The Dopamine D2R Antagonist Triuoperazine Suppresses High-Fat Diet-Induced Hyperglycemia and Hypothalamic Gliosis in Obese Mice

Microglia, the resident macrophages of the central nervous system (CNS), as well as astrocytes, are CNS glia cells to support neurodevelopment and neuronal function. Yet, their activation-associated with CNS inammation is involved in the initiation and progression of neurological disorders. Mild inammation in the periphery and glial activation called gliosis in the hypothalamic region, arcuate nucleus (ARC), are generally observed in the obese individuals and animal models. Thus, reduction in peripheral and central inammation is considered as a strategy to lessen the abnormality of obesity-associated metabolic indices. In this study, we reported that acute peripheral challenge by inammagen lipopolysaccharide (LPS) triggered an upregulation of hypothalamic dopamine type 2 receptor (D2R) expression, and chronic feeding by high fat diet (HFD) caused an increased levels of D2R in the ARC. The in vitro and in vivo studies indicated that a D2R antagonist named triuoperazine (TFP) was able to suppress LPS-stimulated activation of microglia and effectively inhibited LPS-induced peripheral inammation, as well as hypothalamic inammation. Further ndings showed daily peripheral administration intraperitoneally (i.p.) by TFP for 4 weeks was able to reduce the levels of plasma and hypothalamic tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in obese mice receiving HFD for 16 weeks. Moreover, plasma glucose and insulin were effectively decreased by daily treatment with TFP for 4 weeks. In parallel, microglia and astrocytes in the ARC was also inhibited by peripheral administration by TFP. According to our results, TFP has the ability to suppress HFD-induced hyperglycemia, inammation and gliosis in hypothalamus. GFAP body regions was measured. quantied. are


Introduction
Obesity caused by excessive dietary intake is not only a causal factor in the development of cardiovascular disease but also a harmful stimulus that induces neurodegeneration in the central nervous system (CNS) and cognitive dysfunction 1 . The hypothalamus is critical for regulating food intake and energy expenditure 2 . The hypothalamus consists of two centers, including a satiety center and a feeding center, which reciprocally coordinate to maintain a set point for body weight control.
Among hypothalamic nuclei, paraventricular nuclei and arcuate nuclei (ARC) are important for neuroendocrine regulation and secrete hormone-releasing hormone to act on the anterior pituitary gland for hormone release 3 .
Chronic systemic low-grade in ammation is a hallmark of obesity 4,5 . The in ammatory cytokines (i.e., TNF-α, IL-1β, IL-6, CRP, and TLRs) have been detected in the plasma of obese individuals and fat-fed animals. Notably, obesity-associated brain in ammation occurs signi cantly in the hypothalamus 1,6,7 . In a rodent study, hypothalamic in ammation occurred at the onset of high-fat diet (HFD) feeding 8,9 . Gliosis, a reactive change in astrocytes (CNS supporting cell population) and microglia (CNS resident macrophages), also occurs in the hypothalamus of obese rodents and humans 8,9 . An increase in microglia and astrocyte hypertrophy can be observed in the ARC upon exposure to HFD, suggesting that gliosis is involved in HFD-induced leptin and insulin resistance and disturbance of energy homeostasis and TNF-α) were increased in the hypothalamus within 1-3 days after HFD feeding 8, 11 . Similar observations have reported that IL-1β expression was upregulated in the hypothalamus at 3 day after initiation of HFD feeding in mice 9 . However, the expression of these proin ammatory cytokines in rats and mice declines in the weeks after HFD feeding 9 . Nevertheless, prolonged microglial activation and demyelination were observed in the ARC of mice treated with chronic HFD feeding for months 12 . Thus, the inhibition of hypothalamic in ammation is considered an effective strategy for the control of glucose homeostasis 1,13 .
Tri uoperazine (TFP), a prescribed antipsychotic drug that has been used to suppress psychotic activity by inhibiting the postsynaptic dopamine receptor 14 , not only has an immunosuppressive ability to inhibit cytokine production and release in immune cells 15,16 but also shows anticancer effects through different signaling pathways 17 . Thus, the aim of the study was to determine whether TFP has a potential effect on the attenuation of HFD-induced in ammation. Although TFP did not attenuate the body weight of obese mice induced by chronic HFD feeding, the in vivo ndings in conjunction with the in vitro observation that exposure to TFP signi cantly reduced LPS-induced proin ammatory cytokine expression in primary microglia demonstrate that treatment with TFP effectively attenuated HFD-induced elevation in blood glucose, systemic in ammation, and ARC gliosis.

