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Evidence for the presence of a glucosensor in hypothalamus, hindbrain and
Brockmann bodies of rainbow trout
Sergio Polakof1, Jesús M. Míguez1, Thomas W. Moon2, and José L. Soengas1
1
Laboratorio de Fisioloxía Animal, Departamento de Bioloxía Funcional e Ciencias da Saúde,
Facultade de Bioloxía, Universidade de Vigo, 36310 Vigo, Spain.
2
Department of Biology and Centre for Advanced Research in Environmental Genomics,
University of Ottawa, Ottawa, Ontario, Canada K1N 6N5.
Running head: glucosensors in rainbow trout
*Author for correspondence: Dr. José L. Soengas
Laboratorio de Fisioloxía Animal
Facultade de Bioloxía
Edificio de Ciencias Experimentais
Universidade de Vigo
E-36310 Vigo
Spain
Tel. +34-986 812 564
Fax +34-986 812 556
E-mail [email protected]
Copyright © 2006 by the American Physiological Society.
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ABBREVIATIONS
2-DG, 2-deoxy-D-glucose
BB, Brockmann bodies
ELISA, enzyme immunoassay
HK-I, hexokinase I (EC. 2.7.1.11.)
HK-IV, hexokinase IV or glucokinase (EC. 2.7.1.2.)
GAP, glyceraldehyde 3-phosphate
GLUT-2, facilitative glucose carrier type 2
GSase, glycogen synthase (EC. 2.4.1.11.)
ICV, intracerebroventricular
PK, pyruvate kinase (EC. 2.7.1.40.)
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ABSTRACT
The aim of this study was to evaluate the existence of a glucosensor in different
regions of the brain and in the Brockmann bodies (BB) of the rainbow trout, Oncorhynchus
mykiss. Five groups (n = 12) of trout were injected intraperitoneally with saline alone
(control) or saline containing bovine glucagon (100 µg·kg-1), bovine insulin (4 mg·kg-1), 2deoxy-D-glucose (100 mg·kg-1) or D-glucose (500 mg·kg-1) to promote hyperglycemia
(glucagon, D-glucose, 2-deoxy-D-glucose) or hypoglycemia (insulin). Six hours following
injection, samples from four brain regions (hypothalamus, telencephalon, hindbrain and
midbrain) and the entire BB were taken. Our results demonstrate within the BB and both the
hypothalamus and hindbrain a metabolic response different to that observed in other tissues
(midbrain, telencephalon) but similar to that described in tissues known to be glucosensors in
mammals. The metabolic responses of these areas to changes in plasma glycemia were
characterized by parallel changes in GLUT-2 expression, hexokinase–IV or glucokinase
activity and expression, glycolytic potential, and levels of glycogen and glucose. These
changes are similar to those reported in mammalian pancreatic -cells and glucose-excited
(GE) neurons, two cell types containing glucosensors. This study provides evidence for the
presence of glucosensors responsive to hyper- and hypoglycemia in rainbow trout BB,
hypothalamus and hindbrain.
Key words: rainbow trout, glucosensor, hypothalamus, hindbrain, Brockmann bodies
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INTRODUCTION
Feeding behavior and general energy homeostasis are regulated by circulating levels
of nutrients including glucose. In mammals, sensors to detect levels of glucose are found in
pancreatic -cells (31) where phosphorylation of glucose by hexokinase-IV (HK-IV or
glucokinase, GK) plays a central role (27). HK-IV or GK has appropriate kinetics (low
affinity for glucose), lacks end-product inhibition and its selective distribution (5) allows
these cells to increase the rate of glucose phosphorylation in proportion to changes in blood
glucose over the physiological range (12). The glucose sensing mechanism is partially
elucidated in mammalian -cells: following a rise in plasma glycemia, glucose is transported
across the -cell membrane through a GLUT-2 carrier and metabolized by GK to glucose-6phosphate that enters glycolysis and the Krebs cycle. The metabolism of glucose results in
increases in cellular ATP that leads to closure of ATP-sensitive inward rectified K+ channels,
leading to membrane depolarization , Ca2+ influx and insulin release (31). Several mammalian
brain regions including the hypothalamus, medulla oblongata and mesencephalon contain
specialized neurons that utilize glucose as a signalling molecule rather than as an energy
substrate (11). Thus, glucose-excited (GE) neurons increase, whereas glucose-inhibited (GI)
neurons decrease their firing rate as plasma glycemia rises (5). In many ways, GE neurons are
the brain homologs of -cells whereas GI neurons have some similarities to pancreatic -cells.
