Adiponectin Reverses the Hypothalamic Microglial Inflammation during Short-Term Exposure to Fat-Rich Diet

Adiponectin, an adipokine derived from the adipose tissue, manifests anti-inflammatory effects in the metabolically active organs and is, therefore, beneficial in various metabolic diseases associated with inflammation. However, the role of adiponectin in alleviating the hypothalamic inflammation connected to the pathogenesis of obesity has not yet been clearly interrogated. Here, we identified that the systemic administration of adiponectin suppresses the activation of microglia and thereby reverses the hypothalamic inflammation during short-term exposure to a high-fat diet. Additionally, we show that adiponectin induces anti-inflammatory effects in the microglial cell line subjected to an exogenous treatment with a saturated free fatty acid. In conclusion, the current study suggests that adiponectin suppresses the saturated free fatty acid-triggered the hypothalamic inflammation by modulating the microglial activation and thus maintains energy homeostasis.


Introduction
Inflammation is one of the major pathological causes implicated in the development of obesity and its related metabolic disorders [1][2][3]. Chronic inflammation triggered by over-nutrition results in perturbation of the activity of hypothalamic neurons, which govern the whole-body energy balance [4][5][6]. Recent studies report that microglia, the resident macrophages of the central nervous system (CNS), respond to the elevation of saturated free fatty acid (FFA) following consumption of the fat-rich diet and thereby trigger the hypothalamic inflammation [6]. This results in abnormal functioning of the neuronal circuit that controls the energy homeostasis.
Adiponectin, an adipokine derived from adipose tissue, has been reported as beneficial in alleviating multiple physiological disturbances [7][8][9]. It helps in alleviating a variety of metabolic diseases by ameliorating the cellular stresses including inflammation, oxidative stress, and endoplasmic reticulum stress [7,8,[10][11][12]. Although, a recent study identified an anti-inflammatory effect of adiponectin in the CNS [11], the role of adiponectin in rectifying the hypothalamic inflammation during early over-nutrition period remains elusive. Therefore, this study was aimed at identification of the contribution of adiponectin in regulating the hypothalamic inflammation and the microglial function. We evaluated the inflammatory responses and microglial activation utilizing mice model

Adiponectin Reverses the Hypothalamic Inflammation Induced by Short-Term Exposure to High Fat Diet
In order to verify if adiponectin exerts anti-inflammatory effect in the hypothalamus during the early over-nutrition period, we evaluated the pattern of the hypothalamic inflammation after treatment with high fat diet (HFD) for 4 weeks combined with the systemic administration of globular adiponectin, which has a more potent effect on their identified functions [13]. In line with the previous finding that identified a reduction in circulating adiponectin level during short-term exposure to a fat-rich diet [13], we also confirmed a reduction in adiponectin mRNA level in epididymal white adipose tissue (eWAT) 4 weeks after high-fat diet treatment ( Figure 1A). In accordance with the previous reports, we observed that HFD treatment resulted in a drastic increase in the expression levels of mRNA encoding inflammatory cytokines such as IL-1β ( Figure 1B), IL-6 ( Figure 1C), TNF-α ( Figure 1D) as well as Cox-2 gene ( Figure 1E), the rate-limiting enzyme involved in the synthesis of the prostaglandin E2, which controls the cellular inflammatory process. This elevation of mRNA levels was effectively reversed by a systemic treatment with adiponectin for five days. Furthermore, we observed that an elevation in the levels of hypothalamic Iba-1 and CD11b mRNA, the molecular markers of microglia, in HFD-treated group was almost completely rescued by the systemic treatment with adiponectin ( Figure 1F,G). These findings suggest that adiponectin treatment exerts anti-inflammatory effects on the hypothalamus following short-term HFD exposure, at least in part, via targeting the microglial cells. saturated FFA. In crux, the current study unravels the potential contribution of adiponectin in preventing the initiation of inflammatory response in the hypothalamic microglial cells in an early stage of obesity development.

Adiponectin Reverses the Hypothalamic Inflammation Induced by Short-Term Exposure to High Fat Diet
In order to verify if adiponectin exerts anti-inflammatory effect in the hypothalamus during the early over-nutrition period, we evaluated the pattern of the hypothalamic inflammation after treatment with high fat diet (HFD) for 4 weeks combined with the systemic administration of globular adiponectin, which has a more potent effect on their identified functions [13]. In line with the previous finding that identified a reduction in circulating adiponectin level during short-term exposure to a fat-rich diet [13], we also confirmed a reduction in adiponectin mRNA level in epididymal white adipose tissue (eWAT) 4 weeks after high-fat diet treatment ( Figure 1A). In accordance with the previous reports, we observed that HFD treatment resulted in a drastic increase in the expression levels of mRNA encoding inflammatory cytokines such as IL-1β ( Figure 1B), IL-6 ( Figure 1C), TNF-α ( Figure 1D) as well as Cox-2 gene ( Figure 1E), the rate-limiting enzyme involved in the synthesis of the prostaglandin E2, which controls the cellular inflammatory process. This elevation of mRNA levels was effectively reversed by a systemic treatment with adiponectin for five days. Furthermore, we observed that an elevation in the levels of hypothalamic Iba-1 and CD11b mRNA, the molecular markers of microglia, in HFD-treated group was almost completely rescued by the systemic treatment with adiponectin ( Figure 1F,G). These findings suggest that adiponectin treatment exerts anti-inflammatory effects on the hypothalamus following short-term HFD exposure, at least in part, via targeting the microglial cells.

