molecular disruption of hypothalamic nutrient sensing induces obesity

Molecular disruption of hypothalamic nutrient sensinginduces obesity

Wu He, Tony K T Lam, Silvana Obici & Luciano Rossetti

The sensing of circulating nutrients within the mediobasal hypothalamus may be critical for energy homeostasis. To induce

a sustained impairment in hypothalamic nutrient sensing, adeno-associated viruses (AAV) expressing malonyl–coenzyme

A decarboxylase (MCD; an enzyme involved in the degradation of malonyl coenzyme A) were injected bilaterally into the

mediobasal hypothalamus of rats. MCD overexpression led to decreased abundance of long-chain fatty acyl–coenzyme

A in the mediobasal hypothalamus and blunted the hypothalamic responses to increased lipid availability. The enhanced

expression of MCD within this hypothalamic region induced a rapid increase in food intake and progressive weight gain.

Obesity was sustained for at least 4 months and occurred despite increased plasma concentrations of leptin and insulin.

These findings indicate that nutritional modulation of the hypothalamic abundance of malonyl–coenzyme A is required to

restrain food intake and that a primary impairment in this central nutrient-sensing pathway is sufficient to disrupt energy

homeostasis and induce obesity.

The hypothalamus of mammals has developed sensing mechanismsto monitor the body’s nutritional status as a means to initiatecogent behavioral and metabolic responses1–8. Both circulating nutri-ents and hormones have important roles in this adaptation1,5,9–11.Pharmacological and molecular manipulations of hypothalamicnutrient metabolism decrease appetite and body weight6–8 and alsoinhibit liver glucose production7. In various tissues, the cellularabundance of malonyl–coenzyme A (CoA) functions as a sensor fornutrient availability12,13 (Fig. 1a) and has recently been postulated tohave a critical role in the hypothalamic sensing of nutrients14,15. Indeed,hypothalamic malonyl-CoA is markedly induced by feeding andsuppressed by fasting, suggesting its potential role in modulatingfeeding behavior14.

Changes in energy balance result in changes in the circulating levelsof key hormones (for example, leptin and insulin) that in turn signalthe body’s nutritional status to the hypothalamus2,16. The criticalnature of a handful of these hypothalamic homeostatic circuits hasbeen firmly established by the demonstration that their disruption issufficient to induce obesity17–21. For example, a lack of leptin signalingis sufficient to induce severe obesity in rodents17–19 and humans22,23.However, it is currently unknown whether a sustained impairment inhypothalamic nutrient sensing can also lead to obesity. Therefore, wetested whether the sensing of circulating nutrients within the medio-basal hypothalamus (MBH) is critical for the maintenance of energyhomeostasis. We found that a chronic decrease of malonyl-CoA in theMBH induced severe defects in hypothalamic nutrient sensing andin the regulation of liver glucose fluxes and led to hyperphagia andobesity in rats.

RESULTS

AAV-mediated overexpression of MCD in the MBH

To determine whether the overexpression of the malonyl-CoA–degrading enzyme MCD within the MBH is sufficient to alter energyand glucose homeostasis, we constructed the vector pMCD-AAV forexpression of full-length MCD-Flag fusion protein and green fluor-escent protein (GFP) under the control of the cytomegalovirus (CMV)promoter (Fig. 1b). We verified MCD-Flag and GFP expression by real-time polymerase chain reaction (RT-PCR) (Fig. 1c) and by restrictionenzyme digestion (Supplementary Fig. 1 online). The transfection ofthis plasmid into HEK293 cells resulted in the efficient expression ofa functional MCD-Flag fusion protein (Fig. 1d and SupplementaryFig. 1). The recombinant pMCD-AAV plasmid was transfectedtogether with pHelper and pAAV–replication and capsid (pAAV-RC)into HEK293 cells to produce recombinant adeno-associated virus(MCD-AAV). The purified replication-incompetent AAVs were firsttested in HEK293 cells. Cells expressed MCD-Flag protein in both thecytosol and the mitochondria 2 d after viral exposure (Fig. 1e). We theninjected the purified AAVs bilaterally within the MBH of 10- to 12-week-old male Sprague-Dawley rats (Fig. 2a). The hypothalamicexpression of exogenous proteins was demonstrated by the westernblotting of MBH extracts (Fig. 2b) and by immunofluorescence withan antibody to GFP (anti-GFP) (Fig. 2c) 2 weeks after viral injection.

