Supporting Online Material for

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1 Supporting Online Material for Regulation of Pancreatic β Cell Mass by Neuronal Signals from the Liver Junta Imai, Hideki Katagiri,* Tetsuya Yamada, Yasushi Ishigaki, Toshinobu Suzuki Hirohito Kudo, Kenji Uno, Yutaka Hasegawa, Junhong Gao, Keizo Kaneko, Hisamitsu Ishihara, Akira Niijima, Masamitsu Nakazato, Tomoichiro Asano, Yasuhiko Minokoshi, Yoshitomo Oka This PDF file includes: *To whom correspondence should be addressed. Materials and Methods Figs. S1 to S9 References Published 21 November 28, Science 322, 125 (28) DOI: /science

2 Supporting Online Materials METHODS Recombinant adenoviruses Recombinant adenoviruses encoding the constitutively active mutant of the Xenopus MEK1 (1) and the mouse MKK6 (2) genes as well as the control LacZ gene were prepared as reported previously (3) Adenoviruses encoding the dominant negative mutant of the mouse MEK1 gene (4) were kindly provided by Dr. M. Yokoyama (Kobe University). Animals Animal studies were conducted in accordance with Tohoku University institutional guidelines. Male C57BL/6N mice and Akita mice were purchased from Kyudo (Saga, Japan). ob/ob mice (C57BL/6J background) were purchased from Charles River (Yokohama, Japan). These mice were housed in an air-conditioned environment, with a 12-h light-dark cycle, and fed a regular unrestricted diet. The high fat diet-induced obese (HF) mice were fed high-fat chow, as described previously (5). Briefly, after 4 weeks of high-fat-chow loading, beginning at 5 weeks of age, body weight-matched C57BL/6N mice were injected with recombinant adenoviruses. To obtain streptozotocin (STZ)-induced diabetic mice, 15 mg/kg of STZ (Sigma, St. Louis, MO, USA) in citrate sodium buffer (ph 4.5) was administered intraperitoneally to C57BL/6N mice at 8 weeks of age. Glucose and insulin tolerance tests Glucose tolerance tests were performed on mice fasted for 1 hours as previously reported (5). Insulin tolerance tests were performed on ad libitum fed mice. Insulin (.25 U/kg of body weight) was injected into the intraperitoneal space as described previously (6). Plasma insulin levels were measured using a mouse insulin ELISA kit (Morinaga, Tokyo, Japan). Immunoblotting Liver samples obtained from mice that had been fasted for 1 hours were homogenized and subjected to immunoblotting as described previously (3). Antibodies to ERK (#912, Cell Signaling Technology, Danvers, MA, USA), phospho-erk (#4376, Cell Signaling Technology), p38 MAPK (#9212, Cell Signaling Technology) and phospho-p38 MAPK (#9216, Cell Signaling Technology) were commercially obtained. Intensities of the bands were quantified using ImageJ software. Pancreatic insulin and hepatic triglyceride contents Pancreatic insulin (7) and hepatic triglyceride (6) levels were measured as described

3 previously. Islet studies Pancreatic islet isolation and secretion studies were performed as described previously (7). Evaluation of gene expression by RT-PCR Total RNAs were isolated from 5mg of mouse liver and gastrointestinal tissues and approximately 1, islets, as described previously (8). cdnas synthesized from total RNAs as described previously (8) were used as templates for PCR with the following oligonucleotides: Xenopus MEK1 forward, 5 -CAATAAGCTCATACGGTCCT, reverse, 5 -CTCAGCTCCAAATACTCCAC. Mouse MEK1 forward, 5 -CCTCTCCCATGCTTGTCTAT, reverse, 5 -TAGTTCAGCCTGGCATGTGT, PEPCK forward, 5 -TTGCCTGGATGAAGTTTGAT, PEPCK reverse, 5 -GGCATTTGGATTTGTCTTCACT, G6Pase forward, 5 -AAAGAGACTGTGGGCATCAATC, G6Pase reverse, 5 -AATGCCTGACAAGACTCCAGCC. Histological analysis The pancreases were excised on days 3, 9, 15 and 21 after adenoviral treatments, and fixed with 1% formalin and embedded in paraffin. As described previously (9), the entire pancreas was microscopically analyzed in each section (5 μm apart). After insulin staining, islet areas were measured in all sections using Scion Image software (Scion Corporation, Frederick, MD, USA) and islet masses were estimated as previously reported (1). For splanchnic nerve immunohistochemistry, celiac arteries with surrounding tissues were removed from vehicle- and capsaicin-treated mice, followed by fixation with 1% formalin and embedded in paraffin. Vagal nerve immunohistochemistry was performed as previously described (6). The streptavidin-biotin (SAB) method was performed using a Histofine SAB-PO kit (Nichirei, Tokyo, Japan). Immunoreactivity was visualized by incubation with a substrate solution containing 3,3 -diaminobenzidine tetrahydrochloride (DAB). The antibodies to insulin (SIGMA), calcitonin gene-related peptide (BIOMOL International, PA, USA), tyrosine hydroxylase (Chemicon International, CA, USA), S-1 protein (DAKO, Denmark) and GFP (Santa Cruz Biotechnology, CA, USA) were obtained commercially. For brain histology, mice were anesthetized and then perfused transcardially with saline and sequentially with 4% paraformaldehyde in.2m phosphate buffer (PB; ph 7.4). Brains were removed and chilled in.2m PB with 3% sucrose, followed by fixation with 1% formalin and embedded in paraffin. Tissue sections were subjected to