Not surprisingly, mechanisms of glucose activation of GE neurons are similar to those of cells whereas relatively less is known regarding glucose alterations of GI neurons and -cells
(11).
Fish are generally considered to possess poor control over blood glucose levels
(16,40). However, plasma levels of glucose do fluctuate (8) and it is proposed that control of
glucose levels exist at least in those tissues that rely on glucose as a primary fuel such as the
brain (34). All known glucosensors in vertebrates consist of the low-affinity glucose
transporter GLUT-2 and the low affinity glucose-phosphorylating enzyme GK (37). In fish,
GK activities are reported in several species (20,21,22,24,25,36), and are induced by feeding
(10) and dietary carbohydrates (20,21). GK transcripts were also isolated and characterized
from the liver (4,14,20,21) and brain (21,36) of a few fish species but not from white muscle,
heart, gills or kidney (4,21,36). Additionally, experimentally-induced hyperglycemia in some
fish produces an increased glucose uptake in most tissues (3) including brain (2), and Panserat
et al. (23) demonstrated that high levels of GLUT-2 transcript expression occurred in rainbow
trout tissues regardless of nutritional conditions.
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Pancreatic cells in teleost fish are dispersed along the gastrointestinal tract although a
distinct grouping called the Brockmann bodies (BB) exists near to the gall bladder. The BB
are formed by endocrine cells, mainly -cells with fewer - and -cells than found in the
mammalian Islets of Langerhans (44). Little information is reported in fish regarding the
presence of glucosensors in pancreatic cells, and even these studies provide only indirectly
support for their existence. The data include i) the presence of GK activity in the BB of
Atlantic halibut (39), ii) the release of insulin from catfish BB induced by 2-deoxy-D-glucose
treatment (28), iii) the necrosis of -cells in tilapia treated with streptozotocin (41), and iv) the
fact that insulin release is induced by increased levels of plasma glucose in rainbow trout (7).
Several recent studies in rainbow trout suggest the possible existence of brain
glucosensors. These studies found i) that experimentally induced hyperglycemia lead to
decreased food intake whereas increased food intake occurred after ICV treatment of rainbow
trout with 2-deoxyglucose (35), ii) parallel changes between brain glycogen and GK activity
and expression levels and changes in plasma glucose levels (25), and iii) that fish re-fed for 7
days after 14 days of food deprivation displayed in both the hypothalamus and hindbrain
increased GK activity and expression and glycogen levels at the same time than plasma
glucose levels increased (36). Each of these results is consistent with a typical mammalian
glucosensor as found in -cells and GE neurons (12,43).
Thus, the purpose of this study was to obtain direct evidence for the existence of a
glucosensor in BB and specific brain regions (hypothalamus, midbrain, hindbrain and
telencephalon) of the rainbow trout, Oncorhynchus mykiss. To do so, we induced
experimental hyperglycemia with D-glucose, 2-deoxy-D-glucose and glucagon injections or
hypoglycemia with exogenous insulin treatments and assessed in these tissues the responses
of several well known metabolic indicators of the capacity for glucose sensing including GK
activity and expression, GLUT-2 expression, glycolytic potential, and glycogen levels.
MATERIALS AND METHODS
Fish
Rainbow trout (Oncorhynchus mykiss Walbaum) were obtained from a local fish
hatchery (Soutorredondo, Spain). Fish were maintained for 1 month in 100 litre tanks under
laboratory conditions and a natural photoperiod in dechlorinated tap water at 14 °C. Fish mass
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was 95 ± 3 g (n = 60). Fish were fed once daily (09.00 h) to satiety with commercial dry fish
pellets (Dibaq-Diprotg SA, Segovia, Spain; proximate food analysis was 48% crude protein,
6% carbohydrates, 25% crude fat, and 11.5% ash; 20.2 MJ/kg of feed). The experiments
described comply with the Guidelines of the European Union Council (86/609/EU), and of the
Spanish Government (RD 1201/2005) for the use of animals in research. Experiments were
undertaken in February 2006.
