Carotid baroreceptor stimulation in obese rats affects white and brown adipose tissues differently in metabolic protection

The sympathetic nervous system (SNS) regulates the functions of white adipose tissue (WAT) and brown adipose tissue (BAT) tightly. Carotid baroreceptor stimulation (CBS) efficiently inhibits SNS activation. We hypothesized that CBS would protect against obesity. We administered CBS to obese rats and measured sympathetic and AMP-activated protein kinase (AMPK)/ PPAR pathway responses as well as changes in perirenal WAT (PWAT), epididymal WAT (EWAT), and interscapular BAT (IBAT). CBS alleviated obesity-related metabolic changes, improving insulin resistance; reducing adipocyte hypertrophy, body weight, and adipose tissue weights; and decreasing norepinephrine but increasing acetylcholine in plasma, PWAT, EWAT, and IBAT. CBS also downregulated fatty acid translocase (CD36), fatty acid transport protein (FATP), phosphorylated and total hormone sensitive lipase, phosphorylated and total protein kinase A, and PPARγ in obese rats. Simultaneously, CBS upregulated phosphorylated adipose triglyceride lipase, phosphorylated and total AMPK, and PPARα in PWAT, EWAT, and IBAT. However, BAT and WAT responses differed; although many responses were more sensitive in IBAT, responses of CD36, FATP, and PPARγ were more sensitive in PWAT and EWAT. Overall, CBS decreased chronically activated SNS and ameliorated obesity-related metabolic disorders by regulating the AMPK/PPARα/γ pathway.

Therapies targeted at reducing sympathetic activity improve insulin resistance and dyslipidemia in obese subjects (18). The ganglionic blocker, trimethaphan, improves insulin sensitivity and glucolipid metabolism by reducing the SNS efferent to peripheral organs in obese individuals (17). Several recent studies suggested that device-based renal denervation and transcutaneous auricular vagus nerve stimulation exert therapeutic effects on metabolic disorders (19,20).
Carotid baroreceptor stimulation (CBS) has proved efficient in inhibiting SNS activation and has been clinically applied for the treatment of heart failure and resistant hypertension (21,22). Our previous studies showed that CBS exerts anti-arrhythmia effects by decreasing the discharging of the left stellate ganglion (sympathetic ganglion of the heart) in a canine model (23,24). In the current study, we hypothesized that CBS would exert protective effects against obesity, and we aimed to reveal the underlying mechanisms using a high-fat diet (HFD)-induced rat model of obesity.

Animal treatments and study design
Thirty Sprague Dawley rats (male, body weight 210-230 g, 5 weeks old) were purchased from Hunan SJA Laboratory Animal Co., Ltd. (Changsha, Hunan, China). The rats were randomized into three groups: control rats receiving sham operation (CBS device implantation without delivery of stimulation; C-sham, N = 10), obese rats receiving sham operation (O-sham, N = 10), and obese rats receiving CBS device implantation and stimulation (O-CBS, N = 10). All rats were housed in a breeding facility under a 12 h light/dark cycle and had unrestricted access to food and water.
All rats were adaptively fed a control normal diet (CND) for 1 week prior to surgery. The CND contained 10% fat, 20% protein, and 70% carbohydrate (D12450B; Beijing HFK Bioscience, China). One week after the implantation surgery, the C-sham group continued to receive the CND, whereas the O-sham and O-CBS groups were changed to a HFD that consisted of 60% fat, 20% protein, and 20% carbohydrate (D12492; Beijing HFK Bioscience). Food intake, body weight, and core body temperatures (by a rectal digital thermometer, MC-347; OMRON, Japan) were measured weekly between 19:00 and 19:30. All the rats were housed at constant temperature (22 ± 2°C) and the body temperature was maintained between 36°C and 37°C by a heating pad during tissue harvest. All procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (publication no. 85- 23, revised 1996). The study was approved by the Institutional Animal Care and Use Committee at Renmin Hospital, Wuhan University, China (IACUC issue no. WDRM20151210).

Implantation of CBS device
The CBS device (custom-designed and custom-made by Ensense Biomedical Technologies Co., Ltd., Shanghai, China) consists of a battery-powered impulse generator and a bipolar platinum electrode with conductive leads. All the surfaces are coated by biocompatible materials. The device is programmable by a telemeter system and allows the operator to noninvasively modulate the stimulation parameters delivered to the carotid sinus wall. The rats were anesthetized by a gaseous anesthetic system. The pulse generator was implanted on the back subcutaneously, and the conductive leads went across the back and neck subcutaneously. The bipolar platinum electrode was planted around the exterior surface of the right carotid sinus wall adjacent to the right common carotid artery, and the location of the electrode was slightly adjusted to achieve a clear blood pressure response. The stimulation protocol was previously described (20,21). The stimulating frequency was 10 Hz, the pulse duration was 1 ms, and the stimulating cycle was set with an interval of 1 min in each cycle of 5 min. The output of the stimulator was set at a working voltage that was 80% of the threshold voltage. The threshold voltage was defined as the voltage that induced a 10% decrease in blood pressure. Sham surgery was the same as that of CBS device implantation, without delivery of stimulation.

