Download PAPER Acute effects of brain-derived neurotrophic factor on energy

International Journal of Obesity (2001) 25, 1286–1293
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PAPER
Acute effects of brain-derived neurotrophic factor on
energy expenditure in obese diabetic mice
A Tsuchida1, T Nonomura1, M Ono-Kishino1, T Nakagawa1, M Taiji1* and H Noguchi1,2
1
Sumitomo Pharmaceuticals Co. Ltd, Discovery Research Laboratories II, Osaka, Japan; and 2Sumitomo Pharmaceuticals Co. Ltd,
Business Development and Licensing Office, Tokyo, Japan
OBJECTIVE: We recently demonstrated that chronic treatment with brain-derived neurotrophic factor (BDNF) regulates energy
expenditure in obese diabetic C57BL=KsJ-db=db mice. In this study, we investigated the acute effects of BDNF on energy
expenditure.
DESIGN: After BDNF was singly administered to male db=db mice (aged 10 – 12 weeks), their body temperature and whole
body glucose oxidation were measured. Their norepinephrine (NE) turnover and uncoupling protein (UCP) 1 expression in
interscapular brown adipose tissue (BAT) were also analyzed.
RESULTS: Even though the body temperatures of hyperphagic db=db mice dropped remarkably in a 24 h period after food
deprivation, only a single subcutaneous administration of BDNF significantly prevented the reduction of body temperature.
BDNF was also observed to have similar efficacy in cold exposure experiments at 15 C. Respiratory excretion of 14CO2 after
intravenous injection of D-[14C(U)]-glucose was significantly increased by BDNF administration, indicating that BDNF increases
whole-body glucose oxidation. BDNF administered intracerebroventricularly was also able to prevent the reduction of body
temperature of db=db mice. To clarify the BDNF action mechanism we examined NE turnover in BAT. Four hours after a single
administration, BDNF reduced NE content in the presence of the tyrosine hydroxylase inhibitor, a-methyl-P-tyrosine methyl
ester, indicating enhanced NE turnover in BAT. BDNF also increased the expression of the UCP1 mRNA and protein in BAT.
CONCLUSION: These data indicate that BDNF rapidly regulates energy metabolism in obese diabetic animals, partly through
activating the sympathetic nervous system and inducing UCP1 gene expression in BAT.
International Journal of Obesity (2001) 25, 1286 – 1293
Keywords: neurotrophic factor; thermogenesis; glucose metabolism; norepinephrine turnover; uncoupling protein
Introduction
Brain-derived neurotrophic factor (BDNF) is a member of the
neurotrophin family which includes nerve growth factor,
neurotrophin-3 and neurotrophin-4=5.1 – 4 BDNF promotes
neurite outgrowth and provides trophic support to certain
neurons during development and in adulthood.1 The efficacy of BDNF in the treatment of neurological disorders has
also been demonstrated.5 – 7 We have previously shown that
subcutaneous administration of BDNF reduces food intake
and lowers blood glucose to normoglycemic levels in obese
diabetic models such as C57BL=KsJ-db=db mice.8 – 10 This
was the first evidence that BDNF has a pleiotrophic effect
and functions, even though a neurotrophic factor, in the
endocrine system.
*Correspondence: M Taiji, Sumitomo Pharmaceuticals Co. Ltd, Discovery
Research Laboratories II, 3-1-98 Kasugadenaka, Konohana-ku, Osaka 5540022, Japan.
E-mail: [email protected]
Received 31 October 2000; revised 9 February 2001;
accepted 21 February 2001
Since BDNF reduced food intake in obese and hyperphagic
db=db mice, we developed a novel pellet pair-feeding apparatus to control precisely the quantity and timing of feeding
between the control group and BDNF-treated group. With
this apparatus we were able to demonstrate the hypoglycemic action of BDNF independently of food intake. In the
repetitive daily administration of BDNF under such strict
food control, we found that the control pellet pair-fed db=db
mice did not receive sufficient food, resulting in lower body
temperatures than the ad libitum-fed db=db mice. In addition
to its hypoglycemic effect, we demonstrated that daily
administration of BDNF completely ameliorated the alteration of body temperature and oxygen consumption in pairfed db=db mice.11 However, it is not yet known whether
BDNF has a direct effect on the regulation of energy metabolism. There is the possibility that energy expenditure is
being affected by the hypoglycemic and hypophagic effects
of long-term BDNF administration to obese diabetic animals.
