|
| |
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
First published online March 14, 2008
Journal of Experimental Biology 211, 1075-1086 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.014050
Involvement of lactate in glucose metabolism and glucosensing function in selected tissues of rainbow trout
Laboratorio de Fisioloxía Animal, Departamento de Bioloxía Funcional e Ciencias da Saúde, Facultade de Bioloxía, Universidade de Vigo, 36310 Vigo, Spain
* Author for correspondence (e-mail: jsoengas{at}uvigo.es)
Accepted 24 January 2008
 |
Summary |
|---|
 TOP Summary INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
|---|
-cyano-4-hydroxy cinnamate or 10 mmol l–1
D-glucose. The response of parameters assessed (metabolite levels,
enzyme activities and glucokinase expression) in tissues provided evidence for
(1) a role for lactate in the regulation of glucose homeostasis through
changes not only in brain regions but also in liver energy metabolism, which
are further reflected in changes in plasma levels of metabolites; (2) the
possible presence in trout brain of an astrocyte–neuron lactate shuttle
similar to that found in mammals; and (3) the lack of capacity of lactate to
mimic in vitro (but not in vivo) glucose effects in fish
glucosensing regions.
Key words: rainbow trout, glucosensor, lactate, glucose homeostasis, hypothalamus, hindbrain, Brockmann bodies
|
INTRODUCTION |
|---|
|
TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
|---|
). A
primary increase in hypothalamic glucose levels lowers blood glucose through
inhibition of glucose production. This process requires conversion of glucose
to lactate followed by stimulation of pyruvate metabolism, which leads to
activation of ATP-sensitive potassium channels
(Lam et al., 2005). Moreover,
lactate and ketones can serve as alternate energy sources or signalling
molecules to glucose once it is transported into neurons by monocarboxylate
transporter (MCT) (Kang et al.,
2004). Glucose is the major source of lactate formed within the
brain, which can be generated in large amounts under certain conditions. Large
transmembrane fluxes of lactate are characteristic of cultured neurons and,
specially, astrocytes, and lactate production and export rates are high
(Dienel and Hertz, 2001). The
transfer of glucose-derived lactate from astrocytes to neurons is referred to
as the astrocyte–neuron lactate shuttle (ANLS) hypothesis
(Pellerin and Magistretti,
1994). Briefly, blood-borne glucose, which is the major energy
substrate for the brain, enters the brain parenchyma via glucose
transporter 1 (GLUT1; also known as SLC2A1) located on endothelial cells
forming capillaries. It is then available to both neurons and astrocytes
via specific glucose transporters (GLUT3 and GLUT1). In astrocytes,
glucose is stored as glycogen. Owing to basal conversion of glucose into
lactate through glycolysis and its release into the extracellular space,
especially by astrocytes, an extracellular lactate pool is maintained. Lactate
will leave astrocytes by a specific transporter, monocarboxylate transporter 1
(MCT1). Meanwhile, in addition to glucose, activated neurons will take up
lactate from the extracellular pool via their own specific
monocarboxylate transporter, MCT2. Lactate will be converted to pyruvate,
favoured by the preferential expression of lactate dehydrogenase 1 (LDH1)
isoform in neurons, before entering the tricarboxylic acid cycle. Use of
lactate by neurons, in addition to glucose, could have multiple purposes as it
increases redox potential by providing cytoplasmic NADH and it can be used to
generate ATP as well as enter into glutamate synthesis
(Pellerin, 2003).
There have been no studies about ANLS in fish, but there is evidence for
the ability of the brain to use lactate as a fuel in the absence of glucose
(Soengas et al., 1998).
Moreover, in a recent study in rainbow trout
(Polakof et al., 2007a) we
observed that the addition of lactate to the incubation medium decreased
glucokinase (GK; also known as hexokinase IV) activity as the glucose
concentration in the medium increased in hypothalamus and hindbrain but not in
Brockmann bodies [BB; a distinct grouping of pancreatic endocrine cells near
to the gall bladder (Youson et al.,
2006)]. Those results allowed us to hypothesize that lactate may
have a similar metabolic role in brain as that described in mammals
(Marty et al., 2007).
Lactate is known to reduce glucose sensitivity in glucose-inhibited neurons
of suckling rats (Song and Routh,
2006), and brain of suckling rats can use lactate as fuel
(Song and Routh, 2006) in a
way similar to that of fish brain (Soengas
et al., 1998). We have demonstrated in previous studies the
existence of glucosensing mechanisms in hypothalamus, hindbrain and BB of
rainbow trout (Soengas et al.,
2006; Polakof et al.,
2007a; Polakof et al.,
2007b). In hypothalamus and hindbrain incubated in vitro,
lactate treatment elicited a decrease in GK activity
(Polakof et al., 2007a), which
may also support a similar capacity for lactate in glucosensing brain regions
of fish. Therefore, we hypothesized that lactate, under high glucose
concentration, induces an inhibitory effect on the glucosensing machinery,
whereas under low glucose concentrations lactate would be used as an
alternative fuel, thus maintaining the potential of glucosensing
(Polakof et al., 2007b).
Moreover, if the BB does not use lactate as a fuel in a way similar to the
mammalian pancreas (Kang et al.,
2004), no effects of lactate would be expected in BB compared with
brain regions.
Therefore, we hypothesize the existence of central effects of glucose and
lactate on systemic glucose homeostasis in rainbow trout, in a way that
activation of neuronal pyruvate flux is required for hypothalamic glucose
sensing as well as for control of blood glucose levels and liver glucose
metabolism, therefore supporting the existence of ANLS in fish. Therefore, we
have treated fish intracerebroventricularly with lactate or glucose to assess
how glucose metabolism responds to increased levels of metabolites within the
brain. To distinguish these effects from those induced by peripheral increases
of metabolites, we have also carried out intraperitoneal treatments with
lactate. Moreover, we also aimed to elucidate whether or not, in fish, lactate
is able to induce similar responses to those induced by glucose in
glucosensing tissues such as hypothalamus, hindbrain and BB. Thus, those
regions were incubated in vitro with increased concentrations of
lactate in the presence of different agents such as (1) oxamate, an inhibitor
of LDH (Wong et al., 1997),
(2) -cyano-4-hydroxy cinnamate (4-CIN) and
4,4'-Diisothiocyanatostilbene-2,2'-disulfonic acid disodium salt
hydrate (DIDS), inhibitors of lactate transport through MCT
(Cassady et al., 2001), (3)
dichloroacetate (DCA), a stimulator of pyruvate dehydrogenase
(Itoh et al., 2003), (4)
2-deoxy-D-glucose (2-DG), an inhibitor of glucose use, and (5)
glucose.
