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; C2917, Sigma-Aldrich) 594 labeled mouse-speci c secondary antibodies (Invitro- was infused by intra-PVN injection before central admin- gen) for another hour at room temperature. Images were istration of endotoxin-free recombinant mouse FGF21 captured with a fluorescence microscope (IX71; Olympus; (rmFGF21, 0.02 mg/g body weight) (18). QImaging). 4066 FGF21 Mediates Cross Talk of Liver and Brain Diabetes Volume 63, December 2014

Statistical Analysis FGF21 Deficiency Impairs Fasting-Induced Activation All analyses were performed with Statistical Package for of the Hypothalamic-Pituitary-Adrenal Axis and Social Sciences version 11.5 for Windows (SPSS, Chi- Release of Corticosterone in Mice cago, IL). Comparison between groups was performed Glucocorticoids are fasting-inducible hormones that play using one-way ANOVA followed by least-significant a central role in activating gluconeogenic genes and difference post hoc test. In all statistical comparisons, sustaining glucose homeostasis during starvation. Our a P value ,0.05 was used to indicate a statistically sig- recent clinical study showed that FGF21 and cortisol nificant difference. display a similar circadian rhythm in humans (23). There- fore, we next investigated the functional relationship be- RESULTS tween FGF21, corticosterone, and its upstream regulators FGF21 KO Mice Exhibit Hypoglycemia During in mice. In WT mice, serum levels of corticosterone were Prolonged Fasting progressively increased in response to fasting (Fig. 1E). Blood glucose levels were comparable between FGF21 However, the magnitude of fasting-induced elevation of KOandWTmiceinthefedstateandwithin6hoffood serum corticosterone in FGF21 KO mice was much E deprivation (Fig. 1A). However, during the prolonged smaller than WT mice (Fig. 1 ). Likewise, fasting for fasting period (24–48 h), FGF21 KO mice exhibited 24 h caused a marked induction in hypothalamic expres- F a much more rapid decline in blood glucose levels and sion of CRH (Fig. 1 ) and pituitary release of ACTH (Fig. H more severe hypoglycemia as compared with WT mice 1 ), whereas such fasting-induced expression of these (Fig. 1A). Moreover, both the mRNA and protein expres- two corticotropic hormones was severely impaired in F H sion levels of hepatic PGC-1a, a transcriptional coactiva- FGF21 KO mice (Fig. 1 and ), suggesting that FGF21 fi tor that plays a key role in mediating the fasting-induced de ciency causes defective fasting-induced release of cor- expression of gluconeogenic genes (22), were comparable ticosterone possibly by impairing the activation of the between FGF21 KO mice and WT littermates in the fed hypothalamic-pituitary-adrenal (HPA) axis. On the con- state (Fig. 1A and B). However, the amplitude of fasting- trary, the expression of arginine vasopressin (AVP) was induced elevation in PGC-1a expression was significantly comparable between WT and FGF21 KO mice under both G decreasedascomparedwithWTmice,andthischange fed and fasted states (Fig. 1 ), suggesting that these hor- was accompanied by a marked impairment in the fasting- mones do not contribute to the hypoglycemia phenotypes induced expression of G6Pase and PEPCK (Fig. 1A in FGF21 KO mice. Moreover, the difference in fasting and B). The rate of glucose production in FGF21 KO blood glucose between WT and KO mice totally disap- mice, as estimated by pyruvate tolerance test, was peared after the production of corticosterone was blocked I much lower than that in WT littermates in fasted state by adrenalectomy in mice (Fig. 1 ), suggesting that corti- (Fig. 1C). We further assessed whole-body glucose ho- costerone mediates the effects of FGF21 in regulating meostasis in euglycemic glucose clamp studies. During fasting glucose homeostasis. constant hyperinsulinemia in mice after fasting, a higher We next investigated whether the fasting hypoglyce- glucoseinfusionratewasrequiredtomaintainanormal mia