TCPTP Regulates Insulin Signaling in AgRP Neuronsto Coordinate Glucose Metabolism With FeedingGarron T. Dodd,1,2 Robert S. Lee-Young,1,2,3 Jens C. Brüning,4,5,6,7 and Tony Tiganis1,2,3

Diabetes 2018;67:1246–1257 | https://doi.org/10.2337/db17-1485

Insulin regulates glucose metabolism by elicitingeffects on peripheral tissues as well as the brain. Insulinreceptor (IR) signaling inhibits AgRP-expressing neu-rons in the hypothalamus to contribute to the suppres-sion of hepatic glucose production (HGP) by insulin,whereas AgRP neuronal activation attenuates brownadipose tissue (BAT) glucose uptake. The tyrosinephosphatase TCPTP suppresses IR signaling in AgRPneurons. Hypothalamic TCPTP is induced by fastingand degraded after feeding. Here we assessed theinfluence of TCPTP in AgRP neurons in the control ofglucose metabolism. TCPTP deletion in AgRP neurons(Agrp-Cre;Ptpn2fl/fl) enhanced insulin sensitivity, asassessed by the increased glucose infusion rates,and reduced HGP during hyperinsulinemic-euglycemicclamps, accompanied by increased [14C]-2-deoxy-D-glucose uptake in BAT and browned white adiposetissue. TCPTP deficiency in AgRP neurons promotedthe intracerebroventricular insulin-induced repressionof hepatic gluconeogenesis in otherwise unresponsivefood-restricted mice, yet had no effect in fed/satiatedmice where hypothalamic TCPTP levels are reduced.The improvement in glucose homeostasis in Agrp-Cre;Ptpn2fl/fl mice was corrected by IR heterozygosity(Agrp-Cre;Ptpn2fl/fl;Insrfl/+), causally linking the effectson glucose metabolism with the IR signaling in AgRPneurons. Our findings demonstrate that TCPTP controlsIR signaling in AgRP neurons to coordinate HGP andbrown/beige adipocyte glucose uptake in response tofeeding/fasting.

Insulin is secreted by pancreatic b-cells in response toelevated blood glucose levels and signals via the insulinreceptor (IR) in peripheral tissues, including skeletal mus-cle and fat, to promote glucose uptake and storage, and inthe liver to repress hepatic glucose production (HGP) toprevent postprandial hyperglycemia. Insulin can also pro-mote glucose uptake in brown adipose tissue (BAT) (1),where glucose can be stored as glycogen, used for fatty acidesterification and triglyceride synthesis, or converted tolactate by anaerobic glycolysis during nonshivering ther-mogenesis to produce ATP (2,3). The latter may compen-sate for the reduced ATP production that occurs duringthermogenesis where fatty acid oxidation is uncoupledfrom ATP production to generate heat (2,3). The impor-tance of BAT in glucose metabolism is highlighted bystudies demonstrating that greater BAT abundance isaccompanied by decreased glycemic variability (4,5). Be-yond insulin’s roles in the periphery, a large body ofevidence now also supports a role for insulin action inthe central nervous system (CNS) in the regulation ofsystemic insulin sensitivity and glucose homeostasis (6,7).

Mice in which IR was deleted in neurons and astrogliausing the Nestin Cre transgene become obese and developsystemic insulin resistance (8). Although the glucoregula-tory effects of the IR can be ascribed to different regionsof the brain, several nuclei within the hypothalamus areespecially important. Neurons in the arcuate nucleus(ARC) of the hypothalamus, residing at the base of thethird ventricle, are positioned to readily sense peripheral

1Metabolism, Diabetes and Obesity Program, Monash Biomedicine DiscoveryInstitute, Monash University, Clayton, Melbourne, Victoria, Australia2Department of Biochemistry and Molecular Biology, Monash University, Clayton,Melbourne, Victoria, Australia3Monash Metabolic Phenotyping Facility, Monash University, Clayton, Melbourne,Victoria, Australia4Department of Neuronal Control of Metabolism, Max Plank Institute for Metab-olism Research, Cologne, Germany5Center for Endocrinology, Diabetes, and Preventive Medicine (CEDP), UniversityHospital Cologne, Cologne, Germany6Excellence Cluster on Cellular Stress Responses in Aging Associated Diseases(CECAD) and Center of Molecular Medicine Cologne (CMMC), University of Cologne,

Cologne, Germany7National Center for Diabetes Research (DZD), Neuherberg, Germany

Corresponding author: Tony Tiganis, tony.tiganis@monash.edu, or Garron T. Dodd,garron.dodd@monash.edu.

Received 8 December 2017 and accepted 19 April 2018.

This article contains Supplementary Data online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db17-1485/-/DC1.

© 2018 by the American Diabetes Association. Readers may use this article aslong as the work is properly cited, the use is educational and not for profit, and thework is not altered. More information is available at http://www.diabetesjournals.org/content/license.