Glucose tolerance test and insulin tolerance test
Four days before euthanization, a glucose tolerance test (GTT) was conducted after an overnight fast by intraperitoneally injecting 50% D-glucose (2.0 g/kg). Two days before euthanization, an insulin tolerance test (ITT) was performed by intraperitoneally injecting insulin (0.5 units/kg) after a 4 h fast. For the GTT and ITT, small tail cuts were made and blood glucose was measured at 0, 15, 30, 60, and 120 min after the injection with a hand-held glucometer (HEA-230; OMRON Corp., Kyoto, Japan). Areas under the curve (AUCs) of glucose and insulin were calculated by the trapezoidal rule based on the data obtained during the 2 h measurements.

Blood pressure measurement and tissue sample analysis
At the end of the study, body length (from the nose tip to the end of the scrotum) and body weight were measured under isoflurane anesthesia. Thereafter, a catheter was punctured into the left carotid artery and was directly connected to a pressure transducer to obtain carotid artery blood waveforms. LabChart (ADInstruments, Colorado Springs, CO) was used to analyze the waveform data of systolic and diastolic blood pressure. Then, blood samples were taken from the inferior caval vein following an overnight fast. Plasma biochemical parameters were analyzed at the clinical laboratory of Renmin Hospital of Wuhan University using an Advia 2400 automatic biochemical analyzer (Siemens, Germany) and kits for FFAs (A042-2; Nanjing Jiancheng Bioengineering Institute, Nanjing, China), triglyceride (TG) (74023; Siemens), total cholesterol (TCH) (74018; Siemens), LDL (74028; Siemens), and HDL (74027; Siemens). Finally, following decapitation, perirenal WAT (PWAT), epididymal WAT (EWAT), and interscapular BAT (IBAT) were resected and weighed separately.

Immunohistochemistry analysis
PWAT, EWAT, and IBAT were fixed and embedded in paraffin blocks. Tissue sections were stained with anti-CD68 antibody (GB11067; Servicebio, Wuhan, China) following standard procedures. Both macrophages and crown-like structures (CLSs) were counted using a light microscope. CLSs were identified as single adipocytes surrounded by three or more macrophages.

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Oil Red O staining
Liver sections were stained using the Oil Red O work solutions containing 6 ml of Oil Red O stock solution (G1015; Servicebio) and 4 ml of ddH 2 O for 15 min as previously described (25). Integrated optical density (IOD) (IOD = area × average intensity) of Oil Red O staining was quantified by using Image-Pro Plus 6.0 software (Media Cybernetics, Inc., Rockville, MD).

Statistical analysis
Quantitative data were expressed as the mean ± SEM. Homogeneity of variance was analyzed by Levene's test. Means were compared by ANOVA, followed by Tukey's post hoc test. Statistical significance was considered at P < 0.05. SPSS 22.0 was used for analysis.

Obesity induced by 8 weeks of HFD feeding is characterized by glucose intolerance, insulin resistance, and hyperlipidemia
Food intake of the O-sham and O-CBS groups began to be significantly higher than the C-sham group during the fifth week. CBS made no difference in the food intake between the O-CBS and O-sham groups (Fig. 1A). The O-sham and O-CBS groups had significantly elevated body weights compared to the C-sham group in the third week. Body weights of O-CBS group began to be apparently lower than the O-sham group in the sixth week (Fig. 1B). After 8 weeks of HFD feeding, the O-sham group showed higher body weight gain and BMI than the C-sham group and were significantly suppressed in the O-CBS group (Fig.  1C). Core body temperature of the O-sham and O-CBS groups began to be obviously lower than the C-sham group in the seventh week. Core body temperature of the O-CBS group began to be apparently higher than the O-sham group in the ninth week (Fig. 1D). GTT, ITT, and plasma insulin indicated distinct glucose and insulin intolerance in the O-sham group compared with the C-sham group. These abnormities were all partially alleviated in O-CBS group ( Fig. 1E-I).
Plasma FFAs, TG, TCH, and LDL were all evidently higher, whereas HDL was obviously lower in the O-sham group compared with the C-sham group (Fig. 1J). Plasma FFAs were significantly higher and TG was lower in the O-CBS group than in the O-sham group (Fig. 1J). However, CBS had no significant effect on plasma TCH, LDL, and HDL (Fig. 1J).