Adiponectin Suppresses the Microglial Activation Induced by Short-Term Exposure to HFD
To determine whether adiponectin relieves the development of HFD-triggered hypothalamic inflammation by targeting the hypothalamic microglial cells, we identified the presence of adiponectin receptor 1 (AdipoR1) and adiponectin receptor 2 (AdipoR2) in the hypothalamic glial cells utilizing cultured primary microglia and astrocytes (Figure 2A). We also validated the purification of a single cell type determined by a strong expression of Iba-1, a molecular marker for microglia participating in membrane ruffling and phagocytosis in activated microglia ( Figure 2B) [14], or GFAP, a molecular marker for astrocytes ( Figure 2C). Based on the anti-inflammatory effects of adiponectin on hypothalamic inflammation triggered by short-term HFD treatment, we next performed immunohistochemistry (IHC) using an antibody against Iba-1 on brain slices from mice fed either a STD or a HFD combined with adiponectin to evaluate the impact of adiponectin treatment on microglia activation in the hypothalamus. We observed that the HFD treatment resulted in an increase in body weight ( Figure 2D) and WAT weight ( Figure 2E) as compared to the STD-treated group. On the contrary, the systemic administration of adiponectin for 7 days did not alter the increase in the body weight and WAT weight seen in the HFD-treated group ( Figure 2D,E). Consistent with our cellular data, we observed that adiponectin treatment effectively reverses the microglial activation in the hypothalamus characterized by increased in a number of microglia ( Figure 2F,G). In addition, we found that systemic treatment of adiponectin blocked the increase in microglia soma area in the HFD-treated hypothalamus ( Figure 2H). However, adiponectin did not rescue the elevated of Iba-1 intensity seen in the HFD-treated hypothalamus ( Figure 2I). These findings indicate that adiponectin reverses the effects of HFD on the hypothalamic inflammation by ameliorating the microglial activation during the early over-nutrition period.  Results are presented as mean ± SEM. n = 5 mice per group. *** p < 0.001 for effects of adiponectin on HFD-treated group versus effects of adiponectin on STD-treated group or Iba-1 mRNA level in astrocyte versus Iba-1 mRNA level in microglia; * p < 0.05 for GFAP mRNA level in astrocyte versus GFAP mRNA level in microglia. NS, not significant; CTL, control group; ADN, adiponectin-treated group.

Adiponectin Improves Palmitic Acid-Induced Inflammatory Responses in the Microglial Cells
The consumption of fat-rich diet results in an elevation in the levels of circulating free fatty acids which cause the obesity pathogenesis linked to the hypothalamic inflammation [15,16]. Our previous study identified that the levels of saturated free fatty acid were elevated in both the hypothalamus and sera of mice fed a HFD for 4 weeks [17]. Therefore, to verify the anti-inflammatory role of

Adiponectin Improves Palmitic Acid-Induced Inflammatory Responses in the Microglial Cells
The consumption of fat-rich diet results in an elevation in the levels of circulating free fatty acids which cause the obesity pathogenesis linked to the hypothalamic inflammation [15,16]. Our previous study identified that the levels of saturated free fatty acid were elevated in both the hypothalamus and sera of mice fed a HFD for 4 weeks [17]. Therefore, to verify the anti-inflammatory role of adiponectin in alleviating the microglial inflammation induced by over-nutrition, we further evaluated the anti-inflammatory effect of adiponectin in the BV-2 microglial cell line by an administration of palmitic acid, a saturated free fatty acid followed by adiponectin treatment. We confirmed that single treatment of adiponectin did not alter cell viability ( Figure 3A) or IL-1β release ( Figure 3B) in cultured BV-2 microglial cells. However, adiponectin treatment led to a reduction in IL-6 secretion ( Figure 3C). These findings indicated that adiponectin itself did not affect cellular inflammation and degeneration. In accordance with previous reports, palmitic acid treatment induced increase in the mRNA levels of inflammatory cytokines such as IL-1β ( Figure 3D), IL-6 ( Figure 3E) and TNF-α ( Figure 3F), and Cox-2 gene ( Figure 3G) in cultured BV-2 microglial cells. This elevation of inflammatory responses triggered by palmitic acid was completely rescued by an exogenous adiponectin treatment. Moreover, the administration of adiponectin reversed the palmitic acid-induced release of IL-1β ( Figure 3H) and IL-6 ( Figure 3I) in the BV-2 microglial cells. These observations confirmed that adiponectin treatment reverses the development of hypothalamic inflammation and the associated microglial activation.
( Figure 3E) and TNF-α ( Figure 3F), and Cox-2 gene ( Figure 3G) in cultured BV-2 microglial cells. This elevation of inflammatory responses triggered by palmitic acid was completely rescued by an exogenous adiponectin treatment. Moreover, the administration of adiponectin reversed the palmitic acid-induced release of IL-1 ( Figure 3H) and IL-6 ( Figure 3I) in the BV-2 microglial cells. These observations confirmed that adiponectin treatment reverses the development of hypothalamic inflammation and the associated microglial activation.

