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• W2045759524 abstract "Here, we investigated how LDL receptor deficiency (Ldlr−/−) modulates the effects of testosterone on obesity and related metabolic dysfunctions. Though sham-operated Ldlr−/− mice fed Western-type diet for 12 weeks became obese and showed disturbed plasma glucose metabolism and plasma cholesterol and TG profiles, castrated mice were resistant to diet-induced obesity and had improved glucose metabolism and reduced plasma TG levels, despite a further deterioration in their plasma cholesterol profile. The effect of hypogonadism on diet-induced weight gain of Ldlr−/− mice was independent of ApoE and Lrp1. Indirect calorimetry analysis indicated that hypogonadism in Ldlr−/− mice was associated with increased metabolic rate. Indeed, mitochondrial cytochrome c and uncoupling protein 1 expression were elevated, primarily in white adipose tissue, confirming increased mitochondrial metabolic activity due to thermogenesis. Testosterone replacement in castrated Ldlr−/− mice for a period of 8 weeks promoted diet-induced obesity, indicating a direct role of testosterone in the observed phenotype. Treatment of sham-operated Ldlr−/− mice with the aromatase inhibitor exemestane for 8 weeks showed that the obesity of castrated Ldlr−/− mice is independent of estrogens. Overall, our data reveal a novel role of Ldlr as functional modulator of metabolic alterations associated with hypogonadism. Here, we investigated how LDL receptor deficiency (Ldlr−/−) modulates the effects of testosterone on obesity and related metabolic dysfunctions. Though sham-operated Ldlr−/− mice fed Western-type diet for 12 weeks became obese and showed disturbed plasma glucose metabolism and plasma cholesterol and TG profiles, castrated mice were resistant to diet-induced obesity and had improved glucose metabolism and reduced plasma TG levels, despite a further deterioration in their plasma cholesterol profile. The effect of hypogonadism on diet-induced weight gain of Ldlr−/− mice was independent of ApoE and Lrp1. Indirect calorimetry analysis indicated that hypogonadism in Ldlr−/− mice was associated with increased metabolic rate. Indeed, mitochondrial cytochrome c and uncoupling protein 1 expression were elevated, primarily in white adipose tissue, confirming increased mitochondrial metabolic activity due to thermogenesis. Testosterone replacement in castrated Ldlr−/− mice for a period of 8 weeks promoted diet-induced obesity, indicating a direct role of testosterone in the observed phenotype. Treatment of sham-operated Ldlr−/− mice with the aromatase inhibitor exemestane for 8 weeks showed that the obesity of castrated Ldlr−/− mice is independent of estrogens. Overall, our data reveal a novel role of Ldlr as functional modulator of metabolic alterations associated with hypogonadism. Testosterone, a steroid hormone with an important role in male sexual differentiation, maintenance of libido, and erectile function (1Shahidi N.T. A review of the chemistry, biological action, and clinical applications of anabolic-androgenic steroids.Clin. Ther. 2001; 23: 1355-1390Abstract Full Text PDF PubMed Scopus (349) Google Scholar, 2Yassin A.A. Saad F. Testosterone and erectile dysfunction.J. Androl. 2008; 29: 593-604Crossref PubMed Scopus (53) Google Scholar), is also a central modulator of key metabolic processes associated with metabolic syndrome. In humans, testosterone deficiency (TD) alters carbohydrate, lipid, and protein metabolism, thus contributing to oxidative stress, endothelial dysfunction, and increased production of proinflammatory factors (3Traish A.M. Abdou R. Kypreos K.E. Androgen deficiency and atherosclerosis: the lipid link.Vascul. Pharmacol. 2009; 51: 303-313Crossref PubMed Scopus (70) Google Scholar). TD is considered as a primary risk factor for a number of disorders including obesity, metabolic syndrome, dyslipidemia, endothelial cell dysfunction, vascular disease, insulin resistance, and type 2 diabetes mellitus (4Traish A.M. Saad F. Guay A. The dark side of testosterone deficiency: II. Type 2 diabetes and insulin resistance.J. Androl. 2009; 30: 23-32Crossref PubMed Scopus (220) Google Scholar, 5Traish A.M. Saad F. Feeley R.J. Guay A. The dark side of testosterone deficiency: III. Cardiovascular disease.J. Androl. 