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Insulin has been postulated to be a signal that provides negativefeedback to the brain for the long-term regulation of energy bal-ance1–3. According to the ‘lipostatic model’ of the regulation ofenergy balance4, peripheral signals proportional to the size ofenergy stores communicate energy status to brain centers involvedin the regulation of food intake and fuel metabolism4–7. Leptinand insulin are ideal candidates for this lipostatic function,because their levels are closely related to adiposity7,8 and theircentral administration decreases food intake1,7,9,10. Indeed,insulin receptors are expressed in several sites within thebrain11,12, including the medial (containing neuropeptide Y(NPY)-expressing neurons) and lateral (containing pro-opiomelanocortin (POMC)-expressing neurons) portions of thearcuate nucleus of the hypothalamus12. Studies in which insulinis administered into the third cerebral ventricle (ICV) are amongthe strongest evidence to date in support of insulin’s anorecticrole in the central nervous system1,10. These studies showed adecrease in food intake and fat mass after prolonged ICV insulininfusions1,10 and a decrease in the hypothalamic expression ofNPY, a potent orexogenic peptide, after acute ICV injection ofinsulin or insulin mimetics10,13,14. Notably, ICV injections ofinsulin specifically decrease the expression of NPY in the medialportion of the arcuate nucleus12,13,15. However, insulin receptorsare also present in other brain regions, where insulin may haveimportant roles in neuronal growth16, differentiation17 and func-tion18,19. Recent loss-of-function studies have provided strongsupport for a physiological role of brain insulin receptors in thelong-term modulation of energy balance, showing that neuron-

Decreasing hypothalamic insulinreceptors causes hyperphagia andinsulin resistance in rats

Silvana Obici1, Zhaohui Feng1, George Karkanias1, Denis G. Baskin2 and Luciano Rossetti1

1 Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, USA2 VA Puget Sound Health Care System and University of Washington, 1660 S. Columbian Way, Seattle, Washington 98108, USA

Correspondence should be addressed to L.R. (rossetti@aecom.yu.edu)

Published online: 20 May 2002, DOI: 10.1038/nn861

We investigated the role of hypothalamic insulin signaling in the regulation of energy balance andinsulin action in rats through selective decreases in insulin receptor expression in discrete hypothala-mic nuclei. We generated an antisense oligodeoxynucleotide directed against the insulin receptorprecursor protein and administered this directly into the third cerebral ventricle. Immunostaining ofrat brains after 7-day administration of the oligodeoxynucleotide showed a selective decrease ofinsulin receptor protein within cells in the medial portion of the arcuate nucleus (decreased by ~80%as compared to rats treated with a control oligodeoxynucleotide). Insulin receptors in otherhypothalamic and extra-hypothalamic areas were not affected. This selective decrease in hypothala-mic insulin receptor protein was accompanied by rapid onset of hyperphagia and increased fat mass.During insulin-clamp studies, physiological hyperinsulinemia decreased glucose production by 55%in rats treated with control oligodeoxynucleotides but by only 25% in rats treated with insulinreceptor antisense oligodeoxynucleotides. Thus, insulin receptors in discrete areas of the hypothala-mus have a physiological role in the control of food intake, fat mass and hepatic action of insulin.

specific disruption of the insulin receptor (IR) gene throughoutthe central nervous system leads to increases in body fat and inplasma insulin and leptin levels20. The importance of local pop-ulations of neurons expressing insulin receptors in mediating theCNS effects of insulin on energy homeostasis remained to bedelineated, however. To address this question, we generated aselective, transient defect in hypothalamic insulin signaling byinfusion of an antisense oligodeoxynucleotide designed to bluntthe expression of the IR protein in rat hypothalamic areas sur-rounding the third cerebral ventricle (Fig. 1). We found markedand selective decreases in insulin receptor expression in discretehypothalamic nuclei. We then examined the effects on brain IRprotein, food intake, fat mass and insulin action. This rat modelallowed us to assess the possible role of hypothalamic insulin sig-naling in the regulation of energy balance and insulin action.