Results
Tri uoperazine attenuates the production of proin ammatory mediators induced by in ammation challenge An in vitro study using primary microglia prepared from rat pups at PD 1-2 was rst conducted. As shown in Fig. 1A exposure to TFP at 1 mM for 3 h reduced iNOS immunoreactivity in IB4 + -microglia under 10 ng/ml of LPS stimulation. We found that iNOS + -microglia in the culture only treated with LPS displayed an amoeboid shape (Fig. 1A, arrowheads), whereas microglia co-treated with LPS and TFP had a strong IB4 staining exhibited extending cell processes (Fig. 1A, arrows). Moreover, 1 mM of TFP induced the downregulation of proin ammatory cytokines, TNF-a and IL-1b in LPS-stimulated microglia (Fig. 1B). The results from hypothalamic mixed glia stimulated by LPS also indicated that TFP signi cantly inhibited LPS-induced release of the proin ammatory cytokines (i.e. TNF-a and IL-1b) in the cultures (Fig. 1C).
Accordingly, the anti-in ammatory ability of TFP in microglia and mixed glia was validated.
Peripheral acute challenge with 1 mg/kg LPS is able to trigger systemic in ammation and hypothalamic microglial activation 18-20 . Accordingly, further experiments involving the co-administration of LPS with TFP (2 mg/kg) in mice via the i.p. route were designed to examine proin ammatory cytokines in the plasma and hypothalamus, as well as hypothalamic D2R mRNA expression. The plasma levels of TNF-a and IL-1b proteins were signi cantly elevated at 3 h and 6 h post LPS injection ( Fig. 2A). This upregulation of TNF-a and IL-1b at the plasma level was effectively diminished by TFP. In parallel, the results also showed that TFP downregulated LPS-stimulated expression of the two proin ammatory cytokine genes in the hypothalamus at 6 h post injection (Fig. 2B). These data reveal that the peripheral application of TFP suppressed peripheral and central in ammation. Notably, acute LPS injection peripherally increased D2R mRNA expression in the hypothalamus at 6 h ( Fig. 3A). Although the suppression of LPS-induced upregulation of D2R mRNA expression by TFP was not statistically signi cant, hypothalamic D2R mRNA expression in the LPS-TFP group tended to decrease (Fig. 3A).
Tri uoperazine decreases plasma TNF-a and restores blood glucose homeostasis in obese mice Chronic HFD feeding over 2 months acts as the causal regulator to trigger prolonged microglial activation in the hypothalamic ARC 12 . An increase in D2R immunoreactivity in the hypothalamic area, especially the ARC, was detected at 12 weeks after HFD feeding when compared to that examined in the chow group (Fig. 3B, arrows). This nding, in conjunction with the results shown in Fig. 3A, demonstrates that peripheral in ammation either induced by LPS challenge or chronic HFD feeding can trigger D2R expression in the hypothalamus. Accordingly, daily administration of TFP in mice through the i.p. route started at 12 weeks post HFD feeding and continued for 4 weeks (Fig. 4A). The plasma levels of TNF-a and IL-1b were measured at the end of the experiments. As shown in Fig. 4B, chronic HFD feeding increased plasma TNF-a and IL-1b. Plasma TNF-a levels declined e ciently after TFP treatment for 4 weeks, although TFP decreased the amount of plasma IL-1b to a lesser extent. We noticed that the body weight of obese mice was not changed by administration of TFP (Fig. 5A).
Next, we found that chronic HFD feeding for 16 weeks caused hyperglycemia, hyperinsulinemia, and high plasma levels of LDL/VLDL, TG, and HDL in obese mice ( Fig. 5B-F). Moreover, the amount of WAT and BAT was signi cantly increased in obese mice fed a HFD for 16 weeks (Fig. 5G, H). However, daily administration of TFP for 4 weeks effectively diminished the plasma levels of glucose and insulin in obese mice ( Fig. 5B and C). However, TFP application did not in uence the WAT and BAT of obese mice or the plasma levels of LDL/VLDL, triglycerides, or HDL in obese mice. The ndings point to the promising effect of TFP on the regulation of glucose homeostasis in obesity.
Tri uoperazine suppresses HFD-induced gliosis in the hypothalamus Furthermore, we examined microglial activation and astrocyte hypertrophy in the hypothalamic ARC at 16 weeks post HFD feeding and TFP treatment (Fig. 4A). Similar to the results shown in our previous study 12 , activated microglia with a larger cell size were observed in obese mice fed a HFD for 16 weeks when compared to those in the chow group (Fig. 6A, arrowheads); moreover, the activated microglia in the ARC of HFD-fed mice increased in number. However, 4 weeks of treatment with TFP signi cantly reduced the number of ARC microglia and their cell bodies (Fig. 6A, arrows). Chronic HFD feeding for 16 weeks not only caused astrocytic hypertrophy but also increased the intensity of astrocytes in the ARC compared to those observed in the chow-fed group (Fig. 6B, arrows). HFD-induced astrocytic activation was decreased after treatment with daily treatment with TFP for 4 weeks. Note that TFP application for 4 weeks caused insigni cant alterations in microglia and astrocytes in the ARC of chow-fed mice. Altogether, daily treatment with TFP for 4 weeks is effective in suppressing HFD-induced peripheral in ammation and ARC gliosis in obese mice.