Although our previous study showed that BDNF
rapidly enhanced the hypoglycemic action of insulin in
BDNF rapidly increases energy expenditure
A Tsuchida et al
streptozotocin-induced type 1 diabetic mice,11 the acute
effects of BDNF on energy expenditure have not yet been
investigated. In this study we thus decided to determine
whether BDNF immediately affected the regulation of
energy expenditure. We also discuss the analogous effects
of BDNF and leptin on energy and glucose metabolisms.
Methods
Animals
Male C57BL=KsJ-db=db mice and male C57BL=6N mice were
obtained from Clea Japan (Tokyo, Japan). All treatments
were started at 10 – 12 weeks of age. All animals were
housed in group cages and maintained in a daily cycle of
12 h light and 12 h darkness. Food (CE-2, Clea Japan, Tokyo,
Japan) and water were given ad libitum except for mice in the
fasting experiments. All animal experiments were conducted
according to the guidelines of the Sumitomo Pharmaceuticals
Committee on Animal Research.
Administration of BDNF
Human recombinant BDNF (N-terminal methionine-free,
Regeneron Pharmaceuticals, Tarrytown, NY, USA) was administered subcutaneously at a dosage of 20 mg=kg to db=db
mice except for the norepinephrine turnover experiment.
PBS (phosphate-buffered saline) containing 0.01% Tween 80
and 1% mannitol was used as the vehicle. For intracerebroventricular administration, db=db mice were anesthetized
with diethyl ether, the bregma was identified, and 15mg
BDNF=mouse (3 ml=shot) was injected into the lateral ventricle. Artificial cerebrospinal fluid (aCSF — 0.166 g=l CaCl2,
7.014 g=l NaCl, 0.298 g=l KCI, 0.203 g=l MgCl2=6H2O and
2.10 g=l NaHCO3) was used as the vehicle for intracerebroventricular administration.
Body temperature
Body temperature was measured using an electron thermistor (model BAT-12, Physitemp, Clifton, NJ, USA) equipped
with rectal probe (RET-3, Physitemp, Clifton, NJ, USA).
Blood glucose
Blood samples were collected from tail vein, and blood
glucose was measured by the Glucose C II-Test, Wako (Mutarotase-glucose oxidase method, Wako Chemical, Osaka,
Japan).
Glucose oxidation
db=db mice were fasted for 18 h and then given either BDNF
or vehicle subcutaneously. Four hours after BDNF or vehicle
treatment the mice were injected with 5 mCi of D-[14C(U)]glucose (specific activity 3.4 mCi=mmol, NEN Life Science
Products, Boston, MA, USA) in 200 ml of saline via the tail
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vein. Immediately after the tracer injection each mouse was
placed in a metabolic chamber and air was drawn at
200 ml=min through a trap containing 200 ml of 10%
NaOH solution. The NaOH solution was analyzed for
14
CO2 before and at 15, 30, 60, 90 and 120 min after the D[14C(U)]-glucose injection. Radioactive levels in the NaOH
solution were determined with a liquid scintillation analyzer
using the liquid scintillator, HyonicFluor (Packard, Meriden,
CT, USA).
Norepinephrine turnover
The effect of BDNF on norepinephrine turnover was assessed
by the method of Collins et al,12 slightly modified by ourselves. db=db mice were subcutaneously administered with
either BDNF (7, 20, 70, 200 mg=kg) or vehicle at the beginning of a dark cycle and then food was removed. Two hours
after BDNF or vehicle treatment, 250 mg=kg a-methylp-tyrosine methyl ester (AMPT), an inhibitor of tyrosine
hydroxylase, was intraperitoneally injected to block de novo
catecholamine synthesis. The mice were decapitated 2 h after
AMPT injection and the interscapular brown adipose tissue
(BAT) was immediately dissected, weighed and then frozen
in liquid nitrogen. The BAT was homogenized with 0.1 N
perchlonic acid containing 5 mM EDTA. Homogenates were
filtrated through a 0.22 mm mesh membrane to remove
debris. Norepinephrine (NE) content in homogenates was
measured using an HPLC system (LC-10A, Shimadzu Instrumentation, Kyoto, Japan) equipped with a column (CA-5DS,
Eicom, Kyoto, Japan). Based on the results of HPLC analysis
where differing amounts of NE were loaded, NE contents in
homogenates were measured to be in a range wherein the
peak area of eluted NE was proportional to the loaded NE.
