FGF21 Maintains Glucose Homeostasis by Mediating the Cross Talk Between Liver and Brain During Prolonged Fasting

4064 Diabetes Volume 63, December 2014 Qingning Liang,1,2 Ling Zhong,1,2 Jialiang Zhang,1,2 Yu Wang,1,3 Stefan R. Bornstein,4 Chris R. Triggle,5 Hong Ding,5 Karen S.L. Lam,1,2 and Aimin Xu1,2,3 FGF21 Maintains Glucose Homeostasis by Mediating the Cross Talk Between Liver and Brain During Prolonged Fasting Diabetes 2014;63:4064–4075 | DOI: 10.2337/db14-0541 Hepatic gluconeogenesis is a main source of blood glucose Hepatic gluconeogenesis is tightly controlled by counter- during prolonged fasting and is orchestrated by endocrine regulatory hormones such as glucagon, cortisol, and insulin, and neural pathways. Here we show that the hepatocyte- via regulating the expression of key gluconeogenic enzymes, secreted hormone fibroblast growth factor 21 (FGF21) including glucose 6 phosphatase (G6Pase) and phospho- induces fasting gluconeogenesis via the brain-liver axis. enolpyruvate carboxykinase (PEPCK). Fibroblast growth Prolonged fasting induces activation of the transcrip- factor 21 (FGF21), a metabolic regulator mainly secreted tion factor peroxisome proliferator–activated receptor a a from the liver in response to fasting and starvation under (PPAR ) in the liver and subsequent hepatic produc- – tion of FGF21, which enters into the brain to activate the the control of the nuclear receptor peroxisome proliferator hypothalamic-pituitary-adrenal (HPA) axis for release of activated receptor a (PPARa), plays a critical role in main- corticosterone, thereby stimulating hepatic gluconeogene- taining energy homeostasis and insulin sensitivity in both METABOLISM sis. Fasted FGF21 knockout (KO) mice exhibit severe rodents and nonhuman primates (1–6). A therapeutic dose hypoglycemia and defective hepatic gluconeogenesis due of FGF21 decreased blood glucose in diabetic animals with- to impaired activation of the HPA axis and blunted release out causing hypoglycemia (4). FGF21 has also been shown of corticosterone, a phenotype similar to that observed to act as a key downstream effector of PPARa,mediating in PPARa KO mice. By contrast, intracerebroventricular several metabolic adaptation responses to starvation, in- injection of FGF21 reverses fasting hypoglycemia and im- cluding hepatic fatty acid oxidation, ketogenesis, and pairment in hepatic gluconeogenesis by restoring cortico- growth hormone resistance (1,2,7). In addition, FGF21 a steroneproductioninbothFGF21KOandPPAR KO mice, is implicated in hepatic gluconeogenesis, although it whereas all these central effects of FGF21 were abrogated remains controversial whether hepatocytes are a direct by blockage of hypothalamic FGF receptor-1. FGF21 acts action site of FGF21 (8,9). There is an obvious dichotomy directly on the hypothalamic neurons to activate the mitogen-activated protein kinase extracellular signal– between the effects of endogenous FGF21 and pharmaco- related kinase 1/2 (ERK1/2), thereby stimulating the expres- logical actions of the recombinant peptide with respect to sion of corticotropin-releasing hormone by activation of the hepatic metabolism (4,6,9). transcription factor cAMP response element binding FGF21 can cross the blood-brain barrier (10) and is protein. Therefore, FGF21 maintains glucose homeostasis detectable in both human and rodent cerebrospinal fluid during prolonged fasting by fine tuning the interorgan cross (10,11). Continuous intracerebroventricular injection of talk between liver and brain. FGF21 into obese rats increases energy expenditure and 1State Key Laboratory of Pharmaceutical Biotechnology, The University of Hong Received 2 April 2014 and accepted 8 July 2014. Kong, Hong Kong, China This article contains Supplementary Data online at http://diabetes 2 Department of Medicine, The University of Hong Kong, Hong Kong, China .diabetesjournals.org/lookup/suppl/doi:10.2337/db14-0541/-/DC1. 3Department of Pharmacology and Pharmacy, The University of Hong Kong, Hong © 2014 by the American Diabetes Association. Readers may use this article as Kong, China long as the work is properly cited, the use is educational and not for profit, and 4Department of Medicine, University of Dresden, Dresden, Germany the work is not altered. 