Discussion
The ndings from others have indicated that the inhibition of hypothalamic in ammation improves the control of glucose homeostasis 1,13,21 , pointing to the crucial role of hypothalamic in ammation in obesity-associated pathogenesis. Here, we show the anti-in ammatory action of TFP on peripheral and hypothalamic in ammation in obese mice. Although TFP administration did not reduce the body weight of the obese mice, this drug was able to attenuate obesity-associated hyperglycemia and restore plasma insulin levels back to the basal level.
Hypothalamic in ammation induced by HFD feeding has been linked to the development of obesity 8, 9 . Moreover, the selective ablation of microglia in the ARC using the colony-stimulating factor 1 receptor (CSF1R) inhibitor PLX5622 is able to lessen HFD-triggered body weight gain and food intake 22 . However, although microgliosis in the ARC was reduced after the peripheral application of TFP (1 mg/kg) for 1 month, there was no alteration in the body weight of obese mice with or without treatment with TFP. In past studies showing decreased body weight gain by the inhibition of microglial activation 22 , either CSF1R inhibitor-induced selective ablation of microglia or genetic reduction in microglial in ammatory capacity to restrict hypothalamic in ammation was performed at the beginning of HFD feeding. In addition, the approach used to inhibit microglial proliferation for the prevention of central and peripheral in ammation using intracerebroventricular implantation of an Alzet osmotic minipump containing the antimitotic drug arabinofuranosyl cytidine was conducted before HFD feeding 23 . Thus, perhaps TFP treatment should start at an earlier time point rather than 3 months later, since the animals have been through a 3-month course of HFD feeding at the beginning of TFP application. However, diet-induced body weight gain remained when the 4-week paradigm of TFP administration was performed in the beginning at 2 months after HFD feeding (Fig. S1A). The results shed light on the function of a 4-week treatment with TFP in blood glucose homeostasis but not in lipid metabolism.
Given that the effect of chronic HFD feeding on the upregulation of body weight, WAT and BAT was not blocked by a 4-week treatment with TFP, we reason that TFP action for 4 weeks at later time intervals with the continuous stimulation of HFD feeding might not change the animal behavior under HFD consumption conditions. This is supported by the observations that daily administration of TFP in HFDfed mice did not alter their food intake in comparison with the HFD-vehicle group (Fig. S1B). Additionally, neuronal injury in the ARC has been observed in rats receiving a HFD for 1 week 8 . Moreover, decreased cell numbers of proopiomelanocortin-producing neurons, a group of ARC neurons that suppress food intake, have been shown in mice after HFD feeding for 8 months 8 . Perhaps chronic stimulation of HFD feeding in our study might perturb neural circuits that mediate energy expenditure and the food reward system. Thus, anti-peripheral in ammation or anti-gliosis by TFP in obese mice receiving long-term HFD feeding is not satisfactory to promote lipid metabolism and mitigate body weight gain. Dopamine (DA) is well known to mediate immune responses in the periphery through its receptors expressed in immune cells 24 . High levels of brain DA and altered amounts of other neurotransmitters have been detected in obese animals compared to those in the lean group along with an increase in in ammatory mediators and oxidative markers 25 . TFP has been reported to inhibit cytokine release from LPS-stimulated macrophages and dendritic cells and acts as a potential anti-sepsis drug 15 . This is consistent with our observations showing the anti-in ammatory effect of TFP on LPS-stimulated hypothalamic mixed glia and HFD-triggered peripheral in ammation. Moreover, endogenous expression of D2R was found in activated microglia in the infarct site after cerebral ischemia, in conjunction with an in vitro study, pointing to DA function in activated microglia-associated neuroin ammation 26 . Although HFD feeding for 4 weeks did not affect hypothalamic D2R gene expression 27 , our observation in this study indicated that chronic feeding of a HFD for 3 months increased D2R expression in the hypothalamic ARC. This raises the possibility that the inhibitory effect of TFP on the activation of microglia and astrocytes in the ARC of obese mice might occur through D2R action in peripheral immune cells and/or hypothalamic glial cells. Thus, based on our study shown here, D2R indeed can be a therapeutic target for obesity-associated in ammatory pathogenesis in the periphery and in the CNS.
This study shows that a 4-week program of TFP treatment effectively blocked the HFD-induced rise in blood glucose and maintained plasma insulin levels close to the control level. Thus, the results provide signi cant evidence to show the potential treatment paradigm of TFP for obesity-associated hyperglycemia through the inhibition of peripheral in ammation and ARC gliosis (Fig. 7).