2009; 30: 477-494Crossref PubMed Scopus (203) Google Scholar, 6Traish A.M. Guay A. Feeley R. Saad F. The dark side of testosterone deficiency: I. Metabolic syndrome and erectile dysfunction.J. Androl. 2009; 30: 10-22Crossref PubMed Scopus (233) Google Scholar, 7Kalyani R.R. Dobs A.S. Androgen deficiency, diabetes, and the metabolic syndrome in men.Curr. Opin. Endocrinol. Diabetes Obes. 2007; 14: 226-234Crossref PubMed Scopus (150) Google Scholar, 8Kapoor D. Jones T.H. Androgen deficiency as a predictor of metabolic syndrome in aging men: an opportunity for intervention?.Drugs Aging. 2008; 25: 357-369Crossref PubMed Scopus (42) Google Scholar). Likewise, data from the Massachusetts Male Aging Study suggest that obesity leads to decreased levels of total and free testosterone, with follow-up levels lowest among men who remained or became obese (9Derby C.A. Zilber S. Brambilla D. Morales K.H. McKinlay J.B. Body mass index, waist circumference and waist to hip ratio and change in sex steroid hormones: the Massachusetts Male Ageing Study.Clin. Endocrinol. (Oxf.). 2006; 65: 125-131Crossref PubMed Scopus (236) Google Scholar). Additional insights into the role of androgens in maintaining human health have been obtained following androgen deprivation therapy (ADT), which is often used in the treatment of prostate cancer (PCa). Metabolic complications of ADT, such as metabolic syndrome, insulin resistance, and type 2 diabetes mellitus have highlighted key avenues of human pathology associated with this treatment (10Saylor P.J. Smith M.R. Prostate cancer: how can we improve the health of men who receive ADT?.Nat. Rev. Urol. 2009; 6: 529-531Crossref PubMed Scopus (7) Google Scholar). ADT in men with PCa resulted in increased total cholesterol, LDL cholesterol (LDL-C), and TG levels (11Smith M.R. Lee H. Nathan D.M. Insulin sensitivity during combined androgen blockade for prostate cancer.J. Clin. Endocrinol. Metab. 2006; 91: 1305-1308Crossref PubMed Scopus (394) Google Scholar, 12Smith M.R. Finkelstein J.S. McGovern F.J. Zietman A.L. Fallon M.A. Schoenfeld D.A. Kantoff P.W. Changes in body composition during androgen deprivation therapy for prostate cancer.J. Clin. Endocrinol. Metab. 2002; 87: 599-603Crossref PubMed Scopus (548) Google Scholar). Patients undergoing ADT exhibit significantly higher BMI (P < 0.01) and serum cholesterol levels (P = 0.03) than eugonadal subjects (13Braga-Basaria M. Dobs A.S. Muller D.C. Carducci M.A. John M. Egan J. Basaria S. Metabolic syndrome in men with prostate cancer undergoing long-term androgen-deprivation therapy.J. Clin. Oncol. 2006; 24: 3979-3983Crossref PubMed Scopus (461) Google Scholar). On the contrary, testosterone replacement therapy in hypogonadal men resulted in reduced BMI; improved glucose tolerance and insulin sensitivity; decreased total cholesterol, LDL-C, and TG; and increased HDL cholesterol (HDL-C) levels (14Saad F. Gooren L.J. Haider A. Yassin A. A dose-response study of testosterone on sexual dysfunction and features of the metabolic syndrome using testosterone gel and parenteral testosterone undecanoate.J. Androl. 2008; 29: 102-105Crossref PubMed Scopus (106) Google Scholar). The realization that medical castration contributes to metabolic perturbations led to the reevaluation of ADT risks in PCa patients (15Levine G.N. D'Amico A.V. Berger P. Clark P.E. Eckel R.H. Keating N.L. Milani R.V. Sagalowsky A.I. Smith M.R. Zakai N. Androgen-deprivation therapy in prostate cancer and cardiovascular risk: a science advisory from the American Heart Association, American Cancer Society, and American Urological Association: endorsed by the American Society for Radiation Oncology.Circulation. 2010; 121: 833-840Crossref PubMed Scopus (268) Google Scholar) but also emphasized the beneficial role that androgens may play in the general well-being of men. Overall, hypogonadism has been identified as an independent risk factor for cardiovascular morbidity and mortality (16Svartberg J. von Mühlen D. Mathiesen E. Joakimsen O. Bønaa K.H. Stensland-Bugge E. Low testosterone levels are associated with carotid atherosclerosis in men..J. Intern. Med. 2006; 259: 576-582Crossref PubMed Scopus (146) Google Scholar, 17Aversa A. Bruzziches R. Francomano D. Rosano G. Isidori A.M. Lenzi A. Spera G. Effects of testosterone undecanoate on cardiovascular risk factors and atherosclerosis in middle-aged men with late-onset hypogonadism and metabolic syndrome: results from a 24-month, randomized, double-blind, placebo-controlled study.J. Sex. Med. 2010; 7: 3495-3503Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar). Though some cases of human obesity may be caused solely by genetic factors (18Barsh G.S. Farooqi I.S. O'Rahilly S. Genetics of body-weight regulation.Nature. 