|
MATERIALS AND METHODS |
|---|
|
TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
|---|
In vivo experiments
Intraperitoneal injections
Following 1 month acclimation period, fish were randomly assigned to 100 l
experimental tanks, and each tank was randomly assigned to one of four
experimental treatments. Fish were lightly anaesthetized with MS-222 (50 mg
l–1) buffered to pH 7.4 with sodium bicarbonate, weighed and
given an intraperitoneal (IP) injection of 5 ml kg–1 body
mass of Cortland saline alone (control, N=8) or containing different
treatments: L-(+)-lactate (22.5 mg kg–1 body mass;
N=8), L-(+)-lactate (45 mg kg–1 body
mass; N=8) or sodium oxamate (22.5 mg kg–1 body
mass; N=8). Concentrations were selected based on studies carried out
in mammals addressing the role of lactate in energy metabolism
(Cassady et al., 2001;
Lam et al., 2005). Sampling
was carried out 6 h after injection since this time was observed in previous
experiments to elicit changes in the glucosensing system in rainbow trout
(Polakof et al., 2007a). Fish
were fasted for 24 h before injection to ensure basal hormone levels were
achieved.
Intracerebroventricular injections
Fish were randomly assigned to 100 l experimental tanks, and each tank was
randomly assigned to one of three experimental treatments.
Intracerebroventricular (ICV) injections were administered by following
co-ordinates verified for accurate placement into the third ventricle with
Methylene Blue dye and histological examination of brain tissues as described
previously (Sangiao-Alvarellos et al.,
2003; Sangiao-Alvarellos et
al., 2004). Briefly, prior injection, the fish were anaesthetized
as above and placed on a Plexiglas board with Velcro© straps adjusted to
hold them in place. A 29.5 gauge needle attached through a polyethylene
cannula to a 10 µl Hamilton syringe was aligned with the sixth preorbital
bone at the rear of the eye socket, and from this point the syringe was moved
through the space in the frontal bone into the third ventricle. Lack of injury
to the ventricle lining was demonstrated by microscopic evaluation of the
lining following histological processing of the brain. The plunger of the
syringe was slowly depressed to dispense 1 µl 100 g–1 body
mass of Cortland saline alone (control, N=11) or containing
L-(+)-lactate (400 µg µl–1; N=11)
or D-glucose (400 µg µl–1; N=11).
The mass of the fish used in the experiments was very homogeneous resulting in
a homogeneous ICV volume. After injection, fish were returned to their tanks
and allowed to recover. After 6 h fish were removed from the holding tanks and
sampled. Fish were fasted for 24 h before injection to ensure basal hormone
levels were achieved.
After IP or ICV injections, fish were removed from replicate holding tanks,
anaesthetized as above and weighed. Blood was obtained by caudal puncture with
ammonium-heparinized syringes. Plasma was obtained after centrifugation of
blood (1 min at 10 000 g) and divided into two aliquots. One
aliquot was immediately frozen in liquid nitrogen for the assessment of plasma
protein while the second aliquot was deproteinized (6% perchloric acid) and
neutralized (1 mol l–1 potassium bicarbonate) before freezing
in liquid nitrogen and further storage at –80°C until assayed. The
liver and BB were removed, frozen in liquid nitrogen, and stored at
–80°C until assayed. The brain was removed, placed on a chilled
Petri dish and the hypothalamus and hindbrain obtained as described previously
(Soengas et al., 2006), frozen
in liquid nitrogen and stored at –80°C until assayed.
In vitro experiments
In vitro experiments were carried out as described before
(Polakof et al., 2007a). Every
morning of an experiment, fish were dipnetted from the tank, anaesthetized
with MS-222 (50 mg l–1) buffered to pH 7.4 with sodium
bicarbonate, decapitated and weighed. The hypothalamus and hindbrain were
removed and dissected as described previously
(Soengas et al., 2006;
Polakof et al., 2007a;
Polakof et al., 2007b); BB
were also dissected and cleaned of surrounding vessels, bile ducts, and
connective tissue. Tissues were rinsed with modified Hanks' medium (92.56 mmol
l–1 NaCl; 3.63 mmol l–1 KCl, 2.81 mmol
l–1 NaHCO3, 0.85 mmol l–1
CaCl2, 0.55 mmol l–1 MgSO4, 0.4 mmol
l–1 KH2PO4, 0.23 mmol
l–1 Na2HPO4, 7.5 mmol
l–1 Hepes, 50 i.u. ml–1 penicillin and 50 mg
ml–1 streptomycin sulphate, pH 7.0; referred to a basal
medium), sliced in chilled Petri dishes, placed in a chilled Petri dish
containing 100 ml of modified Hanks' medium g–1 tissue, and
gassed with 0.5% CO2/99.5% O2. To ensure adequate mass,
tissues were combined from different fish resulting in pools of three
hypothalami, three to four hindbrains, and three BB. The tissue was finely
minced and mixed and then placed in 48-well culture plates with 100 ml of
modified Hanks' medium g–1 tissue.
Preliminary experiments indicated that the optimal incubation period was 1 h (data not shown) at 15°C. In a second preliminary experiment, tissues were incubated at 15°C for 1 h with modified Hanks' medium containing 0, 0.5, 1, 2, 4 and 8 mmol l–1 lactate (previously neutralized) to determine the linear range of response in parameters linked to changes in lactate concentration. Good linearity was observed at concentrations from 2 to 8 mmol l–1 lactate (data not shown) and these were selected for further experiments.
All subsequent experiments used freshly obtained tissues incubated in
48-well culture plates at 15°C for 1 h with 100 ml g–1
modified Hanks' medium that was gassed with 0.5% CO2/99.5%
O2. Control wells contained medium with 2, 4 or 8 mmol
l–1 L-(+)-lactate (previously neutralized).
Treated wells contained medium at the same lactate concentration and one of
the selected agents related to lactate metabolism. These included (final
concentration): an inhibitor of lactate dehydrogenase (50 mmol
l–1 sodium oxamate), an inhibitor of pyruvate dehydrogenase
complex (1 mmol l–1 sodium dichloroacetate; DCA), inhibitors
of the monocarboxylic acid transporter [1 mmol l–1
-cyano-4-hydroxy cinnamate (4-CIN) and 1 mmol l–1
4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid disodium salt
hydrate (DIDS)], an inhibitor of glucose utilization (10 mmol
l–1 2-deoxy-D-glucose) and D-glucose
(10 mmol l–1). All reagents were dissolved in modified Hanks'
medium, except for DIDS (0.5% dimethylsulphoxide; DMSO) and 4-CIN (0.5%
ethanol). The concentrations of DIDS and 4-CIN were selected based on those
previously used in fish (Soengas and Moon,
1995) and mammals (Philis et
al., 2001) whereas for the other agents, concentrations were
selected based on experiments carried out in mammals
(Wong et al., 1997;
Wender et al., 2000;
Cassady et al., 2001;
Itoh et al., 2003;
Lam et al., 2005). No effects
on the parameters assessed were observed for the vehicle alone (data not
shown). After 1 h incubation, tissues were quickly removed, filtered, rinsed
with modified Hanks' medium, frozen in liquid nitrogen, and stored at
–80°C until assay.