phenotype in FGF21 KO mice can be reversed by glucose level in KO mice than in WT mice (Fig. 1D). supplementation with corticosterone. Injection of the This difference was primarily accounted for by a signifi- synthetic glucocorticoid dexamethasone into FGF21 KO cant reduction in glucose production in KO mice, al- mice reversed fasting hypoglycemia to a level comparable A though whole-body glucose disposal was similar in WT to WT mice (Supplementary Fig. 3 ). The dexamethasone- and KO mice (Fig. 1D). Treatment of mouse liver mediated normalization of fasting glucose homeostasis fi explants with rmFGF21 for up to 24 h had no obvious was associated with signi cantly elevated expression of PEPCK G6Pase effect on the expression of gluconeogenic genes (PGC-1a, PGC-1a and its target genes and (Supple- B–D PEPCK,andG6Pase) and glucose production, although mentary Fig. 3 ). the glucocorticoid receptor agonist dexamethasone led FGF21 Acts in the Brain to Induce Corticosterone to a significant induction of the gluconeogenic program Production and Hepatic Gluconeogenesis (Supplementary Fig. 1A and B). Similarly, dexametha- Consistent with a previous report (10), we found that sone, but not FGF21, increased the expression of the FGF21 was present in the hypothalamus and its hypotha- gluconeogenic genes and glucose production in primary lamic level was markedly elevated in response to fasting rat hepatocytes (Supplementary Fig. 1C and D). Further- (Fig. 2A), despite that there was not any detectable FGF21 more, there was no difference in circulating levels of mRNA expression in this tissue even in the fasted state insulin and glucagon nor the levels of hepatic adrenaline (Fig. 2A). Furthermore, intraperitoneal injection of exog- and noradrenaline between FGF21 KO mice and WT lit- enous rmFGF21 led to its marked accumulation in the termates in either the fed or fasted state (Supplementary hypothalamus region (Fig. 2B), confirming that FGF21 Fig. 2A–D), suggesting that insulin, glucagon, and he- can cross the blood-brain barrier into the hypothalamus. patic sympathetic nerves are not the downstream medi- To investigate whether FGF21 induces fasting gluco- ators of FGF21 in the maintenance of fasting glucose neogenesis through its central actions, rmFGF21 was homeostasis. infused directly into the cerebroventricle of FGF21 KO diabetes.diabetesjournals.org Liang and Associates 4067

Figure 1—Fasting-induced gluconeogenesis and activation of the HPA axis are impaired in FGF21 KO mice. A: Blood glucose levels and the mRNA abundance of hepatic gluconeogenic genes PGC-1a, PEPCK, and G6Pase in FGF21 KO mice and WT littermates in either fed state or after fasting. B: The protein abundance of PGC-1a was determined by Western blot analysis and quantified using scanning densitometry. The results were expressed as fold changes relative to internal control b-actin. C: Pyruvate tolerance test was performed in 24 h–fasted FGF21 KO mice and WT littermate mice by using 2 g pyruvate per kg body weight. D: Glucose infusion rate, glucose disposal rate, and glucose production during a hyperinsulinemic-euglycemic clamp experiment. E–H: The amount of hormones of HPA axis in FGF21 KO mice and WT littermates either in fed or fasted state; the serum levels of corticosterone (E) were determined by ELISA, the mRNA abundance of CRH (F) and AVP (G) in the hypothalamus was determined by qRT-PCR, and plasma ACTH levels (H) were determined by ELISA. I: Blood glucose in FGF21 KO mice and WT littermates receiving adrenalectomy (ADX) or sham surgery in either fed state or various time points after fasting. n = 8 for WT mice and n = 10 for KO mice. *P < 0.05; **P < 0.01. Data were expressed as mean 6 SEM. 4068 FGF21 Mediates Cross Talk of Liver and Brain Diabetes Volume 63, December 2014

