overexpression of neuropeptide y in the dorsomedial hypothalamus causes hyperphagia and obesity in...

Overexpression of Neuropeptide Y in theDorsomedial Hypothalamus CausesHyperphagia and Obesity in RatsFenping Zheng,1,2 Yonwook J. Kim,1 Pei-Ting Chao1 and Sheng Bi1*

We sought to determine a role for NPY overexpression in the dorsomedial hypothalamus (DMH) in obesity etiol-

ogy using the rat model of adeno-associated virus (AAV)-mediated expression of NPY (AAVNPY) in the DMH.

Rats received bilateral DMH injections of AAVNPY or control vector and were fed on regular chow. Five-

week postviral injection, half the rats from each group were switched to access to a high-fat diet for

another 11 weeks. We examined variables including body weight, food intake, energy efficiency, meal

patterns, glucose tolerance, fat mass, plasma insulin, plasma leptin, and hypothalamic gene expression.

Rats with DMH NPY overexpression had increased food intake and body weight and lowered metabolic

efficiency. The hyperphagia was mediated through increased meal size during the dark. Although these

rats had normal blood glucose, their plasma insulin levels were increased in both basal and glucose chal-

lenge conditions. While high-fat diet induced hyperphagia, obesity, and hyperinsulinemia, these effects

were amplified in rats with DMH NPY overexpression. Arcuate Npy, agouti-related protein and proopiome-

lanocortin expression was appropriately regulated in response to positive energy balance. These results

indicate that DMH NPY overexpression can cause hyperphagia and obesity and DMH NPY may have

actions in glucose homeostasis.

Obesity (2013) 21:1086–1092. doi:10.1002/oby.20467

IntroductionThe dorsomedial hypothalamus (DMH) plays an important role in

maintaining energy homeostasis. Lesions of the DMH result in hypo-

phagia, reduced body weight and linear growth (1). Disinhibition of

neurons in the DMH provokes nonshivering thermogenesis and ele-

vates core body temperature (2). Despite these observations, the neural

mechanisms underlying these effects remain incompletely understood.

Within the DMH, a number of neurotransmitters and=or receptors

have been found, and their roles in controlling energy balance have

been investigated (1,3–5). We have recently examined the role of the

orexigenic peptide neuropeptide Y (NPY) in these actions (6,7).

Within the hypothalamus, NPY-containing neurons are primarily

identified in the arcuate nucleus (ARC) and the DMH (8,9). In con-

trast to the well-characterized actions of ARC NPY in energy bal-

ance control (10–12), the importance of DMH NPY in maintaining

energy balance is just being unraveled. DMH Npy overexpression or

induction has been found in certain rodent models with increased

energy demands (8,13,14) and obesity (15–19). Knockdown of NPY

in the DMH via adeno-associated virus (AAV)-mediated RNAi

ameliorates the hyperphagia, obesity, and diabetes of Otsuka Long-

Evans Tokushima Fatty (OLETF) rats (6). NPY knockdown in the

DMH of normally growing rats affects a number of aspects of

energy balance control including food intake, energy expenditure,

thermogenesis, adiposity, and physical activity (7). Overall, these

findings suggest that DMH NPY acts as an important neuromodula-

tor to modulate energy balance and dysregulation of DMH NPY

causes disordered energy balance, leading to obesity and diabetes.

To ascertain a potential causal role for DMH NPY overexpression in

these disorders, we have previously examined the effects of DMH

NPY overexpression on food intake and body weight using the rat

model of AAV-mediated expression of NPY in the DMH. We found

that NPY overexpression in the DMH, particularly within and

around the compact subregion, causes increased food intake and

body weight and enhances high-fat diet-induced obesity (6). In this

study, we sought to more completely characterize the effects of

DMH NPY overexpression on food intake, body weight, adiposity

and blood glucose using the same model.

1 Department of Psychiatry and Behavioral Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA. Correspondence: Sheng Bi([email protected]) 2 Department of Endocrinology, The Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, 310016, China

Disclosure: The authors have no competing interests.

Funding agencies: This work was supported by US National Institute of Diabetes and Digestive and Kidney Diseases Grants DK074269 and DK087888.