2000; 404: 644-651Crossref PubMed Scopus (641) Google Scholar), Western-type diet combined with sedentary lifestyle, physical inactivity, and imbalance in caloric load are the most common contributors to the development of central obesity and metabolic syndrome (19Kopelman P.G. Obesity as a medical problem.Nature. 2000; 404: 635-643Crossref PubMed Scopus (3651) Google Scholar, 20Friedman J.M. Obesity in the new millennium.Nature. 2000; 404: 632-634Crossref PubMed Scopus (629) Google Scholar). Recent studies by us and others have suggested that the lipoprotein metabolic pathway is a pivotal contributor to the development of obesity and related metabolic dysfunctions in experimental mice. Specifically, mice deficient in ApoE, a key protein in the clearance of chylomicron remnants, LDL, and VLDL in plasma, are resistant to diet-induced obesity, insulin resistance, and glucose intolerance (21Karagiannides I. Abdou R. Tzortzopoulou A. Voshol P.J. Kypreos K.E. Apolipoprotein E predisposes to obesity and related metabolic dysfunctions in mice.FEBS J. 2008; 275: 4796-4809Crossref PubMed Scopus (62) Google Scholar, 22Kypreos K.E. Karagiannides I. Fotiadou E.H. Karavia E.A. Brinkmeier M.S. Giakoumi S.M. Tsompanidi E.M. Mechanisms of obesity and related pathologies: role of apolipoprotein E in the development of obesity.FEBS J. 2009; 276: 5720-5728Crossref PubMed Scopus (51) Google Scholar), while knock-in ApoE3 transgenic mice are sensitized toward these conditions (21Karagiannides I. Abdou R. Tzortzopoulou A. Voshol P.J. Kypreos K.E. Apolipoprotein E predisposes to obesity and related metabolic dysfunctions in mice.FEBS J. 2008; 275: 4796-4809Crossref PubMed Scopus (62) Google Scholar). Similarly, expression levels of LDL receptor-related protein 1 (Lrp1), a member of the LDL receptor (Ldlr) superfamily that binds lipoprotein-associated ApoE modulates the development of obesity and its related metabolic perturbations mainly by regulating the flux of dietary TGs to hepatic and adipose tissues and the energy expenditure (EE) of the tested mice (23Hofmann 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). Along the same lines, we have also observed that lack of classical Ldlr-mediated clearance of chylomicron remnants slows down diet-induced obesity following feeding mice with standard Western-type diet (21Karagiannides I. Abdou R. Tzortzopoulou A. Voshol P.J. Kypreos K.E. Apolipoprotein E predisposes to obesity and related metabolic dysfunctions in mice.FEBS J. 2008; 275: 4796-4809Crossref PubMed Scopus (62) Google Scholar), though it has no effect on TG deposition to the liver (24Karavia E.A. Papachristou D.J. Kotsikogianni I. Giopanou I. Kypreos K.E. Deficiency in apolipoprotein E has a protective effect on diet-induced nonalcoholic fatty liver disease in mice.FEBS J. 2011; 278: 3119-3129Crossref PubMed Scopus (35) Google Scholar). Despite the key role of plasma lipoproteins in the management of postprandial lipids and a number of clinical and epidemiological studies indicating that TD is associated with obesity and metabolic syndrome, the functional cross-talk between testosterone and lipoprotein metabolism in the development of diet-induced obesity remains vaguely defined. In the present study, we report that in contrast to studies in wild-type animals, TD in Ldlr−/− mice results in reduced body fat and body weight gain, improved glucose metabolism and plasma TG levels, and increased EE, despite an aggravation in plasma cholesterol profile. Our data suggest that Ldlr is a functional modulator of processes associated with hypogonadism-induced metabolic alterations. The Ldlr−/−, ApoE−/−, and C57BL/6 mice used in our studies were purchased from Jackson Labs (Bar Harbor, ME). Male mice 10–12 weeks old were used in these studies. Mice in each group were caged