Figure 2—FGF21 acts in the brain to activate HPA axis and induce hepatic gluconeogenesis. A: Protein levels of FGF21 in the hypothal- amus and mRNA abundance of FGF21 in the livers and hypothalamus of C57BL/6J mice in fed state or fasted for 24 h. B: FGF21 protein levels in the hypothalamus, which was collected at 30 min after intraperitoneal injection of rmFGF21 (1 mg/kg) or vehicle in FGF21 KO mice. The mice were perfused with 13 PBS by transcardiac perfusion before the collection of hypothalamic tissues. Representative Western blotting analysis and qRT-PCR assays from two independent cohorts run in duplicate are shown (n = 4). C–G: FGF21 KO mice were treated with rmFGF21 (0.02 mg/g, i.c.v.) or vehicle. C: Blood glucose levels and the mRNA abundance of hepatic gluconeogenic genes PGC-1a, PEPCK, and G6Pase in the livers. CRH (D) and AVP (E) mRNA in the hypothalamus, plasma levels of ACTH (F), and serum levels of corticosterone (G) were determined at 3 h after intracerebroventricular injection. *P < 0.05 and **P < 0.01 vs. vehicle control (n = 6). Data were expressed as mean 6 SEM. H: The hypothalamic slices from FGF21 KO mice were incubated with different concentrations of rmFGF21 for 2 h. The culture medium was collected for determination of CRH by ELISA. *P < 0.05 and **P < 0.01 vs. vehicle control (n = 6). Data were expressed as mean 6 SEM. I–J: FGF21 KO mice were pretreated with CRH antagonist a-helical CRH9–41 (anti-CRH, 0.2 mg/g, intra-PVN) for 30 min, followed by injection with rmFGF21 (0.02 mg/g). Serum levels of blood glucose (I) and corticosterone (J) were detected at 3 h after injection. *P < 0.05; n = 6. Data were expressed as mean 6 SEM. K–L: Twelve-week-old FGF21 KO mice were pretreated with RU486 by intraperitoneal injection (20 mg/kg) for 1 h, followed by intracerebroventricular infusion of rmFGF21 (0.02 mg/g) or vehicle for 3 h. K: Blood glucose levels were determined at various time points after treatment. *P < 0.05 and **P < 0.01 vs. vehicle. #P < 0.05 vs. rmFGF21 group. n = 6. Data were expressed as mean 6 SEM. L: The mRNA abundance of hepatic PGC-1a, PEPCK, and G6Pase was determined by qRT-PCR. *P < 0.05 and **P < 0.01 (n = 6). Data were expressed as mean 6 SEM. M: FGF21 KO mice receiving adrenalectomy (ADX) or sham surgery were intracerebroventricularly infused either with rmFGF21 or vehicle. Blood glucose levels were detected at various time points after injection. **P < 0.01 vs. vehicle + sham surgery; #P < 0.05 vs. rmFGF21 + sham surgery. n = 6. Data were expressed as mean 6 SEM. diabetes.diabetesjournals.org Liang and Associates 4069 mice by intracerebroventricular injection via a cannula (Fig. 2E). Furthermore, direct incubation of mouse hypo- preimplanted 1 week before the treatment. The results thalamic slices with rmFGF21 was sufficient to induce the showed that the central infusion of rmFGF21 at 0.02 mg/g CRH expression in a dose-dependent manner (Fig. 2H), body weight, a dose at which there was still no detectable whereas the central effects of FGF21 on stimulation of FGF21 in the peripheral blood of FGF21 KO mice, led to corticosterone release and elevation of blood glucose lev- a significant elevation of blood glucose levels as compared els were blunted by intra-PVN administration of the CRH with the vehicle-treated mice (Fig. 2C). The central admin- antagonist a-helical CRH9–41 (Fig. 2I and J). On the con- istration of rmFGF21 also induced the expression of he- trary, intravenous injection of a low dose of rmFGF21 patic PGC-1a, PEPCK, and G6Pase (Fig. 2C) and increased (0.02 mg/g), which was insufficient to cause any notable the hypothalamic level of CRH (Fig. 2D) and pituitary elevation of FGF21 in the brain of FGF21 KO mice, had release of ACTH (Fig. 2F) and serum corticosterone (Fig. no obvious effect on either blood glucose level or the 2G) but had no effect on hypothalamic expression of AVP expression of the gluconeogenic genes in the liver or the