Northern blot analysis
db=db Mice were subcutaneously injected with either BDNF
or vehicle at the beginning of a dark cycle and then food was
removed. Animals were sacrificed 4 h after BDNF or vehicle
treatment; interscapular BAT was excised and frozen immediately. RNA was prepared from the tissues with Trizol (Gibco
BRL Life Technologies, Rockville, MD, USA) using the manufacturer’s protocol. Yield and purity of RNA were determined by spectrophotometric absorption analysis at
260=280 nm. Three micrograms of total RNA were electrophoresed in a 1% agarose gel containing formaldehyde and
then transferred to GT probe membranes (Bio-Rad Laboratories, Hercules, CA, USA). A 1071-base pair rat uncoupling
protein-1 (UCP1) probe (nucleotides 84-1154 in Genebank
accession no. M11814) was obtained by reverse transcriptasepolymerase chain reaction (RT-PCR) from rat BAT RNA using
0
primers 5 -CCA CAG GAA TTC GAA GTT GAG AGT TCG GTA
0
and 5 -CCC AGC TCT AGA GCC CAG CAT AGG AGC CCA as
reported previously.13 A 349-base pair mouse b-actin probe
(nucleotides 728-1076 in Genebank accession no. M12481)
was obtained by RT-PCR from mouse liver RNA using primers
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BDNF rapidly increases energy expenditure
A Tsuchida et al
1288
0
0
5 -TGG AAT CCT GTG GCA TCC ATG AAA C and 5 -TAA AAC
GCA GCT CAG TAA CAG TCC G. All probes were verified by
sequencing. Probes were randomly labeled using a BcaBest
labeling kit (Takara, Ohtsu, Japan) with [a-32P]-deoxy CTP
(Amersham Pharmacia Biotech, Buckinghamshire, England).
Hybridization was carried out at 65 C in 0.25 M sodium
phosphate (pH 7.2)=7% SDS, and blots were washed twice
with 20 mM sodium phosphate (pH 7.2)=5% SDS and then
with 20 mM sodium phosphate (pH 7.2)=1% SDS. Hybridization signals were quantified using a bio-imaging analyzer
BAS2000 (Fuji Photo Film, Tokyo, Japan).
metabolism. To investigate the effect of BDNF on body
temperature independently of its hypophagic action, food
was removed from db=db mice after BDNF or vehicle treatment. The body temperature of control db=db mice given
vehicle was found to drop dramatically during the first 24 h
period after food deprivation (Figure 1A). In contrast, only a
single administration of 20 mg=kg BDNF significantly prevented this reduction in body temperature (Figure 1A).
During the 24 h experiment, no significant difference was
observed between the BDNF-treated and vehicle-treated
groups in either body weight or blood glucose concentration
(Figure 1B, C).
Western blot analysis
db=db Mice were subcutaneously injected with either BDNF
or vehicle at the beginning of a dark cycle and then food was
removed. Animals were killed 4 h after BDNF or vehicle
treatment; interscapular BAT was excised and frozen immediately. Frozen interscapular BAT were homogenized in icecold lysis buffer (20 mM Tris – HCl (pH 7.4), 0.15 M NaCl, 1%
NP-40, 0.1% sodium deoxycholate, 5 mM EDTA, 10 mM
sodium fluoride, 2 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 10 mg=ml aprotinin and 10 m=ml
leupepsin). Homogenates were centrifuged and protein content of supernatants was determined by BCA method using a
kit (Pierce, Rockford, IL, USA). Equal protein amounts of
whole tissue extracts (20 mg=lane) were resolved by SDS –
PAGE (10=20% gradient polyacrylamide gels), transferred to
Hybond-ECL nitrocellulose membranes (Amersham, Arlington Heights, IL, USA), and subjected to Western blot analysis
using affinity-purified polyclonal rabbit antibody raised
against a 19-amino acid C-terminal sequence of mouse and
rat UCP1 (UCP12-A, Alpha Diagnostics International, San
Antonio, TX, USA). The blots were quantified by densitometry using an chemiluminescent imaging system (DIANA,
M&S Instruments Trading, Tokyo, Japan) and its analysis
software (AIDA, Fuji Photo Film, Tokyo, Japan).