individually (one mouse per cage) and were allowed unrestricted access to food and water under a 12 h light/dark cycle. To ensure similar average cholesterol, TG, and glucose levels and starting body weights, groups of mice were formed after determining the fasting cholesterol, TG, and glucose levels and body weights of the individual mice. In all studies involving high-fat diet, we used the standard Western-type diet (Mucedola SRL, Milano, Italy) that is composed of 17.3% protein, 48.5% carbohydrate, 21.2% fat, and 0.2% cholesterol (0.15% added, 0.05% from fat source) and contains 4.5 kcal/g. The content of the main ingredients expressed as grams per kilogram of diet is the following: casein 195, DL-methionine 3, sucrose 341.46, corn starch 150, anhydrous milkfat 210, cholesterol 1.5, cellulose 50, mineral mix 35, calcium carbonate 4, vitamin mix 10, ethoxyquin, and antioxidant 0.04. For the dietary studies, two groups of mice of the same genotype were formed to have similar starting body weights and plasma cholesterol, TG, and glucose levels. One group was surgically castrated, and the other group was sham operated. Following a recovery period of 4 weeks on chow diet, to allow for testosterone clearance from the circulation of castrated mice, all mouse groups were switched to Western-type diet (week 0) for an additional period of 12 weeks. At the end of each experiment, mice were euthanized, and plasma and tissue samples were collected. Carcasses were stored at −80°C. All animal studies were governed by the European Union guidelines of the Protocol for the Protection and Welfare of Animals. In our experiments, we took into consideration the 3Rs (reduce, refine, replace), and we minimized the number of animal experiments to the absolute minimum. To this date, there is no in vitro system to mimic satisfactorily the lipid and lipoprotein transport system and the in vivo effects of hypogonadism on metabolism, making the use of experimental animals mandatory. The work was authorized by the appropriate committee of the Laboratory Animal Center of the University of Patras Medical School and the Veterinary Authority of the Prefecture of Western Greece. Surgical castration of mice was performed as described previously (25Traish A.M. Park K. Dhir V. Kim N.N. Moreland R.B. Goldstein I. Effects of castration and androgen replacement on erectile function in a rabbit model.Endocrinology. 1999; 140: 1861-1868Crossref PubMed Google Scholar). Briefly, experimental mice were divided into two groups. One group was sham operated by performing only a skin incision. The other group of animals was bilaterally castrated through 1 cm scrotal incisions. Both groups were operated under isofluorane (CP Pharma, Burgdorf, Germany) anesthesia. The animals were allowed to recover from the operation for a period of 4 weeks, and plasma testosterone levels were determined to confirm the success of the operation. Then both groups were switched to Western-type diet for the indicated period of time. For the testosterone replacement study, Ldlr−/− mice were surgically castrated, split into two groups, and switched to Western-type diet. Immediately after, one group was treated with intramuscular administration of 25 mg testosterone (Nebido, Bayer, Frankfurt, Germany) per kg of body weight, once a week, for a period of 8 weeks. The other group was treated with the carrier solution as placebo (a mix of castor oil and benzylbenzoate at a ratio of 4:1; Sigma-Aldrich, St. Louis, MO). Body weights of animals in both groups were monitored weekly. To confirm the success of the experiment, plasma testosterone levels were determined in both groups as described subsequently, immediately prior to castration, 4 weeks after castration (week 0), and then 8 weeks following testosterone supplementation. At the end of each experiment, mice were euthanized, and plasma and tissue samples were collected. Carcasses were stored at −80°C. For exemestane treatment, sham-operated Ldlr−/− mice were split into two groups, and then all mice were switched to Western-type diet. Immediately after, one group was treated with 40 mg of exemestane (Sigma-Aldrich) per kg of body weight (dissolved in carrier solution containing 0.3% w/v hydroxypropyl cellulose; Sigma-Aldrich), once per week, as previously