Melissa L. Borg, Moyra Lemus, Alex Reichenbach, Ahrathy Selathurai, Brian J. Oldfield,Zane B. Andrews, and Matthew J. Watt

Hypothalamic Neurogenesis IsNot Required for the ImprovedInsulin Sensitivity FollowingExercise TrainingDiabetes 2014;63:3647–3658 | DOI: 10.2337/db13-1762

Neurons within the hypothalamic arcuate nucleus (ARC)are important regulators of energy balance. Recent stud-ies suggest that neurogenesis in the ARC is an importantregulator of body mass in response to pharmacologicalstressors. Regular exercise training improves insulinaction, and is a primary treatment modality for obesityand type 2 diabetes. We examined whether exercisetraining causes hypothalamic neurogenesis and whetherthis contributes to exercise-induced improvements ininsulin action. Short-term exercise in adult mice in-duced a proneurogenic transcriptional program involvinggrowth factors, cell proliferation, and neurogenic regu-lators in the hypothalamus. Daily exercise training for 7days increased hypothalamic cell proliferation 3.5-foldabove that of sedentary mice, and exercise-induced cellproliferation was maintained in diet-induced obese mice.Colocalization studies indicated negligible neurogenesis inthe ARC of sedentary or exercise-trained mice. Blockingcell proliferation via administration of the mitotic blockerarabinosylcytosine (AraC) did not affect food intake orbody mass in obese mice. While 4 weeks of exercisetraining improved whole-body insulin sensitivity comparedwith sedentary mice, insulin action was not affected byAraC administration. These data suggest that regularexercise training induces significant non-neuronal cellproliferation in the hypothalamus of obese mice, but thisproliferation is not required for enhanced insulin action.

Obesity dramatically increases the likelihood of the de-velopment of metabolic complications, including dyslipide-mia, fatty liver, glucose intolerance, and cardiovascular

disease, which increase morbidity and mortality (1,2). In-sulin resistance is a prominent metabolic defect in manyobese individuals and contributes to the developmentof type 2 diabetes. “Lifestyle” interventions involvingreduced caloric intake and increased physical activity re-main the cornerstone for the treatment of obesity-relateddiabetes. Laboratory-based studies demonstrate improvedinsulin sensitivity in the muscle and liver after a period ofexercise training (3,4), and exercise reduces the progres-sion from glucose intolerance to type 2 diabetes in at-riskindividuals (5–7). The exercise-induced improvements ininsulin action are mediated by a host of molecular adap-tations that improve metabolic, vascular, and endocrinefunctions in the major peripheral glucoregulatory tissues,which include skeletal muscle, liver, and adipose tissue(for review, see Strasser [8]).

The regulation of plasma glucose was traditionallyviewed as a process controlled by the coordinated actionsof peripheral tissues. The pancreas responds to post-prandial rises in glucose levels by secreting insulin, whichin turn increases glucose disposal into skeletal muscle andother insulin-sensitive tissues, and reduces glucose outputby the liver to restore euglycemia. However, studies in thelast decade have shown that neuronal populations withinthe hypothalamus and hindbrain influence autonomicsystems controlling hepatic glucose and lipid fluxes, pan-creatic insulin secretion, and peripheral glucose uptake. Forexample, central injections of neuropeptide Y (NPY)inhibits hepatic glucose production (9) and stimulates he-patic VLDL-triglyceride secretion (10) via the sympathetic

Department of Physiology, Monash University, Clayton, Victoria, Australia

Corresponding author: Matthew J. Watt, matthew.watt@monash.edu.

Received 19 November 2013 and accepted 30 May 2014.

This article contains Supplementary Data online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db13-1762/-/DC1.

© 2014 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, andthe work is not altered.

Diabetes Volume 63, November 2014 3647

METABOLISM

nervous system. Moreover, insulin signaling in the hypo-thalamus suppresses hepatic glucose production (11) viaphosphoinositide 3-kinase–dependent activation of ATP-dependent potassium channels in agouti-related peptide–expressing neurons within the arcuate nucleus (ARC),which in turn regulate efferent vagal innervation of theliver (12). Insulin action in the central nervous system(CNS) increases muscle glucose uptake, independent ofinsulin signaling in muscle (13,14), and this involves theactivation of ATP-dependent potassium channels. Althoughsomewhat controversial (11,15), it is suggested that theinsulin-dependent CNS pathway may account for morethan half of the insulin-mediated glucose uptake in leanmice (14), making this an important regulatory point forsystemic glucose homeostasis. The deletion of insulinreceptors in the CNS causes insulin resistance (16), andinsulin resistance of the CNS occurs in obese rodents(17–19) and humans (20). These results suggest that themanagement of impaired insulin action in obesity may re-quire restoration of CNS insulin sensitivity. Indeed, suc-cessful treatment of uncontrolled diabetes depends onintact brain insulin action (21), and selective modificationof the CNS in several animal models of insulin resistanceand type 2 diabetes improves peripheral insulin action andreverses hyperglycemia (22).

Emerging evidence shows that new neurons aregenerated within the adult hypothalamus (2,23–25). Insedentary mice, the rate of neuronal turnover is relativelyslow, with new neurons accounting for ;6% of the totalneuronal population (26). Markers of hypothalamic sub-types such as proopiomelanocortin, agouti-related peptide,and NPY are expressed in the newly formed neurons,suggesting involvement in metabolic regulation (2,23,25).Pioneering studies (2) have demonstrated pronounced neu-rogenesis in the energy balance circuitry of the ARC inresponse to central ciliary neurotropic factor (CNTF) ad-ministration. That neurogenesis was required to elicit thesustained and powerful anorexigenic effects of CNTF fur-ther indicates that postnatal neurogenesis may be impor-tant in regulating hypothalamic functions. Consistent withthis notion are the findings that obesity is associated withimpaired neurogenesis in the ARC (24,26), which leads toa relative aging of hypothalamic neuronal populations. Fur-ther, reducing neurogenesis by expressing constitutivelyactive inhibitory kB kinase in hypothalamic neural stemcells (NSCs) causes overeating, weight gain, glucose intol-erance, and hyperinsulinemia (26). Together, these datasuggest that neurogenesis within the hypothalamic cir-cuitry and/or turnover of energy-balance neurons is impor-tant for the maintenance of normal energy balance andglucose metabolism in mice. On the basis of these obser-vations, strategies aimed at enhancing hypothalamic neu-rogenesis may be a viable strategy to combat obesity anddiabetes.

Regular exercise training is commonly used to treatobesity and diabetes and is also known to induce neuro-genesis in several brain regions (27), although exercise-

induced neurogenesis in the hypothalamus is poorlydescribed. In the current study, we tested the hypothesisthat neurogenesis is an adaptive response to exercise thatis important for the weight loss and insulin-sensitizingeffects of regular exercise training in obesity.