Statistical analysis
All data are presented as mean s.d. Differences between
individual groups were analyzed by Student’s t-test or Willliams’ test. Statistical calculations were performed using SAS
software (SAS Institute, Cary, NC, USA). P < 0.05 was considered statistically significant.
Results
Effect of a single administration of BDNF on body
temperature in db=db mice
In our previous study, control pellet pair-fed db=db mice were
observed to have a lower body temperature than ad libitumfed db=db mice. It was shown there that repetitive daily
administration of 20 mg=kg BDNF for 3 weeks ameliorated
this reduction in body temperature.11 In the present study
we have now examined the acute effect of BDNF on energy
International Journal of Obesity
Figure 1 Effect of BDNF on body temperature, body weight and blood
glucose in fasted db=db mice. BDNF (20 mg=kg) or vehicle was administered subcutaneously at 0 h, and thereafter all animals were kept in a
fasting condition. Body temperature (A), body weight (B) and blood
glucose (C) were measured at 0, 3, 6 and 24 h after BDNF or vehicle
administration. Data are shown as means s.d. for seven mice. *P < 0.05;
**P < 0.01 vs vehicle by Student’s t-test.
BDNF rapidly increases energy expenditure
A Tsuchida et al
The effect of BDNF on body temperature was also assessed
in normal C57BL=6N mice. During the first 24 h after food
removal the body temperature of normal mice slightly but
significantly decreased by 1.4 C compared with the ad libitum-fed normal mice. A single 20 mg=kg administration of
BDNF did not affect body temperature in either the fasted or
ad libitum-fed normal mice (data not shown).
Effect of a single administration of BDNF on body
temperature in cold-exposed db=db mice
A cold exposure experiment was also performed to study the
effect of BDNF treatment on the body temperature of db=db
mice. When the animals were placed in a cold room at 15 C
with free access to food and water, body temperatures of the
vehicle-treated db=db mice decreased (Figure 2). In contrast,
only a single administration of 20 mg=kg BDNF significantly
prevented this reduction of body temperature (Figure 2).
Again there were no significant differences in blood glucose
concentration and body weight between the BDNF-treated
animals and vehicle-treated animals (data not shown).
Effect of a single administration of BDNF on glucose
oxidation in db=db mice
Since BDNF ameliorated the reduction of body temperature
in fasted or cold-exposed db=db mice without affecting blood
glucose concentration, it is possible that BDNF increases
whole-body glucose turnover in a manner similar to the
anorexic protein, leptin.14 The production rates of respiratory 14CO2 from uniformly labeled D-[14C(U)]-glucose were
therefore measured in vivo to investigate the effect of BDNF
Figure 2 Effect of BDNF on body temperature of db=db mice during
cold exposure. BDNF (20 mg=kg) or vehicle was administered subcutaneously at 0 h, and thereafter all animals were placed in a cold room at
15 C with ad libitum access to food and water. Body temperature was
measured at the indicated times after BDNF or vehicle administration.
Data are shown as means s.d. for five mice. **P < 0.01 vs vehicle by
Student’s t-test.
on glucose and energy metabolism. D-[14C(U)]-glucose was
given intravenously to fasted db=db mice 4 h after subcutaneous treatment of BDNF (20 mg=kg) or vehicle, and the
accumulative respiratory excretion of 14CO2 from both
groups was analyzed. During the first 30 min after D[14C(U)]-glucose injection, the amount of 14CO2 expired
from the BDNF-treated db=db mice was significantly higher
than the vehicle-treated db=db mice (Figure 3). The BDNFtreated animals maintained a higher 14CO2 expiration rate
throughout the observation period (120 min). There was no
significant difference in blood glucose concentration
between BDNF- and vehicle-treated mice 120 min after the
tracer injection (248 61.7 vs 277 48.9 mg=dl). These observations indicate that BDNF increased whole-body glucose
oxidation and enhanced glucose turnover without affecting
blood glucose concentration.
1289
Intracerebroventricular administration of BDNF on body
temperature in db=db mice
Given the possibility that the acute effect of BDNF on energy
metabolism is due to direct action on the brain, we decided
to investigate the effect of BDNF intracerebroventricular
administration on body temperature in db=db mice. Previously we have shown that repetitive administration of
15 mg BDNF every other day reduced blood glucose concentration effectively11 and thus chose the same dosage (15 mg)
Figure 3 Effect of BDNF on whole-body glucose oxidation in db=db
mice. Five microcurie D-[14C(U)]-glucose was given intravenously to
fasted mice 4 h after subcutaneous treatment of BDNF (20 mg=kg) or
vehicle. The accumulative respiratory excretion of 14CO2 from BDNF or
vehicle treated animals was analyzed at the indicated times after the
tracer dose. Data are shown as means s.d. for five mice. *P < 0.05 vs
vehicle by Student’s t-test.