4 hematoxylin-eosin staining or Nissl staining with toluidine blue. BrdU in-situ detection BrdU in-situ detection was performed as described previously (9). Mice were injected intraperitoneally with 1mg of BrdU, 24 h before pancreas extraction. The labeled cells were immunostained with anti-brdu antibody. BrdU-positive cells in pancreatic islets were counted in each section (5 μm apart) of the whole pancreas. Vagotomy Seven days before adenoviral treatment, mice were subjected to hepatic vagotomy (HV), pancreatic vagotomy (PV) or sham operation. HV was performed as previously described (6). For PV, because the pancreas is innervated by both the ventral trunk and the celiac nerve of the vagus (11), we dissected both of these vagal branches as previously described (11) but with a slight modification. Briefly, a laparotomy incision was made on the ventral midline, the gastrohepatic ligament was severed, and the stomach was gently retracted, revealing the ventral subdiaphragmatic vagal trunk, which was then transected with fine forceps. In addition, the abdominal cavity was exposed, with the intestines, spleen and stomach reflected to the right side, to reveal the celiac nerve branching from the dorsal subdiaphragmatic vagal trunk running along the celiac artery, followed by transection of the celiac vagus using fine forceps. Sham operations were performed using an identical procedure, but with the nerves left intact. Selective blockade of afferent splanchnic nerve Seven days before adenoviral treatment, mice were subjected to selective afferent splanchnic nerve blockade. Splanchnic nerves form a sheath along the celiac artery (12) and contain afferent fibers from the hepatobiliary system (13). Direct topical application of capsaicin to a nerve is an effective means of deafferentation (6, 14, 15). Thus, for selective deafferentation of the spinal nerve from the liver, we applied capsaicin to the splanchnic nerve as previously described (16) with some modifications. A laparotomy incision was made on the ventral midline, and the celiac artery was exposed as described above. The celiac artery was wrapped in gauze immersed with or without capsaicin (Sigma) dissolved in olive oil (5% wt/vol) for 3 min. Because capsaicin treatment of splanchnic nerves significantly increased both food intake and body weight in ob/ob mice (data not shown), these mice were pair-fed following the vehicle- and capsaicin-treatments. The vehicle- and capsaicin-treated ob/ob mice were given the same daily food allotments based on the average consumption by vehicle-treated ob/ob mice. Bilateral midbrain transections Seven days before adenoviral treatment, mice were subjected to bilateral midbrain transaction, as previously reported (17). Briefly, the head was fixed in a stereotaxic