Expression of MCD in the MBH impairs glucose homeostasis

The increased expression of MCD would be expected to reduce thechanges in the hypothalamic abundance of malonyl-CoA that occurin response to dynamic changes in nutritional status. Indeed, the

Received 14 September 2005; accepted 8 December 2005; published online 15 January 2006; doi:10.1038/nn1626

Departments of Medicine and Molecular Pharmacology, Diabetes Research Center, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461,USA. Correspondence should be addressed to L.R. ([email protected]).

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abundance of malonyl-CoA in the MBH was B75% lower in ratstreated with MCD-AAV than in those treated with GFP-AAV (data notshown). Furthermore, because malonyl-CoA is a potent inhibitor ofcarnitine palmitoyltransferase-1 (CPT1; refs. 12 and 24), the rate-limiting enzyme for the entry of long-chain fatty acyl–CoAs (LCFA-CoAs) in the mitochondrion, we postulated that decreasing malonyl-CoA within the MBH could also attenuate the biochemical responses toa sustained elevation in lipid availability (Fig. 3a). To formally test thishypothesis, we generated a physiological increase in the abundance ofcirculating lipid while experimentally controlling the abundance ofglucose and glucoregulatory hormones (Fig. 3a and SupplementaryTable 1 online). Doubling the plasma concentrations of fatty acids(Fig. 3b) markedly increased the abundance of LCFA-CoAs in theMBH of rats injected with GFP-AAV, but not those injected with MCD-AAV (Fig. 3c). These experiments provide strong evidence for thebiochemical impact of the Mlycd transgene, which encodes MCD, onthe in vivo sensing of circulating lipids within the MBH. The hypothal-amic sensing of circulating lipids is required for the maintenance ofglucose homeostasis9. Thus, we next examined whether the increasedMCD activity in the MBH modified whole-body and hepatic glucosefluxes in the presence of increased amounts of lipid (Fig. 3a). Duringinsulin-clamp experiments and lipid infusions, the rate of glucoseinfusion required to maintain steady-state glucose levels was markedlylower in MCD-AAV rats than in GFP-AAV rats (Fig. 3d). Thus, thedepletion of malonyl- and LCFA-CoAs within the MBH was sufficientto induce insulin resistance in the presence of hyperlipidemia.

Expression of MCD in the MBH stimulates liver glucose fluxes

The decreased rate of glucose infusion during the insulin clamp couldbe due to decreased glucose use, increased glucose production or both.Glucose production was increased by 35% in MCD-AAV rats comparedwith GFP-AAV rats, whereas the rate of glucose use was unaffected(Fig. 3e). Similarly, the suppression of glucose production during theinsulin-clamp experiments was markedly impaired in the presence ofMCD overexpression in the MBH (Fig. 3f). Thus, increased lipidavailability rapidly induced hepatic insulin resistance when hypotha-lamic malonyl- and LCFA-CoA were experimentally decreased. Nota-bly, the overexpression of MCD and the pharmacological inhibition ofLCFA esterification within the MBH (ref. 9) had a very similar impacton LCFA-CoA abundance in the MBH and on liver glucose metabolismin the presence of increased amounts of lipid.

Expression of MCD in the MBH alters energy homeostasis

Having demonstrated that the concentration of malonyl- and LCFA-CoAs within the MBH is critical for the hypothalamic sensing ofcirculating lipids and for liver glucose homeostasis, we next postulatedthat MBH malonyl- and LCFA-CoAs also have a major role in theregulation of energy homeostasis. To address this question, we estab-lished the average growth rate and daily food intake in a cohort of malerats (Fig. 4a) and then injected one subgroup of rats with MCD-AAVand another with GFP-AAV in the MBH. The injection of GFP-AAV didnot modify the daily weight gain compared to baseline (Fig. 4b). Incontrast, the growth curve of rats injected with MCD-AAV began todiverge from that of the GFP-AAV rats during the first 10 d after MBHinjection (Fig. 4a,b). Notably, this increase in weight gain was sustainedfor several weeks, and marked differences in body weight persisted forat least 4 months after the MBH viral injections (Fig. 4a). Consistent