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BDNF rapidly increases energy expenditure
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of BDNF for this single intracerebroventricular administration. Since hypophagia was also observed with intracerebroventricular administration, food was removed from db=db
mice after BDNF or vehicle treatment. Although the body
temperature of db=db mice given intracerebroventricular
vehicle dropped during the 24 h period after food deprivation, only a single intracerebroventricular administration of
15 mg BDNF=mouse (approximately 300 mg=kg) significantly
blocked the reduction of body temperature (Figure 4).
Effect of a single administration of BDNF on
norepinephrine turnover
Our finding that intracerebroventricular administration of
BDNF acutely stimulated thermogenesis led us to hypothesize that BDNF activates the sympathetic nervous system and
regulates energy expenditure. We thus analyzed norepinephrine (NE) turnover in BAT, a predominant thermogenic
organ in mice, to assess the effect of BDNF on the sympathetic nervous system. To determine NE turnover we measured the decrease of NE content in BAT before and after
injection of a tyrosine hydroxylase inhibitor, a-methyl-ptyrosine methyl ester (AMPT), which inhibits de novo catecholamine synthesis. db=db mice were given BDNF (7, 20, 70,
200 mg=kg, s.c.) or vehicle and 2 h later injected with AMPT.
Two hours after the AMPT injection, NE content in BAT of
the db=db mice was measured by HPLC. As a zero-time
control, NE content in BAT of db=db mice before AMPT
injection was also measured. NE content in BAT of db=db
mice given vehicle was reduced to 73% of the zero-time
control by AMPT inhibition of catecholamine synthesis
Figure 4 Effect of intracerebroventricular administration of BDNF on
body temperature of fasted db=db mice. BDNF (15 mg=mouse) or vehicle
was administered intracerebroventricularly at 0 h, and thereafter all
animals were kept in a fasting condition. Body temperature was measured at 0, 3, 6 and 24 h after BDNF or vehicle administration. Data are
shown as means s.d. for seven mice. *P < 0.05; **P < 0.01 vs vehicle by
Student’s t-test.
International Journal of Obesity
Figure 5 Effect of BDNF on norepinephrine turnover in brown adipose
tissue. BDNF or vehicle was administered subcutaneously to db=db mice
followed by food removal. Two hours after BDNF or vehicle administration, 250 mg=kg a-methyl-p-tyrosine methyl ester (AMPT) was injected
intraperitoneally. Mice were then decapitated 2 h after AMPT injection
and norepinephrine (NE) content of the interscapular brown adipose
tissue (BAT) was measured by HPLC. As a zero-time control, NE content
in BAT of db=db mice before AMPT injection was also measured. Data are
shown as means s.d. for 10 mice. **P < 0.01 vs vehicle by Willliams’
test. ##P < 0.01 vs zero-time control by Student’s t-test.
(Figure 5). NE content in BAT of db=db mice given BDNF
decreased in a dose-dependent manner. Administration of
BDNF at a dose of higher than 20 mg=kg significantly
reduced NE content compared with that of mice given
vehicle (Figure 5). These results indicate that BDNF acutely
increased NE turnover in BAT.
Effects of a single administration of BDNF on expression
of uncoupling protein 1 mRNA and protein in BAT
To clarify the action mechanism by which BDNF stimulates
the glucose and energy metabolisms, we examined the
expression of UCP1 gene as an indicator of sympathetic
nerve activity in BAT and the effect of BDNF on that. BDNF
(20 mg=kg) or vehicle was administrated subcutaneously to
db=db mice and followed by food removal. Total RNA was
prepared from BAT 4 h after BDNF or vehicle administration.
Northern blot analysis of BAT RNA (3 mg) showed that a
single administration of BDNF significantly recovered
UCP1 mRNA levels, which was 2.6-fold higher than vehicletreated animals without food supply (Figure 6A).
