of adenosine receptor expression in the hypothalami of chow- or HFD-fed mice. β-Actin was used as loading control. (j) Immunofluorescence staining of A 1 R in the PVN of hypothalamus of chow- or HFD-fed mouse. (k) Food intake of mice i.c.v. administered control or A 1 R agonist, CPA. n=9 (Ctrl), 7 (CPA). Data are presented as mean±s.e.m. *P<0.05, two-tailed Student’s t-test (a,c,e,h,k); one-way analysis of variance (ANOVA) with Bonferroni’s post hoc test (g). Full size image

To examine the effect of intracerebroventricularly (i.c.v.) administered adenosine on food consumption, we implanted cannula directed to the third ventricle of C57 BL/6 mice. After the animals were fully recovered, we injected control artificial cerebrospinal fluid (aCSF) or adenosine to the brain. We found that adenosine at doses of 0.5 and 1.0 μg slightly, but significantly increased animal’s appetite (Fig. 1g).

Overexpression of A 1 R in the PVN of DIO mice

Next, we examined the expression of adenosine receptors in the hypothalami of DIO mice. Quantitative reverse transcription PCR (qRT-PCR) result demonstrated that A 1 R, but not other subtypes, was significantly increased in the DIO mice (Fig. 1h). Western blot analysis further confirmed that A 1 R is overexpressed in the hypothalami of these animals (Fig. 1i; Supplementary Fig. 16). To verify these results, we did immunofluorescence staining on mouse brain sections, after confirming the specificity of A 1 R antibody (Supplementary Fig. 1c). Interestingly, we found that there was more aggregated fluorescence of A 1 R in the PVN of DIO mouse (Fig. 1j), indicating higher level of expression in this region. The immunofluorescence results did not reveal any significant change of A 1 R in other hypothalamic nuclei and extra-hypothalamic regions (Supplementary Fig. 2). We also examined the expression of A 2A R, A 2B R and A 3 R in the hypothalamus by immunofluorescence but did not notice any obvious changes (Supplementary Figs 3–5). Lastly, to test whether central A 1 R signalling is involved in the regulation of energy balance, we delivered N6-Cyclopentyladenosine (CPA), a selective agonist of A 1 R, to mouse brains by the i.c.v. route. Mice administered CPA consumed more chow foods than the controls (Fig. 1k), demonstrating that brain A 1 R regulates animal’s appetite.

Overexpression of A 1 R in PVN neurons leads to obesity

To study whether A 1 R is expressed in the neurons of PVN, we performed a double immunofluorescence staining on brain section. Indeed, we found A 1 R was expressed in most of the neurons (Fig. 2a), suggesting it may regulate energy balance via the action in these cells.

Figure 2: Effects of manipulations of A 1 R expression in PVN on systemic energy balance. (a) Double immunofluorescence staining of A 1 R (green) and neuronal marker Hu C/D (red) in mouse PVN. Cell nuclei were counterstained with DAPI (blue). 3V, third ventricle. Scale bar, 50 μm. (b) Expression of EGFP (green) after the injection of control lentivirus (Ctrl-Lenti) into PVN. Cell nuclei were counterstained with DAPI (blue). Scale bar, 50 μm. (c–f) Body weight gain (c), GTT (d), area under the curve (AUC) of GTT (e) and plasma triglycerides (TG) levels (f) were analysed. Mice were injected either Ctrl-Lenti or A 1 R-Lenti virus into the PVN. Ctrl-Lenti, n=6 (c,f), 7 (d,e). A 1 R-Lenti, n=6 (f), 7 (c–e). (g–i) Accumulative food intake (FI) (g), representative infrared images (h) and interscapular temperatures (i) of mice injected either Ctrl-Lenti or A 1 R-Lenti virus into the PVN. Ctrl-Lenti, n=6 (g), 7 (i). A 1 R-Lenti, n=7. (j) qRT-PCR analysis of the expression level of Ucp1 in brown adipose tissue of mice injected Ctrl-Lenti (n=6) or A 1 R-Lenti (n=7) virus. (k) Daily energy expenditure (EE) of mice injected Ctrl-Lenti or A 1 R-Lenti virus. lbm, lean body mass. n=6. (l) Immunofluorescence images showing that A 1 R shRNA-expressing (shA1R-Lenti) lentivirus delivered into the PVN effectively reduced the expression of A 1 R in comparison with control (shCtrl-Lenti). 3V, third ventricle. Scale bar, 20 μm. (m,n) Body weight gain (m) and daily food intake (n) of mice injected either shCtrl-Lenti or shA 1 R-Lenti virus into the PVN. n=7. Data are presented as mean±s.e.m. *P<0.05, **P<0.01, two-tailed Student’s t-test (e,f,i–k,n); two-way analysis of variance (ANOVA) with Bonferroni’s post hoc test (c,d,g,m). Full size image

