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• W2839584865 abstract "Meta-inflammation of hypothalamic areas governing energy homeostasis has recently emerged as a process of potential pathophysiological relevance for the development of obesity and its metabolic sequelae. The current model suggests that diet-induced neuronal injury triggers microgliosis and astrocytosis, conditions which ultimately may induce functional impairment of hypothalamic circuits governing feeding behavior, systemic metabolism, and body weight. Epidemiological data indicate that low circulating HDL levels, besides conveying cardiovascular risk, also correlate strongly with obesity. We simulated that condition by using a genetic loss of function mouse model (apoA-I−/−) with markedly reduced HDL levels to investigate whether HDL may directly modulate hypothalamic inflammation. Astrogliosis was significantly enhanced in the hypothalami of apoA-I−/− compared with apoA-I+/+ mice and was associated with compromised mitochondrial function. apoA-I−/− mice exhibited key components of metabolic disease, like increased fat mass, fasting glucose levels, hepatic triglyceride content, and hepatic glucose output compared with apoA-I+/+ controls. Administration of reconstituted HDL (CSL-111) normalized hypothalamic inflammation and mitochondrial function markers in apoA-I−/− mice. Treatment of primary astrocytes with apoA-I resulted in enhanced mitochondrial activity, implying that circulating HDL levels are likely important for astrocyte function. HDL-based therapies may consequently avert reactive gliosis in hypothalamic astrocytes by improving mitochondrial bioenergetics and thereby offering potential treatment and prevention for obesity and metabolic disease. Meta-inflammation of hypothalamic areas governing energy homeostasis has recently emerged as a process of potential pathophysiological relevance for the development of obesity and its metabolic sequelae. The current model suggests that diet-induced neuronal injury triggers microgliosis and astrocytosis, conditions which ultimately may induce functional impairment of hypothalamic circuits governing feeding behavior, systemic metabolism, and body weight. Epidemiological data indicate that low circulating HDL levels, besides conveying cardiovascular risk, also correlate strongly with obesity. We simulated that condition by using a genetic loss of function mouse model (apoA-I−/−) with markedly reduced HDL levels to investigate whether HDL may directly modulate hypothalamic inflammation. Astrogliosis was significantly enhanced in the hypothalami of apoA-I−/− compared with apoA-I+/+ mice and was associated with compromised mitochondrial function. apoA-I−/− mice exhibited key components of metabolic disease, like increased fat mass, fasting glucose levels, hepatic triglyceride content, and hepatic glucose output compared with apoA-I+/+ controls. Administration of reconstituted HDL (CSL-111) normalized hypothalamic inflammation and mitochondrial function markers in apoA-I−/− mice. Treatment of primary astrocytes with apoA-I resulted in enhanced mitochondrial activity, implying that circulating HDL levels are likely important for astrocyte function. HDL-based therapies may consequently avert reactive gliosis in hypothalamic astrocytes by improving mitochondrial bioenergetics and thereby offering potential treatment and prevention for obesity and metabolic disease. Obesity is associated with a state of moderate but chronic inflammation that is characterized by increased tissue-specific levels, as well as circulating levels, of interleukins and cytokines, a condition that is thought to participate in the development of many obesity-associated comorbidities by inducing cellular dysfunction (1.Hotamisligil G.S. Endoplasmic reticulum stress and the inflammatory basis of metabolic disease.Cell. 2010; 140: 900-917Abstract Full Text Full Text PDF PubMed Scopus (2083) Google Scholar). Emerging evidence indicates that chronic consumption of excess calories induces inflammation in the arcuate nucleus (ARC) of the hypothalamus, a brain area important for the regulation of food intake and energy expenditure (2.Kälin S. Heppner F.L. Bechmann I. Prinz M. Tschöp M.H. Yi C.X. Hypothalamic innate immune reaction in obesity.Nat. Rev. Endocrinol. 