For each experiment, one set of 21 tissue pools was assessed (seven treatments and three lactate concentrations) for enzyme activities (GK and LDH), whereas a separate set of 21 tissue pools was used for the assay of tissue metabolites (lactate, glycogen and glucose levels). The number of independent experiments carried out for enzyme activities was three (N=3) for treatments and ten (N=10) for controls, whereas a similar number of experiments was carried out to assess tissue metabolites.
Assessment of metabolite levels and enzyme activities
Plasma glucose and lactate levels were determined enzymatically using
commercial kits (Spinreact, Barcelona, Spain) adapted to a microplate format.
Plasma total -amino acids were assessed colorimetrically using the
ninhydrin method of Moore (Moore,
1968); alanine was used to develop a standard curve.
Samples used to assess metabolite levels in tissues were homogenized
immediately by ultrasonic disruption in 7.5 vols of ice-cooled 6% perchloric
acid, and neutralized (using 1 mol l–1 potassium
bicarbonate). The homogenate was centrifuged, and the supernatant used to
assay metabolites. Glycogen levels were assessed using the method of Keppler
and Decker (Keppler and Decker,
1974). Glucose obtained after glycogen breakdown (after
subtracting free glucose levels) was determined with a commercial kit
(Biomérieux, Madrid, Spain). Glucose 6-phosphate levels were estimated
by decreases in absorbance of NADH at 340 nm after incubation of sample with
(in mmol l–1) 0.1 Na2HPO4 and 0.1
NaH2PO4 (pH 7.0), 0.2 NADP+, and excess
glucose 6-phosphate dehydrogenase. Di-hydroxiacetone phosphate (DHAP) levels
were assessed by decreases in absorbance of NADH at 340 nm after incubation of
sample with (in mmol l–1) 50 imidazole (pH 7.6), 0.2 NADH,
and excess -glycerophosphate dehydrogenase. Lactate and total
-amino acids were assessed as described above for plasma samples.
Samples for enzyme activities in tissues were homogenized by ultrasonic
disruption with 9 vols ice-cold buffer consisting of 50 mmol
l–1 Tris (pH 7.6), 5 mmol l–1 EDTA, 2 mmol
l–1 1,4-dithiothreitol, and a protease inhibitor cocktail
(Sigma Chemical Co., St Louis, MO, USA; P-2714). The homogenate was
centrifuged and the supernatant used immediately for enzyme assays. Enzyme
activities were determined using a microplate reader SPECTRAFluor (Tecan,
Grödig, Austria) and microplates. Reaction rates of enzymes were
determined by the increase or decrease in absorbance of NAD(P)H at 340 nm. The
reactions were started by the addition of supernatant (15 µl) at a
pre-established protein concentration, omitting the substrate in control wells
(final volume 265–295 µl), and allowing the reactions to proceed at
20°C for pre-established times (3–10 min). Enzyme activities are
expressed per mg protein. Protein was assayed in triplicate in homogenates
using microplates according to the bicinchoninic acid method
(Smith, 1985) with bovine
serum albumin (Sigma) as standard. Enzyme activities were assessed at maximum
rates determined by preliminary tests to determine optimal substrate
concentrations. Fructose 1,6-bisphosphatase, glucose 6-phosphatase (G6Pase),
glycogen synthase (GSase), glycogen phosphorylase, LDH-R (LDH reductase),
LDH-O (LDH oxidase), Low Km hexokinase I (HK), GK and
pyruvate kinase (PK) activities were estimated as described previously
(Soengas et al., 2006;
Polakof et al., 2007b).
RT–PCR analysis of glucokinase (glucokinase gene) expression
Total RNA was extracted from frozen brains using TRIzol reagent as
recommended by the manufacturer (Sigma). The quality and quantity of the
isolated RNA was assessed spectrophotometrically. Total RNA (2 µg) was
reverse transcribed into first-strand cDNA when primed with random primers
(Amersham Biosciences, Barcelona, Spain) using M-MLV reverse transcriptase for
1 h at 37°C by methods recommended by the manufacturer (Promega, Madison,
WI, USA). The expression of single isoforms of glucokinase (GK; via GK gene
expression) were assessed. GK cDNA was PCR-amplified using specific primers
developed for rainbow trout by Panserat et al.
(Panserat et al., 2000):
5'-GATGTTGGTGAAGGTGGGG-3' (forward) and
5'-TTCAGTAGGATGCCCTTGTC-3' (reverse); amplification with these
primers resulted in a 250 bp product. The housekeeping gene used to assess the
relative cDNA levels of GK was rainbow trout 18s, which was amplified by PCR
using specific primers (Kusakabe et al.,
2006): 5'-TCAAGAACGAAAGTCGGAGG-3' (forward) and
5'-GGACATCTAAGGGCATCACA-3' (reverse). The PCR reactions were
carried out using a PTC-200 Peltier thermal cycler (MJ Research Inc, Waltham,
MA, USA) in a final volume of 20 µl, containing cDNA template (8 µl for
GK, and 1 µl for 18s), 1x buffer (50 mmol l–1 KCl,
20 mmol l–1 Tris–HCl and 0.1% Triton X-100), 0.2 mmol
l–1 dNTPs, 1.5 mmol l–1 MgCl2, 2
pmol of each primer (forward and reverse), and 1 i.u. of Taq polymerase
(Ecogen, Barcelona, Spain). The optimal number of cycles for amplification was
established (reactions were terminated in the logarithmic phase of the PCR
reaction). Amplification of cDNA was achieved with an initial denaturation at
94°C followed by 35 cycles of denaturation (94°C for 30 s), annealing
(60°C for 30 s) and extension (72°C for 30 s); a final extension
period of 10 min occurred prior to termination. Negative controls without
reverse transcriptase or cDNAs were performed to ensure observed bands were
not a result of contamination. The PCR products were subjected to
electrophoresis in 1.5% agarose gel. The size of PCR reaction products was
established by comparison with a 50 bp DNA step ladder (Promega).