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METABOLISM

substances, such as insulin, that signal the nutritional andenergy state of the organism (6,7,9). These include twomolecularly defined neuronal populations, the anorexi-genic POMC neurons that repress feeding and increaseenergy expenditure and the orexigenic AgRP/NPY neuronsthat can antagonize the actions of POMC neurons topromote feeding and decrease energy expenditure (6,7,9).Insulin signals via phosphatidylinositol 3-kinase (PI3K)to activate the Ser/Thr protein kinase AKT and othersignaling cascades to elicit discordant effects in POMCand AgRP/NPY neurons (6,7,9). Insulin regulates POMCneuronal excitability and promotes POMC expression,which is processed into the neuropeptide a-melanocyte–stimulating hormone (MSH). a-MSH agonizes postsynapticmelanocortin-4 receptors on neurons in other regions of thebrain to repress feeding and increase ambulatory activityand thermogenesis (6,7,9). By contrast, insulin hyperpolar-izes AgRP neurons and inhibits their firing by opening KATP

channels (10). Insulin also inhibits the expression of theneuropeptide AgRP that functions postsynaptically toantagonize a-MSH/melanocortin-4 receptors interactions(6,7,9). The inhibition of AgRP/NPY neurons by insulin canalleviate inhibitory constraints on POMC neurons and themelanocortin response (6,7,9). However, AgRP neurons canalso elicit melanocortin-independent effects and signalthrough parallel and redundant neural circuits to affectfeeding (11,12). Although the precise neuronal populationsregulating glucose homeostasis remain to be defined, thereis evidence that these can be distinct from those regulatingfeeding. Recent studies, for example, have shown that theacute pharmacogenetic activation of AgRP neurons repressesBAT glucose uptake via projections to the bed nucleus of thestria terminalis that are not involved in the induction offeeding (13).

The infusion of insulin into the brain (lateral ventricle)results in the suppression of HGP and lowers blood glucoseeven in the context of diabetes (14–16). The CNS effectson HPG have been ascribed to AgRP neurons because thespecific deletion of IR in AgRP but not POMC neuronsresults in the defective repression of HGP (10,17). The CNS-mediated repression of HGP is orchestrated by vagal effer-ents and a7-nicotinic acetylcholine receptors (18) thatpromote the expression and release interleukin-6 (IL-6)by Kupffer cells in the liver (15,19). IL-6 in turn acts onhepatocytes via STAT3 to repress the expression of gluco-neogenic enzymes such as glucose-6-phosphatase (encodedby G6pc) and phosphoenolpyruvate-carboxykinase (encodedby Pck1) (20,21). Recent studies using pharmacogeneticapproaches have shown that the acute activation of AgRPneurons promotes systemic insulin resistance by repres-sing the activity of sympathetic fibers supplying BAT andthereby BAT glucose uptake, without affecting HGP (13).These effects appear to be independent of the melanocortinsystem because the acute activation of POMC neurons hasno effect on BAT glucose uptake (13). The extent to whichhormones such as insulin influence BAT glucose uptake viaAgRP neurons remains to be determined.

We previously identified the tyrosine phosphataseTCPTP (encoded by Ptpn2) as a key negative regulator ofIR signaling in the periphery and in the ARC (22–26). TCPTPdephosphorylates the IR and attenuates insulin-inducedPI3K/AKT signaling in AgRP neurons (24,26,27). TCPTPdeletion in AgRP neurons exacerbated the insulin-mediatedinhibition of AgRP neurons to increase the sympatheticoutput to BAT and inguinal white adipose tissue (26). Thisboth increased BAT activity and promoted the conversion ofwhite adipocytes to brown-like or beige adipocytes (brown-ing) and increased their thermogenic activity and energyexpenditure to render mice resistant to diet-induced obesity(26). Importantly, we reported that TCPTP abundance in thehypothalamus exhibited diurnal fluctuations linked to feed-ing (26). Fasting increased hypothalamic TCPTP to repressIR signaling to facilitate AgRP neuronal activation, andfeeding resulted in TCPTP being degraded so that IR sig-naling was exacerbated and AgRP neurons inhibited topromote white adipose tissue browning and energy expen-diture (26). In this study, we explored the role of TCPTP inAgRP neurons on glucose metabolism and the extent towhich TCPTP fluctuations may integrate feeding with theCNS control of glucose homeostasis.

RESEARCH DESIGN AND METHODS

MicePtpn2fl/fl, Agrp-Ires-Cre;Ptpn2fl/fl (AgRP-TC), Npy-humanizedrenilla green fluorescent protein (Npy-GFP), Agrp-Ires-Cre;Ptpn2fl/fl;Npy-GFP (AgRP-TC;Npy-GFP), Pomc-eGFP, andAgrp-Ires-Cre;Ptpn2fl/fl;Insrfl/+ (AgRP-TC-IR) mice havebeen described previously (26,28–30). To generateAgrp-Ires-Cre;Ptpn2fl/fl;Insrfl/+;Npy-GFP (AgRP-TC-IR;Npy-GFP)or Agrp-Ires-Cre;Insrfl/+(AgRP-IRfl/+) mice, AgRP-TC;Npy-GFPor Agrp-Ires-Cre mice were mated with Insrfl/fl mice (31),respectively. Mice were maintained on a 12-h light-darkcycle in a temperature-controlled high barrier facility withfree access to food and water. Mice were fed a standardchow (8.5% fat; Barastoc; Ridley AgriProducts, Melbourne,Victoria, Australia). The Monash University School ofBiomedical Sciences Animal Ethics Committee approvedthe experiments.

ImmunohistochemistryMice were injected intraperitoneally with vehicle or hu-man insulin (0.85, 2.5, 5 mU/g, Actrapid; Novo Nordisk,Bagsvaerd, Denmark), then transcardically perfused with4% weight (w)/volume (v) paraformaldehyde, and thepostfixed brains were then processed for phosphorylated(p)-AKT (Ser-473) or GFP immunohistochemistry, asdescribed previously (26).