Next, we asked whether overexpression of A 1 R in PVN neurons would affect systemic energy balance. Besides, given a recent report showing that A 1 R in the Arc played a role in short-term regulation of food intake mainly by using chemogenetic approach24, we also included the Arc and DMH in our observation. We generated two lentiviral plasmids in which the expression of mouse A 1 R cDNA or enhanced green fluorescent protein (EGFP, as control) was driven by the neuron-specific Synapsin promoter25. These two plasmids were designated as A 1 R-Lenti or Ctrl-Lenti, respectively. We delivered the lentiviruses to mouse PVN, Arc or DMH by utilising a stereotaxic instrument. After confirming the success of surgery (Fig. 2b; Supplementary Figs 6 and 7a,d), we monitored the animal’s body weights and food intakes. We found that mice with ectopic expression of A 1 R in the PVN, but not Arc or DMH, gained more body weights than the controls (Fig. 2c, Supplementary Fig. 7b,e). Mice with overexpression of A 1 R in the PVN were intolerant to glucose challenge (Fig. 2d,e) and hypertriglyceridemic (Fig. 2f).

In addition, mice with overexpression of A 1 R in PVN were hyperphagic (Fig. 2g), whereas overexpression of A 1 R in Arc or DMH did not alter animal’s appetite (Supplementary Fig. 7c,f). Mice injected A 1 R-Lenti in the PVN had less wheel-running activities (Ctrl-Lenti, 6.22 ±0.40 km d−1; A 1 R-Lenti, 3.70±0.75 km d−1. P<0.05, two-tailed Student’s t-test.), lower interscapular temperature and reduced expression of Ucp1 when compared to controls (Fig. 2h–j), suggesting the reduction of energy expenditure. Indeed, calorimetry analysis showed that these mice consumed significantly less O 2 and produced less CO 2 as well as heat than the controls (Fig. 2k; Supplementary Fig. 8a,b). We also extended our observation to arousal and anxiety-like behaviour. However, the data did not reveal any significant changes (Supplementary Fig. 8c–g).

We were also interested to know whether reduced expression of A 1 R in PVN would affect energy balance. To do this, we generated a lentiviral plasmid that expresses short hairpin RNA (shRNA) targeting mouse A 1 R (designated as shA 1 R-Lenti). After confirming the efficiency of knockdown (Fig. 2l), we delivered the shA 1 R-Lenti or control (shCtrl-Lenti) virus to mouse PVN. As expected, mice with reduced expression of A 1 R in PVN consumed significantly less foods and gained less body weights (Fig. 2m,n). Collectively, these data reveal an anabolism-promoting role of A 1 R expressed in the PVN neurons.

Caffeine targets PVN neuronal A 1 R to regulate energy balance

Given that hypothalamic A 1 R is involved in energy balance (Figs 1 and 2), next, we asked whether caffeine, the antagonist of adenosine receptors, would directly regulate the activities of hypothalamic neurons. Initially, we found that i.c.v. administration of caffeine at doses ≥10 μg per mouse significantly reduced animal’s appetite (Supplementary Fig. 9a). Hence, we chose the dose of 10 μg per mouse whenever caffeine is administered via the i.c.v. route, unless otherwise noted. Administration of caffeine into mouse brain significantly increased the numbers of c-Fos+ cells in the PVN, Arc and DMH nuclei (Fig. 3a,b), indicating that caffeine stimulates the activities of neurons in the hypothalamic nuclei involved in energy balance control.