Semi-quantification of PCR products was performed by densitometric analysis of
the bands of interest using the gel electrophoresis documentation and analysis
system EDAS 290 (Kodak, Rochester, NY, USA) of images captured from UV
transiluminated ethidium-bromide-stained gels. Results are shown as arbitrary
units and represent the ratio (%) between GK and 18s expression. To ensure the
bands of interest were in fact trout GK, each band was gel-purified using the
GFX PCR DNA and gel band purification kit (Amersham Biosciences) and cloned
using Pgem-T Vector Systems II (Promega). White colonies were amplified by PCR
using primers T7 and M13 (flanking the insert) and sequenced in both
directions using the dRhodamine terminator cycle sequencing kit (Applied
Biosystems, Foster City, CA, USA). The reactions were run on an Applied
Biosystems automated sequencer model ABI PRISM 310. The resulting sequences
were compared with known sequences in GenBank using BLASTn
(http://www.ncbi.nlm.nih.gov/BLAST/).
Statistics
Data are presented as means ± s.e.m. Comparisons among groups were
performed using SigmaStat (SPSS Inc., Chicago, IL, USA) by one-way ANOVA
(in vivo experiments) or two-way ANOVA (in vitro
experiments, with lactate concentration and treatment as main factors). When
necessary (ratios), data were log transformed to fulfil the conditions of the
analysis of variance. Post-hoc comparisons were made using a
Student–Newman–Keuls test, and differences were considered
statistically significant at P<0.05.
|
RESULTS |
|---|
|
TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
|---|
-amino acid,
glucose 6-phosphate levels, and LDH-R and G6Pase activities, whereas increases
were noticed in DHAP levels and total GSase activity. ICV treatment with
lactate decreased -amino acid levels and LDH-O, PK, G6Pase and total
GSase activities but increased glycogen and glucose 6-phosphate levels and the
percentage GSase a activity.
|
In plasma, glucose levels (Fig.
1A) decreased after ICV lactate treatment but increased when
lactate was administered in IP treatment. Plasma lactate levels
(Fig. 1B) increased when fish
were IP injected with lactate or oxamate, whereas a decrease was observed when
the treatment was administered by ICV injection. Plasma -amino acids
levels (Fig. 1C) showed a
decrease when glucose was administered by ICV injection.
|
|
-Amino acid levels (Fig.
3C) increased in the hypothalamus (L22.5 or 45) after IP lactate
injections, and in hindbrain (L400) after ICV injection. In hindbrain
-amino acid levels increased after IP lactate treatment.
|
|
|
In vitro experiments
Hypothalamus
In hypothalamus, no changes were observed in glucose levels
(Fig. 6A) in control tissues.
No changes were found either after oxamate or 4-CIN treatments. However,
higher glucose levels than controls were detected after D-glucose
and 2-DG treatments. By contrast, a lower glucose concentration was found in
hypothalamus incubated with DCA. Lower levels than controls were observed
after treatment with DIDS at 8 mmol l–1 lactate. Glycogen
levels (Fig. 6B) did not show
any changes in control tissues, whereas lower values than controls were found
after oxamate, 4-CIN or DIDS treatments at 4 mmol l–1
lactate. Lower glycogen levels than controls were observed after incubation
with DCA at 2 and 8 mmol l–1 lactate; but threefold higher
levels were detected at 4 mmol l–1 lactate. Higher glycogen
levels than controls were detected after glucose treatment at 2 and 4 mmol
l–1 lactate, but almost undetectable levels were found at 8
mmol l–1 lactate after 2DG incubations. A clear positive
correlation was found between lactate concentration in the medium and lactate
levels (Fig. 6C). Higher
lactate levels than controls were found after glucose treatments at 2 and 4
mmol l–1 lactate whereas no changes were found in the other
treatments. No changes in GK activity (Fig.
6D) were detected in the control group and lower activities than
controls were observed after DIDS and 4-CIN (all lactate concentrations), 2-DG
(2 and 8 mmol l–1 lactate), DCA (2 mmol l–1
lactate) or oxamate (4 and 8 mmol l–1 lactate) treatments.
However, higher GK activities than controls were found when glucose was added
to the medium at 2 and 4 mmol l–1 lactate concentration.
LDH-R activity in control samples was lower at 8 mmol l–1
than at 2 or 4 mmol l–1 lactate whereas lower activities than
controls were observed after oxamate, DIDS (higher values at 8 mmol
l–1 lactate) or DCA treatments.
|
Hindbrain
In hindbrain, no changes were observed in glucose levels in controls
(Fig. 7A) or after oxamate,
DIDS or 4-CIN incubations whereas higher values than controls were noticed
after 2-DG, glucose or DCA treatments. Glycogen levels in control group
(Fig. 7B) were higher at 2 mmol
l–1 than at 4 or 8 mmol l–1 lactate whereas
no differences among lactate concentrations were found with oxamate, DIDS, DCA
or 4-CIN treatments. However, a peak in glycogen levels was observed at 4 mmol
l–1 lactate in glucose incubations. Except with DCA treatment
(higher values than controls) all treatments showed lower glycogen levels than
controls at 2 mmol l–1 lactate and higher at 4 and 8 mmol
l–1 lactate. A positive correlation between lactate levels
and lactate concentrations in the media
(Fig. 7C) was found in control
treatments. Oxamate in the medium produced an increase in hindbrain lactate
levels at 4 and 8 mmol l–1 lactate, whereas lower values than
controls were found after DIDS or DCA treatments at 2 mmol
l–1 lactate. By contrast, at 8 mmol l–1
lactate, DCA treatment enhanced tissue lactate levels. GK activity in controls
(Fig. 7D) showed a peak at 4
mmol l–1 lactate and lower activities at 8 mmol
l–1 lactate. After DIDS, DCA or 2-DG treatments, GK
activities showed lowest values at 4 mmol l–1 lactate. GK
activity was not affected by glucose in the medium at 2 or 4 mmol
l–1 lactate, but was at very low values at 8 mmol
l–1 lactate. No changes were observed in LDH-R activity
(Fig. 7E) in control samples,
although lower activities than controls were found with all treatments.
|
Brockmann bodies
In BB, no changes were observed in glucose levels
(Fig. 8A) in control samples.
No significant differences were found compared with controls under oxamate,
DIDS, DCA or 4-CIN treatments. However, higher glucose levels than controls
were obtained in tissues incubated with either glucose or 2-DG. No differences
were found in glycogen levels (Fig.
