Article Leptin and Insulin Act on POMC Neurons
to Promote the Browning of White Fat
Garron T.Dodd,1Stephanie Decherf,1Kim Loh,1Stephanie E.Simonds,2Florian Wiede,1Eglantine Balland,2larissa
Troy L.Merry,1Heike Mu¨nzberg,3Zhong-Yin Zhang,4Barbara B.Kahn,5Benjamin G.Neel,6Kendra K.Bence,7
Zane B.Andrews,2Michael A.Cowley,2and Tony Tiganis1,*
1Department of Biochemistry and Molecular Biology,Monash University,Victoria3800,Australia
2Department of Physiology,Monash University,Victoria3800,Australia
3Pennington Biomedical Rearch Center,LSU Systems,Baton Rouge,LA70808,USA
4Department of Biochemistry and Molecular Biology,Indiana University School of Medicine,Indianapolis,IN46202,USA
5Division of Endocrinology,Diabetes and Metabolism,Department of Medicine,Beth Israel Deaconess Medical Center and Harvard Medical School,Boston,MA02215,USA
6Campbell Family Cancer Rearch Institute,Ontario Cancer Institute,Princess Margaret Hospital and Department of Medical Biophysics, University of Toronto,Toronto,ON M5G2M9,Canada
7Department of Animal Biology,School of Veterinary Medicine,University of Pennsylvania,Philadelphia,PA19104,USA
*Correspondence:tony.tiganis@monash.edu
/10.ll.2014.12.022
SUMMARY
The primary task of white adipo tissue(WAT)is the storage of lipids.However,‘‘beige’’adipocytes also exist in WAT.Beige adipocytes burn fat and dissipate the energy as heat,but their abundance is diminished in obesity.Stimulating beige adipocyte develop-ment,or WAT browning,increas energy expendi-ture and holds potential for combating metabolic dia and obesity.Here,we report that insulin and leptin act together on hypothalamic neurons to promote WAT browning and weight loss.D
eletion of the phosphatas PTP1B and TCPTP enhanced insulin and leptin signaling in proopiomelanocortin neurons and prevented diet-induced obesity by increasing WAT browning and energy expenditure. The coinfusion of insulin plus leptin into the CNS or the activation of proopiomelanocortin neurons also incread WAT browning and decread adiposity. Ourfindings identify a homeostatic mechanism for coordinating the status of energy stores,as relayed by insulin and leptin,with the central control of WAT browning.
INTRODUCTION
There are two types of adipo tissue in humans,white adipo tissue(WAT)and brown adipo tissue(BAT).WAT can store vast amounts of chemical energy as triglycerides(TAGs)for uti-lization during periods of fasting or starvation.In contrast,BAT dissipates the chemical energy stored in TAGs as heat to pre-rve core temperature during hypothermia and to counteract obesity(Ron and Spiegelman,2014).Brown adipocytes contain a high density of mitochondria with high amounts of un-coupling protein-1(UCP-1)allowing for the uncoupling of fatty acid oxidation from ATP production to generate heat(Ron and Spiegelman,2014).Although BAT was initially considered to be prent only in infants,it is now established that substantial depots of UCP-1expressing brown-like fat can be detected in the supraspinal,supraclavicular,pericardial,and neck regions of adult humans(Cypess et
al.,2009;van Marken Lichtenbelt et al.,2009;Virtanen et al.,2009).The brown-like fat depots can be induced in respon to cold,but their abundance is diminished in older and obe subjects(Lee et al.,2014;Ouellet et al.,2011).
Brown-like fat is also found in rodents and is compod of beige adipocytes intersperd among white adipocytes(Ron and Spiegelman,2014).Under basal conditions,beige adipo-cytes express little to no UCP-1,but UCP-1induction in respon to cold promotes thermogenesis and energy expendi-ture(Ron and Spiegelman,2014).Interestingly,the interscap-ular BAT in human infants is similar to classical brown fat in rodents(Lidell et al.,2013),whereas the brown-like fat in adult humans has a molecular signature reminiscent of rodent beige fat(Lidell et al.,2013;Wu et al.,2012).Increasing WAT browning in rodents increas energy expenditure and suppress diet-induced obesity and gluco intolerance(Seale et al.,2011). On the other hand,preventing WAT browning by deleting Prdm16,a transcriptional cofactor that increas UCP-1expres-sion,promotes obesity and vere insulin resistance(Cohen et al.,2014;Seale et al.,2011).Understanding the molecular pro-cess governing WAT browning is highly significant,as this may identify novel approaches for increasing energy expenditure and combating obesity and the metabolic syndrome.Efforts to date have focud primarily on the role of factors that act directly on beige pread
ipocytes,such as irisin and FGF21(Bostro¨m et al., 2012;Lee et al.,2014).However,there is mounting evidence that the central nervous system(CNS)control of WAT browning is also important(Plum et al.,2007;Ruan et al.,2014;Williams et al.,2014).