Materials And Methods
Cell culture Primary microglia were collected from mixed glial cultures that were prepared via homogenization of cortical tissues dissected from rat pups at postnatal day (PD) 1-2 28 . The tissues were ltered through a 70-mm nylon lter mesh, and after centrifugation, the cell pellet was re-suspended in DMEM/F-12 medium (Thermo Fisher Scienti c) containing 10% fetal bovine serum (Gibco). The cells were seeded onto poly-D-lysine (Sigma-Aldrich)-coated T-75 culture asks and incubated in 5% CO 2 at 37°C for 7-8 days. After microglia were collected using the shake-off method, these cells were re-plated onto coverslips in 24-well plates at a density of 5 x 10 4 cells/well or in 60-mm Petri dishes at a density of 1 x 10 6 cells/dish in DMEM/F-12 medium containing 10% FBS for 3 h. The cultures were treated with 1 mM TFP (Sigma-Aldrich) in the presence of LPS (10 ng/ml; Sigma-Aldrich) in DMEM containing N1 serum supplement (Thermo Fisher Scienti c) for 3 h to examine isolectin B4 (IB4) and immuno uorescence for inducible nitric oxide synthase (iNOS) or for 6 h to investigate TNF-a and IL-1b mRNA expression. Alternatively, mouse mixed glia were prepared from the hypothalamus of pups at the age of PD 1-2 and re-plated onto PDL-coated coverslips in a 24-well plate at a density of 5 x 10 4 cells per well. After incubation in 5% CO 2 at 37°C for 5 days, the cultures were treated in DMEM/F-12 medium without or with 1 mM TFP in the presence of LPS (10 ng/ml) for 3 h. The cultured media were collected for the measurement of TNF-a and IL-1b levels. Animal use for primary microglia preparation was approved by the National Cheng Kung University Institutional Animal Care and Use Committee, Tainan, Taiwan (IACUC approval number: 106060). The methods were performed in accordance with relevant guidelines and regulations.

Animals
Eight-week-old male C57BL/6 mice were purchased from National Cheng Kung University Laboratory Animal Center (http://www.ncku.edu.tw/animal/eng/nckulac.html), and randomly paired-housed in cages with free access to food and water ad libitum under standard room conditions (room temperature: 23±2°C; humidity: 58±2%; 12-h light/dark cycle). To induce obesity in mice, the animals were fed a HFD containing 61.6% kcal from fats, 18.1% from proteins and 20.3% from carbohydrates (Rodent Puri ed Diet #58Y1; TestDiet, St. Louis, MO, USA). The animals receiving a normal diet (Laboratory Rodent Diet #5001; LabDiet, St. Louis, MO, USA) were referred to as the chow group. All the experiments was performed in compliance with ARRIVE (Animal Research: Reporting In Vivo Experiments) Guidelines (https://www.nc3rs.org.uk/arrive-guidelines) and strictly following the 3Rs (Replacement, Reduction and Re nement) to avoid unnecessary sacri ce. Animal experiments through the performance of the following methods were approved by the National Cheng Kung University Institutional Animal Care and Use Committee, Tainan, Taiwan (IACUC approval number: 106060). The animals were anesthetized and sacri ced by i.p. injection with Zoletil 50 (Virbac Taiwan Co., Ltd.; 5X dilution in saline, 0.05-0.06 ml/10 g). The choice of anesthetics was recommended by the veterinary specialist at the university animal center to effectively reduce pain in animals. The experimental protocol is outlined in Fig. S2.

