Attenuation of Diabetic Hyperphagia in Y–Deficient Mice Dana K. Sindelar,1 Paul Mystkowski,1 Donald J. Marsh,2 Richard D. Palmiter,2 and Michael W. Schwartz1

The combined effects of increased hypothalamic signal- these responses when the stimulus is food deprivation. ing by neuropeptide Y (NPY) and decreased signaling by Moreover, fasting is a more potent stimulus to hypotha- are hypothesized to stimulate food in- lamic AgRP than is STZ-. take when body stores are depleted. To investigate Therefore, central nervous system signal- NPY’s role in the hyperphagic response to uncontrolled ing appears to be suppressed more effectively by fasting diabetes, streptozotocin (STZ) (200 mg/kg intraperito- than by uncontrolled diabetes, which provides a plausi- neally) or saline vehicle was given to NPY-deficient ble explanation for differences in the feeding response :Npy–/–) and wild-type (Npy؉/؉) mice. In Npy؉/؉ mice, to these two stimuli in mice lacking NPY. Diabetes 51) STZ-induced diabetes increased mean daily food intake 778–783, 2002 to plateau values 50% above baseline intake (؉2.0 ؎ 0.6 g/day; P < 0.05), an effect that was not seen in STZ- treated Npy–/– mice (؉0.8 ؎ 0.1 g/day; NS), despite comparably elevated levels of plasma glucose and com- daptive increases of food intake induced by parably decreased levels of body weight, fat content, depleted body energy stores are important for and plasma . Unlike the impaired feeding response to uncontrolled diabetes, Npy–/– mice exhibit intact survival and appear to involve the coordinate hyperphagic responses to fasting (Erickson et al. [1], A regulation of multiple hypothalamic pathways Nature 381:415–418, 1996). To investigate whether dif- that can influence feeding behavior (2). For example, ferences in hypothalamic melanocortin signaling can hypothalamic that contain neuropeptide Y (NPY), explain this discrepancy, we used in situ hybridization a potent stimulator of food intake, are activated when to compare the effects of STZ-diabetes and fasting on body fat content is reduced by energy restriction (3). pro-opiomelanocortin (POMC) and agouti-related pep- Conversely, the hypothalamic production of melanocort- tide (AgRP) mRNA levels in the hypothalamic arcuate ؉ ؉ ins ( with anorexic properties that are cleaved nucleus (ARC) of Npy–/– and Npy / mice. AgRP mRNA levels were increased by both fasting and STZ-diabetes, from pro-opiomelanocortin [POMC]) is reduced in this but the increase in STZ-diabetes was small (50–80%) setting (4). Acute or chonic energy deficits also increase compared with the effect of fasting (ϳ20-fold increase expression of the gene encoding agouti-related of AgRP mRNA). STZ-diabetes also lowered POMC (AgRP), an endogenous antagonist of central nervous -mRNA levels by 65% in the ARC of Npy؉/؉ mice (P < system (CNS) melanocortin receptors (5) that is co-ex –/– 0.05) but by only 11% in Npy mice (NS); fasting pressed with NPY in neurons of the hypothalamic arcuate significantly lowered POMC mRNA levels in both geno- nucleus (ARC) (6). Fasting is an especially potent stimulus types. We conclude that NPY is required for both the to AgRP gene expression in mouse ARC, increasing AgRP increase of food intake and the decrease of hypotha- ϳ lamic POMC gene expression induced by uncontrolled mRNA by 20-fold (6). The combined effects of decreased diabetes. In contrast, NPY is not required for either of melanocortin signaling (due to both increased AgRP and decreased POMC biosynthesis) and increased NPY signal- ing, therefore, comprise an integrated mechanism to me- From the 1Department of Medicine, University of Washington, and Howard diate hyperphagia in response to a sustained energy Hughes Medical Institute, University of Washington, Seattle, Washington; and the 2Department of Biochemistry, University