Figure 3—FGFR1 and bKlotho are expressed in the hypothalamus of mice. The abundance of FGFR1 mRNA and protein (A) and bKlotho mRNA and protein (C) in liver, white adipose tissue (WAT), and hypothalamus (hypo) in 12-week-old male C57BL/6J mice, as determined by real-time PCR and Western blot analysis, respectively. Representative images of mouse hypothalamic slices immunostained for FGFR1 (B), bKlotho (D), and CRH (B). Note that the merged image shows the colocalization between FGFR1 and CRH (B), and the specificity of anti- CRH antibody (catalog no. ab11133, Abcam) was validated by using the CRH blocking peptide (Sigma-Aldrich, catalog no. C3042) to neutralize the antibody. No positive fluorescent signal was detected when the hypothalamic slices were coincubated with anti-CRH antibody and blocking peptides (data not shown). Representative Western blotting analysis and immunostained figures from three independent cohorts are shown; n =6. 4070 FGF21 Mediates Cross Talk of Liver and Brain Diabetes Volume 63, December 2014 hormones of the HPA axis (CRH, ACTH, and corticoste- rone) (Supplementary Fig. 4A–E). Central injection of rmFGF21 did not increase the levels of noradrenaline or adrenaline in the liver (Supple- mentary Fig. 5A and B), suggesting that sympathetic nerves are not involved in FGF21-mediated hepatic glu- cose production. Plasma ketone bodies and hepatic expres- sion of key ketogenic genes and several genes governing lipid metabolism in the liver were not altered by cen- tral treatment of rmFGF21 (Supplementary Fig. 5C and D), indicating that the effects of FGF21 on ketogenesis Figure 4—FGF21 signaling in hypothalamus is blocked by FGFR1 and lipid metabolism are not attributed to its central neutralizing antibody. Twelve-week-old FGF21 KO mice were treated with an anti-FGFR1 monoclonal antibody (AF, 0.2 mg/g body weight) actions. or mouse nonimmune IgG (NI) by a single hypothalamic paraventric- To further confirm the role of the HPA axis in FGF21- ular (intra-PVN) injection, followed by another intra-PVN injection with mediated gluconeogenesis, FGF21 KO mice were pretreated rmFGF21 (0.02 mg/g body weight) or vehicle control. Hypothalamic with the glucocorticoid receptor antagonist RU486 or protein levels of p-ERK and t-ERK were determined by Western blotting analysis after 30 min of injection and were quantified using subjected to adrenalectomy prior to intracerebroventricular scanning densitometry. Representative Western blotting analysis injection of rmFGF21. The results showed that the central from three independent cohorts run in duplicate are shown; n =6. effects of rmFGF21 on elevation of blood glucose and *P < 0.05. Data were expressed as mean 6 SEM. induction of the hepatic gluconeogenic genes were largely abrogated by either pharmacological blockage of the C–F glucocorticoid receptor or depletion of endogenous corti- (Fig. 5 ). On the other hand, intra-PVN administration costerone by adrenalectomy (Fig. 2K–M). of the anti-FGFR1 neutralizing antibody had no obvious effect on glucose levels, hepatic gluconeogenic gene ex- pression, and the HPA activity in FGF21 KO mice without Central Effects of FGF21 on Activation of the HPA Axis A–F and Hepatic Gluconeogenesis Are Mediated by FGF21 treatment (Fig. 5 ). Physiologically, a single intra- Hypothalamic FGFR1 PVN administration of the anti-FGFR1 antibody lowered The metabolic actions of FGF21 are mediated by FGFR1 fasting blood glucose and reduced the expression of hepatic in complex with bKlotho (24). FGFR1 is highly expressed gluconeogenic genes in WT mice to a level comparable to G H in hypothalamic areas such as PVN and supraoptic nuclei those in KO mice (Fig. 5 and ). Furthermore, fasting- (25). Both real-time PCR and Western blot analysis induced elevations in hypothalamic CRH expression and fi showed that the expression level of FGFR1 in the hypo- serum levels of corticosterone were signi cantly dimin- thalamus was comparable to that in white adipose tissue, ished by the neutralizing antibody-mediated blockage of I J a major peripheral target of FGF21 (Fig. 3A). FGFR1 is hypothalamic FGFR1 (Fig. 5 and ). abundantly expressed in the PVN of the hypothalamus FGF21 Mediates PPARa-Induced Corticosterone (Fig. 3B), and a large portion of FGFR1 colocalized with Production and Hepatic Gluconeogenesis in Mice via CRH neurons (Fig. 3B). In addition, both mRNA and pro- Central Nervous System tein expressions of bKlotho were detectable in the hypo- During fasting, the transcription factor PPARa is acti- thalamus, although its expression level was much lower vated in the liver, which in turn promotes hepatic ex- than that in adipose tissues and livers (Fig. 3C). Specifi- pression of FGF21 and its release into the circulation cally, bKlotho was detectable in the PVN of the hypothal- (1–3). Therefore, we investigated whether PPARa activa- amus (Fig. 3D). tion induces corticosterone production by induction of To interrogate the roles of hypothalamic FGFR1 in FGF21. Chronic treatment of WT mice with the PPARa FGF21-induced hepatic gluconeogenisis via the HPA axis, agonist fenofibrate led to a progressive elevation in serum we performed bilateral intra-PVN injection to directly levels of FGF21 as well as corticosterone (Fig. 6A and B). deliver the neutralizing antibody into PVN. The intra- However, the magnitude of the fenofibrate-induced in- PVN injection of anti-FGFR1 antibody largely blocked crease of serum corticosterone was significantly attenuated rmFGF21-induced activation of ERK1/2 in the hypothal- in FGF21 KO mice (Fig. 6B). Likewise, fenofibrate-induced amus (Fig. 4). The central actions of rmFGF21 on ele- hypothalamic expression of CRH was also impaired in vation of blood glucose and induction of hepatic FGF21 KO mice as compared with WT controls (Fig. 6C). gluconeogenic gene expression were abrogated by a single Furthermore, fasting-induced elevation of serum cortico- intra-PVN administration of a neutralizing monoclonal sterone levels and hypothalamic expression of CRH in antibody against FGFR1 (Fig. 5A and B). Furthermore, PPARa KO mice were markedly reduced as compared blockage of hypothalamic FGFR1 by its neutralizing anti- with WT littermates (Fig. 6D and E). On the other hand, body also negated the stimulatory effects of central the impairments in the fasting-induced production of cor- administration with rmFGF21 on hypothalamic CRH ticosterone and CRH were partially reversed by intracere- expression and serum levels of ACTH and corticosterone broventricular injection of rmFGF21 (Fig. 6D and E). diabetes.diabetesjournals.org Liang and Associates 4071

Figure 5—Hypothalamic FGFR1 is obligatory for FGF21-induced activation of the HPA axis and hepatic gluconeogenesis in mice. Twelve- week-old FGF21 KO mice were treated with an anti-FGFR1 monoclonal antibody (AF; 0.2 mg/g body weight) or mouse nonimmune IgG (NI) by a single hypothalamic paraventricular (intra-PVN) injection, followed by another intra-PVN injection with rmFGF21 (0.02 mg/g body weight) or vehicle control. A: Blood glucose levels. B: mRNA abundance of PGC-1a, PEPCK, and G6Pase in the livers. C–F: The amount of hormones of HPA axis. C: mRNA abundance of CRH in the hypothalamus of mice collected 3 h after treatment. D: Representative images of immunofluorescent staining with anti-CRH antibody in the PVN region of the hypothalamus (scale bar, 200 mm). Plasma ACTH levels (E) and serum corticosterone levels (F) were determined at different time intervals. *P < 0.05 and **P < 0.01 vs. vehicle + NI group; #P < 0.05 and ##P < 0.01 vs. rmFGF21 + NI group (n = 5). Data were expressed as mean 6 SEM. G–J: Twelve-week-old WT mice were treated with AF (0.2 mg/g body weight) or NI by intra-PVN injections, followed by fasting for 24 h. Fed WT mice and FGF21 KO mice treated with NI were used as control. Blood glucose levels (G) and the mRNA abundance (H) of PGC-1a, PEPCK, and G6Pase in the livers. mRNA abundance of CRH in the hypothalamus (I) and serum corticosterone levels (J) were determined. *P < 0.05 and **P < 0.01 (n = 5). Data were expressed as mean 6 SEM.