RESULTSICV IR antisense decreased IR protein in hypothalamusWe administered IR antisense (IR-AS) and control (scrambled; IR-SCR) oligodeoxynucleotides (ODNs) by ICV infusion in the thirdcerebral ventricle, and assessed the effects on IR proteins by brainimmunostaining and immunoblotting with anti-IR antibodies.After 7 days of infusion (Fig. 1), we saw a selective downregulationof IR protein in the medial portion of the arcuate nucleus (∼ 80%lower in IR antisense–treated as compared to control mice; Fig. 2a,b and Table 1) and in the adjacent habenular nucleus (Fig. 2i, j andTable 1). This is consistent with the location of these two regionsclose to the third cerebral ventricle and with their high insulin recep-

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firmed that copy number was not affected by IR antisense treatment(Fig. 3d). To rule out the presence of antisense-generated stablemRNA fragments, we quantified the IR mRNA with two separatesets of primers that either spanned the sequence of mRNA targetedby the antisense ODN (Fig. 3d, ‘upstream primers’) or were locatedfurther downstream of the start codon (Fig. 3d, ‘downstreamprimers’). The results obtained with both sets of primers confirmedthat the IR mRNA levels were not changed by IR antisense treat-ment. These findings support the conclusion that the downregula-tion of IR protein in selective arcuate neurons occurs largely by amechanism of ‘hybrid-arrested’ translation and does not involveribonuclease H–dependent degradation of mRNA21.

AGRP and NPY expression in arcuateTo determine whether an attenuation of insulinaction in the hypothalamus affects the expressionof neuropeptides involved in energy balance, weexamined the expression of NPY, agouti-relatedprotein (Agrp) and POMC in the arcuate nuclei ofthe rats treated with IR antisense and control ODNby real-time PCR (Fig. 3e). IR antisense treatment

Fig. 2. Representative images of brain insulin-receptorimmunostaining. Digitized images were produced by confo-cal fluorescence microscopy of immunocytochemical stain-ing for insulin receptors in brains of rats that received thirdventricular infusions of oligonucleotides for 7 d. First andthird columns (a, c, e, g, i, k, m, o; first and third columns),images from rats that received control (scrambled) oligonu-cleotides (IR-SCR); second and fourth columns (b, d, f, h, j,l, n, p), images from rats that received oligonucleotides com-plementary to IR mRNA (IR-AS). Neuronal cell bodies areshown in red pseudocolor (arrows in the first panel), repre-senting fluorescence of the Cy3-labeled secondary antibod-ies used for detection of antibody binding. The abundance ofneurons expressing immunocytochemically detectableinsulin receptors was visually lower in the medial region ofthe arcuate nucleus (ARCm) adjacent to the third ventricle(V) in rats that received IR-AS ODN (b). This effect was notseen in rat that received the control ODN (a) or in the ven-trolateral region of the arcuate nucleus (ARCv) (compare c,d). The abundance of IR-immunoreactive neurons after IRantisense treatment was also lower in the periventricularnucleus (PeVN) (compare e, f) and habenula (HAB) (com-pare i, j). No effect of IR antisense on IR immunostaining wasdetected in the ventromedial hypothalamic nucleus (VMN)(g, h) and dorsomedial hypothalamic nucleus (DMN) (k, l)or in pyramidal cells of the pyriform cortex (PCX) (m, n)and region CA1 of the hippocampus (CA1) (o, p). Scale bar,100 µm (applies to all panels).

Fig. 1. Schematic representation of the experimental procedures. (a) Surgical implantation of ICV cannulae was performed on day 1 (∼ 3weeks before the in vivo study). Body weight and food intake recoveredfully by day 7. Intravenous (i.v.) catheters and osmotic minipumps (forICV infusions of ODNs) were surgically implanted on day 14; ICV infu-sions were continued for 7 d; and body composition and insulin actionwere estimated on day 21. (b) A bolus of tritiated water was given i.v. 3 h before the start of tritiated glucose infusion. At t = 0, a primed-con-tinuous infusion of labeled glucose was initiated and maintained for theremainder of the 4 h study. Pancreatic-insulin clamp study was initiatedat t = 120 min and lasted 120 min. SRIF, somatostatin.

tor densities. In contrast, other hypothalamic areas, the olfactorycortex and the hippocampus were not affected (Table 1 and Fig. 2g,h, k–p). We confirmed the selective inhibition of IR protein in thearcuate nucleus by immunoblot analysis of hypothalamic nucleiisolated by the micro-punch technique. Arcuate nuclei of rats treat-ed with IR antisense ODN for 3 or 7 days contained ∼ 46% ± 9 and∼ 53% ± 13, respectively, of IR protein as compared to controls (Fig.3b lanes 5–8 and Fig. 3c). In contrast, and in agreement with theimmunostaining results, the immunoblot of the lateral hypothal-amic area (LHA) and paraventricular nucleus (PVN) did not showany significant change in IR protein levels (Fig. 3b lanes 1–4).

