Division of Hypothalamic Research, Departments of Internal Medicine and

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1 1 5-HT 2C Rs Expressed by Pro-opiomelanocortin Neurons Regulate Insulin Sensitivity in Liver Yong Xu 1, 3*, Eric D. Berglund 1*, Jong-Woo Sohn 1, William L. Holland 2, Jen-Chieh Chuang 1, Makoto Fukuda 1, Jari Rossi 1, Kevin W. Williams 1, Juli E. Jones 4, Jeffrey M. Zigman 1, Bradford B. Lowell 4, Philipp E. Scherer 2 and Joel K. Elmquist 1. 1 Division of Hypothalamic Research, Departments of Internal Medicine and Pharmacology, The University of Texas Southwestern Medical Center, Dallas, TX USA; 2 Touchstone Diabetes Center, Department of Internal Medicine, The University of Texas Southwestern Medical Center, Dallas, TX USA; 3 Children s Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, Houston, TX USA; 4 Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA USA. * These authors contributed equally to this work. Correspondence should be addressed to J.K.E. Joel K. Elmquist D.V.M. Ph.D. University of Texas Southwestern Medical Center at Dallas 5323 Harry Hines Boulevard, Dallas, Texas Tel: Fax: Joel.Elmquist@UTSouthwestern.edu

2 2 Supplemental Figure 1 Supplemental Figure 1. Re-expression of 5-HT 2C Rs in POMC neurons rescues insulin resistance caused by global 5-HT 2C R deficiency in HFD-fed mice. (a) Insulin tolerance tests in HFD-fed mice (insulin 2 U/kg). N=6-7/genotype. *, P<0.05 between 2C null mice vs WT mice. (b) Glucose tolerance tests in HFD-fed mice (glucose 0.75 g/kg). N=6 7/genotype. (c) Serum glucose at fed or fasted conditions. N=6 7/genotype. (d) Serum insulin in fed or fasted conditions. N=15 19/genotype. (e) Insulin secretion from isolated islets treated with 5 mm glucose (low glucose) or 17.5 mm glucose (high glucose). N=6 12/group. *, P<0.05. (f) Relative levels of POMC mrna in the arcuate nucleus measured by real-time PCR. N=6 8/genotype. *, P<0.05 and **, P<0.01. All data are presented as mean ± s.e.m.

3 3 Supplemental Table 1. Body weight and adiposity in study mice. WT (g) 2C null (g) 2C/POMC (g) Body weight in Fig. 2a 27.2± ± ± 0.6 Body fat in Fig. 2a 10.3± ± ± 1.4 Body weight in Fig. 2b-c (Fasted) 22.4± ± ± 0.9 Body weight in Fig. 2c (Fed) 25.9± ± ± 0.8 Body weight in Fig. 2d (Fed) 26.4± ± ± 0.6 Body weight in Fig. 2d (Fasted) 24.8± ± ± 1.4 Body weight in Fig. 2e 33.8± ± ± 1.0 Body weight in Fig. 2f-h 26.8± ± ± 0.9 Body weight in Fig. 2i (GTT, saline) 34.1± ± ± 1.4 Body weight in Fig. 2i (GTT, mcpp) 34.0± ± ± 1.2 Body weight in Fig. 2j (ITT, saline) 40.0± ± ± 1.4 Body weight in Fig. 2j (ITT, mcpp) 39.0± ± ± 1.2 Body weight in Suppl Fig. 1a, c (Fed) 32.9± ± ± 2.5 Body weight in Suppl Fig. 1b, c (Fasted) 33.0± ± ± 2.1 Body weight in Suppl Fig. 1d (Fed) 33.4± ± ± 0.8 Body weight in Suppl Fig. 1d (Fasted) 30.1± ± ± 1.2 All data are presented as mean ± s.e.m. No statistical significance was detected among age-matched littermates using one way ANOVA.

4 4 Supplementary Methods Generation of 2C null and 2C/POMC mice As described before, 2C null mice were generated by inserting a loxp-flanked transcription blocking cassette 1 into the 5-HT 2C R gene to globally disrupt its expression 2. Crossing 2C null mice with POMC-Cre mice produced 2C/POMC mice, in which expression of endogenous 5-HT 2C Rs was re-activated selectively in POMC neurons by Cre-recombinase 2. In the present study, female 2C null heterozygous mice (backcrossed to the C57BL/6J background for 10 generations) were crossed with male POMC-Cre transgenic mice (backcrossed to the C57BL/6J background for 7 generations). These crosses produced male littermates with one of the 4 genotypes: WT, POMC-Cre, 2C null and 2C/POMC. For most of the studies described below (including GTT, ITT, serum glucose and insulin measures), we included both WT and POMC-Cre mice as controls. Since no phenotypic difference was observed between the WT and POMC-Cre mice (data not shown), only WT data were presented in this paper and only WT mice were used for the rest of the studies (including clamps, mcpp treatments). Additional crosses were set up between female 2C null heterozygous mice with male POMC-Cre transgenic mice, with one of these breeders also carrying a transgenic POMC- EGFP reporter allele 3. The offspring, WT, 2C null and 2C/POMC mice that were also positive for the POMC-EGFP allele, were subject to the electrophysiological experiments described below. Animal care Care of all animals and procedures were approved by the UT Southwestern Medical Center Institutional Animal Care and Use Committees. Mice were housed in a temperature controlled environment in groups of two to five at 22 C 24 C using a 12 hr light/12 hr dark cycle. Animal diets (either 4% fat stand chow or 42% fat HFD, Harlan Teklad,