described (26Goss P.E. Qi S. Cheung A.M. Hu H. Mendes M. Pritzker K.P. Effects of the steroidal aromatase inhibitor exemestane and the nonsteroidal aromatase inhibitor letrozole on bone and lipid metabolism in ovariectomized rats.Clin. Cancer Res. 2004; 10: 5717-5723Crossref PubMed Scopus (112) Google Scholar), for a period of 8 weeks. The other group was treated with only carrier solution. During the course of the experiment, body weights of the animals of both groups were monitored weekly. To confirm the success of the experiment, plasma estradiol levels were determined in both groups as described subsequently, immediately prior to sham operation and 8 weeks following exemestane treatment. At the end of each experiment, mice were euthanized, and plasma and tissue samples were collected. Carcasses were stored at −80°C. Blood was drawn from the tail vein at the indicated times, and serum samples were separated by centrifugation (4,000 rpm, 10 min) and stored at −20°C until used for testosterone and estradiol measurements. Plasma testosterone levels were determined using the Mouse/Rat Total Testosterone Elisa Kit (cat # TE187S-100, Calbiotech, Spring Valley, CA), and estradiol levels were determined using the Mouse/Rat Estradiol Kit (cat # ES180S-100, Calbiotech), according to the manufacturer's instructions. Indirect calorimetry studies were performed using the Calocage small animal colorimetry system (TSE Systems, Frankfurt, Germany). Groups of sham-operated and castrated mice were acclimated in the calocages for 48 h, and then EE was determined based on the carbon dioxide production expressed as a the difference of carbon dioxide exiting from carbon dioxide entering the metabolic cage [VCO2 (ml/h)] and oxygen consumption expressed as the difference of oxygen entering from oxygen exiting the metabolic cage [VO2 (ml/h)] over an additional 24 h period (12 h dark/12 h light). A total of 72 measurements of VCO2 and VO2 were collected. The EE per mouse (expressed in J/h) for each measurement was calculated by the formula EE = (15.818 × VO2) + (5.176 × VCO2) (27Tschöp M.H. Speakman J.R. Arch J.R. Auwerx J. Brüning J.C. Chan L. Eckel R.H. Farese Jr, R.V. Galgani J.E. Hambly C. et al.A guide to analysis of mouse energy metabolism.Nat. Methods. 2012; 9: 57-63Crossref Scopus (542) Google Scholar). Then, the average EE of the last 72 different measurements (1 every 20 min, starting at 7:00 PM on one day and ending at 7:00 PM on the following day) was determined and plotted as a function of body weight, and ANCOVA was performed between the two groups of mice (27Tschöp M.H. Speakman J.R. Arch J.R. Auwerx J. Brüning J.C. Chan L. Eckel R.H. Farese Jr, R.V. Galgani J.E. Hambly C. et al.A guide to analysis of mouse energy metabolism.Nat. Methods. 2012; 9: 57-63Crossref Scopus (542) Google Scholar). Respiratory exchange ratio (RER) was determined as the ratio of VCO2 over VO2. Following a 16 h fasting period (starting at 6:30 PM on the first day and ending at 10:30 AM the next day), plasma samples were isolated from the experimental mice. Plasma cholesterol and TG levels were measured using the Cholesterol Kit (Thermo Fisher Scientific, Waltham, MA) and Triglyceride Determination Kit (Sigma-Aldrich), respectively, according to the manufacturer's instructions and as described previously (28Kypreos K.E. ABCA1 promotes the de novo biogenesis of apolipoprotein CIII–containing HDL particles in vivo and modulates the severity of apolipoprotein CIII–induced hypertriglyceridemia.Biochemistry. 2008; 47: 10491-10502Crossref PubMed Scopus (46) Google Scholar). In the present study, we used density gradient ultracentrifugation, the gold standard for the separation of plasma lipoproteins. Therefore, for the determination of plasma cholesterol and TG levels in various plasma lipoproteins, 0.5 ml of pools of plasma from five mice per group was fractionated by density gradient ultracentrifugation over a 10 ml KBr (Sigma-Aldrich) density gradient in a Beckmann-Coulter Ultracentrifuge (Indianapolis, IN) using an SW41 rotor, as described previously (28Kypreos K.E. ABCA1 promotes the de novo biogenesis of apolipoprotein CIII–containing HDL particles in vivo and modulates the severity of apolipoprotein CIII–induced hypertriglyceridemia.Biochemistry. 