Received: 31 October 2012; Accepted: 12 March 2013; Published online 21 March 2013. doi:10.1002/oby.20467

1086 Obesity | VOLUME 21 | NUMBER 6 | JUNE 2013 www.obesityjournal.org

CommentaryCLINICAL TRIALS: BEHAVIOR, PHARMACOTHERAPY, DEVICES, SURGERY

Obesity

Methods and ProceduresAnimalsMale Sprague-Dawley rats were purchased from Charles River Lab-

oratories and individually housed on a 12:12 h light-dark cycle

(lights on at 0600h) in a temperature-controlled colony room (22–

24�C) with ad libitum access to tap water and standard rodent chow,

except where noted. All procedures were approved by the Institu-

tional Animal Care and Use Committee at the Johns Hopkins

University.

AAV-mediated expression of NPY in the DMHAs described previously (6), we generated a recombinant vector of

AAV-mediated NPY expression (AAVNPY). The vector AAVGFP

served as a control. We first determined the amount of viral par-

ticles injected for AAV-mediated NPY expression in the entire

DMH including both compact and noncompact subregions via

increasing the amount of vectors from the previous dose of

0.3 ll=site (�1 3 109 particles=site) (6) to 0.5 ll=site (�1.7 3 109

particles=site). After verification of AAV-mediated NPY overex-

pression in the DMH using standard in situ hybridization histo-

chemistry (6), 24 male rats weighing 100-110 g were randomly

assigned to bilateral DMH injections of AAVNPY or AAVGFP

(n 5 12 rats=group). As described previously (7), 0.5 ll=site of

AAV vectors were bilaterally injected into the DMH with coordi-

nates: 2.3 mm caudal to bregma, 0.4 mm lateral to midline and

7.6 mm ventral to skull surface. After injection, rats continued to

have ad libitum access to a regular chow diet (RC: 15.8% fat,

65.6% carbohydrate, and 18.6% protein in kcal%, 3.37 kcal=g,

Prolab RMH 1000, PMI Nutrition International, LLC, Brentwood,

MO), designated as AAVNPY-RC and AAVGFP-RC. Five weeks

postviral injection, half the rats from each group were switched to

ad libitum access to a high-fat diet (HF: 60% fat, 20% carbohy-

drate, and 20% protein in kcal%; 5.2 kcal g21; Research Diets;

New Brunswick, NJ), designated as AAVNPY-HF and AAVGFP-

HF, for 11 weeks. Body weights were measured daily and food

intake was recorded weekly. Sixteen weeks postviral injection,

rats were sacrificed in a 2-h fasted state during the light period.

Blood glucose was determined with a FreeStyle glucometer

(Abbott Laboratories, Abbott Park, IL). Trunk blood was taken for

evaluation of leptin and insulin using a rat insulin and leptin radi-

oimmunoassay kit, respectively (Millipore Corporation; Billerica,

MA). Interscapular brown adipose tissue (BAT), epididymal white

adipose tissue (WAT), and subcutaneous inguinal WAT were col-

lected and weighed. Brains were saved for subsequent examina-

tion of hypothalamic gene expression using quantitative real time

RT-PCR.

Analysis of meal patternsAn additional cohort of 12 male rats was used for this study. Rats

received DMH injections of AAVNPY or AAVGFP (n 5 6

rats=group) as described above. Two-weeks postviral injection, rats

were transferred to individual test cages containing computerized

feeding devices (MED Associates, Georgia, VT), which delivered

45-mg chow pellets as previously described (6). Rats had ad libitumaccess to pellets and water. After 7-days adaptation, data for 24-h

food intake were collected and meal patterns were analyzed using a

customized program. A meal was defined as the acquisition of at

least five pellets preceded and followed by at least 15 min of no

feeding (6). Meal size was defined as the number of pellets deliv-

ered during a meal.

Oral glucose tolerance test (OGTT)An additional cohort of 10 male rats received DMH injections of

AAVNPY or AAVGFP (n 5 5 rats=group) as described above. Four-

weeks postviral injection, OGTT was conducted as previously

described (6). Following an overnight fast, rats were administered

oral glucose (2 g kg21) by gavage. Tail blood was sampled before

and 15, 30, 45, 60, and 120 min after giving glucose. Blood glucose

and plasma insulin concentrations were determined as described

above.

Quantitative real-time RT-PCRBrains at the levels of the DMH and the ARC were first sliced via a

cryostat and then individual nuclei of the DMH and the ARC were

punched out. Total RNA was extracted from each sample (punched

hypothalamic nuclei, inguinal WAT, or interscapular BAT) by using

Trizol reagent (Life Technologies, Grand Island, NY). Two-step

quantitative real time RT-PCR was performed for gene expression

determination as described previously (7). b-actin was used as an in-

ternal control for quantification of individual mRNA. A list of

primer sets is shown in Table 1.