2015; 11: 339-351Crossref PubMed Scopus (112) Google Scholar). This process is considered an early and determining factor for the onset of obesity because it occurs prior to body weight gain and participates in the development and progression of metabolic disorders including obesity, diabetes, and CVD. Mitochondrial activity, which is critical for proper functioning of the CNS, is compromised in inflammatory states, and there are documented molecular links among inflammation, impaired insulin signaling, and mitochondrial dysfunction in neurodegenerative diseases and diabetes (3.De Felice F.G. Ferreira S.T. Inflammation, defective insulin signaling, and mitochondrial dysfunction as common molecular denominators connecting type 2 diabetes to Alzheimer disease.Diabetes. 2014; 63: 2262-2272Crossref PubMed Scopus (393) Google Scholar). Novel therapeutic strategies may therefore be developed to target this impairment. Within the hypothalamus, glial cells are key regulators of parenchymal homeostasis and neuronal communication. Glial cells become activated during periods of excess caloric intake, causing them to initiate inflammatory processes known collectively as reactive gliosis. As this occurs, the two main classes of glial cells, astrocytes and microglia, are converted to an activated pro-inflammatory phenotype characterized by cellular hypertrophy and proliferation, increased expression of intermediate filaments, and secretion of inflammatory interleukins and cytokines (2.Kälin S. Heppner F.L. Bechmann I. Prinz M. Tschöp M.H. Yi C.X. Hypothalamic innate immune reaction in obesity.Nat. Rev. Endocrinol. 2015; 11: 339-351Crossref PubMed Scopus (112) Google Scholar). Glial cells can also be activated directly by several pro-inflammatory factors or cytokines, including the metabolic hormone leptin, which stimulate the production of interleukin (IL)-1β, TNFα, and IL-6 within the glial cells (4.Buckman L.B. Ellacott K.L.J. The contribution of hypothalamic macroglia to the regulation of energy homeostasis.Front. Syst. Neurosci. 2014; 8: 212Crossref PubMed Scopus (14) Google Scholar). Activated microglia and astrocytes continue to accumulate in the hypothalamus over months of excess caloric intake, such that hypothalamic inflammation becomes persistent, potentially leading to neuronal dysfunction and, thus, to a chronic impairment of energy homeostasis (5.Thaler J.P. Yi C.X. Schur E.A. Guyenet S.J. Hwang B.H. Dietrich M.O. Zhao X. Sarruf D.A. Izgur V. Maravilla K.R. et al.Obesity is associated with hypothalamic injury in rodents and humans.J. Clin. Invest. 2012; 122: 153-162Crossref PubMed Scopus (1194) Google Scholar). Epidemiological studies have confirmed a strong association between fat intake, plasma cholesterol levels, obesity, and CVD mortality (6.Rees K. Dyakova M. Wilson N. Ward K. Thorogood M. Brunner E. Dietary advice for reducing cardiovascular risk.Cochrane Database Syst. Rev. 2013; 12: CD002128PubMed Google Scholar, 7.Hooper L. Summerbell C.D. Thompson R. Sills D. Roberts F.G. Moore H.J. Davey Smith G. Reduced or modified dietary fat for preventing cardiovascular disease.Cochrane Database Syst. Rev. 2012; 5: CD002137PubMed Google Scholar). One key independent predictor for CVD as well as for Alzheimer's dementia is a low level of HDL particles and their major protein constituent, apoA-I (8.Boden W.E. High-density lipoprotein cholesterol as an independent risk factor in cardiovascular disease: assessing the data from Framingham to the Veterans Affairs High-Density Lipoprotein Intervention Trial.Am. J. Cardiol. 2000; 86: 19L-22LAbstract Full Text Full Text PDF PubMed Scopus (358) Google Scholar, 9.McNeill A.M. Katz K. Girman C.J. Rosamond W.D. Wagenknecht L.E. Barzilay J.I. Tracy R.P. Savage P.J. Jackson S.A. Metabolic syndrome and cardiovascular disease in older people: the cardiovascular health study.J. Am. Geriatr. Soc. 2006; 54: 1317-1324Crossref PubMed Scopus (134) Google Scholar, 10.Stukas S. Robert J. Wellington C.L. High-density lipoproteins and cerebrovascular integrity in Alzheimer's disease.Cell Metab. 