2008; 47: 10491-10502Crossref PubMed Scopus (46) Google Scholar). The cholesterol and TG content of different density fractions was determined as described previously. The experiment was repeated three times with different sets of mice. At the indicated time points during the course of the experiments, mice in each group were briefly anesthetized using isofluor­ane, and their body weight was determined by a Mettler-Toledo® precision microscale (Mettler-Toledo, Columbus, OH). At the end of each experiment, at least six mice from each group were euthanized. Mouse carcasses were weighed to determine wet weight, and then they were dehydrated at 65°C until a constant mass was achieved (dry weight). The dried carcasses were then dissolved completely in 200 ml of ethanolic potassium hydroxide solution (5 M KOH in 50% ethanol; Sigma-Aldrich), the pH of the solution was adjusted to 7, and the final volume was recorded. An aliquot of this solution was used for enzymatic determination of glycerol as a measure of TG content and for total protein determination. Total water content was calculated as wet weight minus dry weight, and lean body mass (LBM) was calculated as wet weight minus total lipid weight. Results are expressed as milligrams of TGs per gram of tissue ± SEM. Food intake was assessed by determining the difference in food weight during a 7 day period to ensure reliable measurements, as described previously (21Karagiannides I. Abdou R. Tzortzopoulou A. Voshol P.J. Kypreos K.E. Apolipoprotein E predisposes to obesity and related metabolic dysfunctions in mice.FEBS J. 2008; 275: 4796-4809Crossref PubMed Scopus (62) Google Scholar, 29Duivenvoorden I. Teusink B. Rensen P.C. Romijn J.A. Havekes L.M. Voshol P.J. Apolipoprotein C3 deficiency results in diet-induced obesity and aggravated insulin resistance in mice.Diabetes. 2005; 54: 664-671Crossref PubMed Scopus (88) Google Scholar). For fasting plasma glucose determination, mice were fasted for 16 h (starting at 6:30 PM on the first day and ending at 10:30 AM the next day). Blood was drawn from the tail vein, and glucose was determined using the ACCU-CHECK AVIVA glucometer (Roche, Nutley, NJ) and comfort curve test strips. To determine the effects of TD on glucose tolerance, we performed a classical glucose tolerance test (GTT). Briefly, mice were fasted overnight (16 h), then dextrose (2 g/kg in PBS; Sigma-Aldrich) was injected intraperitoneally; serum samples were collected at 0, 15, 30, 60, and 120 min postinjection through the tail vein; and glucose levels were measured. Mitochondria from brown adipose tissue (BAT) and white adipose tissue (WAT) were isolated as described previously (30Commins S.P. Watson P.M. Padgett M.A. Dudley A. Argyropoulos G. Gettys T.W. Induction of uncoupling protein expression in brown and white adipose tissue by leptin.Endocrinology. 1999; 140: 292-300Crossref PubMed Scopus (136) Google Scholar). Briefly, BAT and the contralateral fat pad from each depot site were dissected immediately after animals were euthanized. Tissues were minced in ice-cold sucrose buffer [0.25 M sucrose and 5.0 mM N-Tris, i.e., (hydroxymethyl)methyl-2-aminoethanesulfonic acid buffer, pH 7.2; Sigma-Aldrich]; diluted to 10% and 5% (w/v), respectively, in sucrose buffer; and homogenized with a glass-Teflon homogenizer. Homogenates were then centrifuged at 22,500 g for 20 min, and the pellet was resuspended in ice-cold sucrose buffer. After a low-speed spin at 850 g for 10 min, the supernatant, containing crude mitochondria, was decanted to a fresh tube and spun for 20 min at 48,000 g. The pelleted mitochondria were then resuspended in 2 ml solubilization buffer containing 20 mM Tris (pH 8), 1 mM EDTA, 100 Mm NaCl, and 0.9% sodium cholate (Sigma-Aldrich) and incubated on ice for 30 min. Mitochondria samples were then recentrifuged at 48,000 g for 30 min. The pellet was resuspended in solubilization buffer containing 1% Triton X-100 and incubated on ice for 30 min. The suspension was recentrifuged at 48,000 g, and the supernatant was retained for protein assay and Western blotting. The protein concentration of each mitochondrial sample was determined using the detergent-compatible protein assay (cat #500-0006, Bio-Rad Laboratories Inc.). Western blotting for murine ApoE, cytochrome c, uncoupling protein 