Quantitative PCRQuantitative real-time PCR was performed as described pre-viously (25,26). The following TaqMan gene expression assayswere used: Pomc (Mm00435874_m1), Npy (Mm03048253_m1), Agrp (Mm00475829_g1), Gapdh (Mm99999915_g1),Pck1 (Mm01247058_m1), G6pc (Mm00839363_m1), andIl6 (Mm00446190_m1).

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Metabolic MeasurementsUnless otherwise indicated, insulin, glucose, and pyruvatetolerance tests were performed on fasted (fasting from9:00 A.M.) conscious mice by injecting human insulin (0.5mU insulin/g body weight, 4-h fasted), D-glucose (2 mg/gbody weight, 6-h fasted), or sodium pyruvate (1 mg/g bodyweight, 6-h fasted), respectively, into the peritoneal cav-ity. Glucose levels in tail blood were measured usingan Accu-Chek glucometer (Roche, Mannheim, Germany).For the determination of fed and fasted blood glucoseand corresponding plasma insulin levels, submandibularblood was collected at 9:00 A.M. after an overnight fast(food removed at 7:00 P.M. the previous day). Plasma insulinlevels were determined using a Rat Insulin RIA kit (LincoResearch, St. Charles, MO) or an in house ELISA (MonashAntibody Technologies Facility). Body composition was mea-sured by EchoMRI (Echo Medical Systems, Houston, TX) orDEXA, as described previously (25). Lateral ventricle cannu-lations were performed as described previously (25,26).

Hyperinsulinemic-Euglycemic ClampsFor hyperinsulinemic-euglycemic clamps, Ptpn2fl/fl, AgRP-TC, or AgRP-TC-IRmice (8–10 weeks old) were anesthetizedunder 2% (v/v) isoflurane in 250 mL/min oxygen, and theleft common carotid artery and the right jugular vein werecatheterized for sampling and infusions, respectively, aspreviously described (32). Catheters were kept patent byflushing dailywith 10–40mL saline containing 200 units/mLheparin and 5 mg/mL ampicillin. Animals were housedindividually after surgery, and body weights were recordeddaily. On the day of the experiment, food was removed atbetween 7:00 and 8:00 A.M. After 3.5 h fasting, a primed(2 min, 0.5 mCi/min) continuous infusion (0.05 mCi/min)of [3-3H]glucose was administered to measure whole-bodyglucose turnover, as described previously (32). After 5 hfasting, mice received a continuous insulin infusion(4 mU/kg/min), and blood glucose was maintained at basallevels by a variable infusion of a 50% (w/v) glucose solution.Arterial blood samples were collected during steady-stateconditions (Ra = Rd) and at 80, 90, 100, 110, and 120min fordetermination of Rd and Ra, as described above. At 120 min,a 13 mCi bolus of [14C]-2-deoxy-D-glucose was injected intothe jugular vein, and arterial blood was sampled at 122, 125,130, 135, and 145 min. Mice were then culled, and tissueswere extracted and frozen for subsequent gene expressionand glucose uptake determinations.

Statistical AnalysesStatistical significance was determined by a one-way ortwo-way ANOVA with multiple comparisons or repeatedmeasures, or by a two-tailed paired Student t test, as appro-priate. P , 0.05 was considered significant.

RESULTS

TCPTP Regulates AgRP Neuronal Insulin Sensitivityand Glucose MetabolismTo determine the extent to which TCPTP deficiency inAgRP/NPY neurons may influence whole-body glucose

metabolism, we crossed Ptpn2fl/fl mice with Agrp-Ires-Cretransgenic mice to excise Ptpn2 in AgRP-expressing neu-rons (Agrp-Ires-Cre;Ptpn2fl/fl: AgRP-TC). TCPTP deletion inAgRP neurons (26) did not influence the overall number ofAgRP/NPY neurons (Supplementary Fig. 1), as definedby the Npy-GFP reporter that marks .85% of AgRPneurons (33). To specifically assess the influence of TCPTPdeficiency on insulin signaling in AgRP/NPY neurons(marked by Npy-GFP), we monitored for AKT Ser-473phosphorylation (p-AKT) by immunofluorescence micros-copy. In particular, we monitored for differences in p-AKTat intraperitoneal insulin doses where p-AKT predomi-nated in AgRP/NPY neurons as opposed to higher insulindoses where p-AKT was also evident in POMC neurons(Fig. 1 and Supplementary Fig. 2). As noted previously (26),basal p-AKT in AgRP/NPY neurons (as reflected by theincreased number of AgRP/NPY neurons positive forp-AKT) was enhanced in AgRP-TCmice (Fig. 1). Importantly,insulin-induced p-AKT in AgRP/NPY neurons was signifi-cantly increased in AgRP-TC mice (Fig. 1). Thus, TCPTPdeficiency in AgRP/NPY neurons enhances insulin signaling.