Statistical analysisAll values are presented as means 6 SEM. Data were analyzed

using the commercial software Statistica 7 (StatSoft, Tulsa, OK).

Data for body weight and food intake were analyzed using two-way

repeated measures analysis of variance (ANOVA) over the first 5-

weeks postviral injection and three-way ANOVA with one repeated

factor over the next 11 weeks. Data for meal patterns were analyzed

using Student’s t test (two-tailed). Data for cumulative intake, fat

mass, blood glucose, plasma insulin and leptin, and mRNA expres-

sion levels for hypothalamic Npy, agouti-related protein (Agrp) and

proopiomelanocortin (Pomc), inguinal WAT leptin and interscapular

BAT uncoupling protein 1 (Ucp1) were analyzed using two-way

ANOVA. All ANOVAs were followed by pairwise multiple Fisher’s

LSD comparisons. P< 0.05 was considered as a statistically signifi-

cant difference.

ResultsAAV-mediated expression of NPY in the DMHWe verified that the vector AAVNPY infected neurons within the

DMH including both compact and noncompact subregions, and pro-

duced robust Npy overexpression in the DMH area (Figure 1a). Con-

sistent with the previous reports showing the long-lasting effect of

AAV vectors on gene expression (6,7), 16 weeks postviral injection,

Npy mRNA levels in the DMH of AAVNPY rats remained

TABLE 1 A list of primer sets for real time RT-PCR

Forward primer Reverse primer

Neuropeptide Y 50-agagatccagccctgagaca-30 50-aacgacaacaagggaaatgg-30

Agouti-related protein 50-tggcagaggtgctagatcca-30 50-gcacaggtcgcagcaaggta-30

Proopiomelanocortin 50-tccatagacgtgtggagctg-30 50-acgtacttccggggattttc-30

Leptin 50-ccggttcctgtggctttggtcct-30 50-tgaccctctgcctggcggatac-30

Uncoupling protein 1 50-cgttccaggatccgagtcgcaga-30 50-tcagctcttgtcgccgggttttg-30

b-Actin 50-tgtcaccaactgggacgata-30 50-ggatggctacgtacatggct-30

Commentary ObesityCLINICAL TRIALS: BEHAVIOR, PHARMACOTHERAPY, DEVICES, SURGERY

www.obesityjournal.org Obesity | VOLUME 21 | NUMBER 6 | JUNE 2013 1087

significantly increased by 2.5 fold compared to control rats

(AAVNPY-RC vs. AAVGFP-RC, Figure 5).

Effects of DMH NPY overexpression on bodyweightRats with DMH NPY overexpression gained significantly more body

weight than control rats when maintained on regular chow over the

first 5 weeks (P< 0.001, Figure 1b). The weight gain of AAVNPY-

RC rats was increased by 10, 13, and 17% at 3, 4, and 5 weeks

postviral injection respectively relative to AAVGFP-RC rats. Over

the next 11 weeks, there were significant main effects of NPY over-

expression (P< 0.001) and HF (P< 0.001) as well as a significant

interaction between NPY overexpression and HF (P 5 0.008, Figure

1b), indicating that access to HF resulted in greater body weight

gain in AAVNPY rats than in AAVGFP rats. At sacrifice,

AAVNPY-RC rats had sustained increases in body weight and

gained 16% more weight than AAVGFP-RC rats (P 5 0.037, Figure

1b). While HF caused a 15% increase in weight gain in control ani-

mals, HF resulted in a 33% increase in AAVNPY rats (P< 0.05,

Figure 1b). Thus, AAVNPY rats on HF gained 34% more weight

than control rats on HF or 54% more weight compared to control

rats on RC (P< 0.001, Figure 1b).

Effects of DMH NPY overexpression on foodintakeDMH NPY overexpression resulted in increased food intake in rats

on both regular chow and HF diets. The chow intake of AAVNPY-

RC rats was 15% more than that of AAVGFP-RC rats over the 16

weeks (P< 0.001, Figure 1c). When rats were challenged with HF

for 11 weeks, there were significant main effects of NPY overex-

pression (P 5 0.004) and HF (P< 0.001), but no significant interac-

tion between NPY overexpression and HF (P 5 0.272, Figure 1c).

HF induced 29 and 39% increases in 11-week cumulative intake in

AAVGFP (P 5 0.008) and AAVNPY rats (P< 0.001), respectively.

Although the increases in AAVNPY and AAVGFP rats did not sig-

nificantly differ, AAVNPY-HF rats actually ate 23% more food than

AAVGFP-HF rats (P 5 0.007, Figure 1c).