of Washington, Seattle, Wash- deficit. ington. The original description of mice with NPY deficiency Address correspondence and reprint requests to Michael W. Schwartz, –/– Harborview Medical Center, Division of Endocrinology, Box 359757, 325 9th (Npy ) due to targeted NPY gene disruption revealed Ave., Seattle, WA 98104. E-mail: [email protected]. them to have normal levels of daily food intake and body Received for publication 20 June 2001 and accepted in revised form 3 weight and to exhibit normal increases of food intake after December 2001. M.W.S. is on the Scientific Advisory Board of Millennium Pharmaceutical a fast (1). One possible explanation for this finding is that and has received consulting fees for work in this capacity. D.K.S. is currently neural control over feeding under these circumstances is employed as Senior Investigator at Eli Lilly, Inc., a position that he accepted sufficiently redundant that the loss of NPY is compensated after his work on this study was completed. D.J.M. is currently employed as Senior Investigator at Merck Pharmaceuticals, Inc., a position that he ac- for by other responses. Of potential importance in this cepted after his work on this study was completed. context is the marked reduction of melanocortin signaling Current address for D.K.S.: Eli Lilly & Co., Corporate Center, Drop 0545, –/– Indianapolis, IN 46285. Current address for D.J.M.: Merck & Co., Inc., induced by fasting, which may allow Npy mice to RY80T-126, P.O. Box 2000, Rahway, NJ 07065. increase food intake appropriately during refeeding. One AgRP, agouti-related peptide; ARC, hypothalamic ; AUC, approach to test this hypothesis is to identify and study a area under the curve; CNS, central nervous system; Mcr, ; MRS, magnetic resonance spectroscopy; NPY, neuropeptide Y; model in which hyperphagia depends more on increased POMC, pro-opiomelanocortin; STZ, streptozotocin. NPY signaling than on reduced melanocortin signaling. In

778 DIABETES, VOL. 51, MARCH 2002 D.K. SINDELAR AND ASSOCIATES such a model, the ability to increase food intake should be the same assay. Hybridization to NPY, POMC, or AgRP mRNA was performed 33 compromised in NPY-deficient mice. with antisense riboprobes transcribed from cDNA templates using [ P]UTP (5,16). The hybridization signal in the arcuate nucleus of each brain slice was Uncontrolled -deficient diabetes induced by the determined from film autoradiograms using the MCID image analysis system ␤-cell toxin streptozotocin (STZ) is an established model (Imaging Research, St. Catherines, ON, Canada) as previously described (16). of sustained hyperphagia in rodents. Like the response to Both the film density and hybridization image area were measured, and the fasting, diabetic hyperphagia appears to involve increased product of these two measures was calculated as an index of mRNA content. Values for each animal reflect the mean of 6–12 measurements per animal. hypothalamic signaling by NPY (7–10) and is also accom- The mean value of each neuropeptide mRNA level obtained for wild-type panied by increased AgRP mRNA and reduced POMC controls (e.g., vehicle-treated Npyϩ/ϩ mice for the STZ-diabetes study or mRNA expression in ARC neurons (11–13). However, the Npyϩ/ϩ mice fed ad libitum for the fasting study) was considered to represent increase of AgRP mRNA levels induced by STZ-induced 100% of the control value for that study, and all individual hybridization values diabetes in mice appears to be much smaller (ϳ2-fold) from each experiment were normalized to this number. ϳ Body composition. Body fat content was determined post mortem in mice (13) than that induced by fasting ( 20-fold) (5). Diabetic from experiment 1 using magnetic resonance spectroscopy (MRS) as previ- hyperphagia may therefore depend to a greater extent on ously reported (17). Mice were placed within a custom-made radiofrequency NPY than on AgRP when compared to the hyperphagic coil used for both transmitting and receiving the resonant proton signal at response to fasting. 