2014; 19: 574-591Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Low circulating HDL and apoA-I are also a hallmark of obesity in association with insulin-resistance, a pathological precondition associated with T2D (11.ACCORD Study Group Ginsberg H.N. Elam M.B. Lovato C.L. Crouse J.R. Leiter L.A. Linz P. Friedewald W.T. Buse J.B. Gerstein H.C. et al.Effects of combination lipid therapy in type 2 diabetes mellitus.N. Engl. J. Med. 2010; 362: 1563-1574Crossref PubMed Scopus (2203) Google Scholar). HDLs have atheroprotective functions that are independent of their well-known role in lipid transport (12.Kingwell B.A. Chapman M.J. Kontush A. Miller N.E. HDL-targeted therapies: progress, failures and future.Nat. Rev. Drug Discov. 2014; 13: 445-464Crossref PubMed Scopus (262) Google Scholar). In particular, HDLs directly mediate anti-inflammatory reprogramming of immune cells (13.De Nardo D. Labzin L.I. Kono H. Seki R. Schmidt S.V. Beyer M. Xu D. Zimmer S. Lahrmann C. Schildberg F.A. et al.High-density lipoprotein mediates anti-inflammatory reprogramming of macrophages via the transcriptional regulator ATF3.Nat. Immunol. 2014; 15: 152-160Crossref PubMed Scopus (282) Google Scholar), promoting cerebrovascular health (10.Stukas S. Robert J. Wellington C.L. High-density lipoproteins and cerebrovascular integrity in Alzheimer's disease.Cell Metab. 2014; 19: 574-591Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar) and maintaining mitochondrial function [for review see (14.White C.R. Datta G. Giordano S. High-density lipoprotein regulation of mitochondrial function.Adv. Exp. Med. Biol. 2017; 982: 407-429Crossref PubMed Scopus (30) Google Scholar)], activities that may prevent and/or ameliorate metabolic disease. However, it is not known whether circulating HDLs exert an independent effect on the development of obesity and metabolic dysfunction. We therefore investigated the role of genetically reduced HDL levels in the development of hypothalamic inflammation and mitochondrial dysfunction. Using a genetic loss of function mouse model (apoA-I−/−), we report for the first time that low circulating HDL levels specifically promote astrocyte activation and induce mitochondrial dysfunction within the hypothalamic astrocytes that is associated with enhanced adiposity and impaired glucose homeostasis. Treatment of primary astrocytes with apoA-I results in enhanced mitochondrial bioenergetics of primary astrocytes, and administration of reconstituted HDL (rHDL) (CSL-111) in apoA-I−/− mice reverses hypothalamic inflammation by normalizing mitochondrial function markers, suggesting that HDL-based therapies may avert astroglial dysfunction and thus offer a new approach for potential treatment and prevention of obesity and metabolic disease. For phenotyping studies, apoA-I-deficient (apoA-I−/−) male mice on the C57/BL/6J background (Jackson Laboratories, Bar Harbor, ME) were bred with female WT mice in our animal facilities. Age-matched male apoA-I−/− mice and their WT male littermates (apoA-I+/+) (n = 10–14 per group) were 6 months of age at the initiation of the study. For pharmacological studies, reconstituted human HDL (CSL-111) was provided by CSL Behring AG (Bern, Switzerland) and prepared from human plasma as previously described (15.Lerch P.G. Förtsch V. Hodler G. Bolli R. Production and characterization of a reconstituted high density lipoprotein for therapeutic applications.Vox Sang. 1996; 71: 155-164Crossref PubMed Scopus (119) Google Scholar). The preparation was supplied as a lyophilized powder that, upon reconstitution with sterile water, rendered a solution containing 19.5 g/l protein. The molar ratio protein-phospholipid was 1:155. Seven-month-old apoA-I−/− male mice and WT mice (n = 6 per group) were randomized according to body weight and body composition. Mice were treated daily via intraperitoneal injections of CSL-111 (150 mg/kg) for 7 days during the light cycle. Vehicle mice received an equivalent volume of 0.9% saline. All mice were housed in a pathogen-free environment on a 12:12 h light-dark cycle at 22°C with free access to food and water. Mice were fed a regular chow diet (5058 PicoLab Mouse Diet 20; LabDiet, Richmond, IN). Body fat mass was measured in conscious mice using 1H magnetic resonance spectroscopy (EchoMRI-100; Echo Medical Systems). All rodent studies were approved by and performed according to the guidelines of the Institutional Animal Care and Use Committee at the University of Cincinnati and the Helmholtz Center Munich. Animals were fasted for 6 h prior to blood collection from the tail vein. Plasma cholesterol levels were determined using the Infinity cholesterol kit (Thermo Scientific Inc., Rockford, IL) as described (16.Hofmann S.M. Zhou L. Perez-Tilve D. Greer T. Grant E. Wancata L. Thomas A. Pfluger P.T. Basford J.E. Gilham D. et al.Adipocyte LDL receptor-related protein-1 expression modulates postprandial lipid transport and glucose homeostasis in mice.J. Clin. Invest. 