We also studied the UCP1 protein expression in BAT from
the same animals used in Northern blot analysis. The
extracts of whole tissues (20 mg=lane) was analyzed with
SDS – PAGE, followed by Western blotting using anti-UCP1
antibody. Again, BDNF significantly increased the expression
of UCP1 protein in BAT by 1.6-fold (Figure 6B).
BDNF rapidly increases energy expenditure
A Tsuchida et al
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Figure 6 Effect of BDNF on regulation of UCP1 expression in brown
adipose tissue of db=db mice. Mice were treated subcutaneously with
BDNF (20 mg=kg) or vehicle and then fasting was started. Four hours
after BDNF or vehicle administration, interscapular brown adipose tissue
(BAT) was dissected and total RNA and protein isolated. (A) Northern
blot analysis (UCP1 and b-actin probes) of the total RNA (3 mg) was then
performed. Data are shown as means s.d for nine (BDNF) or 10
(vehicle) mice. (B) Western blot analysis of the total protein (20 mg=lane)
using anti-UCP1 antibody was performed. Data are shown as
means s.d. for 10 mice. *P < 0.05; **P < 0.01 vs vehicle by Student’s
t-test. The left panels show representative blots.
Discussion
db=db mice are obese and hyperphagic diabetic animals with
impaired glucose and energy metabolisms.15 – 17 Discoveries
of leptin and its receptor (Ob-R) led to identification of a
mutation in the Ob-R gene of db=db mice.18,19 This was
subsequently determined to be the cause for the obesity
and resistance to leptin treatment.20,21 In our previous studies it was found that BDNF reduces both food intake and
blood glucose in db=db mice.8 – 11 In order to investigate the
effect of BDNF on glucose metabolism independently of
alteration in food intake we developed a novel pellet-pair
feeding apparatus to supply the same quantity of food to
both control mice and BDNF-treated mice. In the 3 week
study of repetitive daily BDNF administration it was found
that pair-fed control db=db mice receiving a restricted quantity of food had significantly lower body temperatures and
oxygen consumption than ad libitum-fed db=db mice.11 This
indicates that db=db mice require excessive food intake to
maintain their body temperature possibly due to reduced
energy expenditure, although the body temperature of ad
libitum-fed db=db mice is still lower than normoglycemic
db=db mice.
In spite of the reduced food intake, to our surprise, db=db
mice receiving BDNF daily could maintain nearly the same
body temperature and metabolic rate as ad libitum-fed db=db
mice.11 These findings indicate that BDNF ameliorated the
erratic energy balance characteristic of db=db mice. However,
we have also demonstrated that daily administration of
BDNF reduces the blood glucose of db=db mice.11 Since the
attenuation of glucose toxicity improves various types of
metabolic dysfunction in diabetic animals,22 we felt that the
improvement of energy expenditure might be due to the
hypoglycemic action of BDNF. Therefore, in order to understand the effect of BDNF on glucose and energy metabolisms
in greater detail, we investigated the acute effect of a single
administration of BDNF in the present study.
During periods of food deprivation or cold-exposure, only
a single administration of BDNF prevented the reduction of
body temperature in db=db mice without affecting blood
glucose concentration. Also, 4 h after BDNF administration,
glucose oxidation was enhanced. These data clearly demonstrate that BDNF has an immediate, direct effect on the
regulation of energy expenditure in diabetic mice. In addition to db=db mice, BDNF reduces food intake and blood
glucose concentration in ob=ob mice and KKAy mice (data
not shown). Furthermore, in our unpublished study, BDNF
prevented reduction of body temperatures in monosodium
glutamate (MSG)-induced obese rats under fasted condition.
Therefore, we consider that BDNF’s effect is not specific for
db=db mice. On the other hand, BDNF did not affect the
body temperature of normoglycemic C57BL=6N mice in
either fed or fasted conditions (data not shown). Furthermore, as we previously reported, BDNF does not reduce
blood glucose concentration in normoglycemic mice.8
Taken together, these results indicate that BDNF may not
affect the energy metabolism of normoglycemic animals.