CBS suppresses the activation of the SNS and the reninangiotensin-aldosterone system in obese rats
Levels of NE in plasma, PWAT, EWAT, and IBAT were distinctly higher in the O-sham group than in the C-sham group ( Fig. 2A, B). Inversely, levels of ACH in plasma, PWAT, EWAT, and IBAT were lower in the O-sham group than in the C-sham group (Fig. 2C, D). Compared with the C-sham group, the O-sham group had higher plasma levels of renin and Ang-II (Fig. 2E, F). All of the above abnormalities were partially ameliorated by CBS ( Fig. 2A-F). Systolic blood pressure and diastolic blood pressure were significantly elevated in the O-sham group compared with the C-sham group. CBS obviously alleviated the increase in systolic blood pressure, but did not affect the diastolic blood pressure (Fig. 2G, H).

CBS inhibits adipocyte hypertrophy, fat deposition, and hepatic steatosis in obese rats
As shown in Fig. 3A, lipids were deposited in a single unilocular droplet in adipocytes of PWAT and EWAT, and in multiple droplets in adipocytes of IBAT. Compared with the C-sham group, the mean areas of adipocytes in PWAT and EWAT, fat accumulation in adipocytes of IBAT, weights and fat mass ratios (vs. body weight) of three adipose tissues, and the amounts of neutral lipids in liver were all significantly higher in the O-sham group. CBS partly ameliorated all the above abnormalities ( Fig. 3A-F).

CBS differentially affects lipid metabolism-related enzymes in adipose tissues
Compared with the C-sham group, protein levels of CD36 and FATP were significantly upregulated in the O-sham group, but were partially suppressed in the O-CBS group compared with the O-sham group (Fig. 4A-C).

Effects of CBS on adipocytokines and inflammation in adipose tissues of obese rats
Leptin levels in plasma, PWAT, EWAT, and IBAT were evidently higher in the O-sham group than in the C-sham group (Fig. 5A, B). In contrast, adiponectin levels in plasma, PWAT, EWAT, and IBAT were obviously lower in the O-sham group than in the C-sham group (Fig. 5C, D). Compared with the O-sham group, all the above abnormalities were partially alleviated in the O-CBS group (Fig. 5A-D).

Effects of CBS on AMPK/PPAR/ signaling in PWAT, EWAT, and IBAT
Compared with the C-sham group, levels of pThr172-AMPK, total AMPK, pThr172-AMPK/total AMPK, and PPAR were significantly downregulated in PWAT, EWAT, and IBAT of the O-sham group and were partly rectified by CBS in the O-CBS group (Fig. 6A, E-I). Levels of pThr198-PKA, total PKA, pThr198-PKA/PKA, and PPAR in PWAT, EWAT, and IBAT of the O-sham group were distinctly higher than in the C-sham group (Fig. 6A-D, H, J). In PWAT and EWAT, the O-CBS group had prominently lower levels of pThr198-PKA, total PKA, pThr198-PKA/total PKA, and PPAR than those of the O-sham group (Fig.  6A-D, H, J). In IBAT, CBS significantly decreased the levels of pThr198-PKA, total PKA, and pThr198-PKA/total PKA, but did not affect the expression of PPAR (Fig. 6A-D, H,  J). Relative mRNA levels of the PPAR target genes, CPT1a and ACOX, were apparently lower, while relative mRNA levels of the PPAR target genes, LPL and aP2, were significantly higher in PWAT, EWAT, and IBAT of the O-sham group than in the C-sham group. CBS partly upregulated CPT1a and ACOX in PWAT, EWAT, and IBAT of the O-CBS group. In the O-CBS group, LPL and aP2 were partially downregulated in PWAT and EWAT, but not in IBAT.

Chronic SNS activation is common in obesity and plays a key role in disease progression
Obesity, especially when accompanied by excessive accumulated visceral fat and high adipokine secretion, critically contributes to sympathetic activation and metabolic disorders (16,26,27). WAT and BAT are both innervated by sympathetic nerves, and the functions of WAT and BAT are tightly regulated by the SNS (8)(9)(10)(11). Physiologically, acute SNS activation is intended to increase lipolysis in WAT and promote thermogenesis in BAT in response to cold (11). However, the responses of the chronically activated SNS in obesity to a variety of metabolism-relevant stimuli are blunted, which contributes to a state of hyperinsulinemia and hyperleptinemia. Hyperinsulinemia and hyperleptinemia promote fat synthesis, thus establishing a vicious circle between sympathetic activation and obesity (11). In short, chronic SNS activation promotes obesity. In this study, obesity was successfully induced by HFD in rats, as indicated by high BMI, dyslipidemia, glucose intolerance, insulin resistance, and adipocyte hypertrophy. CBS exerted significant alleviating effects on these abnormalities. While CBS improved main metabolic disorders, it did not affect plasma TCH, LDL, and HDL in the current study. It is worth further clarification.