Adiponectin Reverses the Palmitic Acid-Induced Alterations in the Intracellular Signaling Molecules Involved in Inflammation
In order to further verify anti-inflammatory functions of adiponectin, we evaluated the phosphorylation of ERK, a molecular component of intracellular signal transduction in the inflammatory signaling pathway [18,19], and the level of IkB-α protein, which inhibits the activity of NF-κB transcription factor regulating the expression of multiple inflammatory cytokines [20]. As shown in the Figure 4, adiponectin reversed the palmitic acid-induced phosphorylation of ERK ( Figure 4A) and degradation of IkB-α ( Figure 4B). These observations suggest that adiponectin treatment suppresses the microglial inflammation in response to saturated FFA, at least in part, via modulating the general signaling molecules linked to the development of cellular inflammation.

Involved in Inflammation
In order to further verify anti-inflammatory functions of adiponectin, we evaluated the phosphorylation of ERK, a molecular component of intracellular signal transduction in the inflammatory signaling pathway [18,19], and the level of IkB-α protein, which inhibits the activity of NF-κB transcription factor regulating the expression of multiple inflammatory cytokines [20]. As shown in the Figure 4, adiponectin reversed the palmitic acid-induced phosphorylation of ERK ( Figure 4A) and degradation of IkB-α ( Figure 4B). These observations suggest that adiponectin treatment suppresses the microglial inflammation in response to saturated FFA, at least in part, via modulating the general signaling molecules linked to the development of cellular inflammation.

Discussion
The present study highlights the beneficial effects of adiponectin in the alleviating the hypothalamic inflammation triggered by the over-nutrition that causes the disturbance in the functioning of hypothalamic neurons that regulate the whole-body energy metabolism.
Multiple lines of evidence have suggested that chronic inflammation in the hypothalamic neuronal circuit, which controls the appetite and energy expenditure, causes a variety of cellular stresses and thereby results in the energy imbalance [4][5][6]. The adipose tissue is a specialized connective tissue that functions as a major storage site for fats in the body [21][22][23]. However, it has been well established that the adipose tissue also acts as an endocrine organ besides regulating the energy balance [22,24,25]. The adipose tissue communicated with the hypothalamus by releasing multiple chemical messengers that systemically propagate the signals reflecting nutrient availability for the homeostatic control of energy homeostasis [21,22,26]. In addition, the adipose tissue also regulates the obesity pathogenesis by secreting a variety of inflammatory cytokines and adipokines