8B) in controls. A positive correlation with lactate in the medium
was observed in tissues incubated with oxamate. In addition, glycogen levels
were higher than controls in tissues incubated with 2-DG (all lactate
concentrations), DIDS (4 and 8 mmol l–1 lactate), glucose or
4-CIN (both at 8 mmol l–1 lactate) treatments. A positive
correlation between lactate concentration in the medium and lactate levels
(Fig. 8C) was found in control
tissues. Lactate levels were lower than controls after glucose (at 2 and 4
mmol l–1 lactate), DIDS (2 mmol l–1
lactate), DCA (2 and 8 mmol l–1 lactate) or DIDS (2 mmol
l–1 lactate) treatments. By contrast, a peak in lactate
levels was evident (maximum at 4 mmol l–1 lactate) when 2-DG
was added to the medium. A negative correlation with lactate in the medium was
found in GK activity (Fig. 8D)
in control tissues whereas a positive correlation was found when glucose was
added to this medium. However, no GK activity was detectable after 2-DG (all
lactate concentrations), DCA or 4-CIN (at 8 mmol l–1 lactate)
or DIDS (4 and 8 mmol l–1 lactate) treatments. When oxamate
was added to the culture medium higher GK activity than controls was observed
at 8 mmol l–1 lactate.
|
|
DISCUSSION |
|---|
|
TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
|---|
), neurons utilize lactate produced by astrocytes to provide
energy (Brown et al., 2001). In
fish, there are many pieces of evidence that indirectly suggest the existence
of the ANLS, such as the fact that rainbow trout brain can oxidize lactate in
the absence of glucose at almost the same rates as glucose
(Soengas et al., 1998) and
recent evidences obtained in vitro in rainbow trout hypothalamus and
hindbrain demonstrating that the presence of lactate in the culture media
induces changes in glucose metabolism in those tissues
(Polakof et al., 2007a).
Moreover, mammalian neurons have developed mechanisms for sensing glucose,
designed to protect them from hypoglycemic injury
(Routh, 2002). In this way,
moderate increases in extracellular hypothalamic glucose levels are sufficient
to lower blood glucose levels through a robust inhibition of liver glucose
production (Lam et al., 2005).
There is no evidence to date in fish literature regarding such a
counteregulatory mechanism.
In the present study, ICV infusion of glucose elicited in hypothalamus and
hindbrain increased glucose levels and GK activity (increasing glucosensing
capacity) addressing a local response of those brain regions to increased
glucose levels, in a way similar to that previously observed after IP infusion
and in vitro experiments (Polakof
et al., 2007a; Polakof et al.,
2007b). Furthermore, the increase in cytosolic glucose levels
after ICV treatment is similar to that observed in mammals
(Lam et al., 2005). Lactate IP
treatment increased both glucose and lactate levels in plasma, but only the
increase in glucose but not lactate levels was blocked by oxamate. The effect
on plasma glucose levels suggests an enhancement of glucose production in
liver through glycogenolysis and/or gluconeogenesis. In fact, both pathways
were activated in the present experiment (data not shown). When glucose
treatment was ICV, no changes were noticed in plasma glucose levels in
contrast to the decreased blood glucose levels occurring in mammals after ICV
glucose infusion (Lam et al.,
2005). However, this counteregulatory response was observed after
lactate injection, resulting in decreased glucose and lactate levels in plasma
in agreement with that observed in mammals
(Lam et al., 2005).
Glucose levels clearly increased after IP lactate treatment in hypothalamus
whereas oxamate generally blocked those responses. This suggests that lactate
is being converted into glucose. Glycogen levels clearly increased in
hypothalamus after IP lactate treatment with no major changes found after ICV
lactate treatment. Again this effect was blocked by oxamate, and was not
apparent in BB. Together with the results observed for glucose levels, these
results suggest that lactate is being used in brain regions as a glycogenic
substrate in a way similar to that suggested in mammalian brain according to
the ANLS hypothesis (Pellerin and
Magistretti, 1994). After ICV injections of lactate, we found an
increase in GK activity in hypothalamus and hindbrain reflecting increased
glucosensing capacity that was similar to that observed under hyperglycemic
conditions in vivo (Polakof et
al., 2007b) in agreement with increased GK expression in mammalian
hindbrain after lactate infusion (Vavaiya
and Briski, 2007). It is possible that neurons in both fish brain
regions interpret this lactate increase as a rise in glucose levels,
activating the glucosensing system and starting a counteregulatory response.
Moreover, other findings of the present study were similar to those in mammals
(Routh, 2002), pointing to the
existence of an ANLS in fish, because: (1) IP lactate treatment induced a rise
in plasma glucose and a subsequent increase in brain glycogen levels
(hypothalamus and hindbrain); (2) ICV lactate treatment decreased plasma
glucose, accompanied in brain regions by decreased glycogen levels and
increased lactate levels and LDH-O activity; (3) IP lactate treatment induced
increased LDH-O activity in brain; and (4) the increase in lactate levels in
the in vitro experiments when glucose (but not 2-DG) is added to the
medium. However, in vitro assays showed that an elevation in lactate
levels (from 2 to 8 mmol l–1) in control conditions (no
glucose) did not stimulate GK activity and did not raise glucose levels, which
is in contrast to the ANLS hypothesis
(Routh, 2002;
Marty et al., 2007).
ICV treatment with glucose or lactate also elicited changes in liver
function, reflecting a functional connection between detection of fuels within
brain and production/release of fuels from liver, in a way similar to that
proposed in mammals (Marty et al.,
2007). Thus, ICV treatment with glucose induced enhanced
glycogenolysis, and a fall of amino acid levels accompanied by decreased
glucose 6-phosphate levels and increased capacity for glycolysis (DHAP and
glyceraldehyde 3-phosphate levels). It seems that the presence of glucose in
brain produces a signal indicative that no production and release of glucose
from liver is needed to sustain plasma glucose levels. This is reflected by
the fact that G6Pase activity actually decreased after glucose treatment and
therefore less glucose is being exported into the plasma. When lactate was
supplied by ICV infusion, changes were observed in liver metabolism that were
different in some cases from those detected after glucose treatment such as
the absence of changes in glycogen levels, the increase in glucose 6-phosphate
levels, and the decrease in LDH-O activity. However, lactate treatment
produced an inhibition of glucose release into the plasma through a fall in
G6Pase activity as well as a decrease in amino acid levels, and glycolytic
potential decreased. Altogether, it seems that ICV lactate treatment is
causing a decrease in glucose release from liver and at the same time is
inducing, in liver, a lower capacity for oxidation of lactate, based on the
decrease observed in LDH-O activity. Therefore, ICV lactate treatment is
inducing some sort of metabolic depression in liver by inhibiting catabolic
pathways (PK and LDH-O activities), reflecting the energy signal (rise in
lactate levels interpreted as a rise in glucose) coming from the brain.
However, other metabolic pathways, such as lipid or amino acid metabolism, may
be not affected by treatment. These results are comparable to those observed
in mammals after similar ICV treatment
(Lam et al., 2005), although
in mammals glucose and lactate elicited quantitatively the same response,
which is not the case in rainbow trout. Interestingly, changes in amino acid
levels were similar, increasing in all brain regions after lactate treatment,
whereas oxamate did produce the converse effect. This is probably indicative
of lactate is being used as carbon skeletons for synthesis of amino acids
within the brain (probably in astrocytes).