200.1 MHz. Body lipid mass was estimated from the area under the curve (AUC) of the lipid peak of the resultant MRS spectra, as previously described Based on this reasoning, we hypothesized that mice (17). For each genotype, the mean AUC for the lipid peak in the nondiabetic lacking NPY would manifest impaired feeding responses group was considered to represent 100% of the control value of relative body to uncontrolled diabetes, despite intact responses to fast- fat content, and individual AUC values for the diabetic groups were expressed ing (which induces a much larger increase of AgRP as a percent of this value. expression). Evidence that the orexigenic response to Plasma assays. Blood samples were collected on heparin and placed ϩ/ϩ immediately on ice in heparinized tubes. Plasma was separated by centrifu- centrally administered AgRP is heightened in Npy mice gation and stored at Ϫ20°C until determination of glucose and leptin concen- (14) provides additional support for this hypothesis. We trations. Plasma glucose was determined by the glucose oxidase method therefore measured food intake and hypothalamic levels (Beckman Instruments, Brea, CA). Plasma leptin levels were determined by of AgRP and POMC mRNA in Npy–/– and Npyϩ/ϩ mice radioimmunoassay (Linco Research, St. Louis, MO). Statistics. All values are reported as group means Ϯ SE. The level of made diabetic with STZ and compared these responses to significance was taken as P Յ 0.05, two-tailed. Data were analyzed for those induced by a 48-h fast in separate groups of each differences from baseline values using one-way ANOVA with repeated mea- genotype. sures, and Fisher’s protected least significance difference test was used for multiple comparisons when significant F ratios were obtained. Interactions between treatment and genotype were analyzed by group mean comparisons RESEARCH DESIGN AND METHODS using two-way ANOVA. Statistical comparisons were made using Statview Animals. Adult mice (C57Bl/6x129Sv/Ev mixed genetic background or pure (Calabasa, CA) software. 129Sv/Ev background) with targeted knockout of the Npy gene (Npy–/–) and wild-type littermate controls (Npyϩ/ϩ) were housed in individual cages in standard vivarium conditions (12 h:12 h light:dark cycle). Standard rodent RESULTS chow (Harlan Teklad Rodent Diet, Purina) was provided ad libitum, except where specified. Free access to drinking water was provided to all animals at Experiment 1: STZ-induced diabetes. Plasma glucose all times. All study protocols were approved by the University of Washington values (Fig. 1A) obtained at the end of the study (day 18) Institutional Animal Care and Use Committee. were significantly and comparably elevated in both geno- Experiment 1: STZ-induced diabetes. Baseline measurements of 24-h food types of STZ-treated mice (Npyϩ/ϩ 332 Ϯ 25; Npy–/– 285 Ϯ intake, water intake, and body weight were obtained for 2 days before 20 mg/dl; P Յ 0.05 vs. respective vehicle-treated controls; administration of STZ (200 mg/kg i.p. in 0.5 mmol/l citrate buffer, pH 4.5) to mice of each genotype (C57Bl/6x129Sv/Ev mixed genetic background) to NS between the two diabetic groups) relative to nondia- ϩ/ϩ –/– create uncontrolled diabetes (n ϭ 6 per genotype). Others of each genotype betic controls of each genotype (Npy 105 Ϯ 6; Npy received injection of the citrate buffer only and remained nondiabetic (n ϭ 7 107 Ϯ 6 mg/dl). Plasma leptin levels did not differ accord- per genotype). Food intake, water intake, and body weight were measured ing to genotype among vehicle-treated animals (Npyϩ/ϩ daily