We further characterized the mode of action of the IR antisenseODN on IR protein through real-time PCR. The IR antisense ODNwas designed to target the translation initiation site of the IR mRNA(Fig. 3a). We assessed the IR mRNA copy number in hypothalamicnuclei isolated by micro-punches through real-time PCR, and con-

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for 3 days increased the prevalence of NPY (by 53% ± 13 versusSCR) and AGRP (by 57% ± 10 versus SCR) transcripts, but didnot affect the expression of POMC. Thus, the selective down-regulation of IR protein in the medial portion of the arcuatenucleus specifically increases the levels of orexogenic peptidesNPY and AGRP.

Hypothalamic insulin, body fat and food intakeBody composition and fat distribution have been assessed by

Fig. 3. Effects of IR antisense in discrete nuclei of thehypothalamus. IR antisense ODN (IR-AS) attenuatesinsulin receptor translation only in the arcuate nucleus,but does not affect its mRNA levels. (a) Schematic illus-tration of the IR mRNA. AUG, putative initiation oftranslation of the IR precursor protein at position +1;UAA, putative stop codon (as predicted by the IRreceptor sequence deposited with GenBank; seeMethods); AS, sequence of the antisense ODNhybridizing to the area spanning the start of translation.Upstream and downstream primers were used to quan-tify IR mRNA expression by RT-PCR. (b) Immunoblotof distinct hypothalamic nuclei after treatment with IR-AS. Lanes 1, 3, 5, 7, IR-SCR treatment; lanes 2, 4, 6, 8,IR-AS treatment. Lanes 1 and 2 are from LHA and lanes3 and 4 from PVN. Lanes 5–8 are from arcuate nucleiafter 3 d (5 and 6) or 7 d (7 and 8), respectively, of anti-sense treatment. (c) Quantification of immunoblots bydensitometry. IR protein levels of arcuate nuclei after 3or 7 d of antisense treatment (�, IR-AS) or controlODN (�, IR-SCR); P < 0.04. (d) IR mRNA quantifica-tion by RT-PCR in arcuate nuclei or LHA of rats treatedwith control (�, IR-SCR) or antisense ODN (�, IR-AS). (e) Expression of NPY, AGRP and POMC in arcu-ate nuclei after control (�, IR-SCR) or antisense ODNtreatment (�, IR-AS); P < 0.01.

tracer experiments and post-mortem dissection of fatdepots22,23. The selective decreasein hypothalamic IR protein in ratsreceiving IR antisense ODN result-ed in rapid onset of hyperphagia(average food intake 25.1 ± 1.2 ver-sus 16.2 ± 1.3 g/day; P < 0.01) andmarkedly increased fat mass (Fig. 4and Table 2). In contrast, the feed-ing behavior of rats treated ICVwith control ODN was quite simi-lar to that of rats treated ICV withvehicle (ref. 24 and data notshown). Notably, this occurreddespite a 65% increase in plasmaleptin levels (Table 2). Plasma lep-tin concentrations were alsoincreased by ∼ 50% in rats treatedICV with IR antisense ODN foronly 3 days (data not shown). Leanbody mass was not affected, and theincrease in fat mass was largelyaccounted for by a marked increasein subcutaneous adipose tissue (Fig. 4); intra-abdominal fat wasincreased only modestly (4.3 ± 0.5versus 3.7 ± 0.4 g; P = 0.09).

Hypothalamic and hepatic insulin action To assess the impact of hypothalamic insulin receptor downreg-ulation on the action of insulin, we performed insulin-clamp (3 mU/kg min) studies in conscious rats23–26. As a result of phys-iological increases in plasma insulin concentrations (to ∼ 40µU/ml), the rate of glucose infusion required to maintain theplasma glucose at basal levels was lower in IR antisense–treated(8.7 ± 1.6) than in control (14.9 ± 1.4 mg/kg min) rats (Fig. 5).Insulin action on glucose metabolism includes stimulation of

Table 1. Immunostaining of brain regions from rats infused ICV with IR antisense (IR-AS) or scrambled (IR-SCR) oligodeoxynucleotides.

Rat ARCmed ARClat PeVN VMH DMH HAB CA1 OCX CPIIIa

IR-AS1 6 30 2 25 9 6 35 41 0.33 7 26 7 28 17 17 38 46 0.36 4 21 4 21 5 5 38 37 0.37 4 17 5 17 13 2 36 0.0Mean 5.3 23.5 4.5 22.8 11.0 7.5 37.0 40.0 0.2s.e.m. 0.6 2.5 0.9 2.1 2.2 2.8 0.7 2.0 0.1IR-SCR2 17 21 9 18 11 36 37 45 2.64 25 21 12 22 14 43 32 45 3.05 24 24 6 26 10 22 32 46 2.38 23 16 11 25 16 34 25 41 2.6Mean 22.3 20.5 9.5 22.8 12.8 33.8 31.5 44.3 2.6s.e.m. 1.6 1.4 1.1 1.6 1.2 3.8 2.1 1.0 0.1t-test P = 0.001 0.405 0.027 1.000 0.578 0.004 0.109 0.162 0.0001aData for choroid plexus represents a staining intensity score, on a 4-level scale from 0 (no staining) to 3(most intense staining seen).