Metabolic efficiency (i.e., energy intake divided by body weight

gain) has been used for evaluation of metabolic rate (20). Analysis

of metabolic efficiency revealed that DMH NPY overexpression

resulted in decreased metabolic efficiency (from 47.5 6 2.1 in

AAVGFP-RC to 34.9 6 2.7 in AAVNPY-RC, P 5 0.001), indicating

that DMH NPY overexpression lowered metabolic rate. Access to

HF caused significant reductions of metabolic efficiency in both

groups (from 47.5 6 2.1 to 26.4 6 1.7 in control rats, P< 0.001,

and from 34.9 6 2.7 to 27.3 6 2.7 in AAVNPY rats, P 5 0.025),

but HF did not produce a further effect in AAVNPY rats (P 5

0.774).

We next examined the effect of DMH NPY overexpression on

meal patterns. AAVNPY rats consumed significantly more pel-

leted chow than AAVGFP rats during the dark period (P 5 0.045),

but the chow intake of AAVNPY and AAVGFP rats did not differ

during the light period (P 5 0.587, Figure 2a). Overall, AAVNPY

rats had a significant increase in 24-h chow intake (P 5 0.039,

Figure 2a). Meal pattern analysis revealed that DMH NPY overex-

pression caused increased meal sizes in the dark (P 5 0.043) and

over the total 24 h (P 5 0.047), but did not affect meal size during

the light period (P 5 0.492, Figure 2b). Meal numbers were not

significantly altered in AAVNPY rats in the total, dark, or light

period (Figure 2c).

Effects of DMH NPY overexpression on fat massand plasma leptin levelsAt sacrifice, we found a significant main effect of DMH NPY over-

expression on inguinal WAT mass (P 5 0.007), but not on epididy-

mal WAT (P 5 0.190) and interscapular BAT mass (P 5 0.208,

FIGURE 1 Effects of NPY overexpression in the dorsomedial hypothalamus (DMH) onfood intake and body weight. (a) 35S-labeled in situ hybridization histochemistryshows adeno-associated virus (AAV)-mediated expression of NPY in the DMH ofrats. AAVNPY or AAVGFP: rats received bilateral DMH injections of the vectorAAVNPY or control AAVGFP; ARC: arcuate nucleus. (b) Body weight gain in AAVGFPand AAVNPY rats on a regular chow (RC) or high-fat (HF) diet, designated asAAVGFP-RC, AAVNPY-RC, AAVGFP-HF, and AAVNPY-HF. (c) Daily food intake inthe four groups of rats. Values are means 6 SEM. n 5 6 rats per group. *P < 0.05 vs.AAVGFP-RC rats, #P < 0.05 vs. AAVNPY-RC rats and §P < 0.05 vs. AAVGFP-HF rats.

Obesity DMH NPY Overexpression Causes Obesity Zheng et al.


Figure 3a), indicating that DMH NPY overexpression produced an

inguinal WAT-specific effect. Inguinal WAT mass was increased

42% in AAVNPY-RC rats compared to AAVGFP-RC rats (Figure

3a). HF induced significant increases in fat weights in all three fat

depots (P< 0.001 in inguinal WAT, P 5 0.001 in epididymal WAT,

P 5 0.005 in interscapular BAT, Figure 3a), but there were no sig-

nificant interactions between NPY overexpression and HF in these

fat depots (P > 0.05, Figure 3a). As compared to AAVGFP-HF rats,

FIGURE 2 Effects of DMH NPY overexpression on patterns of 24-h food intake. (a) Cumulative food intake, (b) mealsize and (c) meal number in AAVNPY and AAVGFP rats. Total, total daily; Dark, the dark period; Light, the light period.Values are means 6 SEM. n 5 6 rats per group. *P < 0.05 vs. AAVGFP.

FIGURE 3 Effects of DMH NPY overexpression on fat mass, plasma leptin, and mRNA levels of interscapular BAT Ucp1 and in-guinal WAT leptin. (a) Fat mass, (b) plasma leptin levels, (c) Ucp1 mRNA levels in interscapular BAT and (d) leptin mRNA levelsin inguinal fat. Values are means 6 SEM. n 5 6 rats per group. *P < 0.05 vs. AAVGFP-RC, #P < 0.05 vs. AAVNPY-RC, and§P < 0.05 vs. AAVGFP-HF.



AAVNPY-HF rats accumulated significantly more fat mass in ingui-

nal WAT, but not other two fat depots (Figure 3a).