RESEARCH DESIGN AND METHODS

Animal Care and HusbandryExperimental procedures were approved by the School ofBiomedical Sciences Animal Ethics Committee (MonashUniversity). Male C57BL/6J mice were purchased fromMonash Animal Services. Mice were fed a standard low-fatlaboratory diet (10% of total energy from fat) or a micro-nutrient matched high-fat diet (HFD; 59% of total energyfrom fat; Specialty Feeds, Glen Forrest, WA, Australia).

Study Design and Analytical Methods

Experiment 1: Transcriptional Responses to Short-termExerciseC57BL/6J mice were familiarized with the treadmill(Columbus Instruments, Columbus, OH) for 3 days beforethe experiment, where mice ran for 30 min at 15 m/minon a 5% grade. Sedentary “control” mice were placed onthe stationary treadmill for the same amount of time.Mice were culled 6 h later, and the hypothalamus wasdissected (defined caudally by the mamillary bodies,rostrally by the optic chiasm, laterally by the optictract, and dorsally by the apex of the hypothalamicthird ventricle).

RT2 Profiler PCR ArrayRNA from the hypothalamus was extracted in Qiazolextraction reagent and was isolated using an RNeasy TissueKit (Qiagen). RNA quality and quantity was determined(NanoDrop p2000 Spectrometer; Thermo Scientific) andreverse transcribed (Invitrogen). Gene products were de-termined by real-time quantitative RT-PCR (ep realplexMastercycler; Eppendorf) using an RT2 profiler PCR Array(SA Biosciences) with targeted expression of genes relatedto neurogenesis and NSC activation. Hspcb was used asa reference gene and did not vary between groups. ThemRNA levels were determined by a comparative CT method.

Experiment 2: Exercise Training and Cell ProliferationAn intracerebroventricular (ICV) cannula and osmoticmini-pump were implanted in 20-week-old C57BL/6Jmice (12 weeks on their respective diet). One day later,mice commenced training, which consisted of 30 min oftreadmill running daily for 7 days at ;12 m/min and a 5%slope (see protocol in Supplementary Table 1). Mice wereperfused transcardially with 4% paraformaldehyde (PFA)24 h or 28 days after the final exercise bout, and brainswere removed for immunohistochemical analysis (Fig. 1A).

Experiment 3: Prolonged Exercise Training and InsulinActionMice were fed an HFD for 4 weeks, then were randomizedinto a vehicle or arabinosylcytosine (AraC) treatment

3648 Exercise-Induced Neurogenesis and Insulin Action Diabetes Volume 63, November 2014

group. Mice were implanted with an ICV cannula attachedto an osmotic mini-pump (see below) and recovered for2 days. Thereafter, mice remained sedentary or commencedexercise training, which consisted of daily treadmill run-ning, five times a week for 4 weeks (Supplementary Table2). Body weight was monitored throughout the training pe-riod, and exercise capacity was assessed after the exercising

training period. Insulin sensitivity was assessed 72 h afterthe final exercise bout (Fig. 2A).

ICV CannulationMice were anesthetized by isoflurane inhalation and stereo-tactically implanted with steel guide cannulae (PlasticsOne, Roanoke, VA) targeted to the right lateral ventricles

Figure 1—Exercise-induced hypothalamic cell proliferation and neurogenesis in lean and obese mice. A: Schematic of the study design.B: Chow-fed mice were exercise trained for 7 days with BrdU ICV (12 mg/day) infusion for 7 days in aCSF at a flow rate of 12 mL/day. Brainswere removed 7 days postsurgery (8 D, representation cell proliferation) in sedentary (Sed) mice or 28 days postsurgery (33 D, representingcell survival) in exercise-trained (Ex) mice. Data are expressed as the average number of BrdU+ cells per section counted (n = 4 mice pergroup). Representative image of hypothalamus depicting BrdU+ cells (red) in sedentary and exercise-trained mice. Scale bar, 50 mm. 3V,third ventricle. Connecting lines indicate P < 0.05 by one-way ANOVA. C: Chow-fed mice were ICV infused for 7 days with BrdU (12mg/day) and/or CNTF (1.2 mg/day). Data are expressed as the average number of BrdU+ cells per section counted (n = 4 mice per group).Representative image of hypothalamus depicting BrdU+ cells (red) in CNTF-treated mice. Scale bar, 50 mm. Connecting lines denote P <0.05 via Student t test. Sed, sedentary. D: Mice fed an HFD were exercise trained for 7 days with BrdU ICV (12 mg/day) infusion for 7 days inaCSF at a flow rate of 12 mL/day. Brains were removed 7 days postsurgery (8 D, representation cell proliferation) in sedentary mice or 28days postsurgery (33 D, representing cell survival) in exercise-trained mice. Data are expressed as the number of BrdU+ cell per sectioncounted (n = 4 per group). Connecting lines denote P < 0.05 by one-way ANOVA. E: Representative image of hypothalamus depictingBrdU+ cells (red) after exercise training. Arrows indicate pairs of BrdU+ cells. Scale bar, 50 mm. F: Aging mice (12 months old) fed eithera low-fat diet (LFD) or HFD were exercise trained (Ex) for 7 days with BrdU ICV (12 mg/day) infusion for 7 days in aCSF at a flow rate of 12mL/day. Brains were removed in sedentary (Sed) mice or 7 days postsurgery (representing cell proliferation) in exercise-trained mice. Data areexpressed as the number of BrdU+ cells per section counted (n = 4 per group). Connecting lines denote P < 0.05 via two-way ANOVA. G:Coimaging of BrdU (red) and NeuN (green) showing negligible colocalization in sedentary (Sed) and exercise-trained (Ex) mice.H: Brain sectionsacross the dentate gyrus showing marked BrdU (red) and NeuN (green) colocalization in exercise-trained (Ex) but not sedentary (Sed) mice.Arrows indicate areas of colocalization of these molecular markers. I: Coimaging of BrdU (red) and GFAP (green) showing negligible colo-calization in sedentary (Sed) and exercise-trained mice. Scale bars in G–I: main image 100 mm; inset 25 mm.

diabetes.diabetesjournals.org Borg and Associates 3649

(20.3 mm anteroposterior, 1.0 mm laterally to bregma, and22.5 mm below the skull).