Figure 1 Overexpression of MCD in HEK293

cells. (a) A hypothesis for the role of malonyl-CoA

in the hypothalamic regulation of energy and

glucose homeostasis. LCFAs are esterified to

LCFA-CoAs by acyl-CoA synthase (ACS) before

they are metabolized by cells. The metabolism of

glucose and other nutrients leads to increased

formation of malonyl-CoA via carboxylation ofacetyl-CoA by the enzyme ACC. Malonyl-CoA in

turn inhibits the CPT1-dependent oxidation of

LCFA-CoA. We proposed that the hypothalamic

malonyl- and LCFA-CoA system is a biochemical

sensor for the availability of multiple nutrients

and that changes in their cellular levels parallel

changes in the body’s nutritional status and lead

to rapid modulation of food intake and glucose

production. (b) Construction of the pMCD-AAV

plasmid (see Methods). (c) Verification of MCD-Flag expression by RT-PCR. (d) Immunofluorescence imaging of the reporter gene GFP expression in the

HEK293 cells transfected with the pMCD-AAV plasmid. Transfection with the pMCD-AAV plasmid led to overexpression of a functional MCD-Flag fusion

protein with cellular MCD enzymatic activity. (e) MCD-AAV transfection in HEK293 cells led to MCD-Flag protein overexpression in both the cytosolic and

mitochondrial compartments. VDAC1 protein expression was used as a mitochondrial marker.

Glucose &other nutrients

LCFAACS

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Figure 2 Hypothalamic (MBH) overexpression of malonyl-CoA decarboxylase

(MCD). (a) Schematic of the experimental design. Surgical implantation of

bilateral MBH cannulae was performed B14 d before in vivo infusion

experiments and/or tissue sampling. Rats were implanted with indwelling

catheters in the jugular vein and carotid artery 1 week later. The rats in these

experiments ate a similar amount of chow and received lipid infusions and

pancreatic insulin clamps 1 week later. Immunofluorescence and western

blots were performed on brain sections sampled at the completion of these

experiments. (b) Representative western blots of MBH obtained from four rats

injected with MCD-AAV and four rats injected with GFP-AAV. GFP proteinexpression was documented in all samples. However, MCD-AAV, but not

GFP-AAV, led to MBH expression of MCD-Flag and to increased levels of

MCD. (c) Representative immunofluorescence with anti-GFP antibody

showing GFP expression within the mediobasal hypothalamus. Green,

GFP; blue, 4,6-diamidino-2-phenylindole (DAPI) nuclear staining.

228 VOLUME 9 [ NUMBER 2 [ FEBRUARY 2006 NATURE NEUROSCIENCE

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with its effect on body weight, the overexpression of MCD in the MBHof MCD-AAV rats also led to a 65% increase in the weight of theepididimal fat pads (5.6 ± 0.9 g) compared with rats injected with theGFP-AAV (3.4 ± 0.6 g; P o 0.05).

Because the first measurable changes in energy balance were detectedwithin days of the viral injection, we next focused our analyses on thechanges in energy homeostasis occurring during the first 14 d after theinjections of the AAVs into the MBH. In these groups of rats, thepreinjection daily food intake and body-weight gain (average of

2 weeks before MBH injections) were well matched across the twogroups (Fig. 4b,c). However, the injection of MCD-AAV into the MBHled to a significant increase (Po 0.05) in the daily gain in body weightthroughout the second week after injection (Fig. 4b). In contrast,weight gain was not affected by the injection of GFP-AAV into theMBH. The progressive weight gain in the rats overexpressing MCD inthe MBH was associated with a 25% increase in average daily foodintake (Fig. 4c). To further assess the time course of the effects of MCDoverexpression in the MBH on daily food intake, we measured daily

Mediobasalhypothalamus

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Figure 3 MCD overexpression in the MBH disrupts central lipid sensing and liver glucose homeostasis. (a) Schematic of the hypothesis and experimental

design. A primary increase in circulating LCFA led to increased levels of LCFA-CoAs in the liver and to the stimulation of hepatic gluconeogenesis, but also

to increased levels of hypothalamic LCFA-CoAs and to decreased hepatic glucose fluxes. We postulated that the overexpression of MCD within the MBH

increased the activity of CPT1 and decreased levels of LCFA-CoA by decreasing hypothalamic malonyl-CoA even in the presence of circulating hyperlipidemia.