In BB, ICV treatment with glucose elicited a glucosensing response characterized by an increase in GK and a decrease in low Km HK activities. This may suggest an increase in plasma insulin levels as part of the system trying to counteregulate the supposed increase in plasma glucose levels elicited by ICV treatment.
GK activity showed dramatic increases after lactate treatment in all
regions assessed (except BB after ICV treatment). In IP treatments, lactate is
probably being converted into glucose by the liver, resulting in higher plasma
levels of glucose, and, as a result, an increase of glucose availability. A
higher GK activity and expression in brain and BB under hyperglycemic
conditions (but not in low Km HK activity) is in agreement
with previous experiments in rainbow trout
(Polakof et al., 2007b). In
ICV treatments, the increased GK activity produced by lactate could be due to
increased glucose production in situ. However, ICV lactate treatment
did not alter GK expression in any of the tissues assessed, in contrast to the
increased GK expression observed in mammalian hindbrain
(Vavaiya and Briski, 2007),
suggesting that changes in activity were due to alosteric/covalent
modification rather than to changes in enzyme expression. However, since gene
expression assessed by conventional RT–PCR is not strictly quantitative,
the mRNA expression data reported in Fig.
2 must be interpreted cautiously. In any case, glucose or lactate
ICV treatments did produce the same effect on GK activity thus resulting in
the same metabolic signal.
Lactate levels decreased in brain regions assessed after IP but not ICV
treatments with lactate, which was not observed after oxamate treatment. Since
plasma lactate levels also increased simultaneously, a higher entry of lactate
in tissues through MCT could result in increased lactate levels. However, a
higher portion of increased lactate levels in plasma is probably not directed
towards the brain but to other tissues. Milligan and Girard
(Milligan and Girard, 1993)
described that the increased lactate levels observed in plasma of rainbow
trout after exercise are mostly directed to white muscle and liver, which in
the case of white muscle is logical considering the size of the tissue.
These results suggest that lactate metabolism in brain can participate in
the regulation of glucose homeostasis through changes not only in brain
regions but also in liver energy metabolism that are further reflected in
changes in plasma levels of metabolites. By contrast, this connection is not
apparently present in BB. This is in agreement with the finding that lactate
does not induce insulin release in the mammalian pancreas
(Ishihara et al., 1999;
Kang et al., 2004) since in
pancreatic cells LDH activity is very low (similar to that observed in BB in
the present study). Furthermore, several findings also provide evidence for
the presence in trout brain of an ANLS similar to that proposed in mammals,
supporting previous data obtained in rainbow trout brain of the use of lactate
as fuel in the absence of glucose (Soengas
et al., 1998).
Evidence for a role of lactate in the activity of glucosensing tissues
Glucosensing neurons in mammals may sense substrates other than glucose,
such as lactate (Himmi et al.,
2001; Vavaiya and Briski,
2007). Moreover, in mammalian glucosensing systems glycolytic
intermediates such as lactate mimic glucose effects
(Yang et al., 1999;
Schuit et al., 2001), and
potentially use astrocyte-derived lactate as an alternate regulator of firing
rate (Borg et al., 2003;
Lam et al., 2005;
Vavaiya and Briski, 2007).
This is different from β-cells that are unable to use lactate to increase
insulin secretion (Kang et al.,
2004).
Since, in previous studies (Polakof et
al., 2007a), we hypothesized that lactate could be also considered
as a metabolic signal in brain glucosensing systems, we aimed to assess the
role of lactate as a metabolic signal in glucosensing tissues such as
hypothalamus, hindbrain and BB. In control fish, incubation with increased
concentrations of lactate resulted in increased lactate levels in all brain
regions assessed, as well as in BB, suggesting a fast transport of lactate.
Blockade of neuronal lactate uptake by 4-CIN in mammals resulted in dramatic
increases in extracellular lactate levels
(Philis et al., 2001). By
contrast, in the present study, the presence of 4-CIN did not alter levels of
lactate in the brain regions assessed. A similar lack of action occurred with
another MCT blocker, DIDS, suggesting either the absence of MCT in brain
regions and/or the presence of different isoforms of MCT
(Poole and Halestrap, 1993).
Strikingly, the presence of oxamate in the media resulted in lactate levels
higher than those of controls only in hindbrain but not in the other regions
assessed. Treatment with glucose did not modify the trend observed in
controls, although levels were higher than in controls in hypothalamus but
lower in BB. The presence of an inhibitor of glucose use, such as 2-DG,
despite minor changes does not generally alter lactate levels in any tissue
assessed. The presence of DCA did not significantly alter lactate levels in
tissues, except for the decrease observed in hindbrain and BB at low lactate
concentrations, which is in agreement with the situation in mammals where DCA
induced in brain decreased lactate levels
(Itoh et al., 2003).
The presence of glucose in the culture media did not alter lactate levels
and LDH activity (except in hypothalamus). This is comparable to the situation
in mammals where glucose concentration did not alter utilization of lactate,
even under hyperglycemia (Bliss and
Sapolsky, 2001). The trend displayed by lactate levels in controls
was different from that of LDH activity, which did not increase with lactate
concentration in any tissue. Oxamate generally inhibited LDH activity in all
tissues assessed, in agreement with its role in mammals
(Wong et al., 1997).
Strikingly, considering the lack of effect of oxamate treatment on lactate
levels (see above), DIDS treatment decreased LDH activity in all tissues
assessed, whereas 4-CIN was effective only in hindbrain. Therefore, in the
tissues assessed, the presence of increased lactate concentrations in the
media did not significantly alter LDH activity. This raises another question
of whether or not parameters related to glucosensing capacity (mainly glucose
levels and GK activity) can be activated by lactate, mimicking the effects of
glucose, as described in mammals (Yang et
al., 1999; Schuit et al.,
2001).
Glucose and glycogen levels in controls were not affected by lactate
concentration in the media in any tissue assessed, confirming that lactate is
not being used as a gluconeogenic substrate, in agreement with the low
gluconeogenic capacity already suggested for those tissues
(Soengas et al., 2006). The
presence of glucose in the media elevated both glucose and glycogen levels
compared with controls in all tissues assessed. The presence of glucose
per se is therefore increasing glucose levels and the levels of other
related metabolites (i.e. glycogen) irrespective of lactate concentration. The
different agents tested elicited very different responses on glucose and
glycogen levels that can be summarized as increased levels of glycogen after
oxamate, DIDS, 4-CIN, 2-DG and DCA treatments (the latter only in hindbrain)
and no major changes in glucose levels, except for the increase after 2-DG
treatment, in all tissues. The increased glycogenic capacity is probably the
result of a blockade of lactate transport and oxidation in neurons. Under
those conditions, the only pathway available for lactate use would be
synthesis of glucose 6-phosphate through gluconeogenesis, to store as
glycogen. In a similar way, the inhibition of glucose use by 2-DG clearly
resulted in increased glucose levels that cannot be further metabolized.