ARCmed, arcuate nucleus medial portion, adjacent to third ventricle, region of NPY neurons; ARClat, ventrolateralarcuate nucleus, away from ventricle and towards the region of predominately POMC neurons; PeVN, periventricu-lar nucleus near the VMH, cells near third ventricle; VMH, main portion of ventromedial hypothalamus; DMH, mainportion of dorsomedial nucleus; HAB, habenular nucleus, adjacent to third ventricle at top of third ventricle cleft;CA1, region of hippocampus with pyramidal cells; OCX, olfactory (pyriform) cortex; CPIII, choroid plexus in thethird ventricle. ICV IR-AS significantly decreased IR protein in ARCmed, PeVN, HAB and CPIII.

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glucose uptake and inhibition of glucose production. Insulinaction on peripheral glucose uptake was similar in IR anti-sense–treated and control rats (19.1 ± 2.0 versus 20.6 ± 1.2 mg/kgmin; Fig. 5 and Table 2). Basal rates of glucose production in thetwo groups were also similar. Physiological increases in plasmainsulin concentration markedly inhibited (by ∼ 55%, to 5.6 ± 0.6mg/kg min) the rate of glucose production in the control rats. Incontrast, glucose production was decreased by only 25% (to 9.6± 0.6 mg/kg min) in IR antisense–treated rats. Thus, hepaticinsulin action was markedly impaired after selective attenuationof hypothalamic insulin receptor expression.

DISCUSSIONInsulin receptors are expressed in several regions of the centralnervous system11,12. Initial analyses have identified pleiotropicfunctions of brain insulin receptors10,16–19, andexperimental evidence supports an important roleof central insulin receptors in the regulation of ener-gy homeostasis1–3,10,13,20. The recent observation thatmice with neuron-specific disruption of the IR genethroughout the brain have increased fat mass andinsulin levels as adult20 demonstrates that insulinreceptors within the CNS have a physiological role

Fig. 5. Selective attenuation of hypothalamic insulin recep-tors impairs hepatic insulin action. The effects of 7-day ICVadministration of IR antisense (�, IR-AS) and IR control(scrambled; �, IR-SCR) ODNs on glucose kinetics duringthe pancreatic-insulin clamp studies are displayed.Treatment with IR-AS resulted in lower rates of glucoseinfusion (P < 0.01; a) and greater endogenous glucose pro-duction (P < 0.01; b) as compared to treatment with IR-SCR. The percentage inhibition of glucose production inresponse to hyperinsulinemia was markedly blunted afterICV IR-AS administration (c), whereas the rate of glucosedisappearance was not significantly affected (d).

in the regulation of energy homeostasis. However, the effects ofinsulin on neuronal growth and differentiation16,17 suggest thataltered brain insulin signaling during development may accountfor some of insulin’s biological consequences. Furthermore, therole of insulin receptors in specific brain regions has been difficultto examine. We describe here a new approach to this question.Central infusion of antisense ODN directed against the insulinreceptor generated a rapid and site-specific decrease in the expres-sion of insulin receptors. By coupling stereotaxic delivery withbrain immunostaining and immunoblotting, we documentedthat infusion of IR antisense ODN into the third cerebral ventri-cle led to a marked decrease in IR protein in nuclei directly adja-cent to the third ventricle, while insulin receptor expression inother brain areas was not affected. This is a useful experimentalmodel that may make it possible to explore additional effects ofhypothalamic insulin receptors on reproductive function20,counter-regulation22 and insulin secretion23. We confirmed theresults of the immunohistochemistry studies with an indepen-dent quantification technique that combines micro-punching ofselective hypothalamic nuclei with immunoblotting. Isolatedarcuate nuclei treated with IR-antisense ODN show ∼ 50% lessIR protein after either 3 or 7 days of IR antisense treatment. Thisdecrease in IR protein is consistent with the results we obtainedby quantification of immunostaining, in which the proportionof cells staining positive for IR protein was markedly decreased(by ∼ 80%) in the medial portion but not in the lateral aspect ofthe arcuate nucleus.