until 18 days postinjection, when retro-orbital blood samples were 2.9 Ϯ 0.4; Npy–/– 4.1 Ϯ 0.8 ng/ml; NS) and were signifi- obtained for plasma glucose and leptin levels between noon and 2:00 P.M. Animals were subsequently killed by exposure to carbon dioxide, and the cantly and comparably decreased in the two diabetic ϩ/ϩ Ϫ/Ϫ brains were removed, frozen on dry ice, and stored at Ϫ70°C for determina- groups (Npy 2.0 Ϯ 0.2; Npy 2.0 Ϯ 0.2 ng/ml; P Յ 0.05 tion of neuropeptide mRNA levels by in situ hybridization. vs. respective vehicle groups) (Fig. 1B). Experiment 2: food deprivation. The effect of a 48-h fast on hypothalamic ϩ/ϩ –/– Baseline (preinjection) 24-h food intake (Fig. 2) in neuropeptide gene expression was determined in male Npy and Npy ϩ/ϩ Ϯ mice (129Sv/Ev background) weighing ϳ25 g. At the onset of the light cycle vehicle-treated Npy mice was 4.1 0.3 g/day, and this (8:00 a.m.), food was removed from a subset of these mice (n ϭ 12 per value did not change over the course of the study, averag- genotype), whereas others were allowed free access to chow (n ϭ 8 per ing 4.0 Ϯ 0.2 g/day for the last 7 days of the experiment. genotype). Forty-eight hours after food removal, animals were killed by CO 2 Baseline 24-h food intake was 3.5 Ϯ 0.2 g/day in vehicle- inhalation and brains were removed for analysis of neuropeptide mRNA. The –/– Ϯ Յ feeding response to food deprivation was determined in a separate group of treated Npy mice and increased to 4.3 0.2 g/day (P ϩ ϩ female Npy / and Npy–/– mice. Food was removed at 8:00 A.M. in a subset of 0.05 vs. baseline food intake) for the last 7 days of the mice of both genotypes and returned 48 h later, with chow intake measured at experiment. After STZ injection, food intake declined 4 h and 24 h thereafter. Food intake was also measured in separate groups of transiently in Npyϩ/ϩ mice before returning to baseline female mice of both genotypes provided with continuous access to chow. levels on day 4 (4.1 Ϯ 0.2 g/day) and increased subse- In situ hybridization. Brains were sectioned in the coronal plane (14 ␮m) ϳ Ϯ Յ with a cryostat, mounted on RNase-free slides, and treated with 4% parafor- quently to values 50% over baseline (to 6.2 0.5 g; P maldehyde, acetic anhydride, ethanol, and chloroform (15). Brain sections 0.05) for the last 7 days of the study. NPY-deficient mice (6–10 per probe) were selected from the mid- to rostral region of the arcuate also experienced a transient decline in food intake from nucleus for hybridization to POMC, NPY, or AgRP mRNA. The anatomical baseline values (3.8 Ϯ 0.5 g) following STZ injection, but equivalence of hypothalamic sections among animals was confirmed by an –/– individual blinded to the study conditions selecting slides (viewed with a unlike wild-type mice, food intake of Npy mice did not dark-field microscope) with the aid of a mouse brain atlas. For each probe, all increase significantly above baseline levels over the re- slides were concurrently prepared for hybridization and were hybridized in mainder of the experiment (4.8 Ϯ 0.3 g/day, days 11–18; NS

DIABETES, VOL. 51, MARCH 2002 779 DIABETIC HYPERPHAGIA AND NPY DEFICIENCY

(FIG. 2. Change in daily food intake in Npy–/– (KO) and Npy؉/؉ (WT mice. Change of daily food intake during baseline (days 0–1) and after injection (days 1–18) of either vehicle or STZ in Npy–/– and Npy؉/؉ mice of experiment 1. Baseline food intake: Npy؉/؉ mice, 4.1 ؎ 0.3 g/day ;(WT-V); 4.1 ؎ 0.2 g/day (WT-D); Npy–/– mice, 3.5 ؎ 0.2 g/day (KO-V) .g/day (KO-D). Data points are means ؎ SE. *P < 0.05 vs 0.5 ؎ 3.8 baseline.

By in situ hybridization, NPY mRNA levels measured in the ARC of Npyϩ/ϩ mice receiving STZ were 230 Ϯ 50% higher than in vehicle-treated controls (P Ͻ 0.05) (Fig. 4A). As expected, NPY mRNA was not detected in Npy–/– mice.