To explore the effect of TCPTP deficiency and thepromotion of IR signaling in AgRP/NPY neurons onglucose metabolism, we used 8- to 10-week-old chow-fed Ptpn2fl/fl and AgRP-TC mice before any overt differ-ences in body weight (Fig. 2A) and whole-body adiposity, asmeasured by DEXA (Fig. 2B). We found that TCPTP de-ficiency in AgRP/NPY neurons enhanced whole-body in-sulin sensitivity and glucose metabolism, as reflected bythe improved glucose handling in response to boluses ofinsulin or glucose (Fig. 2C andD) and by the reduced fastedblood glucose and plasma insulin levels (Fig. 2E and F). Toexplore the extent to which the enhanced insulin signalingin AgRP/NPY neurons in AgRP-TC mice might be respon-sible for the improved whole-body glucose metabolism, webred AgRP-TC mice onto an Insrfl/+ background so that Insrgene expression in AgRP/NPY neurons would be reducedby 50%. We have shown previously that this largelycorrects the otherwise enhanced hypothalamic insulinsignaling, BAT activity, and white adipose tissue browningin AgRP-TC mice (26). We found that the enhanced in-sulin-induced p-AKT in AgRP/NPY ARC neurons inAgRP-TC mice was attenuated by 67% in AgRP-TC-IR(Fig. 3A). Importantly, glucose responses and fasted bloodglucose and insulin levels in AgRP-TC mice were correctedso that AgRP-TC-IR mice resembled Ptpn2fl/fl controls(Fig. 3C–F). By contrast, IR heterozygosity alone (Agrp-Ires-Cre;Insrfl/+:AgRP-IRfl/+) repressed insulin-induced p-AKTsignaling in AgRP/NPY neurons compared with Insrfl/+

controls (Supplementary Fig. 3A). This was not, however,sufficient to overtly affect glucose metabolism (Supple-mentary Fig. 3B–G), precluding any effects of IR hetero-zygosity in AgRP-TC mice being due to baseline effects onglucose homeostasis. TCPTP deficiency and the promotionof IR signaling in AgRP/NPY neurons therefore improveswhole-body glucose metabolism independent of any effectson body weight.

1248 TCPTP in AgRP Neurons Controls Glucose Metabolism Diabetes Volume 67, July 2018

TCPTP Deficiency Improves Systemic InsulinSensitivity

To determine how TCPTP deficiency and the promotion ofIR signaling in AgRP/NPY neurons might improve glucosemetabolism, we used pyruvate tolerance tests as a means ofassessing effects on HGP in the mice; administration of thegluconeogenic substrate pyruvate can increase blood glucoselevels by promoting gluconeogenesis. We found that glucoseexcursions in response to pyruvate (mice fasted for 6 h from9:00 A.M.) were significantly attenuated in AgRP-TC miceand reversed by Insr heterozygosity in AgRP-TC-IR mice(Fig. 4A). Moreover, genes encoding glucose-6 phosphatase(G6pc) and phosphoenolpyruvate-carboxykinase (Pck1),enzymes involved in the rate-limiting steps of gluconeogen-esis, were decreased in AgRP-TC mice (Fig. 4B). The de-creased glucose excursions in response to pyruvate inAgRP-TC mice were corrected by Insr heterozygosity, so

that AgRP-TC-IR mice were indistinguishable from Ptpn2fl/fl

controls (Fig. 4A). These results are consistent with TCPTPdeficiency and the promotion of IR signaling in AgRP/NPYneurons repressing hepatic gluconeogenesis.

Because the transformation of exogenous pyruvate intohepatic glucose is highly dependent on insulin sensitivity,we further explored the effects of TCPTP deficiency inAgRP/NPY neurons on whole-body insulin sensitivity andglucose metabolism using hyperinsulinemic-euglycemicclamps. The glucose infusion rate (GIR) needed to maintaineuglycemia during the clamp was markedly increased inAgRP-TC versus Ptpn2fl/fl mice, consistent with TCPTP de-ficiency in AgRP/NPY neurons improving whole-body in-sulin sensitivity (Fig. 4C and Supplementary Fig. 4). Theimproved insulin sensitivity was accompanied by an in-creased repression of endogenous glucose production(EGP), a measure of HGP (Fig. 4D and E), decreased hepatic

Figure 1—Deletion of TCPTP in AgRP neurons enhances insulin signaling. Male 8- to 10-week-old AgRP-TC;Npy-GFP or Ptpn2fl/fl;Npy-GFPovernight-fasted mice were administered (intraperitoneal) saline (Vehicle) or 0.85 mU/g insulin for 15 min, and paraformaldehyde-fixed brainswere processed for immunofluorescence microscopy monitoring for p-AKT hypothalamic immunoreactivity. Representative images andquantified (means 6 SEM) results are shown for the indicated number of mice. III, 3rd ventricle; 1ve, positive. *P , 0.05; ***P , 0.001.

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expression of the gluconeogenic genes G6pc and Pck1 andincreased hepatic expression of Il6 (Fig. 4F), and an increasedRd (a measure of glucose uptake) (Fig. 4G). Basal endogenousglucose production and glucose disposal were not altered inAgRP-TC versus Ptpn2fl/flmice in keeping with the improvedglucose metabolism being a specific response to insulin(Fig. 4D and G). Furthermore, the increased GIR, decreasedhepatic gluconeogenic gene expression, increased hepaticIl6 expression, decreased EGP, and increased Rd in clampedmice were completely corrected in AgRP-TC-IR mice (Fig.4C–G), causally linking the improved systemic insulin sen-sitivity and glucose metabolism in AgRP-TC mice with thepromotion of insulin signaling in AgRP/NPY neurons.