CBS inhibits sympathetic tone and improves main metabolic disorders in obese rats
In our previous study, CBS was demonstrated to significantly lower the discharging of the left stellate ganglion (sympathetic ganglion of heart) in a canine model (23). As the primary transmitter released by sympathetic nerves, NE is commonly used as the index for sympathetic outflow (28). In this study, increased NE levels in PWAT, EWAT, IBAT, and plasma, as well as increased plasma renin, Ang-II, and systolic blood pressure, were partly reversed by CBS. These findings, along with results in the previous studies above, provide evidence that CBS is able to inhibit SNS activation acutely and chronically. It is widely accepted that adipose tissues are innervated by sympathetic endings, and parasympathetic innervation of adipose pads is also supported by recent findings (29). In our study, decreased ACH levels in the plasma and adipose tissues of obese rats were partly rectified by CBS, which indicates that CBS may also play an active role in the parasympathetic nervous system.
Interestingly, -blockers reportedly increase the propensity for obesity by decreasing insulin sensitivity and energy expenditure (30). Our data suggest that CBS exerts antiobesity effects through an unclear mechanism that is quite different from that of -blockers. An acute increase in SNS outflow is intended to prevent further storage of excess energy as fat via increasing -adrenergic stimulation of thermogenesis. However, chronic SNS activation is demonstrated to actually impair -adrenergic signaling, further reducing -adrenergic responsiveness in peripheral tissue and decreasing energy expenditure (31). Thus, elevated SNS tone initiated to prevent additional storage of body fat ultimately evolves into a mechanism contributing to the development of HFD-induced obesity. Owing to the inhibitory effects on the SNS, CBS may increase -adrenergic stimulation of thermogenesis by ameliorating the desensitization of the -adrenergic signaling pathway in obesity. A recent study demonstrated that a 1°C increase in body temperature is associated with a 10-13% increase in metabolic rate (32). In our study, the O-CBS group showed no significant difference in food intake, but showed a significant increase in core body temperature compared with the O-sham group. This indicates that CBS may increase the energy expenditure of the O-CBS group by sensitizing the -adrenergic signaling pathway. This complex process remains to be elucidated in future studies.

FFA uptake and lipolysis might be the key targets of CBS
Anatomically, WAT comprises two major depots, subcutaneous WAT and visceral WAT. Visceral WAT is closely related to metabolic disorders and various chronic diseases

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Journal of Lipid Research Volume 60, 2019 (33,34). After food intake, FFAs derived from dietary lipids are taken up by white adipocytes and are esterified into TGs in lipid droplets. CD36 is a high-affinity receptor that has been shown to facilitate FFA uptake into adipose tissues and muscle in rodents and humans (35). FATP is an integral membrane protein implicated in the trans-membrane transport of FFAs (36). In obese individuals, insulin resistance induces upregulation of CD36 and FATP, which results in excessive fat synthesis in white adipocytes by promoting FFA uptake (37). In our study, the upregulation of CD36 and FATP in obese rats was partly inhibited by CBS.
When required, TGs are hydrolyzed by lipolysis and released into the circulation as FFAs. ATGL and HSL are responsible for most of the TG hydrolysis in adipose tissues (38). In our study, increased levels of phosphorylated HSL, total HSL, and phosphorylated HSL/total HSL in PWAT, EWAT, and IBAT of obese rats were completely reversed by CBS in the O-CBS group. CBS did not restore total ATGL downregulation, but levels of phosphorylated ATGL and phosphorylated ATGL/ATGL were partly restored in PWAT, EWAT, and IBAT of obese rats. The mechanism involved in the regulation of total ATGL may be different from that of phosphorylated ATGL and HSL in adipocytes (39,40). Total ATGL is mainly upregulated by glucocorticoid (PPAR) agonists (41). During fasting, phosphorylated ATGL and HSL mainly respond to adrenocorticotropic hormone, adrenaline, or some kinases (such as AMPK), and they are all in high relation to the SNS activity (40,42,43). This explains why CBS regulates ATGL and HSL differently in adipose tissues.