FIG. 1. Plasma glucose and leptin levels in Npy–/– (KO) and Npy؉/؉ (WT) mice. Plasma levels of glucose (A) and leptin (B) from vehicle- per genotype) or STZ-diabetic (WT-D 7 ؍ treated (WT-V and KO-V; n per genotype) mice obtained on day 18 of experiment 6 ؍ and KO-D; n 1. *P < 0.05 vs. vehicle-treated controls. vs. baseline). By two-way ANOVA, the change of food intake induced by diabetes differed significantly according to genotype (P Յ 0.05) and was significantly increased only in Npyϩ/ϩ mice (Figs. 2 and 3). Diabetes was also associated with a marked increase of daily water intake in Npyϩ/ϩ mice, from a baseline of 7.1 Ϯ 0.5 ml/day to mean values of 30.9 Ϯ 1.6 ml/day (P Յ 0.05) over the last 7 days of the study. In STZ-treated Npy–/– mice, daily water intake also increased, but to a lesser degree (to 14.4 Ϯ 0.9 ml; P Յ 0.05 vs. all other groups). Among vehicle-treated nondiabetic animals, body weight increased in both genotypes by 0.2–0.9 g over the course of the experiment, whereas it decreased gradually in both diabetic groups, and by day 18, there was no significant effect of genotype on weight loss in STZ-treated mice (Fig. 3). A similar pattern was revealed by MRS- derived measures of relative body fat content. Whereas body fat mass was comparable among vehicle-treated Npyϩ/ϩ and Npy–/– mice, fat content of diabetic Npyϩ/ϩ mice was 44 Ϯ 6% (P Յ 0.05) of vehicle-treated Npyϩ/ϩ values. Similarly, body fat mass of STZ-treated Npy–/– mice was 60 Ϯ 8% (P Յ 0.05) of vehicle-treated Npyϩ/ϩ mice. Like the decreases of body weight, the effect of diabetes FIG. 3. Effect of STZ-diabetes on food intake, body weight, and body fat –/–content by genotype. Data (means ؎ SE) were obtained from Npy on relative fat content did not differ significantly by and Npy؉/؉ mice on day 18 of experiment 1 and are expressed as a genotype (Fig. 3). percent of nondiabetic controls of the same genotype.

780 DIABETES, VOL. 51, MARCH 2002 D.K. SINDELAR AND ASSOCIATES

the response detected in Npy–/– mice, with AgRP mRNA levels being increased by 83 Ϯ 43% over values obtained in vehicle-treated Npy–/– mice. There was no significant effect of genotype on AgRP mRNA levels in either diabetic or vehicle-treated groups. By comparison, POMC mRNA lev- els (Fig. 4C) were reduced in the rostral ARC of diabetic Npyϩ/ϩ mice by 65 Ϯ 7% (P Յ 0.05 vs. vehicle-treated controls) but fell by only 13 Ϯ 1.6% in the ARC of diabetic Npy–/– mice (NS vs. vehicle-treated Npy–/– mice; P Յ 0.05 vs. diabetic Npyϩ/ϩ mice). Among vehicle-treated animals, POMC mRNA levels were not significantly altered by NPY deficiency. Experiment 2: responses to fasting. Consistent with a previous report in male mice (6), food intake increased significantly and similarly in response to a 48-h fast in female mice of both genotypes (Table 1). Four hours after the return of food, intake was increased by 80–120% in both Npyϩ/ϩ and Npy–/– mice compared with controls fed ad libitum, and at 24 h of refeeding, intake was increased by ϳ60% in both genotypes. No difference by genotype was detected in the effect of fasting on food intake (Table 1). In a separate group of Npyϩ/ϩ mice, fasting reduced POMC mRNA levels in the rostral ARC by 52% (P Ͻ 0.05 vs. Npyϩ/ϩ mice fed ad libitum) (Table 1). Hypothalamic POMC mRNA levels were also significantly reduced by fasting in Npy–/– mice, and although the magnitude of this effect (Ϫ33%) was below that detected in wild-type mice, mean levels of POMC mRNA in fasted Npyϩ/ϩ and Npy–/– mice were not significantly different from one another.