TCPTP Deficiency Represses HGPOur studies point toward the improved glucose homeostasisin AgRP-TC mice resulting from both the enhanced repres-sion of HGP and increased Rd. The effects on the liver maytherefore be secondary to the overall improvement in systemicinsulin sensitivity or attributable to exacerbated hypotha-lamic-liver axis responses. To test the influence on the hypo-thalamic-liver axis, overnight fasted Ptpn2fl/fl versus AgRP-TCmice were intracerebroventricularly administered vehicle or

insulin and then processed for pyruvate tolerance tests, or thehypothalamus and liver were extracted and processed forquantitative real-time PCR (Fig. 5A–D). Although glucoseexcursions in response to pyruvate were not altered inPtpn2fl/fl mice at the insulin doses chosen, pyruvate responseswere significantly repressed in AgRP-TC mice (Fig. 5B). More-over, intracerebroventricularly administered insulin signifi-cantly repressed the hypothalamic expression of Agrp andNpy in AgRP-TC mice without altering Pomc expression (Fig.5C) and increased hepatic Il6 expression while repressing Pck1andG6pc expression (Fig. 5D). Although basal hepatic Pck1 andG6pc expression in AgRP-TC mice was increased (Fig. 5D), thiswas likely a compensatory response to prevent overt hypo-glycemia after the overnight fast. Taken together, these resultsare consistent with the enhanced insulin signaling in AgRP/NPY neurons acting through the hypothalamic-liver axis todirectly repress HGP and improve glucose metabolism.

TCPTP Deficiency Increases BAT Glucose UptakeAcute changes in AgRP neuronal activation may eliciteffects on glucose metabolism by specifically influencingglucose uptake in BAT (13). Our studies indicate that theenhanced IR signaling in AgRP/NPY neurons in AgRP-TC

Figure 2—TCPTP deficiency in AgRP neurons improves whole-body glucose metabolism. Body weight (A) and body composition (B) of8-week-old AgRP-TC or Ptpn2fl/fl male mice. BMD, bone mineral density. Insulin (0.5 mU/g) (C) or glucose (2 mg/g) (D) tolerance tests wereperformed in 8-week-old male AgRP-TC or Ptpn2fl/fl mice, and areas under curves (AUC) were determined. Fed and fasted (14 h) plasmainsulin (E ) and blood glucose (F ) levels from 10-week-old AgRP-TC or Ptpn2fl/flmale mice. Results shown are means6 SEM for the indicatednumber of mice. *P , 0.05; **P , 0.01; ***P , 0.001.

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mice increases systemic insulin sensitivity, at least in partby improving Rd. To determine the extent to which thismay involve BAT glucose uptake, we administered mice[14C]-2-deoxy-D-glucose at the end of hyperinsulinemic-euglycemic clamps and assessed uptake in varied tissues(Fig. 4H). Although glucose uptake was not altered in thebrain (hypothalamus), where glucose uptake is not insulinresponsive, or in skeletal muscle or epididymal whiteadipose tissue, where insulin promotes glucose uptake,we found that glucose uptake was overtly increased in theBAT of AgRP-TC mice (Fig. 4H). Glucose uptake was alsoincreased in the inguinal fat pads of AgRP-TC mice (Fig.4H), where we have shown previously there is an increasedabundance of thermogenically active beige adipocytes (26).By contrast, we did not note any increase in glucose uptake

in epididymal white adipose tissue in AgRP-TC mice (Fig.4H). Because the epididymal fat pad does not undergobrowning in AgRP-TC mice (26), these results are con-sistent with TCPTP deficiency in AgRP/NPY neuronsspecifically increasing glucose uptake in brown and beigeadipocytes. Moreover, the selective increase in BAT andinguinal white adipose tissue glucose uptake points to-ward this being a direct response to TCPTP deficiency inAgRP/NPY neurons rather than being an outcome ofthe systemic increase in insulin sensitivity. Importantly,the increased glucose uptake in BAT and inguinal whiteadipose tissue glucose were reduced to normal levels byInsr heterozygosity, so that AgRP-TC-IR mice were indis-tinguishable from Ptpn2fl/fl controls (Fig. 4H). Our studiespoint toward TCPTP deficiency in AgRP/NPY neurons

Figure 3—TCPTP deficiency in AgRP neurons improves whole-body glucosemetabolism by promoting insulin signaling in AgRP neurons.A:Male 8- to 10-week-old Ptpn2fl/fl;Npy-GFP, AgRP-TC;Npy-GFP, or AgRP-TC-IR;Npy-GFP overnight-fasted mice were administered(intraperitoneal) saline or 0.85 mU/g insulin for 15 min, and paraformaldehyde-fixed brains were processed for immunofluorescencemicroscopy monitoring for p-AKT hypothalamic immunoreactivity. B: Body weights in 8-week-old male Ptpn2fl/fl, AgRP-TC, or AgRP-TC-IRmice. Glucose (2 mg/g) (C ) or insulin (0.5 mU/g) (D) tolerance tests were performed in 8-week-old male Ptpn2fl/fl, AgRP-TC, or AgRP-TC-IRmice, and areas under curves (AUC) were determined. Fed and fasted blood glucose (E) and plasma insulin (F) levels in 10-week-oldPtpn2fl/fl,AgRP-TC, or AgRP-TC-IR male mice. Representative images and quantified (means 6 SEM) results are shown for the indicated number ofmice. III, 3rd ventricle; 1ve, positive. *P , 0.05; **P , 0.01; ***P , 0.001.

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promoting IR signaling to improve whole-body glucosemetabolism via the repression of HGP and the promotionof glucose uptake in brown/beige adipocytes.