Consistent with previous reports (26), we found that PPARa KO mice not only led to a significant alleviation of PPARa KO mice exhibited similar blood glucose levels fasting hypoglycemia but also reversed the impairment of with WT controls in the fed state but developed severe fasting-induced expression of several gluconeogenic genes hypoglycemia after 16 h of fasting (Fig. 6F). The hypogly- (PGC-1a, PEPCK, and G6Pase) (Fig. 6F and G). Likewise, cemic phenotype of PPARa KO mice further deteriorated replenishment of PPARa KO mice with dexamethasone after prolonged fasting (24 and 48 h). On the other hand, also resulted in a partial reversal of hypoglycemia and intracerebroventricular administration of rmFGF21 into decreased gluconeogenic gene expression in fasted PPARa 4072 FGF21 Mediates Cross Talk of Liver and Brain Diabetes Volume 63, December 2014

Figure 6—FGF21 mediates PPARa-induced activation of HPA axis and hepatic gluconeogenesis in mice. A–C: FGF21 KO mice and WT littermates were treated with fenofibrate (Feno, 200 mg/kg) or vehicle by daily intraperitoneal injection for 7 days. Serum levels of FGF21 (A) and serum corticosterone (B) and the mRNA abundance of CRH (D) in the hypothalamus were determined at days 3 and 7 after treatment. **P < 0.01 vs. WT mice treated with vehicle. #P < 0.05 vs. WT + Feno group (n =6–8). D and E: PPARa KO mice and WT littermates were treated with rmFGF21 (0.02 mg/g) or vehicle by intracerebroventricular injection every 6 h during 48 h of fasting. D: Serum levels of corticosterone were measured at different time points after treatment. E: mRNA abundance of CRH in the hypothalamus was determined by real-time PCR analysis after 24 h of fasting. *P < 0.05 and **P < 0.01 vs. WT + vehicle group. #P < 0.05 vs. KO + vehicle group (n = 8). F and G: Twelve-week-old PPARa KO mice and WT littermates were treated with rmFGF21 (0.02 mg/kg) or vehicle by intracerebroven- tricular infusion every 6 h during fasting or dexamethasone (DEX, 2 mg/kg) via intraperitoneal injection, followed by fasting for 48 h. F: Blood glucose levels were measured at different time intervals. *P < 0.05 and **P < 0.01 vs. KO + vehicle (n = 5). Data were expressed as mean 6 SEM. G: The mRNA abundance of PGC-1a, PEPCK, and G6Pase in the livers of mice collected at fed state or 24 h after fasting. *P < 0.05 and **P < 0.01 (n = 5). Data were expressed as mean 6 SEM.

KO mice, suggesting that PPARa maintains fasting glu- WT mice (Fig. 7A). However, fasting-induced phosphor- cose homeostasis via activation of the FGF21-glucocorticoid ylation of ERK1/2 and CREB was impaired in FGF21 KO axis (Fig. 6F and G). mice (Fig. 7A). FGF21 Stimulates CRH Production by Activation of the Injection of rmFGF21 into the PVN region of FGF21 ERK1/2-CREB Signaling Axis KO mice induced phosphorylation of both ERK1/2 and CREB is a key transcription factor responsible for trans- CREB, whereas such a stimulatory effect of rmFGF21 was activation of the CRH gene in PVN neurons (27). The completely blocked by pretreatment with the ERK in- B MAP kinase ERK1/2 activates CREB by phosphorylating hibitor PD98059 in the hypothalamic PVN region (Fig. 7 the transcription factor at serine 133 (28). Notably, and Supplementary Fig. 6). Likewise, the central effects of FGF21 induces phosphorylation and activation of ERK1/2 rmFGF21 on induction of hypothalamic CRH expression, both in adipocytes (4) and in whole hypothalamus elevation of serum corticosterone, and blood glucose lev- ofmice(29).Therefore,wenextinvestigatedtheroles els, and stimulation of hepatic gluconeogenic gene expres- of ERK1/2 and CREB in mediating the central actions of sion, were all abrogated by intra-PVN injection of PD98059 FGF21 in mice. Consistent with previous reports (30), (Fig. 7C–F). Furthermore, direct incubation of mouse hypo- we found that phosphorylation of both ERK1/2 and thalamic slices with rmFGF21 was sufficient to induce the CREB was significantly enhanced by fasting for 24 h in phosphorylation of ERK and CREB (Fig. 7G) and to increase diabetes.diabetesjournals.org Liang and Associates 4073