Figure 3: Overexpression of A 1 R in PVN neurons significantly attenuates caffeine’s effect on energy balance. (a) Immunofluorescence staining of c-Fos (red) in the paraventricular (PVN), arcuate (Arc), ventromedial (VMH) and dorsomedial (DMH) nuclei of mice infused with either caffeine (10 μg per mouse) or control. 3V, third ventricle. Scale bar, 50 μm. (b) The number of c-Fos+ cells in the PVN, Arc and DMH nuclei of control or caffeine administered mice. n=7. (c–h) Chow-fed mice were injected Ctrl-Lenti (Ctrl-L) or A 1 R-Lenti (A1R-L) virus into the PVN (c,d), Arc (e,f) or DMH (g,h). Meanwhile, cannula directed to third ventricle were implanted. The mice were then i.c.v. injected control or caffeine (10 μg per mouse), and 24- h food intake (c,e,g) and body weight change (d,f,h) were analysed. Ctrl, control; Caf, caffeine. For PVN, n=7; Arc, n=7 (Ctrl-L, Control), 6 (Ctrl-L, Caffeine), 5 (A 1 R-L); DMH, n=6 (Ctrl-L), 7 (A 1 R-L). (i) Double immunofluorescence staining of c-Fos (red) and A 1 R (green) in the PVN of control or caffeine administered mice. 3V, third ventricle. Scale bar, 50 μm. (j,k) The number of c-Fos+ and A 1 R+ cells (j), as well as the percentage of A 1 R+ cells expressing c-Fos (k) in the PVN of mice administered control (Ctrl) or caffeine. n=3. Data are presented as mean±s.e.m. *P<0.05, two-tailed Student’s t-test (b,j,k); one-way analysis of variance (ANOVA) with Bonferroni’s (c,d,g,h) or Newman–Keuls (e,f) post hoc test. NS, not significant. Full size image

The aforementioned results led us to ask whether caffeine modulates energy balance through its action on hypothalamic A 1 R. To do this, we injected Ctrl-Lenti or A 1 R-Lenti virus into the PVN, Arc or DMH nucleus. In addition, the animals were implanted cannula directed toward third ventricles, and allowed to fully recover from surgeries. We then injected aCSF or 10 μg of caffeine into the brain. Intriguingly, overexpression of A 1 R in PVN, but not Arc or DMH, significantly abolished caffeine’s effects on appetite (Fig. 3c,e,g) and body weight balance (Fig. 3d,f,h), demonstrating that PVN is a critical site for caffeine to regulate energy balance.

Since A 1 R is mainly coupled to G i/o protein, its antagonism will evoke the activity of neurons. Hence, central administration of caffeine would excite A 1 R+ neurons in the PVN. To test this prediction, we i.c.v. administered control or caffeine into mouse brains. After double immunostaining by using antibodies against A 1 R and c-Fos, we found that caffeine readily excites A 1 R+ cells in the PVN, shown by the greater number of cells expressing both c-Fos and A 1 R (Fig. 3i,j), and the increased ratio of double positive cells among A 1 R+ cells (Fig. 3k).

Brain administration of caffeine ameliorates dietary obesity

Given that A 1 R is overexpressed in the PVN of DIO mice (Fig. 1h–j), and PVN is the key hypothalamic region for caffeine to regulate energy metabolism (Fig. 3c–h), it seems intuitive that caffeine might counteract obesity through its action in the PVN. To test this, we performed third ventricle cannulation surgery on HFD-fed mice. After that, we i.c.v. injected aCSF or 10 μg of caffeine to these animals on a daily basis. Mice administered caffeine in the brain gained significantly less body weights than the controls on day 7 of the treatment and thereafter (Fig. 4a). The adipocyte sizes of epididymal white adipose tissue were much smaller than the controls (Fig. 4b–d). In addition, plasma triglycerides (TG) levels of caffeine-infused mice were lower than the controls (Fig. 4e). Glucose tolerance of these mice was also improved (Fig. 4f,g).