TCPTP Regulates Feeding-Associated HepaticGlucose MetabolismWe recently showed that TCPTP levels in the ARC arecoordinated by diurnal feeding rhythms (26). Accordingly,

we asked whether diurnal fluctuations in TCPTPmight alsohelp regulate the hypothalamic control of HGP. To explorethis, we determined whether TCPTP deficiency in AgRPneurons might differentially influence the hypothalamic-liver axis in response to feeding and fasting. To test this weintracerebroventricularly administered vehicle or insulinto mice where food was withheld (food restricted) at thestart of the dark cycle (Fig. 6A–F) versus ad libitum fed

Figure 4—TCPTP deficiency in AgRP neurons enhances the repression of HGP andBAT glucose uptake.A: Pyruvate tolerance tests (1mg/g)were performed in 8-week-old Ptpn2fl/fl, AgRP-TC, or AgRP-TC-IR male mice, and areas under curves (AUC) were determined. B: Male9-week-old Ptpn2fl/fl, AgRP-TC, or AgRP-TC-IR ad libitum fed mice were culled and livers extracted for quantitative PCR. C–H: Hyper-insulinemic-euglycemic clamps were performed in conscious unrestrained 8- to 10-week-old Ptpn2fl/fl, AgRP-TC, or AgRP-TC-IRmale mice.Results are shown for GIR (C ), basal and clamped EGP (D and E), gene expression in extracted livers (F ), and glucose Rd (G). H:Hyperinsulinemic-euglycemic clamped mice were administered a bolus of [14C]-2-deoxy-D-glucose (13 mCi intravenously), and tissue-specific insulin-stimulated 2-deoxy-D-glucose (2DG) uptake was determined in brain (hypothalamus), BAT, gastrocnemius muscle, andepididymal and inguinal white adipose tissue (WAT). Results shown are means 6 SEM for the indicated number of mice. In panel C, #, ##,and ### correspond to AgRP-TC vs. AgRP-TC-IR. *P , 0.05; **P , 0.01; ***P , 0.001; #P , 0.05; ##P , 0.01; ###P , 0.001.

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mice 4 h after the start of the dark cycle (Fig. 6G–K), whenwe have shown previously mice are satiated (26); underthese conditions, hypothalamic TCPTP levels are high andlow, respectively (26). To explore the influence on hepaticglucose metabolism in the mice, we used pyruvate toler-ance tests or extracted livers for gene expression analyses.In food-restricted Ptpn2fl/fl control mice, when hypotha-lamic TCPTP levels were high, we found that intracere-broventricular insulin had no effect on glucose excursionsin response to pyruvate (Fig. 6B and E). By contrast, whenhypothalamic TCPTP levels were low in fed Ptpn2fl/fl

mice, intracerebroventricular insulin effectively repressedpyruvate responses (Fig. 6H and K). Strikingly, pyruvateresponses in food-restricted AgRP-TC mice were not onlylower than in Ptpn2fl/fl controls but reduced further inresponse to intracerebroventricular insulin (Fig. 6C and E),whereas responses in fed mice were similar to those ofcontrols (Fig. 6I and K). Moreover, the precocious intra-cerebroventricular insulin-mediated repression of pyru-vate responses accompanying AgRP TCPTP deficiency inotherwise unresponsive food-restricted mice were cor-rected by Insr heterozygosity (Fig. 6D and E), whereaspyruvate responses in fed mice were not altered by Insrheterozygosity (Fig. 6I and K). Similarly, in food-restrictedPtpn2fl/fl mice, hepatic Il6, Pck1, and G6pc expression wasunaltered by intracerebroventricular insulin, whereas inAgRP-TC mice, intracerebroventricular insulin increasedhepatic Il6 expression and repressed Pck1 and G6pc

expression, and these responses were corrected in AgRP-TC-IR mice (Fig. 6F). These results point toward diurnalfeeding-associated fluctuations in TCPTP in AgRP neu-rons serving to coordinate hepatic glucose metabolism.

DISCUSSION

Gene deletion studies in rodents have established thatinsulin action on AgRP neurons in the hypothalamus isimportant for insulin’s ability to suppress HGP (10).Moreover, recent human studies exploring the utility ofintranasal insulin administration have substantiated insu-lin’s ability to act via the CNS to suppress HGP and promoteglucose uptake in lean but not in overweight individuals(34,35). However, the mechanisms by which hypothalamicinsulin action is coordinated to influence whole-body glu-cose metabolism remain unclear.

We have shown previously that the abundance of theIR phosphatase TCPTP in hypothalamic neurons, includingPOMC and AgRP neurons, is altered by diurnal feedingrhythms in mice (26). Fasting increases TCPTP expression,whereas feeding represses TCPTP expression and promotesits rapid degradation (26). Increases in TCPTP serve toattenuate IR signaling in AgRP neurons after a fast tofacilitate AgRP neuronal activation by hormones such asghrelin (26), whereas the postprandial elimination of TCPTPhelps promote IR signaling in AgRP neurons to facilitateAgRP neuronal inhibition by circulating insulin (26). Wehave shown that the regulation of IR signaling by TCPTP in

Figure 5—TCPTP deficiency in AgRP neurons promotes the intracerebroventricular insulin-mediated repression of HGP. A: Experimentalparadigm schematic: 8-week-old Ptpn2fl/fl or AgRP-TCmalemicewere fasted overnight and administered intracerebroventricular (ICV) saline(Vehicle) or insulin (0.1 mU/animal, five injections over 5 h) as indicated in B, C, and D. Pyruvate tolerance tests (PTT) were performed, andareas under curves (AUC) were determined (B) or the hypothalamus (C) and liver (D) were extracted for quantitative PCR. Results shown aremeans 6 SEM for the indicated number of mice. *P , 0.05; **P , 0.01; ***P , 0.001.