2007; 117: 3271-3282Crossref PubMed Scopus (129) Google Scholar). An intraperitoneal pyruvate tolerance test was conducted by injection of sodium pyruvate (2 g/kg; Sigma-Aldrich, St. Louis, MO) in 0.9% w/v NaCl, as described previously (17.Habegger K.M. Matzke D. Ottaway N. Hembree J. Holland J. Raver C. Mansfeld J. Müller T.D. Perez-Tilve D. Pfluger P.T. et al.Role of adipose and hepatic atypical protein kinase C lambda (PKCλ) in the development of obesity and glucose intolerance.Adipocyte. 2012; 1: 203-214Crossref PubMed Google Scholar). Tail blood glucose levels (milligrams per deciliter) were measured with a TheraSense Freestyle glucometer (Abbott Diabetes Care, Inc., Alameda, CA) before (0 min) and at 15, 30, 45, 60, and 120 min after injection. Samples were analyzed individually except for lipoprotein separation, for which pooled samples were subjected to fast-performance LC, as described previously (16.Hofmann S.M. Zhou L. Perez-Tilve D. Greer T. Grant E. Wancata L. Thomas A. Pfluger P.T. Basford J.E. Gilham D. et al.Adipocyte LDL receptor-related protein-1 expression modulates postprandial lipid transport and glucose homeostasis in mice.J. Clin. Invest. 2007; 117: 3271-3282Crossref PubMed Scopus (129) Google Scholar). Brain cortex and liver lipid content of ad libitum-fed mice was assessed by extraction as described (16.Hofmann S.M. Zhou L. Perez-Tilve D. Greer T. Grant E. Wancata L. Thomas A. Pfluger P.T. Basford J.E. Gilham D. et al.Adipocyte LDL receptor-related protein-1 expression modulates postprandial lipid transport and glucose homeostasis in mice.J. Clin. Invest. 2007; 117: 3271-3282Crossref PubMed Scopus (129) Google Scholar). Briefly, 20 mg of snap-frozen brain cortex and 50 mg of snap-frozen liver were homogenized with 1 ml chloroform. Lipids were extracted by overnight shaking at room temperature. For phase separation, 700 μl of ddH2O were added, samples were centrifuged at 775 g for 20 min at 4°C and the organic layer was collected. For the second extraction, 700 μl of chloroform/methanol (2:1 v/v) were added to the remaining homogenate and lipid extraction, phase separation, and organic-layer collection were performed as described above. Fifteen microliters of both extractions were then transferred to glass tubes, evaporated, and measured with an Infinity cholesterol and triglyceride kit (Thermo Scientific Inc.). Brain cortex tissue was snap-frozen for determination of citrate synthase activity by a colorimetric assay kit (K318-100; Biovision, Germany). Assay was carried out according to the manufacturer's instructions. Briefly, frozen tissues were homogenized with assay buffer and incubated with substrate mix. Enzyme activity was calculated from standard curve. Protein level was determined by a Pierce BCA protein assay kit (Thermo Fisher, Germany). Citrate synthase activity was normalized to protein content. At the termination of the study, mice were decapitated and brains were removed and immerse-fixed in 4% paraformaldehyde at 4°C for 24 h. Brains were then equilibrated for 48 h with 30% sucrose in 0.1 M TBS (pH 7.2). Immunohistochemistry and quantitative analysis for ionized calcium binding adaptor molecule 1 (Iba-1) and glial fibrillary acidic protein (GFAP) were performed on coronal brain sections (30 μm) throughout the hypothalamus as described previously (18.Yi C.X. Al-Massadi O. Donelan E. Lehti M. Weber J. Ress C. Trivedi C. Müller T.D. Woods S.C. Hofmann S.M. Exercise protects against high-fat diet-induced hypothalamic inflammation.Physiol. Behav. 