Plasma leptin levels were not altered in rats with DMH NPY over-

expression (p50.191, Figure 3b) although the rats had increased

body weight and fat mass. Consistent with HF-induced obesity, HF

resulted in significant increases in plasma leptin levels in both

AAVNPY and AAVGFP rats (P< 0.001), but the increases did not

differ between the two groups (P > 0.05, Figure 3b).

Effects of DMH NPY overexpression on Ucp1and leptin gene expressionWe examined Ucp1 gene expression in interscapular BAT and leptin

gene expression in inguinal WAT as DMH NPY has specific effects

on these two fat pads (7). We found significantly decreased expres-

sion of Ucp1 in interscapular BAT of AAVNPY-RC rats relative to

AAVGFP-RC rats (P< 0.05, Figure 3c). HF diet caused increased

expression of Ucp1 in interscapular BAT in both AAVNPY and

AAVGFP rats (P< 0.05), but the changes did not differ between the

two groups (P > 0.05, Figure 3c).

DMH NPY overexpression resulted in downregulation of leptin

expression in inguinal WAT (P< 0.05, Figure 3d) although this

overexpression caused a significant increase in inguinal WAT mass

(Figure 3a). While HF resulted in increased expression of leptin in

inguinal WAT of AAVGFP rats (P< 0.05), NPY overexpression sig-

nificantly reduced this increase, leading to the absence of a signifi-

cant difference between AAVNPY-HF and AAVGFP-RC rats (P 5

0.059, Figure 3d).

Effects of DMH NPY overexpression on glucosehomeostasisAt sacrifice, blood glucose levels were normal in AAVNPY-RC rats

(Figure 4a), but their plasma insulin levels were increased threefold

compared to AAVGFP-RC rats (P< 0.05, Figure 4b), indicating that

AAVNPY rats required more insulin to maintain normal blood glu-

cose. Consistent with HF-induced hyperglycemia and hyperinsulin-

emia, access to HF caused significant increases in blood glucose and

plasma insulin levels in both groups (P< 0.001 in glucose levels,

P 5 0.01 in insulin levels). Although HF-induced hyperglycemia did

not differ between AAVNPY-HF and AAVGFP-HF rats (P > 0.05,

Figure 4a), plasma insulin levels of AAVNPY-HF rats were signifi-

cantly higher than those of AAVGFP-HF rats (P< 0.05, Figure 4b).

To further examine the effect of DMH NPY overexpression on glu-

cose homeostasis, we conducted an OGTT in an additional cohort of

FIGURE 4 Effects of DMH NPY overexpression on glucose homeostasis. (a) Blood glucose levels did not differ betweenAAVNPY and AAVGFP rats, irrespective of diet, but (b) plasma insulin levels were significantly increased in AAVNPY rats com-pared to their counterparts respectively. (c) Oral glucose tolerance test revealed that in response to oral glucose administra-tion, blood glucose levels did not differ between AAVNPY and AAVGFP rats, but (d) plasma insulin levels were significantlyelevated at 0 (fasting), 15, 30, and 45 min in AAVNPY compared to AAVGFP rats. Values are means 6 SEM. n 5 5-6 rats pergroup. *P < 0.05 vs. AAVGFP-RC (or AAVGFP) rats, #P < 0.05 vs. AAVNPY-RC rats and §P < 0.05 vs. AAVGFP-HF rats.



AAVNPY rats with body weight matched with control rats (438 6

16 g in AAVNPY vs. 410 6 18 g in AAVGFP, P 5 0.280) 4-weeks

postviral injection. While fasting glucose levels were normal in

AAVNPY rats (Figure 4c), their basal insulin levels were signifi-

cantly elevated (Figure 4d). In response to oral glucose administra-

tion, blood glucose levels did not differ between AAVNPY and

AAVGFP rats (Figure 4c), but plasma insulin levels were signifi-

cantly elevated at 15, 30, and 45 min in AAVNPY rats compared to

control rats (Figure 4d). These data indicate that AAVNPY rats

required more insulin secretion to clear glucose, suggesting that

DMH NPY overexpression causes insulin insensitivity.