Leptin is produced by adipocytes and is critical for energy homeostasis and body weight control.Leptin receptors (LEPRs)are expresd in distinct regions of the brain,including the arcuate nucleus(ARC)of the hypothalamus(Myers et
al.,
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2008).The ARC contains two opposing neuronal populations: the appetite-suppressing proopiomelanocortin(POMC)and the orexigenic neuropeptide Y(NPY)and agouti-related peptide (AgRP)-neuropeptide-expressing neurons(Cowley et al.,2001; Elias et al.,1999).Leptin acts on POMC and NPY/AgRP neurons to suppress food intake and promote energy expendi-ture(Myers et
al.,2008).One mechanism by which leptin increas energy expenditure is through the promotion of BAT thermogenesis.Leptin action in the hypothalamus in-creas sympathetic nerve activity(SNA)to BAT,increasing both UCP-1expression and BAT activity(Commins et al., 2000;Morrison et al.,2014).Leptin signals via the LEPR to activate Janus-activated kina(JAK)-2,which promotes signaling via effector cascades,including the phosphatidyinosi-tol3-kina(PI3K)/AKT and signal transducer and activator of transcription(STAT)-3pathways,to increa Pomc expression and inhibit Agrp expression(Myers et al.,2008).The principal role of the melanocortin system in body weight control is underscored by the marked obesity in humans and rodents with null mutations in the leptin,LEPR,or POMC genes(Coll et al.,2004).
Another peripheral factor affecting the melanocortin system is insulin(Varela and Horvath,2012).Insulin is relead from pancreatic b cells following a ri in blood gluco and acts via the insulin receptor(IR)tyrosine kina and the PI3K/AKT pathway in liver,muscle,and fat to lower blood gluco levels(Saltiel and Kahn,2001).Insulin also acts in the ARC on POMC and AgRP/NPY neurons to regulate whole-body gluco metabolism and elicit anorectic respons (Benoit et al.,2002;Bru¨ning et al.,2000;Ko¨nner et al.,2007). One prevailing view is that different POMC neurons exist and that leptin and insulin may act on distinct POMC neuronal subts(Hill et al.,2010;Sohn et al.,2011;Williams et al., 2010).
The protein tyrosine phosphatas PTP1B(PTPN1)and TCPTP(PTPN2)regulate body weight and gluco homeosta-sis(Tiganis,2013).PTP1B dephosphorylates JAK2to suppress leptin signaling in hypothalamic neurons,including POMC neurons(Banno et al.,2010;Bence et al.,2006;Tiganis, 2013),whereas TCPTP dephosphorylates STAT3in the hypo-thalamus(Loh et al.,2011).We have taken advantage of mice lacking PTP1B,TCPTP,or both phosphatas in POMC neu-rons to demonstrate that PTP1B and TCPTP lectively regu-late leptin and insulin signaling to affect body weight,energy expenditure,and peripheral gluco homeostasis.We report that the combined inactivation of PTP1B and TCPTP and the promotion of leptin and insulin signaling in POMC neurons in-creas WAT browning and energy expenditure and prevents the development of diet-induced obesity.Ourfindings identify a mechanism in which POMC neurons integrate insulin and leptin feedback to drive WAT browning and maintain energy homeostasis.
RESULTS
Decread Adiposity in POMC-TC Mice
Previous studies have established that PTP1B regulates leptin signaling in POMC neurons(Banno et al.,2010),but the preci neuronal populations in which TCPTP exerts its effects remain unknown.Th
us,we assd TCPTP versus GFP expression in the hypothalami of Pomc-GFP transgenic mice(Figures1A and S1A available online).TCPTP protein was detected in 28.3%±5.9%of all GFP-positive POMC neurons in the ARC with colocalization predominating in the central-caudal ARC (Figure1B),where both LEPR-and IR-responsive POMC neu-rons have been detected(Williams et al.,2010).No TCPTP stain-ing was detected in the rostral ARC(Figures1B and S1B).TCPTP colocalization with GFP-expressing POMC neurons was confirmed byflow cytometry in papain-digested hypothalami (Figure1C).LEPR-responsive POMC neurons are also found in the nucleus of the solitary tract(NTS)in the hindbrain.Although TCPTP expression was evident in the NTS,the majority of TCPTP staining did not colocalize with GFP-expressing POMC neurons(Figure S1C).