5 5 Madison, WI) and water were provided ad libitum. In particular, mice were fed with HFD from week 5 for 2 months to induce DIO. Body weight was monitored weekly and body composition was measured with the Bruker minispec mq10 MRS system. Mice with matched body weight and/or adiposity were chosen for the experiments described below. Importantly, all the mice were either fasted for 2 4 hrs (defined as fed conditions) or for overnight (defined as fasted conditions) prior to experiments. This was to avoid the possible influence from the different meal patterns on glucose/insulin profile. Glucose tolerance tests (GTTs) After an overnight fast, 3 month old mice received intraperitoneal injections of D-glucose (1 g/kg for chow-fed mice or 0.75 g/kg for HFD-fed mice). Blood glucose was measured from tail blood using a glucometer at serial time points as indicated in figures. To examine the effects of mcpp on glucose tolerance, overnight-fasted DIO mice were intraperitoneally injected with saline or mcpp (1.5 mg/kg), and 45 min later GTTs (0.75 g/kg glucose) were performed as described above. Insulin tolerance tests (ITTs) After a 2 hr fast to empty the stomach, 3 month old mice received intraperitoneal injections of insulin (1 U/kg for chow-fed mice or 2 U/kg for HFD-fed mice). Blood glucose was measured from tail blood as described above. To examine the effects of mcpp on insulin sensitivity, DIO mice were intraperitoneally injected with saline or mcpp (1.5 mg/kg), and 45 min later ITTs (1.5 U/kg insulin) were performed as described above. Basal glucose and insulin levels Fed and fasted glucose and insulin levels were measured in mice at 3 months of age. Food was removed from the home cages for 2 hr (fed condition) or overnight (fasted condition), and blood was collected from the tails. Glucose levels were directly measured from tail blood using a glucometer. Blood samples were centrifuged and serum samples

6 6 were collected from the supernatants. Insulin levels were measured using an ELISA kit (Crystal Chem Inc. Downers Grove, IL) according to manufacturer s instruction. Insulin secretion after a glucose load After an overnight fast, 3 month old DIO mice received intraperitoneal injections of D- glucose (0.75 g/kg). Tail blood was collected 30 min after the glucose load, and the samples were processed for insulin measurement as described above. Hyperinsulinemic-euglycemic clamp To identify site(s) of insulin resistance, hyperinsulinemic-euglycemic clamps were performed. Mice at 3 months of age were implanted with a jugular vein catheter that was externalized at the nape of the neck 5 day prior to study. The catheter was flushed daily with sterile saline and only mice returning to within ~10% of preoperative body weight was studied. On the day of study, mice were placed in a sterile cage at ~ 9:00am to begin a 4h fast. At t = 120 min, a primed continuous [3-3 H]glucose infusion (5 Ci bolus followed by 0.05 Ci/min) was given to measure glucose turnover. The clamp was started at t = 0 min with a continuous insulin infusion (4mU/kg/min), and the [3-3 H]glucose was increased to 0.1 Ci/min to minimize changes in specific activity. Blood glucose (3 l) was measured from the cut tail every 10 min. Euglycemia (150 mg/dl) was maintained using a variable glucose infusion rate (GIR). Blood samples (10 l) to determine glucose specific activity were taken at t = 15 and 5 min and every 10 min from t = min. Additional blood samples (20 l) were taken to measure serum insulin at t = 15 and 120 min. Insulin secretion from isolated islets After overnight fasting, 3 months old DIO mice were anesthetised with katemine (45 mg/kg) and xylyzine (5 mg/kg), and the pancreas was perfused and digested with liberase R1 (Roche). Islets were then isolated using Ficoll gradient centrifugation and handselection under a stereomicroscope for transfer to RPMI 1640 medium (11.1 mm glucose)