Furthermore, we hypothesized that the lack of increase in the MBH levels of LCFA-CoAs leads to altered glucose homeostasis in response to increased lipid

availability. A scheme of the modified pancreatic clamp protocol is also shown. SRIF, somatostatin. (b) Lipid infusion increased plasma fatty acids similarly in

MCD-AAV and GFP-AAV rats. (c) MCD-AAV resulted in decreased basal abundance of LCFA-CoAs in the hypothalamus and in a complete lack of increase in

LCFA-CoAs, oleyl-CoAs and palmitoyl-CoAs in response to a doubling in the circulating levels of LCFAs. (d) Rate of glucose infusion during lipid infusion and

pancreatic clamp. (e) Glucose production and glucose use during lipid infusion and pancreatic clamp. (f) Inhibition of glucose production from basal valuesduring lipid infusion and pancreatic clamp. In the absence of lipid infusion (saline controls; not shown), glucose kinetics were similar in the two groups.

*P o 0.05, MCD-AAV (n ¼ 6) versus GFP-AAV (n ¼ 6); �P o 0.05, saline versus lipid infusion. Values are mean ± s.e.m.

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Figure 4 MCD overexpression in the MBH disrupts energy homeostasis. (a) Growth curves in rats before and after the MBH injections of MCD-AAV (n ¼ 11) or

GFP-AAV (n ¼ 11). (b) Time course of the changes in body weight and average daily changes in body weight before and during the first 2 weeks after the MBH

injections of the AAVs (MCD-AAV, n ¼ 11, filled squares; GFP-AAV, n ¼ 11, open squares). (c) Time course of the changes in food intake during the first 2

weeks following the MBH injection of AAVs and average daily changes in food intake during the week before and during the second week after MBH injections

of AAVs (MCD-AAV, n ¼ 6, black bars; GFP-AAV, n ¼ 6, white bars).


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food intake in additional rats receiving either GFP-AAV or MCD-AAVinjections in the MBH. Daily food intake was quite stable and wassimilar in the two groups before the injection of the viruses. However, amarked increase in daily food intake began on day 4 in rats receivingMCD-AAV, whereas daily food intake was unaffected by the MBHinjection of GFP-AAV (Fig. 4c). In general, the onset of hyperphagia inthe MCD-AAV groups preceded and then closely paralleled the initialincrease in weight gain (Fig. 4b,c) and seemed to largely account for theonset of obesity in this model.

Expression of MCD in the MBH alters body composition

We next examined the effect of the overexpression of MCD in the MBHon body composition as assessed by the labeled-water dilution techni-que. Rats were injected with either the GFP- or MCD-AAV 2 weeksbefore the experiment. At the time of the experiment, hyperphagia wasdocumented in MCD-AAV rats for B10 d. As shown (above) in olderrats, the disruption of hypothalamic nutrient sensing led to an B60%increase in the weight of the epididimal fat pads, paralleled by anB50% increase in whole-body fat mass (Fig. 5a). There was also atrend toward increased fat-free mass (Fig. 5a; P ¼ 0.06) as well asincreased body length (8.9 ± 0.1 inches versus 8.6 ± 0.1 inches; P ¼0.09) in MCD-AAV rats compared with GFP-AAV rats. To examinewhether early changes in oxygen consumption, substrate oxidationand/or physical activity also contributed to the rapid changes in energybalance, we measured gas exchanges and movements for a 24-h periodbefore and then during the second week after the viral injections. Atbaseline, oxygen consumption, respiratory quotient and movementswere similar in the two experimental groups(Fig. 5b,c). The overexpression of MCD orGFP in the MBH did not elicit significantchanges in these parameters, though therewas a trend toward decreased oxygen con-sumption (Fig. 5b) and increased movements(Fig. 5c) in the MCD-AAV group. Togetherwith the marked impact of the hypothalamicoverexpression of MCD on feeding behavior(Fig. 4c), these results indicate that the rapidweight gain induced by MBH depletion ofmalonyl-CoA is largely due to hyperphagiarather than to decreases in movements oroxygen consumption.

The expression of MCD in the MBHimpairs leptin action. Because the rapidincrease in food intake and body weight inrats receiving the MBH injection of MCD-AAV was associated with a progressive increasein plasma leptin levels (Supplementary Table2 online), we postulated that the anorecticeffect of leptin would be diminished in theserats. To test this hypothesis, we administeredrecombinant leptin (3 mg) or vehicle (artificialcerebrospinal fluid, ACSF) in the MBH

and then monitored food intake during the following 12 h (Fig. 6a).In rats receiving the GFP-AAV, leptin markedly inhibited food intake(B50%), whereas the vehicle did not have any significant effect (B9%;P = 0.1). In contrast, in rats overexpressing MCD in the MBH, leptindid not decrease food intake significantly more than the vehicle did(B15% versus B9%; P = 0.21). We next examined whether the severeimpairment in the action of leptin on feeding behavior was due todefective leptin signaling through the signal transducer and activator oftranscription 3 (STAT3). Leptin induced STAT3 phosphorylationsimilarly in the MBH of rats overexpressing MCD or GFP (Fig. 6b).Our experimental model supports the idea that the malonyl- and