Peripheral injection of LPS and TFP
Injection of LPS (1 mg/kg) by the i.p. route combined with saline or TFP (2 mg/kg) was performed on 8week-old C57BL/6 mice. Animals that received saline served as the control group (vehicle). Blood samples were collected at 3 h from the retro-orbital sinuses of anesthetized mice using a heparinized capillary tube 29 . Alternatively, under anesthesia, blood samples were collected at 6 h via cardiac puncture using a 26G needle rinsed with 10 ml of heparin (5000 IU/mL; Leo Pharmaceutical, Ltd., Denmark). Animals were then sacri ced, and hypothalamic tissues were removed for measurement of TNF-a and ILb mRNA levels.
TFP (2 mg/kg/injection) administration through the i.p. route began at 12 weeks post HFD feeding, and the daily injection continued for 4 weeks. The animal groups includes chow-vehicle, HFD-vehicle, chow-TFP, and HFD-TFP. Blood samples and brains were collected after animals were anesthetized.

Measurement of cytokines
The mixed glial culture media or blood samples from mice were collected at the different experimental time points as described above. After centrifugation, the supernatant was used to measure TNF-a and IL-1b using a murine TNF-a Quantikine ELISA Kit and murine IL-1b/IL-1F2 Quantikine ELISA Kit following the protocol provided by the vendor (R&D system; Table 1).

Assays for metabolic indices
The blood samples used for the measurement of glucose were collected using a heparinized capillary tube from animals after fasting for 15 h. Roche Accu-Chek ® blood glucose meters were used to measure the amount of blood glucose in the animal groups. In addition, the blood samples collected from the fasting animals were centrifuged, and the supernatant was used to measure plasma insulin (Table 1) and other metabolic markers, including high-density lipoprotein (HDL), low-density lipoprotein/very-lowdensity lipoprotein (LDL/VLDL), and triglyceride (TG), using Quanti cation Colorimetric/Fluorometric Kits ( Table 1).

Collection of adipose tissues
After perfusion, white adipose tissue (WAT) was collected from epididymal adipose tissue, and brown adipose tissue (BAT) was collected from interscapular brown adipose tissue for weight measurement on an electronic analytical balance (ATX224; SHIMADZU, Japan).

Quantitative real-time polymerase chain reaction (QPCR)
Animals were anesthetized with Zoletil 50 (Virbac Taiwan Co., Ltd.; 5X dilution in saline, 0.05-0.06 ml/10 gm) by i.p. injection and then perfused with 0.9% saline in diethylpyrocarbonate (DEPC; Sigma)-treated distilled water. Hypothalamic tissues were removed and homogenized in TRIzol TM (Invitrogen) for RNA extraction. cDNA generation using MMLV reverse transcriptase (Invitrogen) and PCR ampli cation by Fast SYBR® Green Master Mix (Applied Biosystems) were previously described 30 . We used Primer-BLAST software provided by the National Center for Biotechnology Information to design primers that were manufactured by Taiwan

Double immuno uorescence
The cell cultures were xed in PBS containing 4% paraformaldehyde for 10 min and then treated with 0.1% Triton-X-100 in PBS at room temperature for 30 min. Rat microglia were incubated with rabbit anti-iNOS antibody (1:400) and biotin-conjugated IB4 (1:200) in PBS containing 5% horse serum overnight at 4°C, followed by biotinylated anti-rabbit IgG  Table 1.

Brain tissue immunostaining
The brain samples removed from animals were xed overnight in 4% paraformaldehyde and then transferred to tubes containing 30% (w/v) sucrose in PBS until the tissues sank to the bottom of the tube. The tissues were mounted in Tissue Tek optimal cutting temperature compound (Electron Microscopy Sciences, Torrance, CA, USA) and then sectioned at 20-mm thickness. The oating brain sections were treated with 1% Triton-X-100 in PBS at 4°C overnight, followed by incubation with primary antibodies in PBS containing 0.1% Triton X-100 and 1% horse serum at 4°C overnight. The tissue sections were then incubated with biotinylated secondary antibodies for 1 h and with Alexa Fluor 488 or Cy3-avidin (1:200) for another 45 min. The immunostained tissues were observed under an Olympus FLUOVIEW FV1000 confocal laser scanning microscope (FV1000, Japan) at wavelengths of 405, 488 or 594 nm. The primary antibodies used in the study are listed in Table 1. Alternatively, brain sections were permeabilized in 0.3% Triton-X-100 in PBS for 30 min, incubated with rabbit anti-D2R antibody (Table 1) in PBS at 4°C overnight, and treated with biotinylated anti-rabbit IgG (1:200) in PBS for 1 h. The brain sections were then reacted with Vectastain ABC reagent (Vector Labs; Cat# PK-6100) containing 3,3'-diaminobenzidine and nuclear counterstaining by hematoxylin. The immunostained sections were visualized and photographed under a Nikon E800 microscope equipped with a CCD camera.