Experimental protocol
Following the 1 month acclimation period, fish were randomly assigned to 100 litre
experimental tanks, and each tank was randomly assigned to one of 5 experimental
treatments. Fish were lightly anaesthetized with MS-222 (50 mg·l–1) buffered to pH 7.4 with
sodium bicarbonate, weighed and intraperitoneally injected with 5 ml·kg-1 body mass of
Cortland saline alone (control, n = 12) or saline containing the different treatments: glucagon
(100 µg bovine glucagon (Sigma)·kg-1 body mass, n = 12), insulin (4 mg bovine insulin
(Sigma)·kg-1 body mass, n = 12), 2-deoxy-D-glucose (100 mg·kg-1 body weight, n = 12) or Dglucose (500 mg· kg-1 body mass, n = 12). Sampling was initiated 6 h after injection using fish
fasted for 24 h before injection to ensure basal hormone levels were achieved. These
concentrations and times were used as they were shown in other studies to modify glycemia
in rainbow trout (6,7,13,19).
Sampling
Fish were removed from replicate holding tanks at 15:00 h (6 h after injection),
anaesthetized as above and weighed. Four fish per group (2 per tank) were used to assess
mRNA expression in tissues and the remaining 8 fish per group (4 per tank) were used to
assess enzyme activities and metabolite levels.
Blood was obtained by caudal puncture into ammonium-heparinized syringes. Plasma
was obtained after centrifugation of the 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 and cortisol while the second aliquot (for the assessment of plasma metabolites) was
deproteinized (6% perchloric acid, PCA) 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, freeze-clamped in liquid nitrogen, and stored at -80°C until assayed. The brain
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(without the pituitary) was removed, placed on a chilled Petri dish and sectioned into four
regions (hypothalamus, midbrain, hindbrain, and telencephalon) as described previously (36),
and each section freeze-clamped in liquid nitrogen and stored at -80°C until assayed.
Enzyme and metabolite assays
Plasma glucose and lactate levels were determined enzymatically using commercial kits
(Spinreact, Spain) adapted to a microplate format. Plasma protein was measured using the
bicinchoninic acid method with bovine serum albumin as standard (Sigma, USA). Plasma
total -amino acids were assessed colorimetrically using the ninhydrin method of Moore (17);
alanine was used to develop a standard curve. Plasma acetoacetate levels were determined by
decreases in absorbance of NADH at 340 nm after sample incubation with (in mmol·l-1) 0.1
Na2HPO4 and 0.1 NaH2PO4 (pH 7.0), 0.2 NADH, and excess -hydroxybutyrate
dehydrogenase. Cortisol levels were measured using an ELISA validated for rainbow trout
(38).
Frozen tissues were quickly minced on a chilled Petri dish to very small pieces that (still
frozen) were divided into two homogeneous portions to assess enzyme activities and
metabolite levels. The tissue portions used to assess metabolite levels were homogenized
immediately by ultrasonic disruption in 7.5 vol of ice-cooled 6% PCA, and neutralized (using
1 mol·l-1 potassium bicarbonate). The homogenate was centrifuged, and the supernatant used
to assay tissue metabolites. Tissue glycogen levels were assessed using the method of Keppler
and Decker (9). Glucose obtained after glycogen breakdown (after subtracting free glucose
levels) was determined with a commercial kit (Biomérieux, Spain). Tissue glucose 6phosphate 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 NADH,
and excess glucose 6-phosphate dehydrogenase. Tissue glyceraldehyde 3-phosphate 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 and triosephosphate isomerase. Tissue lactate, total -amino acids, and
acetoacetate levels were assessed as described above for plasma samples.