5 instrument. A steel knife (1.5mm wide) was lowered into the brain in a coronal plane, bilaterally.25mm from the midline, 4.mm caudal to the bregma, and 4.75mm ventral to the dura. For sham operation, the skull was exposed and two holes were drilled on both sides of the midline, while the brain was left intact. Behavioral tests were performed one week after surgery as previously reported (18). Briefly, food was removed from the cages 24-hours before testing, then saline or 5 μg/kg of cholecystokinin-sulfated octapeptide (BACHEM, Switzerland) was injected intraperitoneally 1 minute before the start of the test. Numbers and cumulative durations of pauses (in seconds) of behavioral activities were scored. Pauses were defined as complete behavioral inactivity for a minimum 3 second duration. Session length was 5 minutes. DETAILED FUNDING SOURCES This work was supported by Grants-in-Aids for Scientific Research to H. Katagiri. (B2, ), to Y. Oka (A2, ) and to J. Imai (279635) from the Ministry of Education, Culture, Sports, Science and Technology of Japan and a Grant-in-Aid for Scientific Research and to Y. Oka (H19-genome-5) from the Ministry of Health, Labor and Welfare of Japan. This work was also supported by the 21st Century COE Program Comprehensive Research and Education Center for Planning of Drug Development and Clinical Evaluation to H. Katagiri and the Global-COE Program for Network Medicine to Y. Oka from the Ministry of Education, Culture, Sports, Science and Technology of Japan. References 1. T. Miyazaki et al., J Cell Biol 148, 333 (Jan 24, 2). 2. M. Fujishiro et al., Mol Endocrinol 17, 487 (Mar, 23). 3. J. Imai et al., Biochem Biophys Res Commun 326, 42 (Jan 14, 25). 4. T. Ueyama et al., J Mol Cell Cardiol 32, 947 (Jun, 2). 5. Y. Ishigaki et al., Diabetes 54, 322 (Feb, 25). 6. K. Uno et al., Science 312, 1656 (Jun 16, 26). 7. H. Ishihara et al., Hum Mol Genet 13, 1159 (Jun 1, 24). 8. J. Imai et al., Obesity (Silver Spring) 14, 1132 (Jul, 26). 9. Y. Hasegawa et al., Endocrinology (Jan 25, 27). 1. T. Uchida et al., Nat Med 11, 175 (Feb, 25). 11. H. R. Berthoud, A. Niijima, J. F. Sauter, B. Jeanrenaud, J Auton Nerv Syst 7, 97 (Feb, 1983). 12. W. W. Lautt, Prog Neurobiol 21, 323 (1983). 13. H. R. Berthoud, Anat Rec A Discov Mol Cell Evol Biol 28, 827 (Sep, 24).

6 14. S. H. Buck, T. F. Burks, Pharmacol Rev 38, 179 (Sep, 1986). 15. C. A. Maggi et al., Neuroscience 31, 745 (1989). 16. S. Fujita, M. Bohland, G. Sanchez-Watts, A. G. Watts, C. M. Donovan, Am J Physiol Endocrinol Metab 293, E96 (Jul, 27). 17. Y. Date et al., Cell Metab 4, 323 (Oct, 26). 18. J. N. Crawley, J. Z. Kiss, E. Mezey, Brain Res 322, 316 (Nov 26, 1984).

7 Supplemental Figure Legends Figure S1. Phenotypic features of CAM-mice (1) A) Hepatic ERK phosphorylation of CAM-, ob/ob and high fat diet-fed C57BL/6N obese (HF) mice. Liver extracts from C57BL/6N mice on day 3 after injection of PFU/body of recombinant adenovirus containing the CAM gene (CAM), 8 week-old lean C57BL/6N without adenoviral administration (lean), 8 week-old ob/ob and HF mice were subjected to immunoblotting. B) GFP staining of livers from mice with (right) or without (left) administration of adenovirus containing the GFP gene. Liver extracts from 8 week-old lean C57BL/6N mice with or without adenoviral administration were subjected to immunostaining with antibodies against GFP. C) Expressions of exogenous (Xenopus) MEK1 (upper panel) and endogenous (mouse) MEK1 (lower panel) in the stomach (st), duodenum (du), jejunum (je), ileum (ile), colon (co) and liver (liv) of LacZ- and CAM-mice on day 3 after adenoviral administration. After 4 PCR cycles, the RT-PCR samples were subjected to gel electrophoresis. D) Hepatic ERK phosphorylation on day 3 after injection of CAM-adenovirus at a dose of 3 1 7, or PFU/body. Liver extracts from 8 week-old lean C57BL/6N mice administered each dose of adenovirus were subjected to immunoblotting. Liver extracts from 8 week-old lean C57BL/6N mice without adenoviral administration were used as a negative control ((-)).E) Hepatic triglyceride contents of LacZ- (open bars) and CAM- (filled bars) mice on day 3 (left) and day 14 (right) after adenoviral administration. F) Macroscopic and histological images of the livers from LacZ- (left) and CAM- (right) mice on day 44 after adenoviral administration; no tumor formation was detected in livers of CAM-mice (n=7). Representative images are shown The scale bars indicate 1μm. Data are presented as means ± SEM. P<.1 versus LacZ-mice, assessed by unpaired t test. Figure S2. Phenotypic features of CAM-mice (2) A) Glucose stimulated insulin secretion from isolated pancreatic islets of LacZ- (open bars) and CAM- (filled bars) mice on day 3 after adenoviral administration. B) Hepatic expression of phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6 phosphatase (G6Pase) of LacZ- (open bars) and CAM- (filled bars) mice on day 3 after adenoviral administration. C) Insulin staining of pancreases from LacZ- and CAM-mice on day 21 after adenoviral administration. Representative images are shown. The scale bars indicate 1μm. D) Body weights of LacZ- (open bar) and CAM- (filled bar) mice on day 16 after adenoviral administration. Data are presented as means ± SEM. P<.1 versus LacZ-mice, assessed by unpaired t test.