Determination of microglial activation and astrocyte hypertrophy
The measurement of microglia and astrocyte activation was performed as previously described 12,30 . The cell number and cell body size of Iba1 + microglia and the intensity of GFAP immunoreactivity in the ARC were analyzed using National Institutes of Health (NIH) ImageJ analysis software 31 . In general, randomly selected images per brain section were merged and captured in multiple 1-mm-thick steps using an Olympus FLUOVIEW FV1000 confocal laser scanning microscope. Images that were used for quanti cation were captured from 7-9 brain sections for Iba1 immunostaining and 6 brain sections for GFAP immunostaining from three animals per group. The microglial cell numbers are shown as the number of cells in an image with a size of 0.1 mm 2 . The GFAP intensity data were normalized to those of the chow group (100%).

Statistical analysis
The presence of signi cant differences between each group (chow-vehicle, chow-TFP, HFD-vehicle, HFD-TFP) at different time points observed in this study was determined using one-way ANOVA with Sidak's multiple comparisons for the in vivo study or two-tailed Student's t test for in vitro experiments. Data are presented as the mean ± SEM. The statistical signi cance was set as P < 0.05.  TFP inhibits LPS-induced production of TNF-α and IL-1β in the plasma and hypothalamus. Animals were divided into four groups that received a bolus peripheral injection of vehicle, TFP (2 mg/kg), LPS (1 mg/kg), or LPS plus TFP. Blood samples from the four animal groups were collected at 3 and 6 h post injection for TNF-α and IL-1β ELISAs (A), and then hypothalamic RNA isolation was conducted for the determination of TNF-α and IL-1β mRNA expression (B) after animals were sacri ced at 6 h post injection.

Abbreviations
The data are presented as the mean ± SEM (n=5 animals in each group). Samples from ve animals per group were used for most assays, whereas samples from four animals were used for the IL-1β ELISA at 6 h (A). Data are presented as the mean ± SEM (n=5 animals for each group). Each dot represents one animal. *p<0.05, **p<0.01, ***p< 0.001 versus vehicle; #p<0.05, ##p<0.01 versus LPS. . Mice were sacri ced at 12 weeks after feeding with chow or HFD. Brain sections were prepared as described in the Materials and Methods and subjected to immunohistochemistry for D2R expression. The arrows indicate D2R+ cells (brown) in the ARC, which mostly accumulate in HFD-fed mice. The observations were con rmed by quanti cation of D2R immunoreactive intensity in the ARC.
The representative D2R-cells (blue) are shown by arrowheads above the dashed line. The results are presented in A and B as the mean ± SEM (n = 3-4 animals for each group). Each dot represents one animal. *p<0.05 versus vehicle. Scale bar in B, 100 µm.

Figure 4
Decreased levels of plasma TNF-α and IL-1β in obese mice treated with TFP for 4 weeks. (A) The schematic illustration shows that 8-week-old male C57BL/6 mice were fed chow or HFD for 12 weeks and then injected daily with TFP (2 mg/kg) via the i.p. route for another 4 weeks under chow or HFD feeding (chow-vehicle, chow-TFP, HFD-vehicle, HFD-TFP). At 16 weeks post feeding, blood samples were collected from animals without fasting for cytokine assays (B) and with fasting for the analysis of metabolic indices (Fig. 5). The brains were removed for sectioning and immunostaining (Fig. 6). (B) As indicated above, the plasma samples were subjected to TNF-α or IL-1β ELISA. Data are presented as the mean ± SEM (n=5 animals for each group). Each dot represents one animal. *p<0.05, ***p< 0.001 versus chowvehicle; ###p<0.001 versus HFD-vehicle.