Figure 4: Central administration of caffeine reduces the body weights and improves obesity-related syndrome in DIO mice. (a) Daily i.c.v. administration of caffeine (10 μg per mouse) significantly reduced the body weights of DIO mice. Ctrl, aCSF injected mice. n=9 (Ctrl), 11 (Caffeine). (b–d) H&E staining (b), distribution of area (based on 100 cells per mouse) (c), mean area (d) of adipocytes of epididymal white adipose tissue (eWAT) from mice administered control or caffeine (Caf). n=3. (e–g) Post-treatment plasma triglycerides (TG) levels (e), GTT (f) and the AUC of GTT (g) of mice injected control or caffeine. n=6 (e). n=7 (Control), 9 (Caffeine) (f,g). (h) Food intake of mice i.c.v. injected control or caffeine. n=9 (Control), 11 (Caffeine). (i) Distance travelled during the first hour by mice i.c.v. infused control or caffeine. n=6 (Ctrl), 5 (Caffeine). (j) Representative infrared images acquired 4 h post i.c.v. injection. (k) Quantification of the highest 10% temperatures in the interscapular area. n=4 (Control), 5 (Caffeine). (l–n) Changes of O 2 consumption (l), CO 2 production (m) and energy expenditure (EE) (n) of the DIO mice immediately after the i.c.v. injection of control or of caffeine. lbm, lean body mass. n=8. (o,p) Twenty-four hours food intake (o) and body weight change (p) of mice administered control or caffeine (1 μg per mouse) into the PVN. n=9. Data are presented as mean±s.e.m. *P<0.05, **P<0.01, two-tailed Student’s t-test, comparison between caffeine and control groups (d,e,g–i,k,o,p); two-way analysis of variance (ANOVA) with Bonferroni’s post hoc test (a,f,l–n). Full size image

To elucidate the causes of caffeine-related reduction of dietary obesity, we measured both energy intake and expenditure. We found that mice given caffeine consumed significantly less HFD (Fig. 4h) but had more spontaneous wheel-running activities (Fig. 4i). Mice administered caffeine immediately before the dark cycle tended to have shorter duration of food intake, but did not spend more time on non-food intake-related locomotor activities (Supplementary Fig. 9b,c), indicating that the increasing of spontaneous locomotor activity (Fig. 4i) did not directly contribute to the reduction of food intake (Fig. 4h). In addition, after caffeine treatment, the insterscapular temperature and expression levels of thermogenesis-promoting genes in the brown adipose tissue were significantly elevated (Fig. 4j,k; Supplementary Fig. 9d). Indirect calorimetry analysis showed that brain administration of caffeine promoted the consumption of O 2 , and the production of CO 2 as well as heat (Fig. 4l–n). To examine the effect of 11-day caffeine treatment on the expression of adenosine receptors, we performed western blot analysis on hypothalamic samples. The result did not show any significant change between the two treatment groups (Supplementary Fig. 9e).

Since PVN is the major site for A 1 R and caffeine to regulate energy balance (Figs 2 and 3), we were interested to know whether injection of caffeine directly to this nucleus would affect appetite and body weight balance. Caffeine administered at ≤0.5 μg per mouse did not show a significant effect (food intake: control, 3.11±0.26 g d−1; caffeine (0.5 μg), 2.50±0.40 g d−1. P=0.18, two-tailed Student’s t-test). However, the food intakes and body weights of mice given caffeine at the dose of 1 μg were significantly reduced in comparison with the controls (Fig. 4o,p), further confirming PVN is the key brain region for caffeine to counteract obesity.