2012; 106: 485-490Crossref PubMed Scopus (86) Google Scholar). Briefly, after rinsing in 0.1 M TBS, whole-brain sections were incubated with rabbit anti-Iba-1 primary antibody (Synaptic Systems, Goettingen, Germany) at 1:1,000 dilution or rabbit anti-GFAP primary antibody (Dako, Carpinteria, CA) at 1:1,000 dilution overnight at 4°C. Sections were then rinsed and incubated with biotinylated secondary antibody (horse anti-rabbit IgG; Vector Laboratories, Burlingame, CA) for 1 h; subsequently, sections were rinsed and incubated in avidin-biotin complex (Vector Laboratories) for 1 h. The reaction product was visualized by incubation in 1% diaminobenzidine with 0.01% hydrogen peroxide for 1–10 min. Sections were mounted on gelatin-coated glass slides, dried, dehydrated in graded ethanol series, cleared in xylene, and cover-slipped for observation by light microscopy. All quantitative analyses were performed under blinded conditions and confirmed by at least two independent researchers. For each mouse, two to three sections in the middle portion of the ARC within the mediobasal hypothalamus were selected and images were captured by a computerized image analysis system consisting of an Axioskop color video camera (Carl Zeiss International, Thornwood, NY). Both sides of the ARC were manually outlined with an area of 0.04 mm2 on each side. Immunoreactive cells were manually counted throughout the ARC and expressed as number of cells per square millimeter. The average from each mouse was then calculated and expressed as the mean ± SEM from each group. Mice were euthanized by CO2. Brains were subsequently harvested and hypothalami were immediately isolated and stored in liquid nitrogen. At a later time point, RNA was isolated by RNeasy lipid tissue mini kit (Qiagen, Hilden, Germany). After reverse transcription (QuantiTech reverse transcription kit; Qiagen), gene expression was analyzed by real-time PCR (ABI TaqMan 7900; Life Technologies GmbH, Darmstadt, Germany) using the company's reaction system and probes. Hypothalami were isolated from 2-day-old male C57/BL/6J mice and triturated in MEM (Life Technologies GmbH) containing 1% penicillin-streptomycin, 10% FCS (Life Technologies GmbH), and 5.5 mM glucose. Astrocyte isolation was performed as described previously (19.García-Cáceres C. Fuente-Martín E. Burgos-Ramos E. Granado M. Frago L.M. Barrios V. Horvath T. Argente J. Chowen J.A. Differential acute and chronic effects of leptin on hypothalamic astrocyte morphology and synaptic protein levels.Endocrinology. 2011; 152: 1809-1818Crossref PubMed Scopus (84) Google Scholar). The cell suspension was centrifuged for 7 min at 194 g and the pellet was resuspended and seeded in a 175 cm3 cell culture flask. Cells were incubated at 37°C and 5% CO2 for 9 days with a regular medium change every 3 days. Prior to detachment, the flasks were placed in a 37°C shaking incubator at 280 rpm overnight. The cells were then washed and incubated for 2 min at 37°C with a 0.05% trypsin/EDTA solution (Biochrom AG, Berlin, Germany). Trypsinization was blocked with MEM + 10% FCS + 1% antibiotics. After centrifugation for 5 min at 194 g, supernatant was discharged and the cell pellet was resuspended in MEM + 10% FCS + 1% antibiotics and seeded in the XF24 plate with 80,000 cells per well (Seahorse Bioscience, North Billerica, MA). Twenty-four hours after seeding, the medium was removed and primary astrocytes were washed with PBS before overnight incubation with MEM medium containing 20 μg/ml apoA-I (EMD Chemicals, Inc., San Diego, CA) or 20 μg/ml BSA (Sigma-Aldrich) in quintuplicates as reported (20.Lehti M. Donelan E. Abplanalp W. Al-Massadi O. Habegger K.M. Weber J. Ress C. Mansfeld J. Somvanshi S. Trivedi C. et al.High-density lipoprotein maintains skeletal muscle function by modulating cellular respiration in mice.Circulation. 2013; 128: 2364-2371Crossref PubMed Scopus (58) Google Scholar). The next morning, cells were washed with PBS and incubated with XF assay medium containing 5.5 mM glucose for 1 h in a 37°C air incubator. The XF24 plate was then transferred to a temperature-controlled (37°C) Seahorse (extracellular flux) analyzer (Seahorse Bioscience) and subjected to an equilibration period. Respirometry analysis was performed as described previously (21.Keuper M. Jastroch M. Yi C-X. Fischer-Posovszky P. Wabitsch M. Tschöp M.H. Hofmann S.M. Spare mitochondrial respiratory capacity permits human adipocytes to maintain ATP homeostasis under hypoglycemic conditions.FASEB J. 