Effects of DMH NPY overexpression onhypothalamic gene expressionAs mentioned above, the vector AAVNPY-mediated expression of

NPY in the DMH was long lasting. At sacrifice, quantitative real-

time RT-PCR confirmed that Npy mRNA levels in the DMH of

AAVNPY rats remained significantly increased (P< 0.001, Figure

5). Access to HF resulted in significantly decreased expression of

Npy in the DMH in both groups (P< 0.05, Figure 5). Overall, there

was a significant interaction between viral-mediated NPY overex-

pression and HF (P 5 0.001), implying that HF caused a greater

reduction of Npy expression in the DMH of AAVNPY rats. Even

with this, the levels of Npy mRNA in the DMH of AAVNPY-HF

rats remained significantly higher than those of AAVGFP-HF rats

(P 5 0.004, Figure 5).

Within the ARC, we found significant main effects of both viral-

mediated NPY expression in the DMH and HF on Npy or AgrpmRNA levels (P< 0.05, Figure 5). Both Npy and Agrp mRNA lev-

els in the ARC of AAVNPY-RC, AAVNPY-HF, or AAVGFP-HF

rats were significantly lower than those of AAVGFP-RC rats

(P< 0.05, Figure 5), but there were no significant interactions

between viral-mediated NPY expression and HF on these two genes

(P > 0.05). In contrast, Pomc mRNA levels in the ARC were not

significantly altered by viral-mediated expression of NPY in the

DMH (P 5 0.633, Figure 5). Access to HF resulted in significantly

increased expression of ARC Pomc in AAVGFP rats (P 5 0.012),

but not AAVNPY rats (P > 0.05, Figure 5).

DiscussionWe examined the effects of DMH NPY overexpression on food

intake, body weight, adiposity, and blood glucose using the rat

model of AAV-mediated NPY expression in the DMH. Rats with

DMH NPY overexpression had increased food intake and body

weight with decreased metabolic efficiency. These animals had

increased inguinal WAT, decreased leptin expression in inguinal

WAT, and lowered Ucp1expression in interscapular BAT. Although

blood glucose was normal, plasma insulin levels were significantly

elevated in both basal and glucose challenge conditions in rats with

NPY overexpression. While HF induced hyperphagia, obesity, and

hyperinsulinemia in control rats, access to HF amplified these

effects in rats with NPY overexpression. Together, these

results demonstrate that DMH NPY overexpression can produce

hyperphagia and obesity and also provide additional evidence sug-

gesting that DMH NPY plays a role in maintaining glucose

homeostasis.

Previous reports have shown that alterations in DMH NPY result in

a nocturnal and meal size-specific feeding effect. OLETF rats have

disordered feeding behavior characterized by increased meal size

that was proposed to contribute to their hyperphagia and obesity

(21). Analysis of hypothalamic gene expression revealed elevated

expression of Npy in the DMH of OLETF rats (19,22), whereas

DMH NPY knockdown completely normalizes meal size during the

dark period in OLETF rats and significantly ameliorates their hyper-

phagia and obesity (6). Consistent with this view, the present study

identified that DMH NPY overexpression caused increased meal

size during the dark period. Thus, the results from both previous

knockdown and present overexpression of NPY in the DMH of rats

clearly establish a specific role for DMH NPY in the control of

meal size during the dark period, and in this way, modulating over-

all food intake.

A role for DMH NPY in the regulation of adiposity, thermogenesis

and energy expenditure has been implicated. DMH NPY knockdown

promotes brown adipocyte development in inguinal WAT through

sympathetic nervous system and causes elevated expression of ther-

mogenic peptide UCP1 in both inguinal fat and interscapular BAT

(7). This knockdown causes increased temperature in inguinal fat

and interscapular BAT (23) and elevated energy expenditure (7).

In support of this view, DMH NPY overexpression resulted in

lowered metabolic efficiency (or metabolic rate) and decreased

Ucp1expression in interscapular BAT. These data suggest that both

feeding and metabolic effects may contribute to DMH NPY overex-

pression-induced increases in body weight and fat mass. Although

HF-induced elevation of UCP1 in interscapular BAT is consistent

with a proposed role for UCP1 in interscapular BAT in diet-induced

thermogenesis (24), how HF interacts with DMH NPY to affect

BAT thermogenesis or energy expenditure remains to be

determined.

We found that DMH NPY overexpression caused increased inguinal

WAT, but lowered leptin expression in this fat, i.e., increased fat

mass did not lead to increased leptin expression or production.

Although AAVNPY rats were heavier or had more fat than control

rats, plasma leptin levels were not increased in AAVNPY rats.

FIGURE 5 Hypothalamic Npy, agouti-related protein (Agrp) and proopiomelanocortin(Pomc) gene expression in response to viral-mediated Npy overexpression in theDMH and=or access to HF. Values are means 6 SEM. n 5 6 rats per group.*P < 0.05 vs. AAVGFP-RC rats, #P < 0.05 vs. AAVNPY-RC rats, and §P < 0.05 vs.AAVGFP-HF rats.