We also analysed the effect of i.c.v. administered caffeine on anxiety-like behaviour and arousal. In the open field test, mice acutely given caffeine tended to stay longer in the central region (P=0.07, Supplementary Fig. 9f), suggesting an anxiolytic function of caffeine. We did not notice any significant changes in the elevated plus maze and light/dark box tests (Supplementary Fig. 9g,h). It is well recognized that caffeine is a psychoactive agent that promotes wakefulness26. However, considering that the main purpose of our study is to investigate the roles of caffeine and hypothalamic adenosine receptor in energy balance, we performed most of the injections of reagents immediately before the onset of dark cycle, because for C57 BL/6 mice, 67–83% of the amount of food was consumed during this period27. In contrast, studies focusing on arousal were mostly conducted during the light cycle, mainly because mouse is a nocturnal species. Nonetheless, i.c.v. administration of caffeine immediately before the dark cycle did not alter the wakefulness time during the first 4 h of the following light cycle (Supplementary Fig. 9i,j), demonstrating that caffeine administered at this time point did not affect the sleep/wakefulness homeostasis. Taken together, these data demonstrate that central caffeine treatment reduces the body weights and improves obesity-related symptoms in the DIO mice.

Effect of peripheral caffeine treatment on energy balance

Given that caffeine is mostly consumed by the oral route in the human population, next, we asked whether peripheral administration of caffeine would also ameliorate dietary obesity. To begin with, we examined the dose-response effect of peripherally administered caffeine on food intake. Caffeine administered at doses ≥60 mg kg−1 by oral gavage significantly suppressed the appetite of DIO mice (Supplementary Fig. 10a), so we chose the lowest effective dose, that is, 60 mg kg−1 in the present study. Intragastrical infusion of caffeine evidently increased the numbers of c-Fos+ cells in the PVN (Fig. 5a,b), Arc and DMH nuclei (Supplementary Fig. 10b,c), suggesting that peripherally injected caffeine might regulate energy metabolism through the modulation of hypothalamic, in particular PVN, neuronal activities.

Figure 5: Peripheral caffeine treatment ameliorates diet-induced obesity. (a) Peripheral caffeine treatment elicits neuronal activities in the PVN. Immunofluorescence staining of c-Fos (red) in the PVN of mice administered control saline or caffeine (60 mg kg−1) by using oral gavage. Cell nuclei were counterstained with DAPI. 3V, third ventricle. Scale bar, 50 μm. (b) Numbers of c-Fos+ cells in the PVN. n=7 (Ctrl), 6 (Caffeine). (c) Body weight changes of DIO mice administered control (Ctrl) or caffeine (60 mg kg−1) by using oral gavage. n=7. (d–f) H&E staining (d), distribution of area (based on 100 cells per mouse) (e), and the mean area (f) of adipocyte of epididymal white adipose tissue (eWAT) from control or caffeine (Caf) injected mice. Scale bar, 50 μm. n=4. (g–i) Post-treatment plasma triglycerides (TG) levels (g), GTT (h) and the AUC of GTT (i) of mice injected control or caffeine. Control, n=7 (g), 10 (h,i). Caffeine, n=8 (g), 10 (h,i). (j) Daily food intake of control or caffeine administered mice. n=7. (k) Distance travelled in the first hour by mice administered control of caffeine. n=8. (l–n) Changes of O 2 consumption (l), CO 2 production (m) and energy expenditure (EE) (n) of the DIO mice immediately after the administration of control or caffeine. lbm, lean body mass. n=8. Data are presented as mean±s.e.m. *P<0.05, **P<0.01, two-tailed Student’s t-test (b,f,g,i–k); two-way analysis of variance (ANOVA) with Bonferroni’s post hoc test (c,h,l–n). Full size image

To study the effect of peripherally administered caffeine on energy balance, we injected saline or caffeine (60 mg kg−1) to DIO mice by oral gavage. Two-week caffeine treatment significantly reduced the body weights of DIO mice (Fig. 5c). Adipocytes of caffeine-treated mice were much smaller in size (Fig. 5d–f). Plasma TG level was markedly decreased (Fig. 5g), and glucose tolerance was improved (Fig. 5h,i). To interrogate the causes of peripheral caffeine treatment-induced body weight reduction, we measured the food intake and energy expenditure. We found that peripherally administered caffeine significantly reduced the food intakes (Fig. 5j), and increased the wheel-running activities of DIO mice (Fig. 5k). Mice receiving caffeine consumed more O 2 , and produced more CO 2 as well as heat (Fig. 5l–n). We also analysed the expression of adenosine receptors in the hypothalami of 2-week caffeine or saline-treated mice by using western blot, but did not find any significant change (Supplementary Fig. 10d). Together, the results demonstrate that peripheral caffeine treatment ameliorates obesity through both the reduction of food intake and the promotion of energy expenditure.