DISCUSSION To investigate the specific contribution of NPY to the pathogenesis of diabetic hyperphagia, we compared food intake of NPY-deficient and wild-type mice after inducing diabetes with STZ. Whereas food intake increased by 50% within 2 weeks of the onset of diabetes in Npyϩ/ϩ mice, hyperphagia was not detected in diabetic mice that lack NPY. Reduced feeding in response to uncontrolled diabe- tes in Npy–/– mice cannot be attributed to differences in the severity of diabetes, since levels of plasma glucose, plasma leptin, body weight, and relative fat content were FIG. 4. Hypothalamic levels of mRNA encoding NPY (A), AgRP (B), and comparable in the two diabetic groups. Combined with POMC (C) measured by in situ hybridization in the ARC of Npy–/– and ؉/؉ our finding that STZ-diabetes induces increased NPY Npy mice of experiment 1. Values are expressed as a percent of ϩ/ϩ vehicle-treated Npy؉/؉ mice. *P < 0.05 vs. (vehicle-treated) Npy؉/؉ mRNA levels in the ARC of Npy mice, consistent with mice. several earlier reports in both and mice (7–13), these results support the conclusion that increased hypotha- AgRP mRNA expression (Fig. 4B) was also increased in lamic NPY signaling is required for the development of the ARC of diabetic Npyϩ/ϩ mice (by 54 Ϯ 29% relative to diabetic hyperphagia. nondiabetic wild-type mice; P Ͻ 0.05) and was similar to The attenuation of diabetic hyperphagia in Npy–/– mice

TABLE 1 Food intake and hypothalamic POMC and AgRP mRNA levels in response to fasting in Npyϩ/ϩ and NpyϪ/Ϫ mice Food intake (g) Hypothalamic mRNA level Genotype and condition 4 h 24 h POMC AgRP* Npyϩ/ϩ Fed 0.8 Ϯ 0.1 3.5 Ϯ 0.1 100 Ϯ 11% (nϭ 8) 100 Ϯ 40% (nϭ 8) Fasted 1.4 Ϯ 0.1† 5.1 Ϯ 0.9† 48 Ϯ 6%† (nϭ12) 1,908 Ϯ 320%†(nϭ12) NpyϪ/Ϫ Fed 0.6 Ϯ 0.1 3.7 Ϯ 0.2 102 Ϯ 12% (nϭ 7) 122 Ϯ 8.5% (nϭ 8) Fasted 1.4 Ϯ 0.1† 5.6 Ϯ 0.3† 67 Ϯ 8%† (nϭ12) 2,237 Ϯ 301% (nϭ12) Date are means Ϯ SE.*Previously published (14). †pϽ0.05 vs. fed.

DIABETES, VOL. 51, MARCH 2002 781 DIABETIC HYPERPHAGIA AND NPY DEFICIENCY contrasts sharply with the hyperphagic response to fast- cantly reduced in NPY-deficient mice with STZ-induced ing, which is completely intact in mice lacking NPY. Our diabetes. Failure of NPY-deficient mice to suppress mela- current findings of comparable refeeding hyperphagia in nocortin signaling may therefore have contributed to the Npyϩ/ϩ and Npy–/– mice replicate those published previ- attenuation of their hyperphagic response. Moreover, this ously (1) and support the conclusion that NPY is not finding suggests that NPY is required for the effect of required for this response. This finding is at odds with a uncontrolled diabetes to reduce hypothalamic POMC gene recent report showing impaired refeeding responses after expression. Together with evidence that POMC expression fasting in Npy–/– mice (18). The basis for this discrepancy in ARC neurons is, in general, reduced in conditions that is unclear, but our efforts to document abnormal levels of activate ARC NPY neurons (e.g., fasting, uncontrolled intake in Npy–/– mice 24 h after fasting have been consis- diabetes, and genetic leptin deficiency), our results sug- tently unsuccessful. Differences in housing conditions of gest that activation of NPY neurons contributes to the mice before study is a potential factor, since mice in our inhibition of ARC POMC neurons in these conditions. This studies were housed individually before and during all possibility is supported by several findings. Both NPY Y1 experimental interventions, whereas mice from the study and Y5 receptor subtypes are present in the ARC (23,24), by Bannon et al. (18) were group-housed before study. chronic central NPY administration reduces hypothalamic Because Npy–/– mice can have heightened responses POMC mRNA levels in vivo (25), and NPY neurons exert to novel stimuli (1), this difference in housing may have inhibitory effects on the firing of ARC POMC neurons in influenced intake during refeeding. Another possibility is vitro (26). that differences in background strain of mice used in the In contrast to the effect of NPY, leptin administration different laboratories contributed to differences in their increases ARC POMC gene expression in vivo (16) and feeding response. increases electrical activity of these neurons in vitro (26). Our observation of intact refeeding after a 48-h fast in