Experiment 2Cannulae were connected to subcutaneously implantedosmotic mini-pumps (flow rate 0.5 mL/h, 7 days; model1007D; Alzet, Cupertino, CA) via 65-mm-long vinyl tubingfilled with artificial cerebrospinal fluid (aCSF). Pumpswere filled with vehicle solution, vehicle solution contain-ing the known neurogenic agent Axokine (100 ng/mL,1.2 mg/day; a modified CNTF, Regeneron Pharmaceuti-cals), AraC (3.3 mg/mL, 40 mg/day; Sigma-Aldrich, St.Louis, MO; selectively inhibits DNA synthesis), or Axo-kine plus AraC. The vehicle solution was aCSF containing1 mg/mL BrdU (12 mg/day; Sigma-Aldrich). BrdU is in-corporated into the DNA of dividing cells, and is com-monly used for the birth dating and monitoring of cellproliferation (2,24,26).

Experiment 3Cannulae were connected to subcutaneously implantedosmotic mini-pumps (flow rate 0.11 mL/h, 28 days; model1004; Alzet) via 33-mm-long vinyl tubing. Pumps andtheir tubing extensions were filled with vehicle solution orvehicle solution containing AraC (15.2 mg/mL, 40 mg/day;Sigma-Aldrich). The vehicle solution was aCSF containing4.5 mg/mL BrdU (12 mg/day). The different concentrationsof compounds between experiments 2 and 3 ensured thatmice received the same total amount of chemical per day,irrespective of the flow rate. Mice were housed singly andmonitored daily for body weight and food intake for allexperiments.

Tissue Processing and ImmunohistochemistryMice were anesthetized under isoflurane inhalation andperfused transcardially with 0.9% NaCl with 10 mg/Lheparin followed by 4% PFA (Sigma-Aldrich). Brains wereremoved, postfixed by standard methods, and sectionedon a cryostat in the coronal plane. Sections (30 mm thick)were collected in four series. For BrdU immunostaining,after mounting (Superfrost Ultra Plus Slides; Thermo Sci-entific) and drying overnight, sections were fixed with 4%PFA for 10 min at room temperature, rinsed in PBS, thenrinsed with 100% methanol for 20 min, rinsed in PBS,and incubated in 2N HCl for 30 min at 37°C. Sectionswere rinsed in PBS, blocked for 1 h with 5% normal horseserum in PBS/0.02% Triton X-100, and incubated withsheep anti-BrdU antibodies (1:400; Abcam) overnight at4°C. Sections were rinsed and incubated with the appro-priate secondary antibody for 1 h, rinsed in PBS, andcoverslipped. For colabeling analyses, sections first under-went BrdU immunohistochemistry and were then incu-bated with primary antibodies (neuronal nuclei [NeuN]1:1,000; Abcam) or glial fibrillary acidic protein (GFAP;1:1000; Dako) in blocking solution. Sections were rinsedand incubated with the appropriate secondary antibodyfor 1 h, washed in PBS, and coverslipped.

BrdU+ cells in the caudal hypothalamus were countedusing stereological principles of quantifying BrdU+ cellsfrom systematically collected serial sections with the ini-tial section chosen at random. Per animal, every 4th cor-onal section (30 mm thickness) throughout the caudalhypothalamus (21.22 to 22.70 mm from bregma) wasanalyzed by standard fluorescence microscopy. BrdU+

Figure 1—Continued.

3650 Exercise-Induced Neurogenesis and Insulin Action Diabetes Volume 63, November 2014

cells within the hypothalamic parenchyma were countedfor each section analyzed, excluding cells of the uppermostfocal plane to avoid oversampling. The average number ofBrdU+ cells for any given section of the caudal hypothala-mus was calculated as the sum of the counted BrdU+ cellsdivided by the total number of sections counted.

Exercise Capacity TestMice ran on the treadmill at 10 m/min for 2 min (5%grade), and the speed was increased by 2 m/min every2 min until the mouse reached exhaustion.

Insulin Tolerance TestAn insulin tolerance test (ITT) was conducted 72 h afterthe last exercise bout. Mice were fasted for 4 h, andvenous blood glucose was assessed (Accu-Check gluco-menter; Roche) before and after intraperitoneal insulinadministration (1 units/kg body weight; Actrapid; NovoNordisk, Bagsvaerd, Denmark).

Assessment of Tissue-Specific Insulin SensitivityAfter a 3-h fast (0800–1100 h) mice were injected via a tailvein with 0.5 units/kg insulin, 10mCi of 2-[1-3H]deoxyglucose

Figure 2—Hypothalamic cell proliferation does not affect energy balance in obese, sedentary (Sed) mice. A: Schematic of the study design.B: ITT performed after 4 weeks of exercise training in HFD-fed mice (n = 8 per group). Blood glucose level is shown as the percentagedifference from the initial blood glucose level. *P< 0.05 vs. Sed (main effect for treatment) by repeated-measures two-way ANOVA. C: Micewere ICV infused for 7 days with CNTF (1.2 mg/day) and/or AraC (40 mg/day). For all animals, BrdU (12 mg/day) was coadministered.Representative image of hypothalamus depicting BrdU+ cells (red). Scale bar, 50 mm. 3V, third ventricle. D: Mice were fed an HFD for 4weeks, then were ICV infused with or without AraC (40 mg/day) for 4 weeks in aCSF at a flow rate of 2.64 mL/day. Mice were killed 36 h afterthe infusion stopped. Weekly food intake was monitored during the infusion period (vehicle [Veh] and AraC n = 15). E: Body weightassessed at the end of the 4-week infusion (Veh n = 15, AraC n = 14). F: The epididymal fat pad was excised and weighed when themice were culled (Veh n = 7, AraC n = 6). Levels of plasma leptin (Veh n = 9, AraC n = 8) (G) and plasma FFAs (Veh n = 15, AraC n = 14) (H)were assessed after the insulin sensitivity assessment. *P < 0.05 vs. vehicle via Student t test.

diabetes.diabetesjournals.org Borg and Associates 3651

and 2 mCi of [U-14C]glucose. Blood samples were obtainedfrom a cut in the tail at 2, 5, 10, 15, and 20 min. Micewere killed by decapitation, and tissue was collected. 2-[1-3H]Deoxyglucose clearance from the blood and intotissues was performed in mice as described previously(28,29).

Immunoblot AnalysisPhosphorylated Akt (Ser473) and Akt 1:1,000 (catalog#9271 and #4685, respectively; Cell Signaling Technol-ogy) were assessed in hypothalamic lysates by standardmethods (30).

StatisticsResults are expressed as the mean 6 SEM. Statisticalanalysis was performed by using the Student t test, orone-way, two-way, or repeated-measures two-way ANOVAwith Bonferroni post hoc test, as appropriate. Significancewas established a priori at P # 0.05.