These data imply that DMH NPY overexpression may limit leptin

products and the resulting reduction may also contribute to NPY

overexpression-induced disorders. Nevertheless, the functional sig-

nificance of altered leptin in inguinal WAT merits further

investigation.

Previous reports have suggested a role for DMH NPY in glucose ho-

meostasis. DMH NPY knockdown improves glucose intolerance and

ameliorates hyperglycemia and hyperinsulinemia in OLETF rats (6),

an animal model of obesity and diabetes (25), and diet-induced

obese rats (7). Consistent with these reports, DMH NPY overexpres-

sion resulted in increased insulin levels in rats on regular chow and

caused more severe diet-induced hyperinsulinemia. OGTT confirmed

that DMH NPY overexpression caused impaired glucose tolerance

and decreased insulin sensitivity independently of body weight

effect. Together, these results suggest that DMH NPY has additional

actions in glycemic control.

Although NPY-containing neurons have been identified in the ARC

and the DMH, the neural circuits underlying their actions appear to

differ. ARC NPY serves as one of downstream mediators of leptin’s

actions in maintaining energy homeostasis (10,11), but DMH NPY

is not under the control of leptin (8). DMH NPY signaling is

affected by brain cholecystokinin (26) and other yet to be identified

molecules (27). The present findings of downregulation of Npy-Agrp expression and upregulation of Pomc expression in the ARC

of rats with DMH NPY overexpression and=or access to HF were

likely in response to positive energy balance or increases in body

weight and circulating leptin=insulin levels (10–12,28). Although

Npy induction was reported in the DMH of diet-induced obese mice

(17), we did not replicate this result in mice (29). The reason for the

difference is unclear. We actually found a reduction of DMH Npyexpression in rats on HF in both previous (29) and present studies,

implying that this reduction is likely in response to increased energy

intake. Furthermore, DMH NPY neurons project to the brainstem

nucleus of solitary tract (NTS) and produce inhibitory effects on

NTS neurons to modulate food intake (6). Whether this neural path-

way also underlies other effects of DMH NPY such as thermogene-

sis or energy expenditure remains to be determined.

In summary, we provide new evidence demonstrating the specific

role for DMH NPY overexpression in the overall control of energy

balance. DMH NPY overexpression causes increased food intake

and body weight and exacerbates diet-induced hyperphagia and obe-

sity. This overexpression produces a nocturnal meal size-specific

effect that contributes to overall increased food intake. DMH NPY

overexpression also lowers energy efficiency, affects fat mass and

leptin expression in inguinal WAT, and alters Ucp1 expression in

interscapular BAT. Finally, DMH NPY overexpression leads to insu-

lin insensitivity and exaggerates diet-induced hyperinsulinemia.

Overall, these results indicate that DMH NPY is an important neuro-

modulator in modulating energy balance and DMH NPY overexpres-

sion can cause hyperphagia and obesity.

AcknowledgmentWe thank Dr. T.H. Moran for comments and discussions on the

manuscript.

VC 2013 The Obesity Society

References1. Bellinger LL, Bernardis LL. The dorsomedial hypothalamic nucleus and its role in

ingestive behavior and body weight regulation: lessons learned from lesioningstudies. Physiol Behav 2002;76:431-442.

2. Zaretskaia MV, Zaretsky DV, Shekhar A, et al. Chemical stimulation of thedorsomedial hypothalamus evokes non-shivering thermogenesis in anesthetized rats.Brain Res 2002;928:113-125.

3. Bi S. Role of dorsomedial hypothalamic neuropeptide Y in energy homeostasis.Peptides 2007;28:352-356.

4. Dimicco JA, Zaretsky DV. The dorsomedial hypothalamus: a new player inthermoregulation. Am J Physiol Regulat Integr Comp Physiol 2007;292:R47-R63.

5. Morrison SF, Nakamura K. Central neural pathways for thermoregulation. FrontBiosci 2011;16:74-104.

6. Yang L, Scott KA, Hyun J, et al. Role of dorsomedial hypothalamic neuropeptide Yin modulating food intake and energy balance. J Neurosci 2009;29:179-190.

7. Chao PT, Yang L, Aja S, et al. Knockdown of NPY expression in the dorsomedialhypothalamus promotes development of brown adipocytes and prevents diet-inducedobesity. Cell Metab 2011;13:573-583.