The medial aspect of the arcuate nucleus of the hypothala-mus showed ∼ 80% decrease in the number of neurons that con-tained immunoreactive insulin receptors, as detected byimmunostaining. This region is enriched in neurons containingthe orexogenic peptide NPY, whose expression is decreased aftersystemic or central administration of insulin10,13,14. Furthermore,

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these NPY-containing neurons co-express a natural antagonistof the central melanocortin receptors, Agrp, which is also an orex-ogenic peptide. The expression of both peptides is increased by∼ 50% in the arcuate nuclei of IR antisense–treated rats. Thismoderate increase occurred despite concomitant elevations infood intake and leptin levels. These findings suggest that insulinsignaling is required to restrain the expression of these orexo-genic peptides in the arcuate nucleus. The rapid hyperphagiceffect seen here validates an important physiological role ofinsulin receptors within the medial aspect of the arcuate nucleusin the regulation of food intake and body adiposity. Because thesurvival surgery and general anesthesia required for implanta-tion of the indwelling catheters and osmotic minipumps induceda brief period of fasting, in the present study we examined theimpact of hypothalamic insulin signaling during early re-feeding. The pronounced effect of hypothalamic insulin recep-tor status in this phase seems to indicate that under normal cir-cumstances the increase in plasma insulin levels during re-feedingpartially restrains food intake. This observation is also consistentwith the postulated role of NPY in mediating the hyperphagicresponse to fasting3,13,14.

Hyperphagia was associated with a marked increase in sub-cutaneous adipose tissue that entirely accounted for the increasedbody weight. The preferential increase in fat mass over lean bodymass suggests a role of hypothalamic insulin receptors in regu-lating energy storage. Notably, these effects on food intake andbody composition occurred in the presence of moderate increas-es in the plasma leptin concentration. Although leptin and insulinsignaling may share common downstream targets (such as phos-phoinositide-3-kinase)27 in some hypothalamic neurons, it isalso possible that the increase in food intake and fat mass resultsin enhanced hypothalamic leptin signaling. This in turn maypartly antagonize the deficient insulin signaling and may stimu-late the melanocortin pathway, perhaps through increased expres-sion of POMC. The latter effect may account for the lack ofincrease in visceral fat mass24 and for the normalization of foodintake on day 5.

Selective decrease in hypothalamic insulin receptors alsoresults in the rapid onset of hepatic insulin resistance. In fact,the rate of hepatic glucose production was markedly increased inthe presence of equal plasma insulin concentrations. This maybe partly due to the moderate increase in whole body adiposityand/or to the transient increase in nutrient intake. In previousstudies, however, we have not found a significant effect of sim-ilar changes in caloric intake and fat mass per se on hepaticinsulin action28. We have recently reported that bidirectionalchanges in the activity of the melanocortin pathway in the hypo-thalamus rapidly regulate fat distribution and insulin action24. Inparticular, antagonism of hypothalamic melanocortin receptorsinduces hyperphagia, weight gain, selective increase in visceraladiposity and insulin resistance. Here, antagonism of hypothal-amic insulin signaling also resulted in hyperphagia, weight gainand insulin resistance. Notably, the induction of hypothalamicinsulin resistance did not selectively increase visceral adiposity.Taken together, these recent studies support the notion that com-mon hypothalamic pathways are involved in the regulation ofenergy homeostasis and insulin action. Because glucose pro-duction by the liver is the major source of endogenous fuel, it isparticularly intriguing that two central neural circuitries involvedin the regulation of exogenous fuel (food intake) concomitant-ly modulate the rate of endogenous glucose production as well.Although the effect of central melanocortin receptors on glu-cose production are probably due to their potent effects on vis-

ceral adiposity, the effect of hypothalamic insulin receptors seemsto be independent of this parameter.

Understanding the mechanism(s) responsible for suscepti-bility to weight gain and insulin resistance in response toincreased caloric intake is of vital importance to the fight againstprevalent metabolic diseases such as obesity and type 2 diabetesmellitus. The observation that a selective decrease in insulinreceptor expression in a discrete region of the hypothalamus issufficient to rapidly induce several key features of these meta-bolic syndromes indicates that selective hypothalamic insulinresistance may be involved in the pathophysiology of obesity andinsulin resistance. In conclusion, we show that a selective andpartial decrease in IR protein in arcuate neurons containing NPYand Agrp is sufficient to rapidly and markedly impair energy bal-ance and insulin action. This observation suggests that hypo-thalamic insulin resistance may have a role in the susceptibilityto obesity and diabetes mellitus.