2014; 28: 761-770Crossref PubMed Scopus (52) Google Scholar). Briefly, one assay cycle consisted of a 1 min mix, 2 min wait, and 3 min measurement period. After four basal assay cycles, oligomycin (20 μg/ml) was added by automatic pneumatic injection to inhibit ATP synthase for the determination of the proportion of respiration used to drive ATP synthesis for three additional cycles. Subsequently, carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP; 10 μM) was added the same way to determine maximal respiration in mitochondria by uncoupling ATP synthesis from electron transport for another three assay cycles. Finally, rotenone (25 μM) plus antimycin A (25 μM) was added to determine the nonmitochondrial respiratory rate in the next three assay cycles, which was then subtracted from all other rates. Oxygen consumption rate (OCR) traces and a scheme about the different modules that we analyzed are depicted in Fig. 3. Coupling efficiency was calculated as the oligomycin-sensitive fraction of mitochondrial respiratory activity. ATP production from oxidative phosphorylation (OXPHOS) was calculated using a phosphate/oxygen ratio of 2.3 (22.Brand M.D. The efficiency and plasticity of mitochondrial energy transduction.Biochem. Soc. Trans. 2005; 33: 897-904Crossref PubMed Scopus (291) Google Scholar). For analysis of extracellular acidification rates (ECARs) derived from glycolysis, measurements were ended by addition of 2-deoxy-glucose (2DG; 100 mM). ECAR traces and the different modules that we analyzed are depicted in Fig. 4. ECARs were converted into proton production rates (PPRs) by taking into account the buffer capacity. The 2DG-sensitive PPR estimates ATP production from glycolysis with a 1:1 ratio. To normalize respirometry readings to cell number per well, cells were stained with crystal violet after the flux experiment in the Seahorse XF analyzer. Briefly, cells were fixed with 4% paraformaldehyde for 30 min at room temperature. After washing three times with PBS, 200 μl of 0.1% crystal violet were added into each well and incubated for 10 min. After incubation time, crystal violet was removed and the plate was rinsed with distilled water. When the plate was completely dry, 500 μl of 10% acetic acid were added into each well. Absorbance was read at 590 nm.Fig. 4apoA-I reduces glycolytic activity in primary astrocytes and enhances ATP demand fueled by OXPHOS. A: The ECAR was recorded simultaneously with the OCR of astrocytes incubated with BSA or apoA-I (20 μg/ml overnight) with a Seahorse XF96 extracellular flux analyzer, as described in the Materials and Methods. B: Scheme defining cellular ECAR processes. Data after FCCP and rotenone/antimycin A injection were not used for ECAR analysis. C: By inhibiting glycolysis with 2DG (100 mM) as the last step in the measurements, cellular acidification was broken down to nonglycolytic acidification (gray) and acidification due to glycolysis (purple). D: By inhibiting mitochondrial ATP synthesis with oligomycin, the maximal glycolytic capacity was obtained. E: ATP production rates of OXPHOS pathways of BSA-treated (open bar) and apoA-I-treated (closed bar) primary astrocytes. F: ATP production rates of glycolysis as estimated via PPRs of BSA-treated (open bar) and apoA-I-treated (closed bar) primary astrocytes. G: Sum of ATP produced by OXPHOS and glycolysis of BSA-treated (open bar) and apoA-I-treated (closed bar) primary astrocytes. H: Percentage of OXPHOS (closed bars) and glycolytic (open bars) ATP production on total ATP production in BSA- and apoA-I-treated primary astrocytes. Data are expressed as the mean ± SEM of five replicates run in parallel. *P < 0.05, ***P < 0.0005, ****P < 0.00005.