8. Bi S, Robinson BM, Moran TH. Acute food deprivation and chronic foodrestriction differentially affect hypothalamic NPY mRNA expression. Am J PhysiolRegulat Integr Comp Physiol 2003;285:R1030-R1036.

9. White JD, Kershaw M. Increased hypothalamic neuropeptide Y expressionfollowing food deprivation. Mol Cell Neurosci 1990;1:41-48.

10. Elmquist JK, Elias CF, Saper CB. From lesions to leptin: hypothalamic control offood intake and body weight. Neuron 1999;22:221-232.

11. Schwartz MW, Woods SC, Porte D, Jr., et al. Central nervous system control offood intake. Nature 2000;404:661-671.

12. Flier JS. Obesity wars: molecular progress confronts an expanding epidemic. Cell2004;116:337-350.

13. Smith MS. Lactation alters neuropeptide-Y and proopiomelanocortin geneexpression in the arcuate nucleus of the rat. Endocrinology 1993;133:1258-1265.

14. Kawaguchi M, Scott KA, Moran TH, et al. Dorsomedial hypothalamiccorticotropin-releasing factor mediation of exercise-induced anorexia. Am J PhysiolRegulat Integr Comp Physiol 2005;288:R1800-R1805.

15. Kesterson RA, Huszar D, Lynch CA, et al. Induction of neuropeptide Y geneexpression in the dorsal medial hypothalamic nucleus in two models of the agoutiobesity syndrome. Mol Endocrinol 1997;11:630-637.

16. Tritos NA, Elmquist JK, Mastaitis JW, et al. Characterization of expression ofhypothalamic appetite-regulating peptides in obese hyperleptinemic brown adiposetissue-deficient (uncoupling protein-promoter-driven diphtheria toxin A) mice.Endocrinology 1998;139:4634-4641.

17. Guan XM, Yu H, Trumbauer M, et al. Induction of neuropeptide Y expression indorsomedial hypothalamus of diet-induced obese mice. Neuroreport 1998;9:3415-3419.

18. Guan XM, Yu H, Van der Ploeg LH. Evidence of altered hypothalamic pro-opiomelanocortin=neuropeptide Y mRNA expression in tubby mice. Brain Res MolBrain Res 1998;59:273-279.

19. Bi S, Ladenheim EE, Schwartz GJ, et al. A role for NPY overexpression in thedorsomedial hypothalamus in hyperphagia and obesity of OLETF rats. Am JPhysiol Regulat Integr Comp Physiol 2001;281:R254-R260.

20. Winzell MS, Ahren B. The high-fat diet-fed mouse: a model for studyingmechanisms and treatment of impaired glucose tolerance and type 2 diabetes.Diabetes 2004;53 (Suppl 3):S215–S219.

21. Moran TH, Katz LF, Plata-Salaman CR, et al. Disordered food intake and obesityin rats lacking cholecystokinin A receptors. Am J Physiol 1998;274:R618-R625.

22. Schroeder M, Zagoory-Sharon O, Shbiro L, et al. Development of obesity in theOtsuka Long-Evans Tokushima Fatty rat. Am J Physiol Regulat Integr CompPhysiol 2009;297:R1749-R1760.

23. Bi S. Dorsomedial hypothalamic NPY modulation of adiposity and thermogenesis.Physiol Behav 2013.

24. Cannon B, Nedergaard J. Brown adipose tissue: function and physiologicalsignificance. Physiol Rev 2004;84:277-359.

25. Kawano K, Hirashima T, Mori S, et al. Spontaneous long-term hyperglycemic ratwith diabetic complications. Otsuka Long-Evans Tokushima Fatty (OLETF) strain.Diabetes 1992;41:1422-1428.

26. Chen J, Scott KA, Zhao Z, et al. Characterization of the feeding inhibition andneural activation produced by dorsomedial hypothalamic cholecystokininadministration. Neuroscience 2008;152:178-188.

27. Bi S, Kim YJ, Zheng F. Dorsomedial hypothalamic NPY and energy balancecontrol. Neuropeptides 2012;46:309-314.

28. Cone RD. Studies on the physiological functions of the melanocortin system.Endocrine Rev 2006;27:736-749.

29. Bi S, Chen J, Behles RR, et al. Differential body weight and feeding responses tohigh-fat diets in rats and mice lacking cholecystokinin 1 receptors. Am J PhysiolRegulat Integr Comp Physiol 2007;293:R55-R63.



overexpression of neuropeptide y in the dorsomedial hypothalamus causes hyperphagia and obesity in...

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