METHODSDesign of oligodeoxynucleotide (ODN) antisense against IR mRNA.The antisense ODN (IR-AS) was designed to hybridize to the sequencespanning the initiation of translation of the IR precursor mRNA (Fig. 3a). The sequence from IR-AS ODN (5′-CGGAGCCCATAGCAG-3′) was scrambled to obtain a control ODN, IR-SCR (5′-CACACGAGC-CGTAGG-3′). The ODNs were synthesized by Operon Technologies(Alameda, California). All ODNs contained a phosphothiorate bondbetween nucleotides 1 and 2 and between nucleotides 14 and 15.

Primers and real-time PCR. Total RNA was obtained from individual ratsby combining left and right homologous nuclei (that is, arcuate nucleusand PVN). Four rats were analyzed per experimental group. IR mRNAwas quantified using two sets of primers (Fig. 3a). The upstream set ampli-fies a fragment of 191 nucleotides beginning at the start codon of IRmRNA (forward primer (F), 5′-CCTACTGCTATGGGCTCCG-3′; reverseprimer (R), 5′-AGGATCTGCAGATGGCCCTC-3′). The downstream setamplifies a fragment of 491 nucleotides beginning at codon 276 of the IRmRNA (F, 5′-CAGGACTGGCGCTGTGTAAAC-3′; R, 5′-CACAGCTGC-CTCAGGTTCTG-3′). Hypothalamic neuropeptide expression was car-ried out beginning with real-time PCR using the following primers: ratNPY (F 5′-GCCATGATGCTAGGTAACAAACG-3′, R 5′-GTTTCATTTC-CCATCACCACATG-3′); rat POMC (F 5′-CCAGGCAACGGAGATGAAC-

Table 2. Effect of selective decrease of hypothalamicinsulin receptors on food intake, body composition andblood chemistry.

IR-SCR IR-AS P valueBasalCumulative food intake(kcal/5 d) 291 ± 33 443 ± 66* 0.0007BW (g) 297.9 ± 8.5 319.3 ± 19 0.3∆BW (g) –15.3 ± 5.9 +9.6 ± 2.9* 0.004FM (g) 40.3 ± 5.5 75.1 ± 15.0* 0.007FFM (g) 257.7 ± 6.1 244.3 ± 8.4 0.3Leptin (ng/ml) 0.9 ± 0.1 1.5 ± 0.1* 0.02Insulin (µU/ml) 0.75 ± 0.2 1.72 ± 0.5 0.1Glucose (mM) 8.0 ± 0.4 7.8 ± 0.2 0.2GP (mg/kg min) 12.3 ± 0.5 12. 6 ± 0.8 0.9

Insulin clampedGlucose (mM) 7.5 ± 0.3 7.9 ± 0.2 0.7Insulin (µU/ml) 38.6 ± 4 .6 36 ± 7.2 0.8

*, P < 0.01. BW, body weight; FM, fat mass; FFM, fat-free mass; GP, endoge-nous glucose production.

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3′, R 5′-TCACTGGCCCTTCTTGTGC-3′); rat AGRP (F 5′-GCCATGCT-GACTGCAATGTT-3′, R 5′-TGGCTAGGTGCGACTACAGA-3′); and β-actin (F 5′-TGAGACCTTCAACACCCCAGCC-3′ , R 5′-GAGTACTTGCGCTCAGGAGGAG-3′). Total RNA was isolated with Trizol (Invitro-gen, Carlsbad, California) and single-strand cDNA was synthesized withSuperscript (Invitrogen). Real-time PCR reactions were prepared with aLightCycler reaction kit (Roche, Indianapolis, Indiana). A real-time PCRreaction of 20 µl contained 200 nM primers, 1× reaction buffer, 2.3 mMMgCl2, 2 µl SYBR Green, 2 µl cDNA and 2 units Taq DNA polymerase.The reactions were carried out in capillaries in a LightCycler instrument(Roche) and were cycled ∼ 40 times.

Induction of selective attenuation of hypothalamic insulin receptorexpression. We studied 32 10-week male Sprague-Dawley rats (CharlesRiver Breeding Laboratories, Wilmington, Massachusetts). Three weeksbefore brain harvesting or in vivo studies, we placed a chronic catheterin the third cerebral ventricle by stereotaxic surgery25. One week beforeall experimental protocols were performed, we placed additionalcatheters in the right internal jugular and left carotid artery24–26,28–32

and connected osmotic minipumps (Alzet, Palo Alto, California) tothe indwelling catheter placed in the third cerebral ventricle (ICV).The minipump delivered a constant infusion of 0.5 µl/h for 7 d. Foodintake was monitored daily. Rats were randomized into two experi-mental groups. Each group of rats received 3 or 7 d of infusion of eitherIR-AS ODN or IR-SCR (control). All ODNs were purified by HPLCand dissolved in artificial cerebrospinal fluid at a concentration of 2 mM. The solutions were infused ICV for 3 or 7 d with osmoticminipumps at a rate of 1 nmol/h.