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Quantitative data are presented as mean ± SEM. Values were analyzed for statistically significant differences applying two-tailed unpaired t-tests for comparisons between untreated apoA-I+/+ and apoA-I−/− mice and one-way or two-way ANOVA tests for CSL treatment comparisons between apoA-I+/+ and apoA-I−/− mice, as recommended by GraphPad Prism for optimal analysis. Data homogeneity was analyzed by Bartlett's test and F test, accordingly. P < 0.05 was considered significant (GraphPad Prism, GraphPad Software, La Jolla, CA; SigmaStat, Systat Software, San Jose, CA). To determine whether circulating HDL levels modulate hypothalamic inflammation, we used a genetic loss of function mouse model for apoA-I (apoA-I−/−), the main protein component of HDL. As expected, fast-performance LC analysis of apolipoprotein profiles revealed a severely reduced circulating HDL cholesterol concentration in apoA-I−/− mice compared with WT littermates (apoA-I+/+; Fig. 1A, B). We found that in the absence of apoA-I, mRNA levels of the inflammatory IL-6 were significantly increased in the hypothalamus (Fig. 1C). GFAP is an intermediate filament protein that reliably identifies reactive astrocytes in association with cell process expansion (23.Mrak R.E. Griffin W.S.T. Glia and their cytokines in progression of neurodegeneration.Neurobiol. Aging. 2005; 26: 349-354Crossref PubMed Scopus (526) Google Scholar, 24.Pekny M. Nilsson M. Astrocyte activation and reactive gliosis.Glia. 2005; 50: 427-434Crossref PubMed Scopus (1242) Google Scholar). Using immunohistochemical staining of brain sections for GFAP, we found that astrocytic gliosis is markedly increased specifically in the ARC of apoA-I−/− compared with apoA-I+/+ mice (Fig. 1D). Consistent with increased reactive astrogliosis, we also found that the GFAP-labeled cells were enlarged in size and had more extensive processes, thus exhibiting the typical pro-inflammatory morphology. In parallel, we determined whether ARC microglial cells were activated by immunohistochemistry using the well-established macrophage/microglia-specific marker, ionized calcium-binding adapter molecule 1 (Iba-1) (25.Imai Y. Kohsaka S. Intracellular signaling in M-CSF-induced microglia activation: role of Iba1.Glia. 2002; 40: 164-174Crossref PubMed Scopus (281) Google Scholar). Iba-1 is specifically expressed in activated microglia and is thus regarded as a specific surrogate parameter for microgliosis. It has been commonly used to detect and compare activated microglia in numerous species (26.Drake C. Boutin H. Jones M.S. Denes A. McColl B.W. Selvarajah J.R. Hulme S. Georgiou R.F. Hinz R. Gerhard A. et al.Brain inflammation is induced by co-morbidities and risk factors for stroke.Brain Behav. Immun. 2011; 25: 1113-1122Crossref PubMed Scopus (153) Google Scholar). Our quantitative analysis of brain sections revealed no differences in microglial activation in the ARC between the two genotypes (Fig. 1E). As an additional sign of microglial activation, Iba-1-positive cells display the typical pro-inflammatory morphology of enlarged cell bodies and thickened processes. Herein, we observed no difference in morphology of microglia in the hypothalami of apoA-I−/− compared with apoA-I+/+ mice. In other regions of the brain, including the hippocampus or the cortex, we did not observe differences in astroglial and microglial activation (supplemental Fig. S1A, B). In conclusion, our results indicate that circulating HDLs associate with reactive astrogliosis in the ARC, an area known to play a pivotal role in the regulation of energy balance. To understand whether the increased reactive astrogliosis observed in apoA-I−/− mice is associated with early markers of metabolic dysfunction, we determined body composition and circulating glucose levels in apoA-I−/− and apoA-I+/+ mice. Although apoA-I−/− mice had similar body weights as apoA-I+/+ mice when fed a regular chow diet, we confirmed previous results by us and others showing that absence of apoA-I in mice results in increased fat mass (27.Han R. Lai R. Ding Q. Wang Z. Luo X. Zhang Y. Cui G. He J. Liu W. Chen Y. Apolipoprotein A-I stimulates AMP-activated protein kinase and improves glucose metabolism.Diabetologia. 2007; 50: 1960-1968Crossref PubMed Scopus (120) Google Scholar) (Fig. 2A, B). Hepatic triglyceride content was also markedly increased in apoA-I−/− compared with apoA-I+/+ mice (Fig. 2C), indicating that the excess body fat mass in apoA-I−/− mice results, in part, from an increased triglyceride accumulation within the liver. We next asked whether the hepatic steatosis in apoA-I−/−" @default.
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• W2839584865 title "Circulating HDL levels control hypothalamic astrogliosis via apoA-I" @default.
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