Discussion
The present study highlights the beneficial effects of adiponectin in the alleviating the hypothalamic inflammation triggered by the over-nutrition that causes the disturbance in the functioning of hypothalamic neurons that regulate the whole-body energy metabolism.
Multiple lines of evidence have suggested that chronic inflammation in the hypothalamic neuronal circuit, which controls the appetite and energy expenditure, causes a variety of cellular stresses and thereby results in the energy imbalance [4][5][6]. The adipose tissue is a specialized connective tissue that functions as a major storage site for fats in the body [21][22][23]. However, it has been well established that the adipose tissue also acts as an endocrine organ besides regulating the energy balance [22,24,25]. The adipose tissue communicated with the hypothalamus by releasing multiple chemical messengers that systemically propagate the signals reflecting nutrient availability for the homeostatic control of energy homeostasis [21,22,26]. In addition, the adipose tissue also regulates the obesity pathogenesis by secreting a variety of inflammatory cytokines and adipokines that deteriorate the cellular stresses such as inflammation, endoplasmic reticulum stress, and oxidative stresses [21,22]. Although majority of the adipokines act as proinflammatory factors, adiponectin displays anti-inflammatory properties that potentially improve the dysfunction of metabolic controls in the body [8,27,28]. In the CNS, adiponectin inhibits neuronal degeneration by ameliorating the cellular stresses including inflammation [10,11,29].
A growing body of evidence suggested that the microglial cells essentially participates in the development of the neuronal inflammation [16,30,31] and the reactive gliosis is an important cellular event for both acute and chronic inflammation triggered by the over-nutrition [32,33]. Therefore, it is not surprising that the anti-inflammatory role of adiponectin in the microglial cells is coupled to the improvement of metabolic abnormalities. Since adiponectin binds via adiponectin receptors, AdipoR1 and AdipoR2 in the microglial cells, we confirmed the presence of these receptors in the microglial cells.
It has been quite well established that the short-term exposure of HFD results in an increased hypothalamic inflammation accompanied by the reactive gliosis of both microglia and astrocytes [5,6,34].
Intriguingly, mice having HFD for a couple of days displayed a significant increase in the inflammatory cytokines expression in the hypothalamus [5,6]. In line with this notion, we verified the role of adiponectin in ameliorating the hypothalamic inflammation during the consumption of the short-term fat-rich diet. Furthermore, we verified that adiponectin successfully reverses the inflammatory responses in the hypothalamic microglia during early over-nutrition period by observing the reduced microgliosis as determined by the accumulation of Iba-1 protein and the altered number and morphology of microglial cells.
A variety of substances are involved in the obesity-related pathogenesis. Among them, saturated FFAs are critical substances that trigger the inflammation in brain during early obesity period [16]. We observed that adiponectin effectively rescues the palmitic acid-induced microglial inflammation via regulation of the intracellular signaling molecules that mediate the inflammatory responses. Although there are substantial evidences highlighting the pathogenic factors associated with obesity and their impact on the development of metabolic dysfunction [35,36], the pathogenic substances involved in the initiation of hypothalamic inflammation during early over-nutrition period remain unexplored. Thus, it is also valuable to investigate the cellular and molecular responses following induction or prevention of the hypothalamic inflammation during early over-nutrition period. Indeed, previous studies indicated that adiponectin levels are slightly elevated during short-term exposure to HFD [37]. Therefore, we suggest that the anti-inflammatory effects of adiponectin might be due to the homeostatic response to suppress the hypothalamic inflammation induced by the multiple humoral factors such saturated FFAs and adipokines. However, the beneficial effects of adiponectin treatment on the severe obesity associated with the chronic inflammation need to be determined. Therefore, further studies are required to identify the beneficial effects of adiponectin in alleviating the cellular stresses following long-term treatment of adiponectin to the obesity model accompanied by chronic hypothalamic inflammation and the cellular lipotoxicity. Collectively, the current study identifies a reversible effect exerted by adiponectin treatment on the initiation of the hypothalamic inflammation during early over-nutrition period and provides novel insight into the strategies to prevent early disruption of the energy homeostasis.

Animals
Seven-week-old C57B/L6 mice (Dae Han Bio Link, Eumseong, Korea) were maintained under specific pathogen-free conditions at 22 • C and given access to food and water ad libitum. To examine the effects of adiponectin on early obesity stage-induced hypothalamic inflammation, mice were adapted for a week, randomly divided into two groups and fed either a standard diet (STD, 10% calories from fat, Research Diet Inc., New Brunswick, NJ, USA) or a high-fat diet (HFD, 60% of calories from fat, Research Diets Inc.) for four weeks. The components of the HFD and STD are indicated in Table 1. For adiponectin treatment, mice were given intraperitoneal (i.p) injections of globular adiponectin (3 mg/kg, Lugen Sci, Bucheon, Korea) for five days. Brains and serum were collected 3 h after last injection. In addition, body weight and epididymal fat weight were measured when tissue was harvested. The experimental procedure is described in a flowchart ( Figure 5). All the animal care and experimental procedures were performed in accordance with the protocols approved by the Institutional Animal Care and Use Committee (IACUC) at the Incheon National University (permission number: INU-2016-001). collected 3 h after last injection. In addition, body weight and epididymal fat weight were measured when tissue was harvested. The experimental procedure is described in a flowchart ( Figure 5). All the animal care and experimental procedures were performed in accordance with the protocols approved by the Institutional Animal Care and Use Committee (IACUC) at the Incheon National University (permission number: INU-2016-001).

Cell Culture and Treatments
The murine microglial BV-2 cells were maintained in Dulbecco s modified Eagle medium (DMEM) with high glucose (Gibco BRL, Grand Island NY, USA), containing 5% (v/v) fetal bovine serum (Gibco BRL, Grand Island NY, USA) and incubated at 37 • C in humidified 5% CO 2 . For gene expression assay, cells were seeded at a density of 5 × 10 5 cells/well in 12-well plate. After 24 h, the attached cells were pre-treated with adiponectin (100 ng/mL or 1000 ng/mL) for 1 h followed by the treatment with 200 µM