8 Figure S3. Long-term phenotypes of CAM-mice A) Blood glucose (left) and plasma insulin (right) levels during glucose tolerance tests performed on day 42 after adenoviral administration. Open and closed circles indicate LacZ- and CAM-mice, respectively. B) Pancreatic insulin content of untreated15 week-old mice (black bar) and of LacZ- (white bar) and CAM-mice (gray bar) on day 44 after adenoviral administration. The scale bars indicate 1μm. Data are presented as means ± SEM. * P<.5 versus LacZ-mice, assessed by unpaired t test. Figure S4. p38 MAPK is not involved in inter-organ communication A) Hepatic p38 MAPK phosphorylation on day 3 after injection of PFU/body of recombinant adenovirus containing LacZ (LacZ) or the constitutively active mutant of the MKK6 (CAMKK6) gene. B) Blood glucose (left) and plasma insulin (right) levels during glucose tolerance tests performed on day 3 after adenoviral administration. Open and closed circles indicate LacZ- and CAMKK6-mice, respectively. C) Pancreatic insulin content of LacZ- (open bar) and CAMKK6-mice (filled bar) on day 16 after adenoviral administration. D) Hepatic p38 MAPK phosphorylation in high fat diet-induced obese mice on day 7 after injection of recombinant adenovirus containing LacZ (LacZ) or the dominant-negative mutant of MEK1 (DNM) gene.data arepresented as means ± SEM. * P<.5, P<.1 versus LacZ-mice, assessed by unpaired t test. Figure S5. Pharmacological deafferentation of the splanchnic nerves was confirmed by immunohistochemistry A) Body weights of mice, subjected to sham operation (open bar) and pancreatic vagotomy (filled bar), on day 16 after CAM-adenoviral administration. B) Calcitonin gene-related peptide (CGRP) (upper) and tyrosine hydroxylase (lower) staining of splanchnic nerve fibers (arrows) from vehicle-treated (left) and capsaicin-treated (right) mice. C) CGRP (upper) and S1 (lower) staining of subdiaphragmatic vagal trunks from vehicle-treated (left) and capsaicin-treated (right) mice. Representative images are shown. The scale bars indicate 2μm. Data are presented as means ± SEM. Figure S6. Dissection of the celiac branch of the vagal nerve did not inhibit the insulinotropic effects observed in CAM-mice A) Blood glucose and plasma insulin levels during glucose tolerance tests performed on day 3 after adenoviral administration, following dissection of the celiac vagus. Open and closed circles indicate LacZ- and CAM-mice, respectively. B) Pancreatic insulin content