These effects are likely mediated, at least in part, via direct Npy–/– mice, therefore, suggests that factors that stimulate stimulatory effects of leptin, since a majority of these food intake after fasting must differ from those involved in neurons express the signaling form of the diabetic hyperphagia. That plasma glucose levels are in- (OB-Rb) (27,28). Thus, conditions of negative energy bal- creased in diabetes and reduced by fasting raises the ance such as fasting can inhibit POMC neurons in the ARC possibility that differences in CNS glucose delivery con- via at least two mechanisms: decreased input from leptin tributed to differences in feeding in response to the two and activation of adjacent NPY neurons (a response also conditions in NPY-deficient mice. By comparison, both triggered in part by leptin deficiency). In the current fasting and uncontrolled diabetes lower circulating insulin studies, we found that although the effect of fasting to and leptin levels and induce comparable increases of lower ARC POMC mRNA levels was less pronounced in hypothalamic NPY gene expression. Because reduced Npy–/– than in Npyϩ/ϩ mice, these differences did not melanocortin signaling is implicated in the hyperphagic achieve statistical significance. Thus, unlike the situation response to both fasting and diabetes, and because mela- with uncontrolled diabetes, NPY is not required for the nocortin-producing neurons are sensitve to input from effect of a 48-h fast to lower hypothalamic POMC gene NPY, we determined the effect of these two conditions on expression, although it may contribute to this response. hypothalamic expression of POMC and AgRP mRNA in NPY-independent mechanisms may therefore play a more wild-type and Npy–/– mice. Although neuropeptide mRNA important role to inhibit POMC neurons during fasting levels are an indirect measure of the activity of hypotha- than they do in STZ-deficient diabetes, and additional lamic neurons, their use in assessing the response to studies are required to identify these factors. Fasting- hormonal and metabolic stimuli is supported by a large induced decreases of leptin signaling are unlikely to literature (5,7–13,15,16). explain reduced expression of POMC mRNA in this set- The hypothalamic melanocortin system is made up of ting, since fasting and STZ-diabetes lower leptin levels neurons that synthesize melanocortin receptor (Mcr) ago- comparably. nists, such as ␣-melanocyte–stimulating hormone In data from a previously published study, fasting in- (␣-MSH), a product of POMC, and those that produce the duced marked (ϳ20-fold) increases of AgRP mRNA in the Mcr antagonist, AgRP. Both POMC- and AgRP-containing ARC of both Npyϩ/ϩ and Npy–/– mice (Table 1 and cell bodies are located in the ARC, with AgRP being reference 14). Thus, NPY deficiency did not significantly co-localized with NPY in this brain area. Since Mc4r alter the effect of either diabetes or fasting on hypotha- antagonists stimulate feeding (19,20), and since genetic lamic AgRP mRNA levels, but the increase of hypotha- deletion of either POMC or Mc4r causes hyperphagia and lamic AgRP gene expression induced by fasting is much in mice (21,22), defective CNS melanocortin sig- greater that that induced by STZ-diabetes in both geno- naling is a potent stimulus to increase food intake. Evi- types. Although AgRP is co-localized with NPY in ARC dence that STZ-diabetes increases AgRP and decreases neurons (5,14,29), our results suggest that NPY is not POMC mRNA in rodent suggests that re- required for the effect of fasting or diabetes to induce duced Mcr signaling might, along with increased NPY AgRP gene expression. However, the relatively modest signaling, contribute to diabetic hyperphagia (12,13). Our effect of diabetes to increase of hypothalamic AgRP ex- current finding that POMC mRNA levels were markedly pression (50–80%) was evidently inadequate to increase reduced by uncontrolled diabetes in the ARC of Npyϩ/ϩ food intake in NPY-deficient mice. Because the AgRP mice is consistent with this hypothesis and is in agreement response to uncontrolled diabetes was small in compari- with data obtained in diabetic rats (12). son to that induced by fasting, it can be concluded that By comparison, POMC mRNA levels were not signifi- NPY-deficient mice respond to fasting with a much greater