Tissue portions used to estimate enzyme activities were homogenized by ultrasonic
disruption with 9 vol of ice-cold-buffer consisting of (in mmol·l-1) 50 Tris (pH 7.6), 5 EDTA,
2 1,4-dithiothreitol, and a protease inhibitor cocktail (Sigma, USA; P-2714). The homogenate
was centrifuged and the supernatant used immediately for enzyme assays. Enzyme activities
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were assessed spectrophotometrically using a microplate reader (SPECTRAFluor; Tecan,
Grödig, Austria). Reaction rates followed increases or decreases in absorbance of NAD(P)H
at 340 nm. The reactions were started by the addition of homogenates (15 µl), at a preestablished protein concentration, omitting the substrate in control wells (final volume 265295 µl), and allowing the reactions to proceed at 20°C for pre-established times. Low Km
hexokinase (HK, EC 2.7.1.1) and high Km hexokinase-IV (HK-IV, EC 2.7.1.2) or
glucokinase (GK) activities were estimated as described by Panserat et al. (20) by coupling
ribulose-5-phosphate formation from glucose 6-phosphate to the reduction of NADP at 340
nm. GK activities were corrected for glucose dehydrogenase (GlDH, EC 1.1.1.47) activities.
Thus, GK activity is calculated as total HK activity minus low Km HK activity minus 1/3 of
GlDH activity (see also 36). The specific conditions for the remaining enzyme assays were
described previously (25,29,30). Enzyme activities are presented as units (µmoles·min-1) per
mg tissue protein. Protein was assayed in homogenates using the bicinchoninic acid method
as noted above.
RT-PCR analysis of HK-I, HK-IV/GK, and GLUT-2 gene expression
Total RNA was extracted from frozen brains using TRI reagent as recommended by
the manufacturer (Sigma, USA). 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 5´-pd(T)12-18-3´ (Amersham Biosciences Barcelona, Spain) using M-MLV
reverse transcriptase for 1 h at 37 ºC by methods recommended by the manufacturer
(Promega, Madison, WI, USA).
We have assessed the expression of single isoforms of three different genes: HK-I,
HK-IV/GK, and GLUT-2. GK cDNA was PCR-amplified using specific primers developed
for rainbow trout by Panserat et al. (20): 5´-TGATGTTGGTGAAGGTGGGG-3´ (forward)
and 5´-TTCAGTAGGATGCCCTTGTC-3´ (reverse); amplification with these primers
resulted in a 250 bp product. HK-I cDNA was PCR-amplified using specific rainbow trout
primers reported in Soengas et al. (36): 5’-GGCTTCTCAGAGAGGGGATT-3´ (forward)
and 5’-TTTCGAAAATGCCTCTGGTC-3´ (reverse); amplification with these primers
resulted in a 245 bp product. GLUT-2 cDNA was PCR-amplified using specific primers
developed for rainbow trout by Panserat et al. (23): 5´-CGTCTTTACCATGGTGTCG-3´
(forward) and 5´-CCACAATGAACCAGGGGATG-3´ (reverse); amplification with these
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primers resulted in a 222 bp product. The housekeeping gene used to assess the relative
cDNA levels of HK-I, GK, and GLUT-2 was rainbow trout -actin. Accordingly, -actin
cDNA was amplified by PCR using specific primers: 5´-ACCCTCAGCTCGTTGTAGA-3´
(forward) and 5´-ACGGATCCGGTATGTGCAA-3´ (reverse) designed using the -actin
mRNA sequence for rainbow trout (Gen Bank accession number AJ438158); amplification
with these primers resulted in a 253 bp product.
The PCR reactions were carried out using a PTC-200 Peltier thermal cycler (MJ
Research Inc, Waltham, USA) in a final volume of 20 µl containing cDNA template (8 µl for
HK-I, GK and GLUT-2, and 2 µl for -actin), 1 x buffer (50 mM KCl, 20 mM Tris-HCl and
0.1 % Triton X-100), 0.2 mM dNTPs, 1.5 mM MgCl2, 2 pmol of each primer (forward and
reverse), and 1 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
94ºC followed by 35 cycles of denaturation (94ºC for 30 s), annealing (60ºC for 30 s) and
extension (72ºC 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. Size of PCR reaction products was established by comparison to 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) of images captured from UV transiluminated
ethidium bromide stained gels. Results are shown as arbitrary units and represent the ratio (%)
between HK-I/GK/GLUT-2 and -actin expression. To ensure the bands of interest were in
fact trout GK, HK-I, and GLUT-2, 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). The reactions were run on an Applied Biosystems automated
sequencer model ABI PRISM 310. The resulting sequences were compared with known
sequences on GenBank using BLASTn (http://www.ncbi.nlm.nih.gov/BLAST/).