Next,we examined the localization of PTP1B in POMC neu-rons in the ARC and its coincidence with TCPTP(Figures1D–1F and S1A).PTP1B was detected in74.8%±11.2%of all GFP-positive POMC neurons in the ARC,with PTP1B/GFP co-localization predominating in the central-caudal ARC(Figure1E). In contrast to TCPTP,PTP1B was also expresd in the rostral ARC(Figure S1B).Moreover,PTP1B expression in the central-caudal ARC was not restricted to medial POMC neurons,where it largely coincided with TCPTP(Figure1F)but was also found in lateral POMC neurons,where TCPTP expression was not evident(Figures1B,1E,and1F).Also,TCPTP,but not PTP1
B, was expresd in a subt of medial-caudal POMC neurons(Fig-ure1F).Thus,TCPTP and PTP1B are found in both overlapping and distinct POMC neuronal subts in the ARC.
To determine whether TCPTP might function in the melano-cortin pathway,we crosd Ptpn2fl/flmice(Loh et al.,2011) with Pomc-Cre transgenic mice to exci Ptpn2(Pomc-Cre; Ptpn2fl/fl:POMC-TC)in POMC-expressing neurons(Figures S1D and S1E).We compared the mice to PTP1B POMC neuronal cell-specific knockout mice(Pomc-Cre;Ptpn1fl/fl: POMC-1B),generated as described previously(Banno et al., 2010).To visualize POMC cell-specific Cre-mediated recombi-nation,we crosd POMC-TC mice to Z/EG reporter mice that express GFP after Cre-mediated recombination(Figure1G). Z/EG;POMC-TC mice expresd GFP in the ARC and this over-lapped with92%of POMC-expressing cells(Figures1G and1H); no GFP staining was evident in non-POMC cells.As reported for POMC-1B mice(Banno et al.,2010),no differences were evident in the number of hypothalamic POMC neurons in POMC-TC mice(Figure1I).POMC is expresd in the ARC,as well as the pituitary and the NTS in the hindbrain.In addition,ARC POMC neurons project to the lateral reticular nucleus in the brainstem.Consistent with this,the recombined Ptpn2allele (D Ptpn2)in POMC-TC mice was evident in whole-brain,hypo-thalamic,pituitary,and hindbrain DNA extracts,but not in liver extracts(Figure S1D).As expected,differences in TCPTP protein were not obrved in the
hypothalamic extracts of POMC-TC versus control mice(Figure S1E)becau POMC neurons constitute only a small proportion of the total hypothalamic cell population(Cowley et al.,2001).
Body weights were not altered in POMC-TC or POMC-1B versusfloxed controls(Figures2A,2B,and S2A).Nevertheless, Cell160,88–104,January15,2015ª2015Elvier Inc.89
Figure1.TCPTP and PTP1B in POMC Neurons
mcts(A)Immunostaining for TCPTP in GFP-positive ARC POMC neurons.
(B)Rostral-caudal immunostaining quantification of GFP-and TCPTP-expressing ARC cells.
sheet什么意思
(C)Papain-digested hypothalami of Pomc-GFP mice were analyzed byflow cytometry for GFP and TCPTP.
(D)Immunostaining for PTP1B in GFP-positive ARC POMC neurons.
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临时翻译
epididymal and subcutaneous fat pad weights were decread in POMC-TC mice and variably decread in POMC-1B mice; only epididymal fat was significantly decread in POMC-1B mice(Figures2A and2B).Dual-energy X-ray absorptiometry (DEXA)revealed that whole-body adiposity was significantly decread in POMC-TC mice,but not in POMC-1B mice(Fig-ures2A and2B).Neither bone mineral densities nor lean mass were altered(Figures2A and2B),and body le
ngths and liver weights were similar in both groups(Figures S2B and S2C).Previous studies have established that PTP1B dele-tion in the pituitary(Cga-Cre;Ptpn1lox/lox)does not impact on body weight(Banno et al.,2010).Similarly,we found that TCPTP deletion in the pituitary(Cga-Cre;Ptpn2lox/lox)did not result in overt differences in body weight or adiposity(Figures S2D–S2H).The differences in adiposity in POMC-TC and POMC-1B mice could not be ascribed to alterations in food intake,energy expenditure,or activity,and there were no differ-ences in fuel utilization(assd by respiratory exchange ratio, RER)in either group(Figures S2I and S2J).Thefindings are consistent with previous studies reporting that LEPR or IR de-ficiencies in POMC neurons do not alter food intake or RER (Balthasar et al.,2004;Berglund et al.,2012;Hill et al.,2010; Ko¨nner et al.,2007).
