3700 Diabetes Volume 64, November 2015 Anne-Laure Poher,1 Christelle Veyrat-Durebex,2 Jordi Altirriba,1 Xavier Montet,3 Didier J. Colin,4 Aurélie Caillon,1 Jacqueline Lyautey,1 and Françoise Rohner-Jeanrenaud1 Ectopic UCP1 Overexpression in White Adipose Tissue Improves Insulin Sensitivity in Lou/C Rats, a Model of Obesity Resistance Diabetes 2015;64:3700–3712 | DOI: 10.2337/db15-0210 Brown adipose tissue (BAT), characterized by the pres- epidemic is long-term dysregulation of energy balance. In view ence of uncoupling protein 1 (UCP1), has been described oftherelativelackofdrugssuppressing appetite, approaches as metabolically active in humans. Lou/C rats, originat- to increase energy expenditure are viewed as potential new ing from the Wistar strain, are resistant to obesity. We therapeutic options to treat obesity and metabolic complica- previously demonstrated that Lou/C animals express tions. Along this line, brown adipose tissue (BAT), now known UCP1 in beige adipocytes in inguinal white adipose tissue to be present in adult humans (2–4),isthefocusofmajor (iWAT), suggesting a role of this protein in processes interest, due to its role in inducing thermogenesis (5). The such as the control of body weight and the observed mitochondria of brown adipocytes are characterized by the b improved insulin sensitivity. A 3 adrenergic agonist was presence of uncoupling protein 1 (UCP1), which uncouples administered for 2 weeks in Wistar and Lou/C rats to oxidative phosphorylation from ATP synthesis, resulting in activate UCP1 and delineate its metabolic impact. The heat production (6). This process consumes substantial treatment brought about decreases in fat mass and METABOLISM amounts of free fatty acids (FFAs) and glucose (7). improvements in insulin sensitivity in both groups. In BAT, UCP1 expression increased similarly in response to In rodents, brown adipocytes are found in discrete areas, the treatment in the two groups. However, the intervention such as interscapular, cervical, peri-renal, and intercostal “ ” induced the appearance of beige cells in iWAT, associated depots (8), which are referred to as classical BAT depots. In with a marked increase in UCP1 expression, in Lou/C white adipose tissue (WAT), brown-like cells, called beige or fi rats only. This increase was correlated with a markedly brite cells, express UCP1 (9). The existence of a speci cpre- enhanced glucose uptake measured during euglycemic- cursor, different from classical white or brown adipocytes, hyperinsulinemic clamps, suggesting a role of beige cells arising from smooth muscle cells that would differentiate in this process. Activation of UCP1 in ectopic tissues, such into beige adipocytes in WAT has been proposed (10). More- as beige cells in iWAT, may be an interesting therapeutic over, some studies suggest that, under specificconditions, approach to prevent body weight gain, decrease fat mass, most or all of white adipocytes transdifferentiate into beige and improve insulin sensitivity. adipocytes (11). Interestingly, whatever their developmental origin, white, beige, and brown adipocytes seem to greatly differ Obesity has reached epidemic proportions worldwide and in their function. As mentioned above, BAT is the effector has become a major global public health problem in recent organ of nonshivering thermogenesis that, by utilizing decades (1). It represents a considerable risk factor for the large quantities of glucose and lipids from the circulation, development of several comorbidities, among which is type can promote negative energy balance. Moreover, as recently 2 diabetes (1). Globally, the key component of the obesity reviewed by Peirce and Vidal-Puig (12), the role of BAT 1Laboratory of Metabolism, Division of Endocrinology, Diabetology, Hypertension Corresponding author: Anne-Laure Poher, [email protected]. and Nutrition, Department of Internal Medicine Specialties, Faculty of Medicine, Received 13 February 2015 and accepted 20 July 2015. University of Geneva, Geneva, Switzerland This article contains Supplementary Data online at http://diabetes 2Department of Cell Physiology and Metabolism, Faculty of Medicine, University .diabetesjournals.org/lookup/suppl/doi:10.2337/db15-0210/-/DC1. of Geneva, Geneva, Switzerland 3Division of Radiology, Faculty of Medicine, University of Geneva, Geneva, Switzerland © 2015 by the American Diabetes Association. Readers may use this article as 4MicroPET/SPECT/CT Imaging Laboratory, Centre for BioMedical Imaging, University long as the work is properly cited, the use is educational and not for profit, and Hospital of Geneva, Geneva, Switzerland the work is not altered. diabetes.diabetesjournals.org Poher and Associates 3701 activation might be broader than solely promoting negative administered. Blood samples were collected by tail nicking energy balance. Indeed, such activation was described to and were used for further analyses of plasma glucose and exert anti–type 2 diabetic effects (13,14), associated with insulin levels. improvements of dyslipidemia (rev. in 15). These