7 7 supplemented with 10% (v/v) heat-inactivated fetal bovine serum, 100 IU/ml penicillin, and 100 g/ml streptomycin (Invitrogen). After recovering for overnight (37 C, 5% CO 2 ), islets (6/well of 24-well culture plate) were incubated in Secretion Assay Buffer (containing M NaCl, 4.7 mm KCl, 1.2 mm KH 2 PO 4, 1.16 mm MgSO 4, mm NaHCO 3, 25 mm CaCl 2, 20 mm HEPES and 0,2% BSA, ph 7.4) without glucose for 1 hr, and then were stimulated with 5 or 17.5 mm glucose for 1 hr. Insulin levels in media were measured using an ELISA kit (Crystal Chem Inc.) according to manufacturer s instruction. Real-time PCR analysis of POMC expression in the ARC To measure POMC mrna levels in the ARC, 12-week old chow-fed male mice were decapitated after anesthesia (hydrochloride, 1 g/kg, i.p.) and the ARC was microdissected and quickly stored at 80ºC. Total mrnas from the ARC were extracted using an RNeasy kit (Qiagen) and reverse-transcribed to cdnas using SuperScript III First- Strand cdna Synthesis kit (Invitrogen, Carlsbad, CA) according to the manufacturer s instructions. Quantitative real-time PCR assays was performed according to published protocols 4. Tissue mrna levels were measured with an ABI 7900HT Sequence Detection System. We used pre-developed Taqman assays purchased from Applied Biosystems for POMC and 18S (as the housekeeping gene). Normalized mrna levels were expressed in arbitrary units obtained by dividing the averaged, efficiency-corrected values for sample mrna expression by that for 18S RNA expression for each sample. The resulting values were expressed as fold change above average WT levels. Electrophysiological studies To validate the loss of 5-HT 2C Rs in 2C null mice and the re-expression of 5-HT 2C Rs in POMC neurons in 2C/POMC, we examined the effect of mcpp (4 M) on the membrane potential of identified POMC neurons in WT, 2C null and 2C/POMC mice, respectively. As described previously 5-6, 4- to 10-week old mice were deeply anesthetized with chloral

8 8 hydrate and transcardially perfused with a modified ice-cold ACSF (described below), in which an equiosmolar amount of sucrose was substituted for NaCl. The mice were then decapitated, and the entire brain was removed and immediately submerged in ice-cold, carbogen-saturated (95% O 2 and 5% CO 2 ) ACSF (126 mm NaCl, 2.5 mm KCl, 1.2 mm MgCl 2, 2.4 mm CaCl 2, 1.25 mm NaH 2 PO 4, 26 mm NaHCO 3, and 10 mm glucose). Coronal sections (250 M) were cut with a Leica VT1000S Vibratome and then incubated in oxygenated ACSF at 32 C for at least 1 h before recording. Slices were transferred to the recording chamber and allowed to equilibrate for min prior to recording. The slices were bathed in oxygenated ACSF (32 C 34 C) at a flow rate of approximately 2 ml/min. Epifluorescence was briefly used to target fluorescent cells, at which time the light source was switched to infrared differential interference contrast imaging to obtain the whole-cell recording (Zeiss Axioskop FS2 Plus equipped with a fixed stage and a Hamamatsu C charged-coupled device camera). Membrane potentials were recorded in the current clamp mode using an Axopatch 700B amplifier (Axon Instruments), low-pass filtered at 1 khz, digitized at 10 khz and analyzed offline on a PC with pclamp programs (Axon Instruments), Origin (Microcal) or IgorPro (Wavemetrics). Recording electrodes had resistances of M when filled with the K-gluconate pipette solution which contains 120 mm K-gluconate, 10 mm KCl, 10 mm HEPES, 5 mm EGTA, 1 mm CaCl 2, 1 mm MgCl 2, and 5 mm Mg-ATP adjusted to ph 7.3. Statistical analysis Data were presented as mean ± s.e.m. Statistical analyses were carried out with SigmaStat software. For glucose and insulin levels (basal or after a glucose load), and real-time PCR data, results were analyzed by one-way ANOVA analysis, followed by the post-hoc Student-Newman-Keuls test when the ANOVA analysis indicated significant differences. For GTTs, ITTs and hyperinsulinemic-euglycemic clamp, results were analyzed by two-

9 9 way ANOVA with repeated measurements, followed by the post-hoc Student-Newman- Keuls test. For isolated islet study, results were analyzed by two-way ANOVA, followed by the post-hoc Student-Newman-Keuls test. P < 0.05 indicated statistical significance.

10 10 References 1. Zigman, J.M., et al. Mice lacking ghrelin receptors resist the development of dietinduced obesity. J Clin Invest 115, (2005). 2. Xu, Y., et al. 5-HT2CRs expressed by pro-opiomelanocortin neurons regulate energy homeostasis. Neuron 60, (2008). 3. Parton, L.E., et al. Glucose sensing by POMC neurons regulates glucose homeostasis and is impaired in obesity. Nature 449, (2007). 4. Bookout, A.F. & Mangelsdorf, D.J. Quantitative real-time PCR protocol for analysis of nuclear receptor signaling pathways. Nucl Recept Signal 1, e012 (2003). 5. Hill, J.W., et al. Acute effects of leptin require PI3K signaling in hypothalamic proopiomelanocortin neurons in mice. J Clin Invest 118, (2008). 6. Williams, K.W., et al. Segregation of acute leptin and insulin effects in distinct populations of arcuate proopiomelanocortin neurons. J Neurosci 30, (2010).