GK activity in controls did not differ significantly at the different
lactate concentrations assessed, except in hindbrain in which there was
increased activity at 4 mmol l–1 lactate. It is interesting
that in mammals increased lactate concentration has been shown to inhibit
glucose utilization in brain regions under normo- and hypo- but not
hyperglycemic conditions (Bliss and
Sapolsky, 2001) in a comparable situation to that herein reported.
DIDS or 4-CIN treatment decreased GK activity at all concentrations of lactate
assessed in all tissues, which is again striking considering that those
molecules did not affect lactate levels (see above). Since GK is not inhibited
by glucose 6-phosphate and therefore no product inhibition can occur, this
probably indicates that at least part of the glucose being phosphorylated by
GK resulted from lactate synthesis. DCA treatment decreased GK activity in all
tissues, which could be the result of the effect of DCA on glycogen metabolism
(stimulating glycogen storage) in a way similar to that observed in mammalian
liver (Kato-Weinstein et al.,
1998). Comparing this response with that previously observed when
the same tissues were incubated with glucose alone
(Polakof et al., 2007a) this
suggests that the presence of lactate is altering the action of glucose on GK
activity possibly because it is replacing at least part of the glucose to be
used as fuel.
Altogether, it seems that lactate is not able to induce similar metabolic
changes to those elicited by glucose in the glucosensing regions assessed
in vitro, and therefore does not support the hypothesis of lactate
mimicking glucose effects in fish glucosensing regions
(Polakof et al., 2007a), in
contrast to that occurring in mammals
(Yang et al., 1999;
Schuit et al., 2001). However,
several lines of evidence obtained in vivo such as the similar
increase in GK activity after either glucose or lactate ICV treatments may
suggest an involvement of lactate in the activity of glucosensing tissues, in
a way similar to that proposed in mammals
(Song and Routh, 2005).
In summary, data obtained in the present study provide several pieces of
indirect evidence allowing us to suggest the presence in trout brain of an
astrocyte–neuron lactate shuttle similar to that proposed in mammals
(Routh, 2002;
Marty et al., 2007),
reinforcing previous data obtained in rainbow trout brain of the use of
lactate as fuel (Soengas et al.,
1998). However, since other findings were contradictory,
additional studies should be carried out to obtain direct evidence for an
ANLS, such as pharmacological disruption of metabolic coupling between neurons
and astrocytes followed by histological analysis of neural activation.
Furthermore, lactate metabolism is also apparently involved in glucose
homeostasis through changes in plasma glucose levels and glucose production in
liver. Finally, evidence obtained in the present study in vitro do
not support our previous hypothesis regarding a mimicking effect of lactate
(compared with glucose) in fish glucosensing regions
(Polakof et al., 2007a).
However, evidence obtained in vivo are compatible with such a role,
making necessary more studies to clearly assess the role of lactate in
glucosensing tissues.
LIST OF ABBREVIATIONS
-cyano-4-hydroxy cinnamate
|
Acknowledgments |
|---|
|
References |
|---|
|
TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
|---|
Bliss, T. M. and Sapolsky, R. M. (2001). Interactions among glucose, lactate and adenosine regulate energy substrate utilization in hippocampal cultures. Brain Res. 899,134 -141.[CrossRef][Medline]
Borg, M. A., Tamborlane, W. V., Shulman, G. I. and Sherwin, R.
S. (2003). Local lactate prefusion of the ventromedial
hypothalamus suppresses hypoglycemic counterregulation.
Diabetes 52,663
-666.
Brown, A. M., Wender, R. and Ransom, B. R. (2001). Metabolic substrates other than glucose support axon function in central white matter. J. Neurosci. Res. 66,839 -843.[CrossRef][Medline]
Cassady, C. J., Philis, J. W. and O'Regan, M. H. (2001). Further studies on the effects of topical lactate on amino acid efflux from the ischemic rat cortex. Brain Res. 901,30 -37.[CrossRef][Medline]
Dienel, G. A. and Hertz, L. (2001). Glucose and lactate metabolism during brain activation. J. Neurosci. Res. 66,824 -838.[CrossRef][Medline]
Himmi, T., Perrin, J., Dallaporta, M. and Orsini, J.-C. (2001). Effects of lactate on glucose-sensing neurons in the solitary tract nucleus. Physiol. Behav. 74,391 -397.[CrossRef][Medline]
Ishihara, H., Wang, H., Drewes, L. R. and Wollheim, C. B. (1999). Overexpression of monocarboxylate transporter and lactate dehydrogenase alters insulin secretory responses to pyruvate and lactate in beta cells. J. Clin. Invest. 104,1621 -1629.[Medline]
Itoh, Y., Esaki, T., Shimoji, K., Cook, M., Law, M. J., Kaufman,
E. and Sokoloff, L. (2003). Dichloroacetate effects on
glucose and lactate oxidation by neurons and astroglia in vitro and
on glucose utilization by brain in vivo. Proc. Natl. Acad. Sci.
USA 100,4879
-4884.
Kang, L., Routh, V. H., Kuzhikandathil, E. V., Gaspers, L. D.
and Levin, B. E. (2004). Physiological and molecular
characteristics of rat hypothalamic ventromedial nucleus glucosensing neurons.
Diabetes 53,549
-559.
Kato-Weinstein, J., Lingohr, M. K., Orner, G. A., Thrall, B. D. and Bull, R. J. (1998). Effects of dichloroacetate on glycogen metabolism in B6C3F1 mice. Toxicology 130,141 -154.[CrossRef][Medline]
Keppler, D. and Decker, K. (1974). Glycogen. Determination with amyloglucosidase. In Methods of Enzymatic Analysis (ed. H. U. Bergmeyer), pp.1127 -1131. New York: Academic Press.
Kusakabe, M., Nakamura, I., Evans, J., Swanson, P. and Young,
G. (2006). Changes in mRNAs encoding steroidogenic acute
regulatory protein, steroidogenic enzymes and receptors for gonadotropins
during spermatogenesis in rainbow trout testes. J.
Endocrinol. 189,541
-554.
Lam, T. K. T., Gutierrez-Juarez, R., Pocai, A. and Rossetti,
L. (2005). Regulation of blood glucose by hypothalamic
pyruvate metabolism. Science
309,943
-947.
Marty, N., Dallaporta, M. and Thorens, B.
(2007). Brain glucose sensing, counterregulation, and energy
homeostasis. Physiology
22,241
-251.