RESULTS

Short-term Exercise Upregulates Genes Involved inHypothalamic Neurogenesis and Cell ProliferationShort-term exercise increased the expression of genesinvolved in cell proliferation, including Egf, Fgf2, Il3, andVegfa (Table 1). Proneurogenic growth factors, includingArtn, Bdnf, Bmp15, Bmp2, and Ndp, were induced by exercise.Levels of regulators of the cell cycle and known transcrip-tional regulators of neurogenesis, including Arnt2, Hey1,Mef2c, Neurod1, Notch2, Pax3, and Pax6, were also increasedafter exercise (Table 1). No gene was downregulated by

exercise. Thus, short-term exercise promotes a proneuro-genic transcriptional program in the hypothalamus of adultmice.

Regular Exercise Induces Hypothalamic CellProliferation in Lean MiceTo determine whether regular exercise training induceshypothalamic cell proliferation, mice ran at a moderateintensity on a treadmill for 30 min/day for 1 week. Micereceived a continuous ICV infusion of BrdU to label dividingcells and were killed 1 day after the last exercise bout.Hypothalamic cell proliferation in sedentary mice averaged31 6 5 BrdU+ cells per 30-mm section, and this numberincreased 3.5-fold in exercise-trained mice (Fig. 1B). Thiswas substantially less than in mice treated with the neuro-genic factor CNTF, which was implemented as a positivecontrol (Fig. 1C). The number of BrdU+-labeled cells wasreduced by 45% in exercise-trained mice at 33 vs. 8 days(Fig. 1B), indicating that approximately half of the new-born cells survived. One caveat to this interpretation isthat cells newly labeled with BrdU cannot be distin-guished from cells that have incorporated BrdU and thendivided. Thus, actual “survival” rates are semiquantitative.Together, these findings show that exercise training increasescell proliferation in the hypothalamus of mice.

Examination of coronal sections of sedentary andexercise-trained mice show that BrdU+ cells were diffuselydistributed throughout the hypothalamus but were con-centrated mostly in regions;200–300 mm from the thirdventricle, including the ARC and the ventromedial and

Table 1—Exercise-induced changes in the expression of neurogenic genes in the hypothalamus

Gene Gene name Fold change* P value

Regulators of cellproliferation

Egf Epidermal growth factor 1.7 6 0.2 0.007Fgf2 Fibroblast growth factor 2 2.1 6 0.2 0.003Il3 Interleukin 3 1.5 6 0.3 0.015Vegfa Vascular endothelial growth factor A 3.2 6 0.5 0.004

Growth factorsArtn1 Artemin 2.7 6 0.4 0.005Bdnf Brain-derived neurotrophic factor 3.1 6 0.5 0.004Bmp15 Bone morphogenetic protein 15 3.4 6 0.6 0.008Bmp2 Bone morphogenetic protein 2 2.5 6 0.3 0.004Gpi1 Glucose phosphate isomerase 1 1.6 6 0.1 0.004Ii3 Interleukin 3 1.8 6 0.2 0.015Inhba Inhibin, beat a 1.8 6 0.3 0.024Nrg1 Neuregulin 1 1.5 6 0.2 0.009

Transcription regulatorsof neurogenesis

Apbb1 Amyloid b (A4) precursor protein-binding, family B, member 1 4.0 6 0.7 0.003Ascl1 Achaete-scute complex homolog 1 (Drosophila) 1.9 6 0.3 0.011Ep300 E1A binding protein p300 1.5 6 0.2 0.039Heyl Hairy/enhancer-of-split related with YRPW motif-like 2.8 6 0.4 0.001Mef2c Myocyte enhancer factor 2C 2.5 6 0.3 0.001Ncoa6 Nuclear receptor coactivator 6 1.7 6 0.3 0.020Notch2 Notch gene homolog 2 (Drosophila) 2.2 6 0.3 0.021Pax6 Paired box gene 6 2.6 6 0.4 0.012Pou3f3 POU domain, class 3, transcription factor 3 2.3 6 0.2 0.002

Values are expressed as mean 6 SEM. n = 6 for rest, n = 6 for exercise. *Fold change is reported relative to rest.

3652 Exercise-Induced Neurogenesis and Insulin Action Diabetes Volume 63, November 2014

dorsomedial hypothalamic nuclei (Fig. 1B). Very few BrdU+

cells were present in the ependymal lining of the third ven-tricle. Pairs of newly divided BrdU cells were distributedextensively within the hypothalamic parenchyma (Fig. 1E),which is consistent with the notion that cell proliferation inthe hypothalamus is not restricted to a specific zone ornuclear region.

Exercise-Induced Hypothalamic Cell Proliferation IsNot Impaired in ObesityCell proliferation was not impaired in obese sedentarymice compared with lean sedentary mice, with an averageof 43.4 6 6.6 and 31.5 6 4.5 BrdU+ cells per section,respectively (Fig. 1D). Exercise increased the number ofBrdU+ cells by 3.3-fold above that observed in obese sed-entary mice, and there was no significant reduction inBrdU+ cells after 28 days in the obese exercise-trainedmice (Fig. 1D), suggesting that the majority of the pro-liferating cells survived. Thus, the exercise-induced pro-liferation and survival rates in obese mice were notimpaired when compared with lean mice.

Given that aging has been associated with reducedneuroregenerative capacity, comparison was made betweengroups of aged (;52 weeks) and relatively young(;18 weeks) mice that were exercise trained for 1 week.These experiments showed that hypothalamic cell prolifer-ation is reduced by 2.2-fold in aged mice compared withyoung mice and that exercise increases cell proliferation by1.5-fold in aged mice, even after 1 year of high-fat feeding(Fig. 1F). The level of cell proliferation in exercise-trainedaged mice was similar to that in sedentary young mice.

Limited Neurogenesis and Generation of Astrocytes inthe ARC After Exercise TrainingWe next determined the neural contribution to cellproliferation in the ARC by assessing colocalization ofBrdU with neuron (NeuN) and astrocyte (GFAP) markers.Only ;0.5% of the BrdU+ cells were colabeled with NeuNin exercise-trained mice (33 days), and there was noBrdU/NeuN colabeling in sedentary mice (Fig. 1G). Weapplied this same approach to the subgranular zone ofthe hippocampal dentate gyrus, an area in which neuro-genesis is well established. The majority of BrdU-labeledcells were colocalized with NeuN in exercise-trained mice,confirming marked neurogenesis. BrdU/NeuN colabelingwas less apparent in sedentary mice (Fig. 1H). There wasalso limited colocalization of BrdU and GFAP in sedentaryand exercise-trained mice, indicating restricted produc-tion of new astrocytes (Fig. 1I). Because negligible neuro-genesis was detected in the ARC of lean mice (0.5% ofproliferating cells), we rationalize that major changes inthe neurochemical phenotype of the proliferating cells isunlikely in obesity and have not conducted these analyses.