Statistics
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Data are presented as means ± SEM. Comparisons among groups were performed
using one-way ANOVA (SigmaStat; SPSS Inc., Chicago, IL). When necessary 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
No differences existed between control and un-injected fish for any parameter
assessed (data not shown). Changes in plasma metabolic parameters and cortisol levels are
presented on Figure 1. Plasma glucose levels significantly decreased in insulin-treated fish
and increased in glucagon, 2-DG and glucose-treated fish. There were no other significant
differences with control fish for the other parameters assessed in plasma.
No significant differences were noted for HK-I expression in any tissue assessed (data
not shown). Relative GK and GLUT-2 expression are presented on Figure 2. No significant
differences existed between treatments in midbrain and telencephalon GK expression (data
not shown). GK expression significantly decreased in hypothalamus, hindbrain and BB in
insulin-treated trout, and the expression in this group was always lower than in the other
treatment groups. Relative GK expression was enhanced after glucagon (hypothalamus,
hindbrain and BB), 2-DG (hindbrain and BB), and glucose (hypothalamus and hindbrain)
treatments. Relative GLUT-2 expression significantly decreased after insulin treatment
(hinbdbrain and BB) and increased with glucagon (BB), 2-DG (hindbrain and BB) and
glucose (hindbrain and BB). No changes were noted in GLUT-2 expression in hypothalamus
(Fig. 2) or in the other brain regions assessed (data not shown).
GK activities decreased in hypothalamus, hindbrain and BB after insulin treatment
whereas an increase occurred in the same tissues after glucagon, 2-DG or glucose treatments
(Fig. 3). Low Km HK activities did not change in the brain regions studied, whereas in BB
insulin treatment resulted in a significant decrease in activity (Fig. 3).
Glycogen synthetase (GSase) activities in hypothalamus (Table 1) and hindbrain
(Table 2) paralleled changes in glycogen levels in these tissues (Fig. 4); no significant
differences were noted in the other brain regions (data not shown) and inadequate tissue mass
was available to assess activities in BB. Pyruvate kinase (PK) activities in hypothalamus
(Table 1) and hindbrain (Table 2) decreased with insulin treatment, whereas the activity ratio
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(activities at sub-saturating phosphoenol pyruvate (PEP) concentration to activities at
saturating PEP concentration) of this enzyme increased with glucose and 2-DG treatments. No
significant differences were noted in PK activities in the other brain regions assessed (data not
shown); again inadequate tissue was available for the BB for these estimates.
Tissue glucose, glycogen and glucose 6-phosphate levels are presented on Figure 4.
Glucose levels in hindbrain, midbrain and BB significantly decreased after insulin treatment
and increased in hypothalamus, hindbrain and midbrain after glucose treatment. Glycogen
levels decreased after glucagon treatment in the BB and insulin in hypothalamus, hindbrain,
and midbrain whereas levels increase with 2-DG in hypothalamus, hindbrain and BB and with
glucose in hypothalamus, hindbrain, and BB. Glucose-6 phosphate levels decreased with
insulin treatment in hindbrain and increased with glucose in hypothalamus and BB.
No significant differences were noted after any treatment for acetoacetate and lactate
levels in the brain regions (data not shown). -Amino acid levels were increased only in the
hypothalamus after treatment with 2-DG and glucose (Table 1). Glyceraldehyde 3-phosphate
levels in hypothalamus (Table 1) and hindbrain (Table 2) increased after glucagon, glucose
and 2-DG treatments and decreased after insulin treatment whereas no significant differences
were noted in the other brain regions assessed (data not shown). Inadequate tissue was
available to assess those metabolite contents in the BB.
DISCUSSION
Since gene expression assessed by conventional RT-PCR is not strictly quantitative,
the mRNA expression data reported in Fig. 2 must be interpreted cautiously.
Plasma levels of cortisol displayed no significant differences among treatments, and in
all cases values were close to baseline values for this species (38), supporting that changes in
metabolic parameters were not linked to any stress induced by our experimental conditions.