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Figure 6 Altered regulation of food intake in rats with MCD overexpression in the MBH. (a) MBH

overexpression of MCD blunted the anorectic effect of leptin. MBH injection of recombinant leptin

markedly decreased food intake in rats injected with GFP-AAV (n ¼ 6), but not in rats injected with

MCD-AAV (n ¼ 8). (b) MBH leptin equally increased STAT3 phosphorylation in both MCD-AAV– and

GFP-AAV–injected rats. (c) MBH overexpression of MCD increased the expression of AgRP and NPY

in the MBH. *P o 0.01, MCD-AAV versus GFP-AAV. Values are mean ± s.e.m.

Figure 5 Effect of MCD-AAV on body composition and energy metabolism.

(a) A 2-week injection of MCD-AAV (n ¼ 6) versus GFP-AAV (n ¼ 5) in the

MBH increased fat pad and fat mass (FFM), but only slightly increased fat-

free mass (FM) (P ¼ 0.06). (b) Oxygen consumption (VO2) and respiratory

quotient (RQ) in rats before and after the MBH injections of MCD-AAV

(n ¼ 4) or GFP-AAV (n ¼ 4). (c) Analysis of movements during a 24-h

period before and after the MBH injections of AAVs. Gray, MCD-AAV; black,

GFP-AAV. *P o 0.001, MCD-AAV versus GFP-AAV. Values are mean ± s.e.m.


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LCFA-CoA biochemical sensor within the MBH is important in theregulation of feeding behavior. To begin investigating the mechanismsresponsible for the onset of hyperphagia in rats receiving MCD-AAV inthe MBH, we analyzed the gene expression of key MBH neuropeptides.Quantitative analyses by RT-PCR revealed a marked increase in agouti-related protein (AgRP) and neuropeptide Y (NPY) mRNA in thearcuate nuclei of rats treated with MCD-AAV in the MBH comparedwith those treated with GFP-AAV (Fig. 6c). The expression of pro-opiomelanocortin (POMC) in the MBH was not significantly alteredby the administration of MCD-AAV (Fig. 6c). The effects of MCDoverexpression on the expression of MBH orexigenic neuropeptides isparticularly noteworthy because it occurred in the presence of hyper-phagia, hyperleptinemia and increased body weight.

Fasting and MBH expression of MCD decrease LCFA-CoAs

Notably, the induction of AgRP and NPY expression in rats injectedwith MCD-AAV was paralleled by marked decreases in LCFA-CoAswithin the arcuate nuclei (Fig 7a). To examine whether this hypo-thalamic nutrient-sensing pathway is modulated under physiologicalconditions, we analyzed the effect of fasting on the amount of LCFA-CoAs in the arcuate nuclei (Fig. 7b). Consistent with a previous reporton the regulation of malonyl-CoA levels in the hypothalamus14, theamount of LCFA-CoA in the arcuate nuclei of the hypothalamus alsodecreased with fasting (Fig. 7b). Thus, changes in the rat’s nutritionalstatus resulted in congruent changes in the hypothalamic abundance ofmalonyl-CoA and LCFA-CoA, extending their well-established bio-chemical link at this anatomical site and supporting their participationin the sensing of nutrients within the hypothalamus.