Hypothalamic Cell Proliferation Does Not AffectEnergy Balance in Sedentary or Exercise-Trained MiceGiven the well-recognized impact of regular moderateexercise on improved metabolic performance and the ob-servation that exercise promotes marked cell proliferation

in the hypothalamus (Fig. 1B), the link between thesetwo factors was tested in groups of HFD-fed mice thatwere exercise trained and exposed to ICV AraC (Fig. 2A).Four weeks of training was selected because this was theminimum volume of training required to detect improve-ments in whole-body insulin action in high-fat–fedC57BL/6J mice (Fig. 2B). A range of metabolic parame-ters was measured with and without AraC treatment totest the involvement of cell proliferation in the beneficialeffects of exercise training on energy balance and insulinaction. AraC blocked cell proliferation in the hypothala-mus of mice treated with CNTF, a powerful neurogenicstimulator (Fig. 2C), and in exercise-trained mice (blankimages not shown). Blocking cell proliferation did notaffect food intake (Fig. 2D) (P = 0.77), body mass (Fig.2E) (P = 0.23), epididymal fat mass (Fig. 2F) (P = 0.31),plasma leptin levels (Fig. 2G) (P = 0.49), or plasma freefatty acid (FFA) levels (Fig. 2H) (P = 0.36) in sedentarymice.

Exercise training improved the endurance exercisecapacity of mice by 2.2-fold compared with untrainedage-matched mice (Fig. 3A). The endurance capacity wasreduced in exercise-trained mice treated with AraC com-pared with vehicle-treated mice (Fig. 3A) (P = 0.0002).Blocking cell proliferation did not affect food intake(Fig. 3B) (P = 0.44), or body mass (Fig. 2C) (P = 0.48)during exercise training. Epididymal fat mass (Fig. 2D)(P = 0.22), plasma leptin levels (Fig. 2E) (P = 0.78), andplasma triglyceride levels (vehicle: 0.65 6 0.07 vs. AraC0.56 6 0.07, n = 16 per group, P = 0.39) were not differ-ent between groups, while plasma FFA levels were mildlyincreased (Fig. 2F) (P = 0.04).

Hypothalamic Cell Proliferation Is Not Required for theExercise Training Improvements in Insulin SensitivityTo test whether hypothalamic cell proliferation (includingneurogenesis) contributes to the improvements in insulinaction after exercise training, insulin was coadministeredintravenously with 3H-2-deoxyglucose (3H-2DG) and14C-glucose tracers into conscious mice. Whole-body insulin-stimulated glucose uptake was not affected by AraCadministration in sedentary mice (Fig. 4A). Exercise train-ing improved whole-body insulin sensitivity, as demon-strated by a 45% increase in whole-body 14C-glucoseclearance (Fig. 4A) (main effect P = 0.04). 14C-glucoseclearance (Fig. 4A) (P = 0.34) and the reduction in bloodglucose levels (Fig. 4B) (P = 0.29) were not different be-tween vehicle-treated and AraC-treated groups of exercise-trained mice. 3H-2DG clearance in the mixed quadriceps(Fig. 3C) (P = 0.74), heart (Fig. 3D) (P = 0.38), brownadipose tissue (Fig. 3E) (P = 0.98), and liver (vehicle17.8 6 5.1 vs. AraC 17.8 6 6.7 K 3 1,000, P = 0.91)was not different between groups, whereas 3H-2DG clear-ance in white adipose tissue tended to be decreased inAraC-treated mice (Fig. 3F) (P = 0.06). Insulin-mediated14C-glucose accumulation in liver lipids was also unaffectedby AraC treatment (Fig. 3G) (P = 0.80). Finally, AraC treat-ment did not affect Akt Ser473 phosphorylation in

diabetes.diabetesjournals.org Borg and Associates 3653

hypothalamic lysates (Fig. 3H) (P = 0.59), indicating thatthere was no improvement in insulin sensitivity. To-gether, these data demonstrate that cell proliferation isnot required for the exercise-mediated enhancement ininsulin action in obese mice.

DISCUSSION

Adult neurogenesis is most prominent within the sub-ventricular zone of the lateral ventricles and the sub-granular zone of the dentate gyrus in the hippocampus(31), and impaired neurogenesis is implicated in the eti-ology of several neurodegenerative diseases including Par-kinson disease, Alzheimer disease, and amyotrophic lateralsclerosis (32). Notably, enhancing neurogenesis throughpharmacological or physiological stimuli (e.g., environmen-tal enrichment and voluntary physical activity) attenuatesneurodegenerative disease progression and improves cog-nitive function (27,33–36). Together, these results link themaintenance of adult neurogenesis to normal CNS functionand suggest a therapeutic strategy for treating neurodegen-erative conditions. The questions are then as follows:to what extent does neurogenesis impact metabolic

phenotypes and can regular exercise training modulatethis response?

In this study, we have observed cell proliferation in thehypothalamus of lean mice that is most abundant in theregion immediately surrounding the third cerebral ven-tricle. Newly formed cells, identified by the incorporationof BrdU, were not localized to anatomically distinct nucleibut were graded in abundance with distance from theventricle, although there were instances where isolatedcells were present deep in the lateral hypothalamus. Whilewe have demonstrated that exercise upregulates a subsetof transcripts involved in cell proliferation and neuro-genesis, very few newborn cells take a neuronal fate,indicating limited hypothalamic neurogenesis with exer-cise training. Hypothalamic cell proliferation was notdysregulated in diet-induced obese mice and was sub-stantially increased after exercise training. While exercise-induced cell proliferation occurs in the hypothalamus,a CNS region essential for the control of energy balanceand insulin action, our data do not support an importantrole for hypothalamic cell proliferation in the exercise-induced improvements in insulin sensitivity and energy

Figure 3—Hypothalamic cell proliferation does not affect energy balance in obese, exercise-trained mice. A: After high-fat feeding, micewere exercise trained for 4 weeks with ICV infusion with or without AraC (40 mg/day) for 4 weeks in aCSF at a flow rate of 2.64 mL/day. Anexercise endurance test was performed after the 4 weeks of exercise training (n = 6 per group). *P < 0.05 vs. vehicle (Veh) exercise by two-way ANOVA. B: Weekly food intake during the exercise-training period (Veh n = 17, AraC n = 16). C: Body weight assessed at the end ofexercise training (Veh n = 16, AraC n = 17). D: The epididymal fat pad was excised and weighed when the mice were culled (Veh n = 4, AraCn = 6). Levels of plasma leptin (n = 8 Veh, n = 6 AraC) (E) and plasma FFAs (n = 17 Veh, n = 16 AraC) (F ) were assessed after the insulinsensitivity assessment. *P < 0.05 vs. vehicle by Student t test.