1 (Ucp1), and cytochrome c oxidase subunit 4 (Cox4) was performed using IgG primary rabbit anti-mouse antibodies (cat# K23100R, Meridian, Memphis, TN; cat# 4272, Cell Signaling, Danvers, MA; cat# GTX10983, Acris, Herford, Germany; cat# Ab165056, AbCam, Cambridge, UK, respectively). Briefly, mitochondrial samples from BAT (3 μg/lane) and WAT (10 μg/lane) were resolved by SDS-PAGE (12.5% acrylamide and 0.51% N,N'-diallyltartardiamide), transferred to polyvinylidene difluoride membranes, and probed with the corresponding primary antibodies. Semiquantitative determination of the relative protein amounts was performed by Image J free software. Total RNA was extracted from fresh frozen liver tissue using the NucleoSpin RNA/Protein kit from Macherey-Nagel (cat# 740933.50, Dueren, Germany) according to the manufacturer's instructions. Reverse transcription was performed using the PrimeScriptTM RT reagent kit from Takara Bio Inc. (cat# RR037A, Otsu, Shiga, Japan). Real-Time PCR for ApoE, Lrp1, and the housekeeping gene Rps18 was performed in a MicroAmp® 96-well reaction plate from Applied Biosystems (cat# 4346906, Foster City, CA) using the KAPA SYBR® FAST Universal qPCR kit from Kapa Biosystems (cat# KK4601, Woburn, MA), in an Applied Biosystems® StepOne™ cycler. Primers used are shown in Table 1. Primers were synthesized by Invitrogen-Life Technologies (Renfrewshire, Scotland). Data were normalized for Rps18 expression.TABLE 1Primers used in the real-time PCR analysis of ApoE and Lrp1 gene expressionPrimer NamePrimer SequenceMurine Lrp1 forward5′-GAC CAG GTG TTG GAC ACA GAT G-3′Murine Lrp1 reverse5′-AGT CGT TGT CTC CGT CAC ACT TC-3′Murine ApoE forward5′-CTG ACA GGA TGC CTA GCC G-3′Murine ApoE reverse5′-CGC AGG TAA TCC CAG AAG C-3′Murine Rps18 forward5′-GTA ACC CGT TGA ACC CCA TT-3′Murine Rps18 reverse5′-CCA TCC AAT CGG TAG TAG CG-3′ Open table in a new tab Data are reported as mean ± estimated SEM (* P < 0.05, ** P < 0.005; n indicates the number of animals tested in each experiment). Comparison of data from two groups of mice was performed using the Student's t-test. Analysis of metabolic data was performed by ANCOVA, a statistical test that in our case evaluates whether population means of EE (our dependent variable) differ between mice of different genotype (our independent variable), while statistically controlling for the effects of body weight on EE (our covariate). The F value is the ratio of variability between groups (which in our case relates to genotype differences), divided by the variability within group (which in our case relates to body weight differences), and is a measure of the difference in EE between groups. The higher the F value, the higher the difference between groups when corrected for body weight, while P value indicates the statistical significance of the observed differences. All statistical tests were performed using SPSS software (released 2009, PASW statistics for Windows, Version 18.0; SPSS Inc., Chicago, IL). Twelve mice deficient for the classical Ldlr and 12 C57BL/6 mice were either surgically castrated or sham operated as described in Materials and Methods. Following a period of 4 weeks on chow diet, mice were placed for an additional 12 weeks on Western-type diet, and then biochemical and metabolic analyses were performed. In agreement with our previous data (21Karagiannides I. Abdou R. Tzortzopoulou A. Voshol P.J. Kypreos K.E. Apolipoprotein E predisposes to obesity and related metabolic dysfunctions in mice.FEBS J. 2008; 275: 4796-4809Crossref PubMed Scopus (62) Google Scholar), sham-operated Ldlr−/− mice showed a considerable increase in their body weight after consuming Western-type diet for 12 weeks. However, to our great surprise castrated animals remained lean during the course of the experiment, and thus resistant to diet-induced body weight gain (Fig. 1A). Specifically, the sha" @default.
• W2045759524 created "2016-06-24" @default.
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• W2045759524 date "2014-07-01" @default.
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• W2045759524 title "The low density lipoprotein receptor modulates the effects of hypogonadism on diet-induced obesity and related metabolic perturbations" @default.
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