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Figure 6—TCPTP in AgRP neurons inhibits the intracerebroventricular insulin-mediated repression of HGP in fasted mice. A and G:Experimental paradigm schematics. B–F: Male 8- to -10-week-old Ptpn2fl/fl, AgRP-TC, or AgRP-TC-IR mice were food restricted (just beforelights out, 6:30 P.M.) and administered intracerebroventricular (ICV) saline (Vehicle) or insulin (0.1 mU/animal, five injections over 5 h) asindicated. Pyruvate tolerance tests (PTT) were performed, and areas under curves (AUC) were determined (B–E ) or livers were extracted forquantitative PCR (F ). H–K: Male 8- to 10-week-old Ptpn2fl/fl, AgRP-TC, or AgRP-TC-IR mice were ad libitum fed until satiated (4 h after lightsoff) and administered intracerebroventricular saline or insulin (0.1 mU/animal, five injections over 5 h) as indicated. Pyruvate tolerance tests(1mg/g) were performed, and areas under curves (AUC) were determined. Results shown aremeans6SEM for the indicated number of mice.*P , 0.05; **P , 0.01; ***P , 0.001.

1254 TCPTP in AgRP Neurons Controls Glucose Metabolism Diabetes Volume 67, July 2018

AgRP neurons coordinates the browning of white adiposetissue and the expenditure of energy with feeding, so thatfasting and AgRP neuronal activation repress browning andfeeding and AgRP neuronal inhibition promotes browningand the expenditure of energy (26). In this way, diurnalfluctuations in hypothalamic TCPTP associated with feedingand fasting help maintain energy balance.

Here we report that the regulation of IR signaling byTCPTP in AgRP neurons is also important in coordinatingwhole-body glucose metabolism. We demonstrate thatTCPTP deletion in AgRP neurons (emulating the fed statewhen hypothalamic TCPTP is eliminated) promotes IRsignaling to enhance whole-body insulin sensitivity andglucose homeostasis. Mice lacking TCPTP in AgRP neuronsshow improved responses to glucose, pyruvate, and in-sulin, reduced fasted blood glucose and plasma insulinlevels, and reduced GIRs during hyperinsulinemic-eugly-cemic clamps, independent of any differences in bodyweight/adiposity. Importantly, the enhanced glucosemetabolism could be corrected by Insr heterozygosity inAgRP neurons, which largely corrected insulin-inducedPI3K/AKT signaling in AgRP neurons. The improved glu-cose metabolism was partly attributable to the enhancedsuppression of HGP. The enhanced suppression of HGPwas a direct consequence of the hypothalamic-liver axis(7,10,15,16,18–21), because the CNS insulin-induced pro-motion of hepatic Il6 expression and STAT3 signaling, andconsequent suppression of gluconeogenic genes and glu-cose excursions in response to pyruvate, were exacerbatedby TCPTP deficiency in AgRP neurons. Although TCPTPdeficiency in AgRP neurons attenuated hepatic gluconeo-genic gene expression, we cannot rule out a contribution ofglycogenolysis to the overall suppressed HGP in AgRP-TCmice, because previous studies have indicated that AgRPneurons may regulate glycogenolysis (36).

The extent to which feeding-/fasting-associated TCPTPfluctuations in AgRP neurons might help coordinate HGPwas highlighted by the lack of any overt effect of intra-cerebroventricular insulin on pyruvate-induced glucoseexcursions and hepatic gluconeogenic gene expression infood-restricted control mice, where hypothalamic TCPTPlevels would be high (26). This was contrasted by the strikingability of TCPTP deletion in AgRP neurons to reinstatesuch responses. By comparison, ad libitum fed and satiatedmice readily responded to intracerebroventricular insulinby repressing hepatic gluconeogenic gene expression andpyruvate-induced glucose excursions, and these responseswere unaltered by TCPTP deletion. Our results point towardthe control of IR signaling by TCPTP in AgRP neuronsserving to coordinate hepatic glucose metabolism so thatfasting is accompanied by elevated HGP to prevent hypo-glycemia. In obesity, where we have shown hypothalamicTCPTP levels are high and remain elevated even afterfeeding (26,28), the resulting sustained repression of IRsignaling in AgRP neurons would be expected to contributeto the elevated HGP and hyperglycemia characteristic of theobese and insulin-resistant state. However, the decreased

weight gain evident in high fat–fed AgRP-TC mice pro-hibited us from testing this directly.