Matschinsky, F. M., Magnuson, M. A., Zelent, D., Jetton, T. L.,
Doliba, N., Han, Y., Taub, R. and Grimsby, J. (2006). The
network of glucokinase-expressing cells in glucose homeostasis and the
potential of glucokinase activators for diabetes therapy.
Diabetes 55,1
-12.
Milligan, C. L. and Girard, S. S. (1993). Lactate metabolism in rainbow trout. J. Exp. Biol. 180,175 -193.[Abstract]
Moore, S. (1968). Amino acid analysis: aqueous dimethyl sulfoxide as solvent for the nynhidrin reaction. J. Biol. Chem. 1242,6281 -6283.
Panserat, S., Médale, F., Blin, C., Brèque, J., Vachot, C., Plagnes-Juan, E., Gomes, E., Krishnamoorthy, R. and Kaushik, S. (2000). Hepatic glucokinase is induced by dietary carbohydrates in rainbow trout, gilhead seabream, and common carp. Am. J. Physiol. 278,R1164 -R1170.
Pellerin, L. (2003). Lactate as a pivotal element in neuron-glia metabolic cooperation. Neurochem. Int. 43,331 -338.[CrossRef][Medline]
Pellerin, L. and Magistretti, P. J. (1994).
Glutamate uptake into astrocytes stimulates aerobic glycolysis: a mechanism
coupling neuronal activity to glucose utilization. Proc. Natl.
Acad. Sci. USA 91,10625
-10629.
Philis, J. W., Ren, J. and O'Regan, M. H. (2001). Studies on the effects of lactate transport inhibition, pyruvate, glucose and glutamine on amino acid, lactate and glucose release from the ischemic rat cerebral cortex. J. Neurochem. 76,247 -257.[CrossRef][Medline]
Polakof, S,., Míguez, J. M. and Soengas, J. L. (2007a). In vitro evidences for glucosensing capacity and mechanisms in hypothalamus, hindbrain and Brockmann bodies of rainbow trout. Am. J. Physiol. 293,R1410 -R1420.
Polakof, S., Míguez, J. M., Moon, T. W. and Soengas, J. L. (2007b). Evidence for the presence of a glucosensor in hypothalamus, hindbrain, and Brockmann bodies of rainbow trout. Am. J. Physiol. 292,R1657 -R1666.
Poole, R. C. and Halestrap, A. P. (1993). Transport of lactate and other monocarboxylates across mammalian plasma membranes. Am. J. Physiol. 264,C761 -C782.[Medline]
Routh, V. H. (2002). Glucose-sensing neurons: are they physiologically relevant? Physiol. Behav. 76,403 -413.[CrossRef][Medline]
Sangiao-Alvarellos, S., Bouça, P., Míguez, J. M. and Soengas, J. L. (2003). Intracerebroventricular injections of noradrenaline affect brain energy metabolism of rainbow trout. Physiol. Biochem. Zool. 76,663 -671.[CrossRef][Medline]
Sangiao-Alvarellos, S., Lapido, M., Míguez, J. M. and Soengas, J. L. (2004). Effects of central administration of arginine vasotocin on monoaminergic neurotransmitters and energy metabolism of rainbow trout brain. J. Fish Biol. 64,1313 -1329.[CrossRef]
Schuit, F. C., Huypens, P., Heimberg, H. and Pipeleers, D.
G. (2001). Glucose sensing in pancreatic β-cells. A
model for the study of other glucose-regulated cells in gut, pancreas, and
hypothalamus. Diabetes
50, 1-11.
Smith, P. K. (1985). Measurement of protein using bicinchoninic acid. Anal. Biochem. 150, 76-85.[CrossRef][Medline]
Soengas, J. L. and Moon, T. W. (1995). Uptake and metabolism of glucose, alanine and lactate by red blood cells of the American eel Anguilla rostrata. J. Exp. Biol. 198,877 -888.[Medline]
Soengas, J. L., Strong, E. F. and Andrés, M. D. (1998). Glucose, lactate, and β-hydroxybutyrate utilization by rainbow trout brain: changes during food deprivation. Physiol. Zool. 71,285 -293.[Medline]
Soengas, J. L., Polakof, S., Chen, X., Sangiao-Alvarellos, S. and Moon, T. W. (2006). Glucokinase and hexokinase expression and activities in rainbow trout tissues: changes with food deprivation and refeeding. Am. J. Physiol. 291,R810 -R821.
Song, Z. and Routh, V. H. (2005). Differential effects of glucose and lactate on glucosensing neurons in the ventromedial hypothalamic nucleus. Diabetes 54, 15-22.[Medline]
Song, Z. and Routh, V. H. (2006). Recurrent hypoglycemia reduces the glucose sensitivity of glucose-inhibited neurons in the ventromedial hypothalamus nucleus. Am. J. Physiol. 291,R1283 -R1287.
Vavaiya, K. V. and Briski, K. P. (2007). Caudal hindbrain lactate infusion alters glucokinase, SUR1, and neuronal substrate fuel transporter gene expression in the dorsal vagal complex, lateral hypothalamic area, and ventromedial nucleus hypothalamus of hypoglycemic male rats. Brain Res. 1176,62 -70.[CrossRef][Medline]
Wender, R., Brown, A. M., Fern, R., Swanson, R. A., Farrell, K. and Ransom, B. R. (2000). Astrocytic glycogen influences axon function and survival during glucose deprivation in central white matter. J. Neurosci. 200,6804 -6810.
Wong, C., Rodríguez-Páez, L., Nogueda, B., Pérez, A. and Baeza, I. (1997). Selective inhibition of the sperm-specific lactate dehydrogenase isozyme-C4 by N-isopropyl oxamate. Biochim. Biophys. Acta 1343,16 -22.[CrossRef][Medline]
Yang, X.-J., Kow, L.-M., Funabashi, T. and Mobbs, C. V. (1999). Hypothalamic glucose sensor, similarities to and differences from pancreatic β-cell mechanisms. Diabetes 48,1763 -1772.[Abstract]
Youson, J. H., Al-Mahrouki, A. A., Anemiya, Y., Graham, L. C., Montpetit, C. J. and Irwin, D. M. (2006). The fish endocrine pancreas: research update and future directions in ontogenic and phylogenetic development. Gen. Comp. Endocrinol. 148,105 -115.[CrossRef][Medline]
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati 
Twitter What's this?
This article has been cited by other articles:
|
|
 |
|
  S. Polakof, S. Panserat, E. Plagnes-Juan, and J. L. Soengas Altered dietary carbohydrates significantly affect gene expression of the major glucosensing components in Brockmann bodies and hypothalamus of rainbow trout Am J Physiol Regulatory Integrative Comp Physiol, October 1, 2008; 295(4): R1077 - R1088. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
significantly different from control (dotted line) in ICV
treatments (P<0.05).