DISCUSSION

The hypothalamus can gather information on the body’s nutritionalstatus by integrating multiple signals1–8. These include potent hormo-nal signals such as leptin17, but also direct metabolic signals6,7,9,25.Understanding the biochemical steps involved in hypothalamic nutri-ent sensing is a difficult and important task. The malonyl- and LCFA-CoA signaling pathway is ideally positioned to integrate informationon the cellular availability of multiple nutrients12,15,24. Consistent withthis notion, the increased cellular levels of malonyl- and LCFA-CoAwithin the MBH may be signal of nutrient abundance, whereas theirdepletion with fasting may be a signal nutrient deficiency (Supple-mentary Fig. 2 online). In this regard, the whole-body deficiency of theenzyme acetyl-CoA carboxylase-2 (ACC-2) led to marked hyperphagia(25% increase in daily food intake) that could have been a result of adecreased formation of malonyl-CoA in certain hypothalamic neu-rons26. However, the increased caloric intake in this model occurred inthe presence of marked stimulation of whole-body energy expenditureand fat oxidation, leading to a lean phenotype26. In contrast, a decreasein the MBH malonyl-CoA pool occurs with fasting14 and maycontribute to the associated increases in appetite and liver gluconeo-genesis. However, the hypothalamic malonyl-CoA pool markedlyexpands with re-feeding14, and this increase may be required to restrainfood intake and liver glucose production (Supplementary Fig. 2).Because it is quite likely that the MCD overexpression affected multiplepopulations of neuronal and glial cells within the MBH, the presentstudy cannot identify a subset of cells responsible for hypothalamicnutrient sensing. Genetic strategies designed to specifically targetselective populations of MBH cells should provide a means to achievethis important goal.

Our findings also suggest that the anorectic action of leptin withinthe MBH required the cellular accumulation of malonyl-CoA. In fact,the overexpression of MCD in the MBH blunted the acute effects ofleptin on feeding behavior. These results are consistent with a recentreport suggesting that leptin decreases AMP kinase activity in the MBHand that this effect is required for its anorectic action8. Because adecrease in AMP kinase activity results in the activation of the malonyl-CoA–generating enzyme ACC, it is conceivable that its effects would beantagonized by the overexpression of the malonyl-CoA–degradingenzyme MCD.

Our results provide strong support for the idea that hypothalamicmalonyl-CoA has an important role in the regulation of energyhomeostasis, as a molecular intervention designed to chronicallydecrease the amount of malonyl-CoA in the MBH was sufficient tomarkedly increase food intake and to cause a sustained increase in bodyweight in rats. Modulation of the hypothalamic abundance of malonyl-and LCFA-CoAs may represent a promising strategy for the treatmentof obesity and the underlying metabolic syndrome by resetting abiochemical pathway that is central to the sensing of nutrients withinthe hypothalamus.

METHODSConstruction of the pMCD-AAV. We cloned the full-length rat MCD encoding

sequence in-frame with the 3�Flag sequence at the C terminus into the pAAV-

IRES-hrGFP plasmid, under the control of the CMV promoter, and followed by

the internal ribosome entry site (IRES) and GFP sequences, which express GFP

from the same transcript as MCD-Flag. MCD-Flag fusion protein expression

was verified by RT-PCR with three pairs of primers. P1 and P2 were designed

for a 248-bp fragment in the MCD sequence, P1 and P3 for a 378-bp MCD-

Flag fusion region and P4 and P5 for the complete MCD-Flag insert. The pri-

mer sequences are as follows: P1, 5¢-GCCTGGTACCTTTACGGTGA-3¢; P2, 5¢-GCTACCAGGCTGAGGATCTG-3¢; P3, 5¢-CACTACTTGTCATCGTCATCC-3¢;

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Pal

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Figure 7 Nutritional regulation of hypothalamic LCFA-CoA levels. (a) MCD-

AAV, but not. GFP-AAV, lowered LCFA-CoA, palmitoyl-CoA and palmitoleyl-

CoA levels in the arcuate nucleus of the hypothalamus. (b) Overnight fasting

decreased the concentration of LCFA-CoA, palmitoyl-CoA and palmitoleyl-CoA

in the arcuate nucleus of the hypothalamus. *P o 0.05, MCD-AAV

(n ¼ 6) versus GFP-AAV (n ¼ 6). Values are mean ± s.e.m.


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P4, 5¢-ATGAGAGGCTTGGGGCCAAG-3¢; and P5, 5¢- TCACTACTTGT

CATCGTCATCC-3¢.

Viral production and purification. Viral production was accomplished using

the AAV Helper-Free System (Stratagene), and purified with standard density

ultracentrifugation as well as column chromatography methods. Briefly,

HEK293 cells were cultured in ten 150 mm � 25 mm cell culture dishes and

cotransfected with the recombinant pMCD-AAV, pHelper and pAAV-RC

plasmids (Stratagene) using a standard calcium phosphate method. Cells were

collected, pelleted and resuspended in freezing buffer (0.15 M NaCl and 50 mM

Tris, pH 8.0) 66–70 h after transfection. After three freeze-thaw cycles,

benzonase was added (250 U ml–1) and the mixture was incubated at 37 1C

for 30 min. The lysate was added to a centrifuge tube containing a 15%, 25%,

40% and 60% iodixanol step gradient. The gradient was spun at 350,000g for

60 min at 18 1C. The 40% fraction was collected, applied to a heparin affinity

column, washed with 0.1 M NaCl and eluted with 0.4 M NaCl. Elution buffer

was exchanged with 1� phosphate-buffered saline (PBS) using Amicon

BioMax 100-kDa nominal molecular weight limit (NMWL) concentrators.