Immunostaining of rat brains. Immunostaining was performed after 7 dof administration of either IR-AS or IR-SCR ODNs. Rat brains were per-fused in situ through a cardiac cannula with 100 ml of 4% paraformalde-hyde in 0.1 M sodium phosphate buffer (PB), pH. 7.2. After removalfrom the cranium, the brains were immersed in PB containing 25%sucrose for 48 h, then frozen and sectioned at 14 µm with a cryostat.Slide-mounted coronal sections of the hypothalamus were collectedbetween stereotaxic levels –2.2 and –4.0 mm caudal to bregma33, whichincluded the region targeted by the injector cannula tip.

Immunocytochemistry. Slides containing sections of hypothalamus at thelevel of the cannula tip, as well as up to 1 mm anterior and posterior to it,were immunostained by standard procedures34. Briefly, slides wereimmersed 1 h at 20°C in 0.01 M PB containing 5% normal goat serum and1% BSA, followed by anti-IR primary antiserum diluted 1:1,000 in 0.1 MPB with 1% BSA overnight at 6°C. The rabbit polyclonal primary anti-serum (provided by J.N. Livingston, Bayer Corp.) was generated againstthe C-terminal 14-amino-acid sequence of the human IR β-subunit. The specificity of the antiserum for rat IR was verified by immuno-precipitation and western immunoblots. The antiserum recognizes bandsof appropriate molecular size for IR β-subunits respectively in immunoblotsof rat liver and hypothalamus homogenates separated by SDS-PAGE. Theprotein immunoprecipitated from brain homogenates and detected by theantiserum migrated on the gel slightly more quickly than that from liver,consistent with the smaller molecular size of this subunit in brain tissue.In addition, hepatic portal vein injection of insulin resulted in the tyro-sine phosphorylation of the protein immunoprecipitated by this antiserum,consistent with the known behavior of the IR β-subunit tyrosine kinase(data not shown). After incubation with primary antiserum, sections werewashed in PBS and immersed 1 h at 20°C in goat anti-rabbit IgG conju-gated with Cy3 (Jackson ImmunoResearch Laboratories, West Grove, Penn-sylvania) following standard protocols34. Controls included samples with(i) primary antibody omitted, (ii) secondary antibody omitted and (iii)normal rabbit serum (diluted 1:1,000) substituted for the rabbit anti-insulinantibody35. Immunofluorescence was absent in all controls.

Microscopy and analysis. Images of immunostained brain sections (Fig. 2) were captured with a Leica (Solm, Germany) TCS-SP ConfocalLaser Scanning Microscope. Images were scanned with a 40× objectivewith a krypton laser at 568 nm, using a 150-µm pinhole and an emis-

sion filter setting of 582–625 nm. Each field recorded was scanned as aseries of 10 z planes that were separated by 1 µm for a total thickness of10 µm. The final image at each z plane was frame-averaged three timesand all 10 z planes were projected into a single image for analysis andrecording as tiff files. All images were captured using the same pinhole,laser intensity and photomultiplier tube sensitivity settings.

Images for quantification (Table 1) were captured with a Hamamatsu(Bridgewater, New Jersey) cooled CCD camera and the MCID imageanalysis program (Imaging Research, St. Catharines, Ontario, Canada).The data shown in Table 1 for all brain areas (except choroid plexus) rep-resent mean counts of IR-immunopositive neurons on three sections ofeach brain region, measured in 400× fields. The data from the three slideswere averaged to obtain a mean value per rat (n = 4 per group). For thearcuate nucleus, ‘medial’ images and counts were obtained adjacent tothe third ventricle, whereas the ‘lateral’ images and counts were made inthe ventrolateral region of the arcuate nucleus.