3654 Exercise-Induced Neurogenesis and Insulin Action Diabetes Volume 63, November 2014

balance. By extension, our data question the importanceof hypothalamic cell proliferation and neurogenesis in theregulation of energy balance and insulin action in thesetting of obesity.

While new hypothalamic cells are produced duringadulthood, their relevance to physiology remains unclear.The genesis for this, and previous work in the field, wasderived from the seminal observation that CNTF ad-ministration into the CSF of mice leads to rapid and

pronounced weight loss that is maintained for at least1 month after the cessation of CNTF administration.Importantly, when neurogenesis was blocked by the co-administration of AraC, the sustained weight loss in CNTF-treated mice was abrogated, indicating that neurogenesiswas required for the sustained anorectic effects of CNTF(2). These long-lasting effects were attributed to neurogen-esis within the hypothalamic feeding circuits, specificallyNPY-expressing and proopiomelanocortin-expressing

Figure 4—Insulin sensitivity is enhanced by exercise training in obese mice and does not require hypothalamic cell proliferation. A: Afterhigh-fat feeding, mice were exercise trained for 4 weeks with ICV infusion with or without AraC (40 mg/day) for 4 weeks in aCSF at a flowrate of 2.64 mL/day. Insulin sensitivity was assessed with an intravenous ITT and glucose tracers. Whole-body clearance of 14C-glucoseduring the intravenous ITT in sedentary and exercise-trained mice (vehicle [Veh] Sedentary n = 15, Veh Exercise n = 14, AraC Sedentary n =11, AraC Exercise n = 14). *P< 0.05, main effect for exercise training via two-way ANOVA. B: Blood glucose levels during intravenous ITT inexercise-trained mice. Blood glucose level is shown as the percentage difference from the initial blood glucose level (Veh n = 15, AraC n =17). Insulin-stimulated 3H-2DG uptake into the quadriceps (C), cardiac muscle (D), brown adipose tissue (E ), and epididymal adipose tissue(F ) of exercise-trained mice (Veh n = 12, AraC n = 14). Statistics are determined via Student t test. G: Insulin-stimulated 14C -glucose uptakeinto the liver of exercise-trained mice (Veh n = 13, AraC n-15). H: Insulin-stimulated Akt phosphorylation (Ser473) in the hypothalamus ofexercise-trained mice. All groups analyzed on the same exposure from the same immunoblot. Membranes were stripped and reprobed fortotal Akt (Veh n = 7, AraC n = 8). pAkt, phosphorylated Akt.

diabetes.diabetesjournals.org Borg and Associates 3655

neurons, with each playing crucial antagonistic roles inthe regulation of energy balance. We asked whether cellproliferation, and by extension neurogenesis, could beenhanced by a physiological stimulus and whether thiswas critical for metabolic regulation. We then exploitedtwo well-known phenomena (that obesity induces insulinresistance and that regular exercise training improves in-sulin action) to test the hypothesis that cell proliferationis important in homeostatic control of metabolism. Block-ing cell proliferation in exercise-trained mice did notaffect fasting blood glucose levels, whole-body insulin-stimulated glucose uptake, or tissue-specific insulin sensi-tivity. In addition, hypothalamic insulin signaling wasunaffected by AraC administration, indicating that hy-pothalamic insulin signaling is not impacted despiteimprovements in whole-body insulin action. While exer-cise training reduces body mass gain in HFD-fed mice(37), the blocking of cell proliferation has no effect onbody mass or food intake, thereby indicating no effecton daily energy expenditure. The simplest conclusionfrom our studies is that cell proliferation, and byextension neurogenesis, does not play a major role inmodulating insulin action and energy balance in obesemice. However, regular exercise training may induce otherchanges to the hypothalamic circuitry to improve insulinaction independent of cell-proliferative effects.

Obesity induced by high-fat feeding results in alteredsynaptic plasticity in mice (38), which is postulated tolead to a relative aging of the hypothalamic neuronal pop-ulation (39). Such a disruption in hypothalamic circuitrymay be predicted to impair the sensitivity of ARC neuronsto nutrient and hormonal signals associated with meta-bolic challenges and to compromise energy homeostasis.While previous studies have reported neurogenesis in theARC of the hypothalamus in lean mice, it is currentlyunclear whether neurogenesis is actually compromisedin obesity or whether neurogenesis impacts the develop-ment of obesity and its related disorders. On the onehand, both high-fat feeding (24,26) and inhibitory kBkinase/nuclear factor-kB activation in NSCs lead to re-duced proliferation and survival of new-born hypotha-lamic neurons, which is associated with overeating andinsulin resistance (26). Conversely, neurogenesis in themedian eminence of the hypothalamus is enhanced byhigh-fat feeding in young mice and is associated withenergy overconsumption and increased fat mass (23).The same authors (23) also showed that localized ir-radiation in the mediobasal hypothalamus of HFD-fedmice reduced neurogenesis, which was associated withincreased energy expenditure and decreased weight gain.While, these conflicting data relating to the effect of diet-induced obesity on hypothalamic neurogenesis are diffi-cult to reconcile, there are a number of variables acrossthe studies that are likely to impact outcomes. These in-clude the age of mice, the timing of the dietary interven-tion, the timing of BrdU administration and subsequentanalyses, the hypothalamic localization of progenitors

that would invariably impact the specific neurogenic mi-croenvironment (e.g., median eminence ependymal layer[23]), discrete zones of the mid–third ventricle wall (40),ARC, mediobasal hypothalamus (2,23), and the cell oforigin being examined (e.g., b2-tanycytes vs. NSCs). De-spite the lack of clear direction from previous studies, thepresent work shows that high-fat feeding does not impactcell proliferation in the hypothalamus of adult mice, a re-sult that is in keeping with data derived from rats (41).