Figure 7—FGF21 increases CRH production via ERK-CREB-CRH signaling pathways. A: The hypothalamic tissue lysates of 12-week-old FGF21 KO mice and WT littermates in either fed or fasted (24 h) state were subjected to immunoblotting analysis with antibodies against p-ERK, t-ERK, p-CREB, or t-CREB. B–F: FGF21 KO mice were treated with the ERK inhibitor PD98059 (2 mg/mouse) or vehicle control (DMSO) by a single intra-PVN injection, followed by another intra-PVN injection with rmFGF21 (0.02 mg/g body weight) or vehicle control. B: Hypothalamic protein levels of p-ERK, t-ERK, p-CREB, and t-CREB were determined by Western blotting analysis after 30 min of injection. Blood glucose (C) and the mRNA abundance of PGC-1a, PEPCK, and G6Pase in the livers (D), the mRNA abundance of CRH in the hypothalamus (E), and serum corticosterone levels (F) were measured after 3 h of injection. G–I: The hypothalamic slices from FGF21 KO mice were preincubated with PD98059 (20 mmol/L) for 30 min, followed by treatment with rmFGF21 (1 mg/mL). G: Hypothalamic protein levels of p-ERK, t-ERK, p-CREB, and t-CREB were determined by Western blotting analysis after 15 min of incubation with rmFGF21. The mRNA abundance of CRH in the hypothalamus (H) and the CRH levels in culture medium (I) were determined after 3 h of incubation with rmFGF21. Representative Western blotting analysis and qRT-PCR assays from three independent cohorts run in duplicate are shown. *P < 0.05 (n = 6). Data were expressed as mean 6 SEM.

the mRNA expression and protein release of CRH (Fig. 7H role in controlling hepatic fatty acid metabolism, PPARa and I). However, all these effects of rmFGF21 on hypotha- is also a critical mediator of fasting-induced hepatic glu- lamic slices were blocked by PD98059 (Fig. 7G–I). Taken coneogenesis in mice (26,31). PPARa KO mice exhibit together, these findings suggest that FGF21 stimulates hy- severe fasting hypoglycemia despite normoglycemia in pothalamic CRH production via the ERK1/2-CREB signaling the fed state (26). However, PPARa is not a direct tran- cascade, which in turn triggers the release of corticosterone scriptional activator of the key gluconeogenic genes, sug- for induction of gluconeogenesis (Fig. 8). gesting that it induces hepatic gluconeogenesis via an indirect mechanism(s) (32). In the current study, we DISCUSSION found that both PPARa and FGF21 KO mice exhibited Appropriate counter-regulatory hormone responses to a similar degree of hypoglycemia due to defective hepatic hypoglycemia are critical for maintaining blood glucose gluconeogenesis during prolonged fasting, whereas hypo- levels within a narrow range. However, the molecular glycemia in fasted PPARa KO mice and FGF21 KO mice can events that coordinate this process remain poorly defined. be rectified by central administration of FGF21, suggesting In both rodents and humans, fasting-induced hepatic an obligatory role of the PPARa-FGF21 axis in inducing production of FGF21 is mediated by PPARa, which is hepatic gluconeogenesis during metabolic adaptation to a master regulator coordinating metabolic adaptions to prolonged fasting. In support of this notion, transgenic fasting and starvation (1–3). In addition to its central expression of FGF21 was sufficient to induce hepatic 4074 FGF21 Mediates Cross Talk of Liver and Brain Diabetes Volume 63, December 2014

stimulates CRH expression in the hypothalamus via ERK1/2- mediated activation of CREB, suggesting that CRH neu- rons are the direct target of FGF21. Consistent with our observations in PPARa KO mice, administration of the PPARa agonist fenofibrate has been shown to elevate fasting serum levels of corticosterone, enhance hepatic gluconeogenesis, and increase blood glu- cose levels (36). These findings support the notion that PPARa coordinates metabolic adaptation responses to starvation through a bipartite mechanism: direct trans- activation of the genes involved in hepatic fatty acid ox- idation and ketogenesis (26,31) and indirect activation of the HPA axis via induction of FGF21. The synergic effect of this dual action of PPARa is to provide glycerol, fatty acids, ketones, and alanine as sources for gluconeogenesis to maintain glucose homeostasis. In peripheral tissues, adipocytes have been identified as a main target that confers the pleiotropic metabolic actions of FGF21 (4,19). In white adipocytes, adiponectin expression and secretion is induced by FGF21 and medi- ates several metabolic benefits of FGF21 treatment Figure 8—A working model by which FGF21 maintains glucose (18,37). These findings together with our present report homeostasis during fasting via mediating the cross talk