Beyond influencing glucose production, our studiesindicate that the regulation of IR signaling in AgRP neu-rons might also affect glucose clearance by specificallyinfluencing glucose uptake in BAT and inguinal whiteadipose tissue, where browning in mice predominates(37–39). Activated brown and beige adipocytes containhigh amounts of the uncoupling protein-1 (UCP-1), allow-ing for the uncoupling of fatty acid oxidation from ATPproduction to generate heat (2,3,39). Although largelyignored as a tissue involved in glucose homeostasis, earlyhyperinsulinemic-euglycemic clamp studies in rodents high-lighted BAT as a major insulin-responsive depot for glucoseuptake, exceeding on a per unit mass basis glucose uptake inmuscle or white adipose tissue (1). Consistent with this, wefound that when normalized for tissue mass, BAT was moreeffective thanmuscle or epididymal adipose tissue in taking upglucose under hyperinsulinemic-euglycemic conditions. Inhumans, brown and beige adipocytes are found interspersedin different white fat depots, including the supraclaviculardepot as well as in the supraspinal, pericardial, and neckregions (2,39–45). Implantation of human brown/beige adi-pocytes into chow-fed, high fat–fed, or glucose-intolerantNOD-scid IL2(null) mice dramatically enhances systemic glu-cose tolerance (46). Moreover, variables such as high BMI,increased age, or type 2 diabetes have been shown to correlatewith attenuated brown/beige glucose uptake at least in somestudies (41,47–49). Noteworthy, Lee et al. (5) recently high-lighted the importance of brown/beige fat in humans insystemic glycemic control, demonstrating that higherbrown/beige fat activity results in lesser glycemic variabil-ity. Moreover, the same study demonstrated that the ther-mogenic activity of brown/beige fat in humans was increasedin response to a glucose challenge, which increases circulatinginsulin (5). Our recent studies have shown that enhancedleptin plus insulin signaling in POMC neurons or insulinsignaling in AgRP neurons can function to promote BATactivity and the browning of inguinal white adipose tissuein rodents (25,26). In particular, we reported that in fed/satiatedmice when TCPTP was degraded, the enhancement ofinsulin signaling and resultant inhibition of AgRP neuronsincreased the sympathetic innervation and browning of whiteadipose tissue to promote the expenditure of energy (26).Mice lacking TCPTP in AgRP neurons were remarkably re-sistant to weight gain due to the increased white adiposetissue browning as well as BAT activity (26). In this study weassessed the influence of TCPTP loss and the promotion ofinsulin signaling in AgRP neurons on glucose metabolism. Forthese studies, we used 8- to 10-week-oldmice before any overtdifferences in adiposity/body weight resulting from the in-creased browning and BAT activity. Our studies indicate thatthe promotion of insulin signaling and inhibition AgRPneurons by TCPTP deletion at least partly improves glucosemetabolism through the promotion of BAT and beige adipo-cyte glucose uptake. On the other hand, Steculorum et al. (13)reported that the pharmacogenetic activation of AgRP

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neurons promotes insulin resistance by repressing BAT glu-cose uptake.

Our results demonstrate that the deletion of TCPTP andinhibition of AgRP neurons (26) promotes systemic insulinsensitivity accompanied by increased glucose uptake in BATand beige adipocytes in inguinal fat depots. Our findingsare consistent with the TCPTP control of AgRP neuronalactivation being instrumental in coordinating BAT/beigeadipocyte activity with glucose uptake. Consistent with thisassertion, Lee et al. (5) reported that BAT glucose uptake inhumans correlates with thermogenesis (as assessed by mea-suring heat productionwith infrared thermography). Becausefatty acid oxidation is essential for BAT thermogenesis(50–53), glucose likely indirectly contributes to BAT/beigeadipocyte activity by promoting lipogenesis and/or support-ing ATP production during thermogenic responses throughanaerobic glycolysis (2,3). By contrast, other studies arguethat BAT activity and glucose uptake can be dissociated.Blondin et al. (49), for example, reported recently that despiteglucose uptake in BAT being diminished in older men withtype 2 diabetes, cold-induced BAT oxidative metabolism andthermogenesis were not altered. However, this does notpreclude feeding-induced beige adipocyte thermogenesis/WAT browning (26) normally being coordinated withglucose metabolism. This would provide an effective mech-anism for coordinating both the removal and utilization ofglucose by beige adipocytes to prevent of postprandialhyperglycemia.

Our results underscore the critical role of CNS insulinsignaling in coordinating peripheral glucose metabolismthrough effects on both HGP and BAT/beige adipocyteglucose uptake. Taken together with our previous findings(26), our results point toward feeding-/fasting-associatedalterations in hypothalamic TCPTP integrating the sys-temic control of glucose metabolism and energy expendi-ture with the nutritional state of the organism to maintainglucose and energy homeostasis. Thus, the promotion ofCNS insulin sensitivity is likely to provide an importantmeans by which to concomitantly promote weight lossand improve whole-body glucose metabolism and glycemiccontrol in obesity and type 2 diabetes.

Acknowledgments. The authors thank Sunena Bhandari for technicalsupport (Department of Biochemistry and Molecular Biology, Monash University,Clayton, Melbourne, Victoria, Australia) and Herbert Herzog (Neuroscience Di-vision, Garvan Institute of Medical Research, St. Vincent’s Hospital, Sydney, NewSouth Wales, Australia) for access to Insrfl/fl mice.Funding. This work was supported by the Diabetes Australia Research Trust(Y16G-DODG to G.T.D.) and by the National Health and Medical Research Council ofAustralia (APP1100240 to T.T.). T.T. is a National Health and Medical ResearchCouncil Principal Research Fellow.Duality of Interest. No potential conflicts of interest relevant to this articlewere reported.Author Contributions. G.T.D. and R.S.L.-Y. performed investigations. G.T.D.,R.S.L.-Y., J.C.B., and T.T. contributed to the methodology and wrote, reviewed,and edited the manuscript. G.T.D. and T.T. conceptualized the study and wrote theoriginal draft of the manuscript. T.T. is the guarantor of this work and, as such, had

full access to all the data in the study and takes responsibility for the integrity of thedata and the accuracy of the data analysis.

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