While the majority of hypothalamic neurons aregenerated during embryogenesis, it is thought that post-natal neurogenesis is essential for maintaining functionalplasticity and adaptability in adulthood. The questionarises as to what are the determinants of such neurogenicactivity. Voluntary physical activity increases neurogene-sis in several brain regions in mice (42–44), and as suchthe current study sought to determine whether structuredregular exercise training, similar to what would be pre-scribed for maintaining healthy weight, would increasehypothalamic neurogenesis. We observed a generalizedupregulation of transcripts involved in cell proliferationand survival, transcriptional regulation of neurogenesis,and growth factors in the hypothalamus after a shortbout of exercise. Thus, the hypothalamic niche is rapidlyaltered after exercise to promote neurogenesis. Despitethese molecular responses and the finding of marked hy-pothalamic cell proliferation after exercise training inboth lean and obese mice, exercise did not increase neuro-genesis per se, and there was evidence of limited astrocytegeneration. The absence of hypothalamic neurogenesis isperplexing, especially in light of the coordinated pro-neurogenic transcriptional response to exercise andthe marked hippocampal neurogenesis observed in thesame exercise-trained mice (using the same BrdU/NeuNcolabeling approach), essentially acting as a positive con-trol. The negligible hypothalamic neurogenesis could beexplained by slow neuronal differentiation of NSCs(24,26) or proliferation of NSCs that is matched by de-creased survival (24). Further studies that delineate theupstream drivers and requisite molecular signaling thatfacilitates the proliferation and differentiation of NSCswill provide valuable insights into why ARC neurogenesisis severely limited when compared with the dentate gyrusof the hippocampus. In addition, the phenotype of thenew exercise-generated ARC cells remains undetermined,but, based on the differentiation potential of NSCs (26),these BrdU+ cells might be NSCs undergoing self-renewal.Alternatively, previous studies of brain regions other thanthe hippocampus (45,46), such as the substantia nigraand cerebral cortex, report an abundance of cells witholigodendrocytic precursor characteristics that differenti-ate into mature oligodendrocytes with exercise. In thisregard, oligodendrocytes constitute the majority (;55%)of newly generated cells in the medial prefrontal cortexwith exercise training, with evidence of limited neurogen-esis (;5%) (47). Moreover, the majority of BrdU+ cells in thehypothalamus of sedentary mice are of oligodendrocyte

3656 Exercise-Induced Neurogenesis and Insulin Action Diabetes Volume 63, November 2014

lineage, and only a small percentage of these become neu-rons (;8%) (48,49).

In conclusion, these data demonstrate that exerciseinduces a rapid transcriptional response in the hypothal-amus that drives substantial cell proliferation, even inobesity and aging. In contrast to studies using pharma-cological doses of mitogens, exercise does not inducesignificant neurogenesis in the hypothalamus, and block-ing cell proliferation does not change feeding, energybalance, or insulin action in the setting of rodent obesity.These results question whether physiological cell pro-liferation and neurogenesis are major regulators of insulinaction and metabolic phenotypes.

Acknowledgments. The authors thank Clinton Bruce (Deakin University,Burwood, Victoria, Australia) and Vanessa Haynes (Monash University) for tech-nical assistance.Funding. These studies were supported by grant APP1005053 from theNational Health and Medical Research Council (NHMRC) of Australia (to B.J.O.,Z.B.A., and M.J.W.). M.L.B. was supported by an Australian Postgraduate Award.Z.B.A. is supported by a Future Fellowship from the Australian Research Council(FT100100966), and B.J.O. and M.J.W. are supported by research fellowshipsfrom the NHMRC.Duality of Interest. No potential conflicts of interest relevant to this articlewere reported.Author Contributions. M.L.B. and M.J.W. researched the data andwrote the manuscript. M.L., A.R., and A.S. researched the data. B.J.O. contrib-uted to the discussion, and reviewed and edited the manuscript. Z.B.A.researched the data, contributed to the discussion, and reviewed and editedthe manuscript. M.J.W. is the guarantor of this work and, as such, had fullaccess to all the data in the study and takes responsibility for the integrity ofthe data and the accuracy of the data analysis.

References1. Flegal KM, Kit BK, Orpana H, Graubard BI. Association of all-cause mortalitywith overweight and obesity using standard body mass index categories: a sys-tematic review and meta-analysis. JAMA 2013;309:71–822. Kokoeva MV, Yin H, Flier JS. Neurogenesis in the hypothalamus of adultmice: potential role in energy balance. Science 2005;310:679–6833. DeFronzo RA, Sherwin RS, Kraemer N. Effect of physical training on insulinaction in obesity. Diabetes 1987;36:1379–13854. Dela F, Larsen JJ, Mikines KJ, Ploug T, Petersen LN, Galbo H. Insulin-stimulated muscle glucose clearance in patients with NIDDM. Effects of one-legged physical training. Diabetes 1995;44:1010–10205. Lindström J, Ilanne-Parikka P, Peltonen M, et al.; Finnish Diabetes Pre-vention Study Group. Sustained reduction in the incidence of type 2 diabetes bylifestyle intervention: follow-up of the Finnish Diabetes Prevention Study. Lancet2006;368:1673–16796. Pan XR, Li GW, Hu YH, et al. Effects of diet and exercise in preventingNIDDM in people with impaired glucose tolerance. The Da Qing IGT and DiabetesStudy. Diabetes Care 1997;20:537–5447. Knowler WC, Barrett-Connor E, Fowler SE, et al.; Diabetes PreventionProgram Research Group. Reduction in the incidence of type 2 diabetes withlifestyle intervention or metformin. N Engl J Med 2002;346:393–4038. Strasser B. Physical activity in obesity and metabolic syndrome. Ann N YAcad Sci 2013;1281:141–1599. van den Hoek AM, van Heijningen C, Schröder-van der Elst JP, et al. In-tracerebroventricular administration of neuropeptide Y induces hepatic insulinresistance via sympathetic innervation. Diabetes 2008;57:2304–2310