Blocking Oxt attenuates caffeine’s effect on energy balance

We have shown that caffeine regulates energy balance mainly through its action on A 1 R in the PVN (Fig. 3c–h). It is well recognized that in the PVN there are several types of peptidergic neurons, such as Oxt, AVP, TRH and corticotropin-releasing hormone (CRH)12,18,28. Next, we investigated the type(s) of neurons that express A 1 R in the PVN by double immunofluorescence staining with antibodies against A 1 R and Neurophysin I (NP-Oxt), Neurophysin II (NP-AVP), TRH or CRH. NP-Oxt and NP-AVP are carrier proteins that are specifically associated with Oxt (Supplementary Fig. 11a) or AVP, respectively29,30. The data demonstrated that A 1 R was expressed in both the Oxt (NP-Oxt+) and AVP (NP-AVP+) neurons (Fig. 6a). A 1 R was also expressed in very few TRH, but not CRH neurons (Supplementary Fig. 11b,c).

Figure 6: Oxt mediates caffeine's effect on energy balance in the DIO mice. (a) Double immunofluorescence staining of A 1 R (red) and Neurophysin I (NP-Oxt, green) or Neurophysin II (NP-AVP, green), which is co-expressed with Oxt or AVP in the PVN, respectively. Cell nuclei were counterstained with DAPI (blue). 3V, third ventricle. Scale bar, 50 μm. (b–g) HFD-fed mice were i.c.v. administered aCSF or IgG as control (Ctrl), and 2 μg of Oxt receptor (OTR) antagonist (anta), L-368,899 (b,c), or 0.5 μg of antibody against AVP (d,e) or TRH (f,g). An hour later, mice were i.c.v. injected control or 10 μg of caffeine (Caf or C). Twenty-four hours food intake (b,d,f) and body weight change (c,e,g) were then measured. ab, antibody. In OTR antagonist experiment, n=10 (Ctrl+Ctrl), 11 (Ctrl+Caf), 9 (OTR anta+Ctrl), 13 (OTR anta+Caf); AVP antibody, n=11 (IgG+Ctrl), 9 (IgG+Caf), 7 (AVP ab+Ctrl), 14 (AVP ab+Caf); TRH antibody, n=7 (IgG+Ctrl), 7 (IgG+Caf), 8 (TRH ab+Ctrl), 10 (TRH ab+Caf). (h) Single-cell RT-PCR analysis of A 1 R expression in Oxt, AVP or TRH-expressing cells isolated from the PVN of chow or HFD-fed mice. Gapdh was used as an internal control. Data are presented as mean±s.e.m. *P<0.05, one-way analysis of variance (ANOVA) with Bonferroni’s (b,d,e,f) or Newman–Keuls (c,g) post hoc test. NS, not significant. Full size image

To identify the PVN neuropeptide(s) that mediate caffeine’s effects on energy balance, we performed the following pharmacological study. We i.c.v. administered Oxt receptor (OTR) antagonist, L-368,899, or antibodies against AVP or TRH to mouse brains. An hour later, aCSF or 10 μg of caffeine was infused into mouse brains via the same route. The results demonstrated that pre-treatment with Oxt receptor antagonist L-368,899, but not AVP or TRH antibody, significantly attenuated caffeine’s effects on food intake and body weight (Fig. 6b–g), indicating Oxt is the key mediator of caffeine’s effect on energy metabolism.

We were also interested to find out the type(s) of neurons in which the expression of A 1 R was elevated in the DIO mice (Fig. 1j). To do this, we employed single-cell RT-PCR to examine the messenger RNA levels of A 1 R in specific neurons isolated from PVN. Interestingly, the messenger RNA level of A 1 R was significantly elevated in the Oxt, but not the other two types of neurons (Fig. 6h), suggesting A 1 R might play a role in DIO through its action in the Oxt neuron.