Brain stereotaxic micro-punches of individual hypothalamic nuclei.Brain micro-punches of individual hypothalamic nuclei were preparedby a modification of a method previously described36. Briefly, rats werekilled by decapitation and the brains rapidly removed and frozen inisopentane on dry ice at –15°C for 5 min. The brain were then implant-ed frozen to a pedestal, placed in the cryostat and maintained at –15°C.Brain sections 500 µm thick were made and mounted on glass slides.Using anatomical landmarks from a rat brain stereotaxic atlas33, indi-vidual hypothalamic nuclei were identified and punched out with a stain-less steel needle under a stereomicroscope. The brain punch was expelledwith a stainless steel insert into an Eppendorf tube on dry ice and thenstored at –80°C until assayed. The reproducibility of micro-punches wasestablished by thawing the sections and inspecting the topography of theholes by transillumination under a low-power light microscope. Addi-tionally, the complete removal of arcuate nuclei was validated by mea-suring arcuate-specific genes (POMC and AGRP) in punches immediatelylateral to the arcuate nuclei. In the latter samples, these transcripts wereundetectable by highly sensitive real-time PCR.

Western blot analysis. Protein analysis was performed on individual ratsby combining left and right homologous nuclei (that is, arcuate nucleusand PVN). Each sample was mixed with 100 µl of Laemmli buffer (50 mM Tris-HCl, pH 6.8, 100 mM dithiothreitol, 2% SDS, 0.1% bro-mophenol blue, 10% glycerol) and immediately boiled for 10 min. Thesamples were vortexed and centrifuged for 10 min at 4°C and 5,000 g, andthe supernatants were fractionated on 10% SDS-PAGE and blotted ontonitrocellulose. Filters were incubated with antibodies against the C terminalof the IR β-subunit (Transduction, San Diego, California) and against glyc-eraldehyde phosphate dehydrogenase (anti-GAPDH, a gift of E.R. Stanley).

Body composition. On day 8 after the start of ICV infusions, we initiatedin vivo studies in conscious rats fasted for ∼ 6 h24–26,28–30. To ensure a sim-ilar postabsorptive state, on the night preceding the clamp study weassigned a fixed allotment of chow (∼ 12 g) to all rats.

We administered an intra-arterial bolus injection of 20 µCi of tritiat-ed water (3H2O; New England Nuclear, Boston) to the rats, and obtainedplasma samples at 30 min intervals for 3 h26,28. Steady-state conditions forplasma 3H2O specific activity were achieved within ∼ 40 min in all stud-ies. Five plasma samples obtained between 1 and 3 h after the injectionwere used in the calculation of the whole-body distribution space ofwater. This was obtained by dividing the total radioactivity injected (ind.p.m.) by the steady-state specific activity of plasma water (in d.p.m./ml).Plasma was assumed to be 93% water. Fat-free mass was calculated asthe whole-body water distribution space divided by 0.73, and fat massas the difference of body weight and fat-free mass. Epididymal, peri-renaland omental fat depots were dissected and weighed at the end of eachexperiment. Visceral adiposity was calculated as the sum of epididymal,peri-renal and omental fat depots and subcutaneous adiposity as the dif-ference between total fat mass and visceral adiposity.

Measurements of in vivo glucose kinetics. Briefly, a primed-continuousinfusion of HPLC-purified [γ-3H]glucose (New England Nuclear, Boston;40 µCi bolus, 0.4 µCi/min) was administered for the duration of the

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572 nature neuroscience • volume 5 no 6 • june 2002

articles

study24–26,28–30. Two hours after the basal period, a primed-continuousinfusion of somatostatin (1.8 µg/kg min) and regular insulin (3 mU/kgmin) were administered, and a variable infusion of a 25% glucose solu-tion was started at time zero and periodically adjusted to clamp the plasmaglucose concentration at ∼ 7.5 mM for the rest of the study. Samples fordetermination of [3H]glucose specific activity were obtained at 10-minintervals throughout the infusions.

All values are presented as the mean ± s.e.m. Comparisons amonggroups were made by unpaired Student’s t-test. The study protocol wasreviewed and approved by the Institutional Animal Care and Use Com-mittee of the Albert Einstein College of Medicine.

GenBank accession number. Insulin receptor, 204953.

AcknowledgmentsWe thank J.N. Livingston for the gift of the IR antiserum and J. Murphy for

performing the immunostaining. This work was supported by grants from the

National Institutes of Health to L.R. (DK 48321 and DK 45024), from the AECOM

Diabetes Research & Training Center (DK 20541), the Union of Washington

Diabetes Endocrinology Research Center (DK 17047) and the Career Scientist and

Merit Review Research Programs of the Department of Veteran Affairs. S.O.

received a postdoctoral fellowship from the American Diabetes Association.

Competing interests statementThe authors declare that they have no competing financial interests.

RECEIVED 25 MARCH; ACCEPTED 23 APRIL 2002

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