10. Bruinstroop E, Pei L, Ackermans MT, et al. Hypothalamic neuropeptideY (NPY) controls hepatic VLDL-triglyceride secretion in rats via the sympatheticnervous system. Diabetes 2012;61:1043–105011. Obici S, Zhang BB, Karkanias G, Rossetti L. Hypothalamic insulin signalingis required for inhibition of glucose production. Nat Med 2002;8:1376–138212. Könner AC, Janoschek R, Plum L, et al. Insulin action in AgRP-expressingneurons is required for suppression of hepatic glucose production. Cell Metab2007;5:438–44913. Perrin C, Knauf C, Burcelin R. Intracerebroventricular infusion of glucose,insulin, and the adenosine monophosphate-activated kinase activator, 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside, controls muscle gly-cogen synthesis. Endocrinology 2004;145:4025–403314. Coomans CP, Biermasz NR, Geerling JJ, et al. Stimulatory effect of insulinon glucose uptake by muscle involves the central nervous system in insulin-sensitive mice. Diabetes 2011;60:3132–314015. Pocai A, Lam TK, Gutierrez-Juarez R, et al. Hypothalamic K(ATP) channelscontrol hepatic glucose production. Nature 2005;434:1026–103116. Brüning JC, Gautam D, Burks DJ, et al. Role of brain insulin receptor incontrol of body weight and reproduction. Science 2000;289:2122–212517. De Souza CT, Araujo EP, Bordin S, et al. Consumption of a fat-rich dietactivates a proinflammatory response and induces insulin resistance in the hy-pothalamus. Endocrinology 2005;146:4192–419918. Posey KA, Clegg DJ, Printz RL, et al. Hypothalamic proinflammatory lipidaccumulation, inflammation, and insulin resistance in rats fed a high-fat diet. AmJ Physiol Endocrinol Metab 2009;296:E1003–E101219. Spanswick D, Smith MA, Mirshamsi S, Routh VH, Ashford ML. Insulin ac-tivates ATP-sensitive K+ channels in hypothalamic neurons of lean, but not obeserats. Nat Neurosci 2000;3:757–75820. Anthony K, Reed LJ, Dunn JT, et al. Attenuation of insulin-evoked responsesin brain networks controlling appetite and reward in insulin resistance: the ce-rebral basis for impaired control of food intake in metabolic syndrome? Diabetes2006;55:2986–299221. Gelling RW, Morton GJ, Morrison CD, et al. Insulin action in the braincontributes to glucose lowering during insulin treatment of diabetes. Cell Metab2006;3:67–7322. Sandoval DA, Obici S, Seeley RJ. Targeting the CNS to treat type 2 diabetes.Nat Rev Drug Discov 2009;8:386–39823. Lee DA, Bedont JL, Pak T, et al. Tanycytes of the hypothalamic medianeminence form a diet-responsive neurogenic niche. Nat Neurosci 2012;15:700–70224. McNay DE, Briançon N, Kokoeva MV, Maratos-Flier E, Flier JS. Remodelingof the arcuate nucleus energy-balance circuit is inhibited in obese mice. J ClinInvest 2012;122:142–15225. Pierce AA, Xu AW. De novo neurogenesis in adult hypothalamus as a com-pensatory mechanism to regulate energy balance. J Neurosci 2010;30:723–73026. Li J, Tang Y, Cai D. IKKb/NF-kB disrupts adult hypothalamic neural stemcells to mediate a neurodegenerative mechanism of dietary obesity andpre-diabetes. Nat Cell Biol 2012;14:999–101227. Zhao C, Deng W, Gage FH. Mechanisms and functional implications of adultneurogenesis. Cell 2008;132:645–66028. Cooney GJ, Caterson ID, Newsholme EA. The effect of insulin and nor-adrenaline on the uptake of 2-[1-14C]deoxyglucose in vivo by brown adiposetissue and other glucose-utilising tissues of the mouse. FEBS Lett 1985;188:257–26129. Hoy AJ, Bruce CR, Turpin SM, Morris AJ, Febbraio MA, Watt MJ. Adiposetriglyceride lipase-null mice are resistant to high-fat diet-induced insulin re-sistance despite reduced energy expenditure and ectopic lipid accumulation.Endocrinology 2011;152:48–5830. Borg ML, Andrews ZB, Watt MJ. Exercise training does not enhance hy-pothalamic responsiveness to leptin or ghrelin in male mice. J Neuroendocrinol2014;26:68–7931. Gage FH. Mammalian neural stem cells. Science 2000;287:1433–1438

diabetes.diabetesjournals.org Borg and Associates 3657

32. Winner B, Kohl Z, Gage FH. Neurodegenerative disease and adult neuro-genesis. Eur J Neurosci 2011;33:1139–115133. Kim SE, Ko IG, Kim BK, et al. Treadmill exercise prevents aging-inducedfailure of memory through an increase in neurogenesis and suppression of ap-optosis in rat hippocampus. Exp Gerontol 2010;45:357–36534. van Praag H, Christie BR, Sejnowski TJ, Gage FH. Running enhancesneurogenesis, learning, and long-term potentiation in mice. Proc Natl Acad SciU S A 1999;96:13427–1343135. van Praag H, Schinder AF, Christie BR, Toni N, Palmer TD, Gage FH.Functional neurogenesis in the adult hippocampus. Nature 2002;415:1030–103436. Leuner B, Gould E, Shors TJ. Is there a link between adult neurogenesis andlearning? Hippocampus 2006;16:216–22437. Borg ML, Omran SF, Weir J, Meikle PJ, Watt MJ. Consumption of a high-fatdiet, but not regular endurance exercise training, regulates hypothalamic lipidaccumulation in mice. J Physiol 2012;590:4377–438938. Thaler JP, Yi CX, Schur EA, et al. Obesity is associated with hypothalamicinjury in rodents and humans. J Clin Invest 2012;122:153–16239. Lee EB, Ahima RS. Alteration of hypothalamic cellular dynamics in obesity.J Clin Invest 2012;122:22–2540. Pérez-Martín M, Cifuentes M, Grondona JM, et al. IGF-I stimulates neuro-genesis in the hypothalamus of adult rats. Eur J Neurosci 2010;31:1533–154841. Rivera P, Romero-Zerbo Y, Pavón FJ, et al. Obesity-dependent cannabinoidmodulation of proliferation in adult neurogenic regions. Eur J Neurosci 2011;33:1577–1586

42. van Praag H, Kempermann G, Gage FH. Running increases cell pro-liferation and neurogenesis in the adult mouse dentate gyrus. Nat Neurosci1999;2:266–27043. Pereira AC, Huddleston DE, Brickman AM, et al. An in vivo correlate ofexercise-induced neurogenesis in the adult dentate gyrus. Proc Natl Acad SciU S A 2007;104:5638–564344. Clark PJ, Brzezinska WJ, Puchalski EK, Krone DA, Rhodes JS. Functionalanalysis of neurovascular adaptations to exercise in the dentate gyrus of youngadult mice associated with cognitive gain. Hippocampus 2009;19:937–95045. Klaissle P, Lesemann A, Huehnchen P, Hermann A, Storch A, Steiner B.Physical activity and environmental enrichment regulate the generation of neuralprecursors in the adult mouse substantia nigra in a dopamine-dependentmanner. BMC Neurosci 2012;13:13246. Simon C, Götz M, Dimou L. Progenitors in the adult cerebral cortex: cellcycle properties and regulation by physiological stimuli and injury. Glia 2011;59:869–88147. Mandyam CD, Wee S, Eisch AJ, Richardson HN, Koob GF. Methamphet-amine self-administration and voluntary exercise have opposing effects on medialprefrontal cortex gliogenesis. J Neurosci 2007;27:11442–1145048. Yamauchi T, Tatsumi K, Makinodan M, et al. Olanzapine increases cellmitotic activity and oligodendrocyte-lineage cells in the hypothalamus. Neuro-chem Int 2010;57:565–57149. Robins SC, Trudel E, Rotondi O, et al. Evidence for NG2-glia derived, adult-born functional neurons in the hypothalamus. PLoS One 2013;8:e78236

3658 Exercise-Induced Neurogenesis and Insulin Action Diabetes Volume 63, November 2014