Oxt neuron-specific knockdown of A 1 R mitigates DIO

To further explore the Oxt neuron-specific role of A 1 R in DIO, we constructed a Cre-inducible, A 1 R-targeted shRNA lentiviral expression plasmid based on the pSico vector (Supplementary Fig. 12a)31. We designated this and the control plasmids as shA 1 R-pSico and shCtrl-pSico, respectively. Double immunofluorescence staining revealed that, after the transduction of shA 1 R-pSico lentivirus, expression of A 1 R in PVN Oxt neurons reduced ∼40% (Supplementary Fig. 12b,c). We then injected the shA 1 R-pSico or control lentivirus into the PVN of Oxt-Cre mice32, respectively. The results showed that Oxt neuron-specific knockdown of A 1 R significantly reduced the food intakes and body weight gains of mice under HFD treatment (Fig. 7a,b).

Figure 7: Caffeine and A 1 R regulate the PVN Oxt release. (a,b) HFD intake (a) and body weight gain (b) of Oxt-Cre mice injected either shCtrl-pSico or shA 1 R-pSico lentivirus into the PVN. n=6 (shCtrl-pSico), 7 (shA 1 R-pSico). (c) Double immunofluorescence staining of c-Fos (red) and NP-Oxt (green) in the PVN of mice i.c.v. administered control or caffeine (10 μg per mouse). Cell nuclei were counterstained with DAPI (blue). Arrows indicate c-Fos and NP-Oxt co-expressing cells. Scale bar, 50 μm. (d,e) Number of c-Fos+ and NP-Oxt+ cells (d), as well as the percentage of NP-Oxt+ cells expressing c-Fos in the PVN (e). n=3 (Ctrl), 4 (Caffeine). (f) PVN slices of 12 weeks HFD-fed mice were dissected from the brains. Basal and caffeine (2 mmol l−1) elicited Oxt release were measured. n=8. (g) Chow-fed mice were injected Ctrl-Lenti (Ctrl-L) or A 1 R-Lenti (A1R-L) virus into the PVN. The animals were allowed to recover from surgeries, and then spontaneous (Basal) and high K+ (KCl) elicited Oxt release of PVN slices were examined. n=6 (Ctrl-L), 8 (A 1 R-L). (h) Chow-fed mice were injected Ctrl-L or A 1 R-L virus into the PVN, and cannulas directed to third ventricle were implanted. The mice were then i.c.v. administered control or 1 μg of Oxt, and food intake was measured. n=7 (Control), 6 (Oxytocin). Data are presented as mean±s.e.m. *P<0.05, two-tailed Student’s t-test (a,d,e,f); two-way analysis of variance (ANOVA) with Bonferroni’s post hoc test (b); or one-way ANOVA with Newman–Keuls post hoc test (g,h). Full size image

Effect of caffeine or A 1 R on Oxt release from the PVN

Previously, we have shown that impaired Oxt release from PVN is involved in the pathogenesis of DIO18. Next, we asked whether caffeine or A 1 R would regulate the releasing of Oxt. We first examined the Oxt neuron activity after the mice had been i.c.v. administered caffeine or aCSF. We were able to find that caffeine greatly stimulated the activities of these neurons in the PVN (Fig. 7c–e), suggesting that it may evoke Oxt release. We then performed an Oxt release assay to examine this possibility. When PVN slices dissected from DIO mice were treated with caffeine (2 mmol l−1), the ex vivo Oxt releasing rate was significantly increased (Fig. 7f). Indeed, caffeine seemed to augment Oxt-induced OTR signalling in the mouse brain (Supplementary Fig. 13), although A 1 R did not interact directly with OTR (Supplementary Fig. 14). Moreover, when we induced overexpression of A 1 R in the PVN of chow-fed mice by the injection of A 1 R-Lenti virus, the rates of spontaneous and high potassium evoked Oxt release were significantly attenuated (Fig. 7g). The modulation of Oxt release by A 1 R does not seem to be a direct effect, because double immunofluorescence staining of NP-Oxt and A 1 R did not reveal any significant co-localization in the nucleus of solitary tract (Supplementary Fig. 15), a region heavily innervated by Oxt neurons.