PTP1B,but Not TCPTP,Regulates Leptin Sensitivity
The deletion of TCPTP or PTP1B in neural and glial cells by Cre-LoxP recombination using the Nes-Cre transgene en-hances leptin nsitivity and protects mice from diet-induced obesity(DIO)(Loh et al.,2011).Accordingly,we assd whether the decread adiposity in chow-fed POMC-TC and POMC-1B mice might be ascribed to enhanced leptin nsi-tivity.POMC-TC,POMC-1B,orfloxed control mice were administered leptin intraperitoneally(IP)for3days,and body weights and overnight f
ood intake were recorded.Surprisingly, deletion of TCPTP in POMC neurons did not alter the leptin-mediated attenuation of food intake or decrea in body weight (Figures2C and S2K),despite the fact that86.5%±6.5%of LEPR-positive ARC POMC neurons express TCPTP(Fig-ure S1E).In addition,plasma leptin levels(Figure2D),leptin-induced STAT3Y705phosphorylation(p-STAT-3;Figures S2L and S2M),and leptin-induced hypothalamic Pomc expression (Figure2G)were not altered by TCPTP deficiency,which is consistent with unaltered leptin nsitivity.As reported previ-ously(Banno et al.,2010),PTP1B deficiency enhanced the leptin-mediated repression of body weight(Figure2E)without overt changes in food intake(Figure S2K),reduced fed plasma leptin levels(Figure2F),and significantly incread leptin-induced hypothalamic p-STAT-3and Pomc expression(Figures 2G,S2N,and S2O);as expected,there were no differences in Agrp and Npy expression(Figure S2P).Thus,the reduced adiposity in POMC-TC mice is independent of changes in leptin nsitivity.TCPTP,but Not PTP1B,Regulates Insulin Signaling
PTP1B and TCPTP dephosphorylate the IR and attenuate insu-lin signaling in the periphery(Tiganis,2013).As the CNS effects of leptin and insulin overlap,and hypothalamic IR activation alters peripheral lipid and gluco metabolism(Marino et al., 2011;Plum et al.,2006),we monitored the effects of TCPTP versus PTP1B deficiency on hypothalamic insulin signaling.First,we
laborday>gaudydetermined whether PTP1B versus TCPTP deficiency in POMC neurons enhanced insulin-induced PI3K/ AKT signaling in the ARC by monitoring AKT Ser-473phos-phorylation(p-AKT)by immunohistochemistry.TCPTP,but not PTP1B,deficiency enhanced p-AKT staining in the ARC in respon to insulin(Figures2H–2J).In keeping with the lective effects on p-AKT signaling,insulin-induced hypotha-lamic Pomc expression was significantly enhanced in POMC-TC,but not in POMC-1B mice(Figure2K).To independently asss TCPTP’s capacity to regulate insulin signaling in POMC neurons,we also took advantage of a highly specific TCPTP inhibitor,compound8(Zhang et al.,2009).This inhibitor is highly lective for TCPTP over PTP1B and intracerebroven-tricular(ICV)compound8administration enhances leptin signaling and nsitivity in wild-type mice,but not neuronal cell-specific TCPTP knockout mice(Loh et al.,2011).Com-pound8or aCSF(artificial cerebrospinalfluid)vehicle control were administered ICV into fasted C57BL/6mice that were subquently injected with insulin and hypothalami extracted for analysis by real-time PCR.Administration of compound8 incread insulin-induced Pomc expression by 2.5-fold(Fig-ure2L).Taken together,the results demonstrate that TCPTP attenuates insulin signaling in POMC neurons.
Decread Adiposity and Incread Energy Expenditure in DKO Mice
last but not leastOur results indicate that PTP1B and TCPTP differentially contribute to leptin and insulin signaling in POMC neurons. Hence,we generated POMC-TC and POMC-1B double-knockout(DKO)mice to examine whether the combined in-crea in IR and LEPR signaling in POMC neurons affects body weight and gluco metabolism.DKO mice had a modest reduction in body weight at10weeks of age,but body length, liver weight,lean mass,and bone density were unaltered(Fig-ures3A–3C).Differences in body weight could be explained by reduced whole-body adiposity(Figures3B and3C).The reduction in body weight and adiposity in DKO mice was accompanied by incread dark-pha energy expenditure without significant changes in ambulatory activity or RER or changes in food intake or feeding efficiency(Figure3D).Further-more,leptin nsitivity,as assd by the effects of leptin on body weight(Figure3E)or inferred by the reduced fed plasma leptin levels,was improved in DKO mice(Figure3F).This was accompanied by incread leptin-induced hypothalamic Pomc expression in DKO mice(Figure3G).Finally,insulin-induced
(E)Rostral-caudal immunostaining quantification of GFP-and PTP1B-expressing ARC cells.
(F)Immunostaining for PTP1B and TCPTP in GFP-positive POMC neurons in the ARC.
(G–I)(G)Immunostaining and(H and I)quantification of ARC GFP and POMC colocalization and the to
tal number of ARC POMC neurons in Z/EG;POMC-TC mice. Reprentative images of three or more experiments are shown.Data are means±SEM for the indicated number of mice(30ctions/mou)or(C)experimental repeats.
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