In our previous study, both a hypoglycemic effect and
appetite reduction were also observed with repetitive intracerebroventricular administration of BDNF in db=db mice.11
This suggests the possibility that the hypoglycemic effect is
due to the direct action of BDNF on the central nervous
system. Furthermore, this study showed that a single central
administration of BDNF at a dose too low to have an effect
when delivered peripherally was able to immediately ameliorate the reduction of body temperature. Since the BDNF
receptor, trk B, has been shown to be expressed in hypothalamic nucleuses,23 it seems reasonable to speculate that
BDNF administrated peripherally enters the hypothalamus
by crossing the blood – brain barrier (BBB) and regulates
energy metabolism and appetite at this region. In fact, it
has been reported that BDNF in the peripheral circulation
crosses the BBB and reaches the central nervous system.24,25
In addition to these pharmacological profiles of BDNF, it has
been reported that mice that are heterozygous for targeted
disruption of the BDNF gene exhibit hyperphagia and obesity.26,27 This suggests the physiological significance of BDNF
in weight control and glucose metabolism.
Our findings thus reveal that the actions of BDNF are
strikingly similar to those of leptin in their effect on body
temperature, oxygen expenditure and food intake. The role
of leptin in the regulation of energy metabolism and food
intake has been well documented.20,21,28 Since the leptin
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BDNF rapidly increases energy expenditure
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1292
receptor, Ob-R, is expressed in the hypothalamus, it has been
suggested that leptin secreted peripherally from adipose
tissue acts on the hypothalamus,29 – 31 the regulatory center
of autonomic nervous system controlling both food intake
and energy expenditure.32 Although BDNF receptor, trk B is
also found in the hypothalamic region of rat brain,23 it has
not been reported so far whether some hypothalamic neurons may share expression of trk B and leptin receptor or not.
The autonomic nervous system is involved in processes
where brain controls energy metabolism.33 For example,
activation of the sympathetic nervous system leads to
increases of gluconeogenesis and glycogenolysis in liver,
glucose uptake in skeletal muscles, and thermogenesis and
glucose uptake in BAT.33 In our present study BDNF
decreased NE content in BAT of db=db mice given the blocker
of catecholamine synthesis, AMPT. On the other hand BDNF
did not affect NE content in BAT without AMPT injection
(our unpublished data). These results have clearly shown
that BDNF does not block de novo catecholamine synthesis,
but that BDNF increases NE turnover in BAT. It has been
reported that NE turnover is enhanced either by leptin
administration or electrical stimulation of the ventromedial
hypothalamic nucleus (VMH).12,34 Therefore, the observed
increase in energy metabolism in BDNF-treated db=db mice
could be the result of adrenergic activation in BAT. This,
taken together with our finding that intracerebroventricular
administration of BDNF also regulates energy metabolism,
strongly supports our hypothesis that BDNF modulates
energy metabolism through its effect on hypothalamic
nuclei and the autonomic nervous system which in turn
activate neuronal regulation of the energy metabolism.
Uncoupling protein (UCP) is a mitochondrial protein
which regulates energy expenditure including thermogenesis. Among the several UCPs known so far, UCP1 is
expressed specifically in BAT and has a significant role in
maintaining body temperature in rodents. It has been
reported that 4 h after leptin treatment the expression of
UCP1 mRNA was rapidly increased in BAT.35 It has also been
shown that treatment with b3-adrenergic agonists resulted in
increase of UCP1 mRNA levels in BAT.36 Our results indicate
that BDNF also increased UCP1 mRNA and UCP1 protein
expression in BAT. Expression level of UCP1 in BAT of db=db
mice was reported to be comparable with that of lean control
mice under ad libitum-fed condition.37 Since body temperatures of db=db mice given vehicle dropped after food removal
in our present study, it is likely that sympathetic nerve
activity was suppressed and UCP1 expression in BAT was
reduced. BDNF may compensate such suppression and normalize energy expenditure of db=db mice.
In addition to its effect on energy metabolism and food
intake, it has been reported that leptin also shows a hypoglycemic effect in some disease models.38,39 This lends more
evidence to support our belief that the efficacy of leptin is
very similar to BDNF. Further studies, similar to those done
with leptin, will be needed to evaluate the molecular
mechanism by which BDNF regulates energy expenditure
International Journal of Obesity
and glucose metabolism. In conclusion, we have shown that
BDNF is an immediate and direct modulator of energy
expenditure and glucose metabolism in obese diabetic animals. This supports our belief that BDNF may be a potentially useful therapeutic agent for diabetic mellitus and
obesity.
Acknowledgements
The authors thank Mr Masayuki Satoh (Sumitomo Chemical,
Osaka, Japan) for conducting the glucose oxidation analysis.
This study was supported by Sumitomo Pharmaceuticals Co.
Ltd, Osaka, Japan.
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