9 of LacZ- (open bar) and CAM- (closed bar) mice subjected to celiac vagus dissection on day 16 after adenoviral administration. Data are presented as means ± SEM. * P<.5, P<.1 versus LacZ-mice, assessed by unpaired t test. Figure S7. Bilateral midbrain transection was confirmed by behavioral tests and immunohistochemistry A) Cumulative pause duration (left) and number of pauses (right) of behavioral activities following intraperitoneal injection of saline (sal) or cholecystokinin (CCK) into sham operated (SO) and bilateral midbrain transected (MBT) mice. B) Hematoxylin-eosin (HE) or Nissl (NI) staining of sagittally sectioned brains from SO and MBT mice. The arrow indicates the lesion made by a knife cut. The scale bars indicate 5μm. Data are presented as means ± SEM. P<.1 versus saline treated mice, # P<.1 versus sham operated mice with CCK administration, assessed by unpaired t test. Figure S8. Effects of DNM-adenovirus administration and pancreatic vagotomy in mouse obesity models A) Hepatic ERK phosphorylation in high fat diet-induced obese (HF) mice on day 7 after administration of PFU/body of recombinant adenovirus containing LacZ (LacZ) or the dominant-negative mutant of the MEK1 (DNM) gene. B) Pancreatic insulin content of HF mice on day 8 after LacZ- (white bar) or DNM- (gray bar) adenovirus administration. Pancreatic insulin content of 9 week-old C57BL/6N mice fed high fat diet for 4 weeks were used as baseline controls (black bar). C) Blood glucose (left) and plasma insulin (right) levels during glucose tolerance tests performed on day 12 after pancreatic vagotomy in ob/ob mice. Open and closed circles indicate sham operated and pancreatic vagotomized ob/ob mice, respectively. Data are presented as means ± SEM. * P<.5, P<.1 versus LacZ-adenovirus-treated HF-mice (B) and sham operated ob/ob mice (C), assessed by unpaired t test. Figure S9. Glucose metabolism of CAM-adenovirus-treated insulin-deficient murine models A) Blood glucose (left) and plasma insulin (right) levels of LacZ- (open circles) and CAM- (filled circles) adenovirus-treated STZ-mice during glucose tolerance tests performed on day 16 after adenoviral administration. B) Blood glucose levels of LacZ- (open circles) and CAM- (filled circles) adenovirus-treated STZ-mice after intraperitoneal insulin injection performed on day 22 after adenoviral administration. Data are presented as percentages of the blood glucose levels immediately before insulin

10 loading. C) Blood glucose (left) and plasma insulin (right) levels of LacZ- (open circles) and CAM- (filled circles) adenovirus-treated Akita mice during glucose tolerance tests performed on day 14 after adenoviral administration. D) Blood glucose levels of LacZ- (open circles) and CAM- (filled circles) adenovirus-treated Akita mice after intraperitoneal insulin injection performed on day 1 after adenoviral administration. Data are presented as percentages of the blood glucose levels immediately before insulin loading. Data are presented as means ± SEM. * P<.5, P<.1 versus STZ-LacZ- (A), or Akita-LacZ- (C and E) mice, assessed by unpaired t test.

11 fig. S1 A B GFP (-) GFP (+) CAM lean ob/ob HF p-erk ERK C Xenopus MEK1 Mouse MEK1 LacZ CAM st du je ile co liv st du je ile co liv D p-erk ERK (-) E Hepatic triglyceride content (µg/mg protein) day day 14 F LacZ CAM

12 fig. S2 A Insulin secretion (% of contents) mM glu 15mM glu B Arbitray Unit PEPCK Arbitray Unit G6Pase C D 25 LacZ CAM Body weight (g)

13 fig. S3 A 2 Blood glucose (mg/dl) Plasma insulin (ng/ml) B Pancreatic insulin content (ng/mg pancreas) 2 1 *

14 fig. S4 A LacZ CAMKK6 p-p38 MAPK p38 MAPK B Blood glucose (mg/dl) Plasma insulin (ng/ml) * C Pancreatic insulin content (ng/mg pancreas) 2 1 D LacZ DNM p-p38 MAPK p38 MAPK

15 fig. S5 A 25 Body weight (g) B VEH splanchnic nerve CAP CGRP Ty-H VEH vagal nerve CAP CGRP S-1

16 fig. S6 A 2 Blood glucose (mg/dl) * * Plasma insulin (ng/ml) 1 * * B Pancreatic insulin content (ng/mg pancreas) 3 2 1

17 fig. S7 A Cumulative pause duration (seconds) /5min 2 1 # Number of pauses /5min # sal CCK sal CCK sal CCK sal CCK SO MBT SO MBT B SO MBT HE NI

18 fig. S8 A p-erk ERK LacZ DNM B Pancreatic insulin content (ng/mg pancreas) 3 * * * HF (baseline) HF-LacZ HF-DNM C Blood glucose (mg/dl) * Plasma insulin (ng/ml) *

19 fig. S9 A Blood glucose (mg/dl) Plasma insulin (ng/ml) * B 1 % of basal 8 6 C Blood glucose (mg/dl) Plasma insulin (ng/ml) * D 1 % of basal