782 DIABETES, VOL. 51, MARCH 2002 D.K. SINDELAR AND ASSOCIATES increase of AgRP and a greater decrease of POMC gene basal hypothalamus-preoptic area of streptozotocin-diabetic rats with and expression in the ARC. These observations suggest that without insulin substitution therapy. Endocrinology 126:192–198, 1990 fasting inhibited CNS melanocortin signaling to a greater 10. McKibbin PE, McCarthy HD, Shaw P, Williams G: Insulin deficiency is a –/– specific stimulus to hypothalamic neuropeptide Y: a comparison of the extent than did STZ diabetes in Npy mice, which pro- effects of insulin replacement and food restriction in streptozotocin- vides a plausible explanation for the intact hyperphagic diabetic rats. Peptides 13:721–727, 1992 response to fasting in these animals. This hypothesis is 11. Mizuno TM, Makimura H, Silverstein J, Roberts JL, Lopingco T, Mobbs CV: strengthened by evidence that the orexigenic response to Fasting regulates hypothalamic neuropeptide Y, agouti-related peptide, AgRP is heightened in Npy–/– mice (14). and in diabetic mice independent of changes in leptin or insulin. Endocrinology 140:4551–4557, 1999 In conclusion, our results support a major role for NPY 12. Havel PJ, Hahn TM, Sindelar DK, Baskin DG, Dallman MF, Weigle DS, in the stimulation of food intake triggered by uncontrolled Schwartz MW: Effects of streptozotocin-induced diabetes and insulin diabetes. Unlike wild-type mice, diabetic mice lacking NPY treatment on the hypothalamic melanocortin system and muscle uncou- also fail to suppress hypothalamic expression of POMC pling protein 3 expression in rats. Diabetes 49:244–252, 2000 mRNA. Combined with evidence that diabetes-associated 13. Qu S, Yang YK, Li J, Zeng Q, Gantz I: Agouti-related protein is a mediator of diabetic hyperphagia. Reg Peptides 98:69–75, 2001 increases of AgRP mRNA in the ARC are much smaller 14. Marsh DJ, Miura GI, Yagaloff KA, Schwartz MW, Barsh GS, Palmiter R: than those induced by fasting, our results support a model Effects of neuropeptide Y deficiency on hypothalamic agouti-related in which failure to suppress hypothalamic melanocortin protein expression and responsiveness to melanocortin analogues. Brain signaling contributes to the attenuation of diabetic hy- Res 848:66–77, 1999 perphagia in mice lacking NPY. By comparison, food 15. Schwartz MW, Seeley RJ, Weigle DS, Woods SC, Campfield LA, Burn P, deprivation appears to inhibit hypothalamic melanocortin Baskin DG: Leptin increases proopimelanocortin (POMC) mRNA expres- –/– sion in the rostral arcuate nucleus. Diabetes 46:2119–2123, 1997 signaling more effectively than STZ-diabetes in Npy 16. Schwartz MW, Baskin DG, Bukowski TR, Kuijper JL, Foster D, Lasser G, mice, a mechanism that may help explain why these Prunkard DE, Porte D Jr, Woods SC, Seeley RJ, Weigle DS: Specificity of animals exhibit intact hyperphagia during refeeding but leptin action on elevated blood glucose levels and hypothalamic neuropep- not in response to uncontrolled diabetes. tide Y gene expression in ob/ob mice. Diabetes 45:531–535, 1996 17. Mystkowski P, Shankland E, Scheyer SA, LeBoeuf RC, Schwartz RS, Cummings DE, Kushmerick M, Schwartz MW: Validation of whole-body ACKNOWLEDGMENTS magnetic resonance spectroscopy as a tool to assess murine body com- This work was supported by National Institutes of Health position. Int J Obes Relat Metab Disord 24:719–724, 2000 (NIH) Grants DK 52989, NS 32273, and DK12829 (M.W.S.). 18. Bannon AW, Seda J, Carmouche M, Francis JM, Norman MH, Karbon B, D.K.S. is supported by National Research Service Award McCaleb ML: Behavioral characterization of neuropeptide Y knockout mice. Brain Res 868:79–87, 2000 (NRSA) fellowship DK 09932-02 from the NIH. MRS anal- 19. Fan W, Boston B, Keterson R, Hruby V, Cone R: Role of melanocortinergic yses were performed by the Body Composition Core neurons in feeding and the agouti obesity syndrome. Nature 385:165–168, Laboratory of the Clinical Nutrition Research Unit in the 1997 laboratory of Dr. Martin Kushmeric. 20. Grill HJ, Ginsberg AB, Seeley RJ, Kaplan JM: Brainstem application of The authors recognize the excellent technical help re- melanocortin receptor ligands produces long-lasting effects on feeding and body weight. J Neurosci 18:10128–10135, 1998 ceived from Ruth Hollingworth, Vicki Hoagland, Jira 21. 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