The final purified virus was then titered by quantitative RT-PCR for genomic

titer (DNase-resistant particles) using primers within viral inverted terminal

repeats (ITR) and specific primers spanning the MCD-Flag fusion region.

MCD activity assay. The reaction cocktail contained 0.1 M Tris-Cl buffer (pH

8.0), 0.5 mM dithioerythritol, 0.01 M L-malate, 0.5 mM b-nicotinamide

adenine dinucleotide and 0.1 mM b-nicotinamide adenine dinucleotide

hydrate. To this we added 5 ml of 1 U ml–1 malic dehydrogenase and 5 ml of

0.17 U ml–1 citrate synthase. We incubated the mixture for 2 min, then added

20 ml of 2 mM malonyl-CoA and 5 ml of the MCD preparation. We measured

the absorbance change at 340 nm at 1, 2, 3, 4 and 5 min in the kinetic mode.

The initial linear velocity was used for the enzyme activity.

Experimental model. We studied 54 10-week-old male Sprague-Dawley rats

(Charles River Breeding Laboratories). Rats were housed in individual cages

and subjected to a standard light-dark cycle. We implanted chronic catheters

bilaterally within the MBH by stereotaxic surgery9,11 2 weeks before the in vivo

experiments. In rats subjected to the pancreatic insulin-clamp protocols,

additional catheters were placed in the right internal jugular and left carotid

artery 1 week before the study27.

Clamp procedure in rats. The in vivo infusion experiments lasted a total of 6 h,

and the experiments were carried out in rats that had their food removed B5 h

before the experiments (Fig. 2a). We infused intravenous 20% intra-

lipid (Baxter Healthcare) mixed with 20 IU ml–1 of heparin at a rate of

0.4 ml h–1) to elevate the concentration of free fatty acid in the plasma in rats

2 weeks after the MBH injection of the MCD-AAV and GFP-AAV. A primed-

continuous infusion of [3-3H]glucose (New England Nuclear; 40 mCi bolus;

0.4 mCi min–1) was initiated at 120 min and maintained throughout the study

to assess glucose kinetics. Samples for the determination of [3-3H]glucose

specific activity were obtained at 10-min intervals. Pancreatic insulin-clamp

experiments were performed as previously described28. A near-basal insulin

clamp was performed in the final 2 h of the study (that is, from 240 to

360 min). In brief, a continuous infusion of insulin (1.5 mU kg–1 min–1) and

somatostatin (3 mg kg–1 min–1) was administered, and a variable infusion of a

25% glucose solution was started and periodically adjusted to clamp and

maintain the plasma glucose concentration at B8 mM. Plasma samples for

determination of plasma free fatty acid, insulin, adiponectin and glucagon

concentrations were obtained at 30-min intervals during the study. At the end

of the infusion experiments, rats were anesthetized and tissue samples were

freeze-clamped in situ with aluminum tongs precooled in liquid nitrogen. All

tissue samples were stored at –80 1C for subsequent analysis.

Feeding experiments. This experimental protocol was designed to examine the

acute effect of MBH leptin and vehicle on food intake in rats 2 weeks after the

injection of the MCD-AAV and GFP-AAV. On day 0 (Fig. 4b), rats were given

an MBH bolus injection of either leptin (3 mg) or vehicle at a rate of 0.2 ml min–1

using a gas-tight syringe (Hamilton) 1 h before the start of the dark cycle. Food

consumed during the following 16 h (overnight) was measured.

Body composition. Body composition was assessed as previously described29.

Briefly, rats received an intra-arterial bolus injection of 20 mCi of tritiated

water (3H2O; New England Nuclear) and plasma samples were obtained at

30-min intervals for 3 h. Steady-state conditions for plasma 3H2O specific

activity were achieved within 45 min in all experiments. Five plasma

samples obtained between 1 and 3 h were used in the calculation of the

whole-body distribution space of water. This was obtained by dividing the

total radioactivity injected (d.p.m.) by the steady-state specific activity of

plasma water (d.p.m. ml–1). Plasma was assumed to be 93% water. Fat-free

mass was calculated as the whole-body water distribution space divided by

0.73 and fat mass as the difference of body weight and fat-free mass.