Different effects of CBS on adipocytokines and inflammation in adipose tissues of obese rats
In our study, obese rats had significant fat accumulation and high levels of TNF- in plasma and adipose tissues, as well as other increased inflammatory markers, including CRP, IL-1, IL-6, and MCP-1, in adipose tissues. In addition, obese rats showed more macrophages and CLSs in adipose tissues. These data correspond to the recent points that obesity is always accompanied by chronic low-intensity inflammation and macrophage accumulation in expanding adipose tissues (51,52). CBS partially alleviates the abnormal secretion of leptin and adiponectin. It is interesting that CBS significantly decreased the content of IL-6 and MCP-1 in PWAT, EWAT, and IBAT, but not the levels of TNF-, CRP, and IL-1. Meanwhile, CBS obviously decreased macrophage infiltration in PWAT and EWAT, but not in IBAT. CBS also made no difference on the number of CLSs in PWAT, EWAT, and IBAT. These results indicate that the effects of CBS on inflammatory reaction and macrophage accumulation in adipose tissues vary with the type of inflammatory markers and adipose tissue.

CBS attenuates metabolic disorders of adipose tissue by regulating the AMPK-PPAR/ pathway
PPAR and PPAR are both ligand-activated transcription factors that are involved in the differentiation of adipocytes and lipid metabolism. In adipose tissues, PPAR increases FFA -oxidation and insulin sensitivity, whereas PPAR promotes lipid synthesis and endocrine function (53,54). AMPK, a serine/threonine protein kinase, acts as an efficient sensor of the cellular energy state and is a critical modulator for PPAR and PPAR (55)(56)(57). AMPK is regarded as a master switch in the regulation of lipid metabolism and is involved in mediating the beneficial effects of the autonomic nervous system (57)(58)(59). In addition, circulating adipokines, such as adiponectin, increase insulin sensitivity and enhance fatty acid oxidation by upregulating AMPK in the peripheral tissues (57,58) In our study, CBS upregulated phosphorylated AMPK, total AMPK, and phosphorylated AMPK/total AMPK and may consequently have upregulated PPAR and downregulated PPAR in adipose tissues, which in turn increased FFA -oxidation and decreased FFA uptake. This is supported by the alterations in the enzymes mediating FFA oxidation (ATGL, HSL, and UCP-1) and uptake (CD36 and FATP) in adipocytes. Herein, we furthermore explored the effects of CBS on the upstream and downstream of the AMPK/PPAR/ pathway. Our data correspond to the recent study in which PKA negatively modulated AMPK (60). The modulations of CBS on PPAR target genes (CPT1a and ACOX) and PPAR target genes (LPL and aP2) are accorded with the effects of CBS on PPAR and PPAR, respectively. Taken together, these novel findings demonstrate that CBS exerts beneficial effects against obesity via the AMPK/PPAR/ pathway in adipose tissues.

BAT and WAT differentially respond to HFD and CBS
In our study, responses of NE, ACH UCP-1, phosphorylated ATGL, phosphorylated HSL, total HSL, phosphorylated PKA, total PKA, phosphorylated AMPK, and PPAR to both HFD and CBS were less sensitive in PWAT and EWAT  than those in IBAT. This may be explained by the fact that IBAT is innervated by more nerve endings than WAT, and the regulating proteins above are highly regulated by the SNS (40,(61)(62)(63)(64)(65)(66). The expression of CD36, FATP, and PPAR responded more sensitively to both HFD and CBS in PWAT and EWAT than in IBAT. This may be because CD36, FATP, and PPAR are largely involved in FFA uptake in PWAT and EWAT, whereas HSL, UCP-1, and PPAR are mainly involved in FFA -oxidation in IBAT (53,54). The differences of leptin and UCP-1 expression among different adipose tissues and the contribution of HFD to their expression have been reported. Van Harmelen et al. (67) showed that leptin secretion is positively correlated with adipocyte size, and the subcutaneous adipose tissue has a higher leptin secretion than that of visceral adipose. This point was further confirmed by our study in which PWAT and EWAT are similar in cell size, and leptin levels in plasma and adipose tissues are respectively approximate. In a recent study, HFD was indicated to increase UCP-1 expression in BAT generally, but exerted inconsistent effects on UCP-1 expression in WAT (68). Our data agree with the points that PWAT and EWAT may play more critical roles in secreting several adipokines, whereas IBAT plays more important roles in UCP-1-mediated thermogenesis (44,69).

CONCLUSIONS
Based on the results of this study, we draw three conclusions: 1) CBS exerts protective effects against obesity by suppressing the chronic activation of the SNS in obese rats; 2) CBS exerts anti-obesity effects likely by regulating the AMPK/PPAR/ pathway; and 3) WAT and BAT differentially respond to HFD and CBS.