effects are partly interrelated but can also be dissociated and EHC exerted by different UCP1-expressing adipocyte types. Rats were overnight fasted and anesthetized with pentobar- The Lou/C rat is a model of age- and diet-induced bital (50 mg/kg i.p.; Abbott Laboratories, Chicago, IL). The obesity resistance, which also exhibits a lower body fat glucose infusion rate (GIR) required to maintain euglycemia mass, increased leptin sensitivity, and improved insulin under insulin-stimulated conditions (18 mU/kg/min; Actrapid sensitivity compared with Wistar animals (16–18). We HM, Novo Nordisk, Bagsvaerd, Denmark) was determined also demonstrated that the most striking differences as previously described (19,20). At the end of the EHC, the between Lou/C and Wistar rats were the presence, in in vivo insulin-stimulated glucose utilization index inguinal WAT (iWAT) of the Lou/C group, of Ucp1 (16,17) of individual tissues was measured, using 2-deoxy-D- 3 m and of a marked Adrb3 overexpression (17). [1,2- H]glucose (30 Ci/rat, NET 328A; PerkinElmer, In the current study, we used the Lou/C rat as a model Schwerzenbach, Switzerland) (20). Rats were killed by – to investigate the impact of UCP1 activation on glucose decapitation and tissues stored at 80°C. The 2-deoxy-D- 3 – fi metabolism. To this end, various groups of Wistar and Lou/C [1,2- H]glucose speci c activity was measured in deproteinized rats were subcutaneously infused for 2 weeks with a b3 blood samples. Determination of tissue concentration of 3 adrenoreceptor agonist (CL-316243), and UCP1 in BAT and 2-deoxy-D-[1,2- H]glucose-6-phosphate allowed for the cal- WAT depots, as well as the insulin-stimulated glucose utili- culation of the in vivo glucose utilization index of individ- zation rate, of different tissues was determined. ual tissues and was expressed in nanograms of glucose per milligram of tissue per minute (21,22). RESEARCH DESIGN AND METHODS Plasma Measurements Animals and Diets Plasma glucose levels were measured by the glucose Two month-old male Lou/C and Wistar rats were pur- oxidase method (Glu; Roche Diagnostics GmbH, Rotkreuz, chased from Harlan UK Limited (Oxon, U.K.) and Charles Switzerland). FFA and triglyceride (TG) levels were deter- ’ River (L Arbresle, France), respectively. They were housed mined using a Wako Chemicals GmbH (Neuss, Germany) – in pairs under controlled conditions (22°C; light on 7:00 A.M. and a Biomérieux (Marcy l’Etoile, France) kit, respectively. 7:00 P.M.) and were allowed free access to water and Plasma leptin (Linco Research, Inc., St Charles, MO) levels diet (RMI; Hersteller, Essex, U.K.). Osmotic minipumps were measured using a double antibody radioimmuno- b (Alzet, Cupertino, CA) delivering a 3 agonist (CL-316243, assay kit. Plasma insulin levels were determined using 1 mg/kg/day; Tocris, Bristol, U.K.,) or NaCl 0.9% for 14 days an ELISA commercial kit (10-1250; Mercodia, Uppsala, were placed subcutaneously at the age of 12 weeks. One week Sweden). before and during the treatment, body weight and food in- take were measured daily (9:00 A.M.). A first colony was sub- Tissue Processing and RT-PCR mitted to a glucose tolerance test (GTT) at day 9 and a body RNA was reverse transcribed (M-MLV-RT; Invitrogen, fat composition analysis by MRI (Echo-MRI) at day 14. A Basel, Switzerland) and quantitative PCR (qPCR) was second colony was submitted to a computed tomography performed using the SYBR green PCR Master Mix (Applied (CT) scan on day 12 and a euglycemic-hyperinsulinemic clamp Biosystems, Warrington, U.K.) on a Stepone Plus machine (EHC) on day 14. A third colony underwent a positron emis- (Applied Biosystems). Primers (Supplementary Table 1) sion tomography (PET) scan coupled to a micro-CT on day were designed with the PrimerExpress software (Applied 14. A fourth colony was housed in pairs at 22°C or at Biosystems). Results were normalized to the expression 30°C in a climatic chamber (Meditest H 1300L; Froilabo, levels of the housekeeping gene, ribosomal protein S29 Meysieu, France). (Rps29). Except for EHC experiments, all rats were killed between 9:00 A.M. and 1:00 P.M.,usingisoflurane (Halocarbon Labora- Mitochondrial DNA Copy Number tories, River Edge, NJ) anesthesia and rapid decapitation. Quantification of mitochondrial DNA (mtDNA) copy num- Trunk blood was collected to measure the concentration of ber was achieved by qPCR. Briefly, DNA was extracted from various hormones and metabolites. Tissues were freeze iWAT using the DNeasy Blood and Tissue kit (Qiagen, clamped and stored at –80°C for determination of gene Düsseldorf, Germany). Nuclear and mtDNA copy numbers and protein expressions. The procedures were approved by were assessed by real-time PCR using primers targeted the ethics committee of our university and were in accor- toward the Cox1 gene (for mtDNA) and nuclear RNAseP dance with the Swiss guidelines for animal experimentation. (for nuclear DNA). GTT Western Blotting Wistar and Lou/C rats were food deprived for 4 h (from Frozen tissues were homogenized in ice-cold RIPA buffer.