Epididimal, perinephric and mesenteric fat depots were dissected and weighed

at the end of each experiment.

Neuropeptide expression experiments. For the analysis of AgRP, NPY and

POMC gene expression7, we studied two groups of rats receiving MBH

injections of MCD-AAV or GFP-AAV. Total RNA was isolated from mediobasal

hypothalamic wedges with Trizol (Invitrogen). AgRP, NPY and POMC gene

expression were measured by quantitative RT-PCR as described before9. The

following primers were used for the PCR: forward primer 5¢-TGGCA

GAGGTGCTAGATCCA-3¢ and reverse 5¢-GCACAGGTCGCAGCAAGGTA-3¢for AgRP; forward 5¢-CCGCCATGATGCTAGGTAAC-3¢ and reverse 5¢-TGTCGCAGAGCGGAGTAGTA-3¢ for NPY; forward 5¢-ACCTCCGAGAA

GAGCCAGAC-3¢ and reverse 5¢-GGCCTTGGAGTGAGAAGACC-3¢ for

POMC; and forward 5¢-TGAGAGGGAAATCGTGCGTG-3¢ and reverse 5¢-GTACTTGCGCTCAGGAGGAGCA-3¢ for b-actin. The copy number of each

transcript was measured against a copy number standard curve of cloned target

templates. Expression of each transcript was normalized to the copy number

for b-actin.

Western blot analyses. Frozen tissue samples were sonicated, boiled in 1%

SDS, separated by 10% SDS-PAGE electrophoresis and transferred to 0.2-mm

photomodified polyvinylidene difluoride (PVDF) membranes (Bio-Rad). The

membranes were immunoblotted with primary antibodies to the Flag peptide

(Sigma), voltage-dependent anion-selective channel protein 1 (VDAC1, Santa

Cruz), phosphoSTAT3 (Tyr705), total-STAT3 (Cell Signaling), GFP (Molecular

Probes), b-tubulin (Covance) and MCD (rabbit antiserum to MCD peptide

KEYGRNELFTDSEC, Covance). After incubation with horseradish peroxidase–

conjugated secondary antibodies, chemiluminescence was detected with

Western Lightning Plus reagent (Perkin-Elmer). Blots were quantified by

densitometry using NIH Image 1.61 software. The mitochondria isolation kit

was from Pierce.

Immunofluorescence staining. Rats were anesthetized with pentobarbital

followed by transcardial perfusion of PBS for 5 min and 4% paraformaldehyde

in 1� PBS for 20 min. Brains were removed and post-fixed overnight in 4%

paraformaldehyde and cryoprotected for at least 4 h in 30% sucrose in 1� PBS.

Brains were frozen on dry ice and sectioned at 5 mm on a sliding microtome.

For GFP immunostaining, sections were blocked with 3% normal goat

serum with 0.3% Triton X-100 for 1 h and incubated overnight with mouse

anti-GFP antibody (Molecular Probes). After washing and incubation with

fluorescein-conjugated goat anti-mouse immunoglobin antibody (Jackson

ImmunoResearch), the slides were viewed under fluorescence microscope.

LCFA-CoA and malonyl-CoA measurements. The mediobasal hypothalamus

was sampled9, and the LCFA-CoAs and malonyl-CoA were extracted from the

mediobasal hypothalamus and measured by high-performance liquid chroma-

tography (HPLC) as previously described9,30,31.

All values are presented as the mean ± s.e.m. Comparisons among groups

were made using analysis of variance or unpaired Student’s t-test as appro-

priate. The study protocol was reviewed and approved by the Institutional

Animal Care and Use Committee of the Albert Einstein College of Medicine.

Note: Supplementary information is available on the Nature Neuroscience website.

ACKNOWLEDGMENTSWe wish to thank C. Baveghems, B. Liu, H. Zhang and S. Gaweda for experttechnical assistance. This work was supported by the US National Institutesof Health, the American Diabetes Association and the Skirball Foundation.


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COMPETING INTERESTS STATEMENTThe authors declare competing financial interests (see the Nature Neurosciencewebsite for details).

Published online at http://www.nature.com/natureneuroscience/

Reprints and permissions information is available online at http://npg.nature.com/

reprintsandpermissions/

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molecular disruption of hypothalamic nutrient sensing induces obesity

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