5Department of Pharmacology, Weill Cornell Medical College in Qatar, Doha, Qatar See accompanying article, p. 4013. Corresponding author: Aimin Xu, [email protected], or Karen S.L. Lam, [email protected]. diabetes.diabetesjournals.org Liang and Associates 4065 insulin sensitivity (12). More recently, FGF21 has been Biochemical and Immunological Analysis shown to act on the central nervous system to increase Serum levels of insulin and FGF21 were quantified with systemic glucocorticoid levels, suppress physical activity, immunoassays according to the manufacturer’s instruc- and alter circadian behavior (13). Furthermore, FGF21 tions (Antibody and Immunoassay Services, The Univer- acts on the hypothalamus to suppress the vasopressin- sity of Hong Kong). Mouse FGF21 assay was based on an kisspeptin signaling cascade, thereby mediating starvation- affinity-purified rabbit polyclonal antibody specificto induced infertility of female mice (14). However, the mouse FGF21 and did not cross-react with other members physiological roles of FGF21 and its central actions in reg- of the FGF family. The intra- and interassay variations ulating glucose metabolism during adaptive starvation were 4.2 and 7.6%, respectively. Mouse insulin assay responses remain unknown. was based on a monoclonal antibody pair raised using In this study, we show that FGF21 is a key metabolic recombinant mouse insulin as an antigen, with intra- regulator essential for maintaining glucose homeostasis, and interassay variations of 5.3 and 6.2%, respectively. by sending the starvation signal from the liver to the Serum levels of corticosterone (Enzo Life Sciences) and brain, where it acts on the hypothalamic neurons to induce glucagon (Millipore) and plasma levels of adrenocortico- phosphorylation of the mitogen-activated protein (MAP) tropic hormone (ACTH; MD Bioproducts) and CRH kinase extracellular signal–related kinase 1/2 (ERK1/2), (Phoenix Pharmaceuticals) were determined using immu- which in turn stimulates the production of corticotropin- noassays according to the manufacturer’s instructions. releasing hormone (CRH) and subsequent release of adre- For analysis of adrenaline and noradrenaline in the liver, nal corticosterone by activation of cAMP response element frozen tissue was extracted with 0.1 N HCl and analyses binding protein (CREB), thereby leading to enhanced he- performed according to the supplier’s instructions (Labor patic gluconeogenesis. Diagnostika Nord, Nordhorn, Germany). RESEARCH DESIGN AND METHODS Ex Vivo Studies The livers of male 12-week-old C57BL/6 mice were iso- Animal Studies lated, and primary hepatocytes were prepared from male All animal experimental protocols were approved by the Wistar rats (200 g) and used to determine glucose pro- Animal Ethics Committee of The University of Hong duction as previously described (19). The mouse hypotha- Kong. PPARa knockout (KO) mice in C57BL/6N back- lamic explants were prepared as previously described (20). ground were originally obtained from The Jackson Labo- The hypothalamic slices were pretreated without or with ratory (Sacramento, CA). FGF21 KO mice in C57BL/6J PD98059 for 30 min, followed by treatment with background were described previously (15). The mice rmFGF21 for different periods. were housed in a room under controlled temperature (23 6 1°C) with free access to water and standard RNA Extraction and Real-Time PCR chow. Male mice at the age of 12–14 weeks were used Total RNA was extracted from mouse tissues and rat in all animal experiments. Mice were studied by hyper- hepatocytes, cDNA was synthesized, and the mRNA fi insulinemic-euglycemic clamp to assess endogenous glu- expression levels were quanti ed with real-time PCR as cose production as previously described (16), except previously described (21). that mice were fasted 24 h before the clamp and so- Western Blot matostatin was not infused. Bilateral or sham adrenal- Total cellular proteins in liver and hypothalamic lysates fl ectomy was conducted under iso urane anesthesia 1 week were separated by SDS-PAGE and probed with a rabbit before various fasting experiments. Plasma corticoste- polyclonal antibody against peroxisome proliferator– fi rone levels were measured to con rm the completeness activated receptor-g coactivator 1a (PGC-1a;Abcam), of adrenalectomy. FGF21 (18), phosphorylated ERK (p-ERK), total ERK (t-ERK), p-CREB, t-CREB (Cell Signaling Technology), Intracerebroventricular and Intraparaventricular FGFR1, bKlotho (Santa Cruz), or b-actin (Sigma-Aldrich). Nucleus Injection in Mice Male FGF21 KO and PPARa KO mice and their respective Immunohistochemistry wild-type (WT) littermates at the age of 12–14 weeks Paraformaldehyde-fixed brains were frozen in liquid were chronically implanted with 26G stainless cannula nitrogen and stored at 280°C until sectioning with a cryo- (Plastics One, Inc., Roanoke, VA) in the cerebral ventricle stat (CM1950, Leica) at 220°C. Slides were washed and or the paraventricular nucleus (PVN) region of the brain then blocked in 10% goat serum with 3% BSA in TBS for as previously described (17). A neutralizing monoclonal 1 h at room temperature, followed by incubation with antibody against FGF receptor 1 (FGFR1, 0.2 mg/g body primary antibody overnight at 4°C. On the 2nd day, the weight; NBP2–12308, Novus) or CRH antagonist (a-helical slides were further incubated with Alexa Fluor 488 or – fi CRH9–41, 0.2 mg/g body weight;

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