between brain and liver. FFA, free fatty acids; HSL, hormone-sensitive further support the notion that FGF21 exerts its diverse lipase; TG, triglycerides. metabolic effects indirectly, by modulating the production of other hormones such as adiponectin and corticoste- rone. The central and peripheral actions of FGF21 on activation of the HPA axis and its peripheral actions on promoting adipose secretion of adiponectin appear to gluconeogenesis in the fed state to a level normally attained have opposite effects on hepatic glucose production. How- during prolonged fasting, whereas FGF21 KO mice showed ever, these two actions of FGF21 may occur under differ- impaired hepatic gluconeogenesis after 24-h fasting (9). By ent physiological conditions. FGF21 secretion in adipose contrast, Badman et al. (33) found that fasting glucose tissue is elevated in the fed state, which in turn serves as levels were comparable between WT and FGF21 KO mice an autocrine signal to induce PPARg activity and adipo- generated in their laboratory. This discrepancy may be due nectin secretion, thereby leading to enhanced hepatic in- to different ages of mice or different fasting schedules. sulin sensitivity (18). Conversely, the central effect of Nevertheless, they also showed impaired gluconeogenic FGF21 on hepatic glucose production consequent to in- gene induction (PGC-1a and PEPCK) in their FGF21 KO creased release of corticosterone occurs only during pro- mice, which is consistent with our present study. longed fasting. Such a dual effect of FGF21 may enable The hypothalamus has emerged as a key player in the the control of blood glucose within a narrow range in regulation of glucose homeostasis, by sensing hormones different nutritional states. and nutrients to initiate metabolic responses (34). Hor- In conclusion, our results demonstrate FGF21 as mones such as insulin, glucagon, and leptin have been a critical hormonal regulator of glucose homeostasis shown to regulate hepatic glucose metabolism through during prolonged fasting, by coupling hepatic PPARa ac- both central and peripheral actions (34,35). In this study, tivation to corticosterone release via stimulation of the we provide several lines of evidence demonstrating that HPA axis, thereby leading to enhanced hepatic gluconeo- the stimulatory effect of FGF21 on hepatic gluconeogen- genesis (Fig. 8). This novel liver-PPARa-FGF21-brain- esis is mainly mediated by its central actions on hypotha- corticosterone circuit can fine tune the cross talk between lamic neurons. First, fasting hypoglycemia and impaired brain and liver to avoid hypoglycemia during nutrient expression of hepatic gluconeogenic genes in both FGF21 deprivation or other adverse clinical events. Interestingly, KO and PPARa KO mice can be reversed by intracere- a novel “hepato-adrenal syndrome” has recently been sug- broventricular injection of FGF21 at a low dose that is gested, demonstrating a defect in HPA activation in not detectable in the peripheral blood. Second, FGF21- patients with severe liver disease (38). Hypoglycemia is induced corticosterone release and hepatic gluconeogene- a common clinical problem in patients with a defect in sis can be completely blocked by central administration of HPA regulation such as hypopituitarism and Addison dis- a neutralizing antibody against its receptor FGFR1 or ease. It is also frequently seen in diabetic patients treated a pharmacological inhibitor of ERK1/2. Furthermore, with insulin or other medications improperly. Unraveling both our animal and ex vivo studies showed that FGF21 the liver-adrenal circuit with FGF21 as key player may diabetes.diabetesjournals.org Liang and Associates 4075 open new avenues for the treatment of these metabolic 15. Hotta Y, Nakamura H, Konishi M, et al. Fibroblast growth factor 21 regulates diseases with hypoglycemia unawareness. lipolysis in white adipose tissue but is not required for ketogenesis and tri- glyceride clearance in liver. Endocrinology 2009;150:4625–4633 16. Combs TP, Berg AH, Obici S, Scherer PE, Rossetti L. Endogenous glucose Acknowledgments. The authors thank N. Itoh and M. Konishi (University production is inhibited by the adipose-derived protein Acrp30. J Clin Invest 2001; – of Kyoto) for FGF21 KO mice. 108:1875 1881 The Mouse Brain in Stereotaxic Coordinates Funding. 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