Plasma glucose levels of treated fish displayed changes that clearly validated the experimental
design since both hypoglycemia (insulin) and hyperglycemia (glucose, 2-DG, and glucagon)
were achieved. Changes observed in glycemia are consistent with those previously observed
in fish after treatment with 2-deoxy-D-glucose (6), D-glucose (7), insulin (19) and glucagon
(13). Further validation of this experimental design was achieved by assessing the effect of
these treatments on several parameters in liver (glycogen levels, GSase and PK activity, etc.)
known to change under similar experimental designs. In general, the results obtained in liver
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(data not shown) agree with those previously observed in other studies providing additional
support for the validity of our experimental design (6,7,19).
Brockmann bodies
Increased plasma glucose levels induce GK gene expression in -cells of the
mammalian pancreas (37). We therefore predicted changes in GK expression and activity in
rainbow trout under altered glycemic conditions. In fact, GK activity and relative mRNA
expression in the trout BB closely followed changes in plasma glucose levels, as observed in
mammals (31). This is the first study, as far as we are aware, in which GK gene expression
was assessed in piscine BB although previous studies did evaluate GK activity (39). The fact
that low Km HK activity and HK-I mRNA expression did not follow plasma glycemia
reinforces the important role of GK in the trout BB. Thus, changes in the relative expression
and activity of GK not the low Km HK (HK-I) may provide the capacity for trout BB to
detect changes in plasma glucose levels in a way similar to that observed in mammals (31).
This is also the first study, in which GLUT-2 expression was assessed in fish
pancreatic cells, as the only available fish study (23) did not examine pancreatic cells.
Relative GLUT-2 mRNA expression decreased under hypoglycemic and increased under
hyperglycemic conditions. Again, this response is similar to that reported in mammalian
pancreatic -cells where GLUT-2 mRNA levels changed directly with blood glucose (18),
and fasting reduced expression of GLUT-2 and GK whereas re-feeding enhanced expression
of both transcripts (45). Interestingly, hyperglycemia induced by glucose did not induce GK
mRNA expression in the trout BB but it did induce GLUT-2 expression. This implies distinct
regulatory signals governing GK and GLUT-2 expression and that glucose per se may not be
the only inducer in this species. In fact, studies in fish (15) find that amino acids like arginine
are better insulin secretagogues than glucose.
Another important component supporting the existence of a glucosensor in the
mammalian -cell model is that glycogen levels change in parallel with plasma glucose and
GK activity as glucose phosphorylation is a key step in the activation of glycogen synthesis
through increases in intracellular glucose 6-phosphate leading to allosteric activation of
glycogen synthase (GSase) or covalent activation or translocation of GSase (26,33). In the
present study, levels of glycogen generally paralleled plasma glucose and GK activities
(except for glucagon treatment where a decrease rather than an increase occurred). Changes
observed in tissue levels of glucose and glucose 6-phosphate also agree in general with those
observed for glycogen levels. Due to the small mass of the BB there was inadequate mass for
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the assessment of other metabolic parameters as in the brain regions. Therefore, we cannot
compare the available mammalian data with respect to changes in NADH production (42) or
glycolytic potential (32) following changes in glycemia with those of fish in the present study.
These results support the existence of a metabolic response in BB comparable with
that reported in mammalian -cells, providing evidence that in rainbow trout the BB act as a
glucosensor and contribute to the increased release of insulin reported under hyperglycemic
conditions in rainbow trout (7).
Brain regions
Increased plasma glycemia induce GK gene expression in the mammalian
hypothalamus (43). We therefore predicted changes in GK expression and activity in those
brain regions that in trout could act as glucosensors when submitted to different glycemic
conditions. Values of GK activity and relative mRNA expression in the brain regions assessed
were similar to those previously reported in this species after food deprivation and re-feeding
(36). GK activity and expression in the hypothalamus and hindbrain closely followed changes
in plasma glucose levels in a way similar to that observed in mammalian GE neurons (11);
similar correlations were not found in the remaining brain regions assessed (midbrain and
telencephalon). As noted for the BB, no differences were noted in the low Km HK activity or
relative HK-I mRNA expression in any tissue assessed, in agreement with our previous study
in fasted and re-fed rainbow trout (36). These results demonstrate that GK in the
hypothalamus and hindbrain respond to changes in plasma glucose levels, which is a key
component of the glucose-sensing machinery.
Panserat et al. (23) was unable to detect GLUT-2 mRNA expression in the whole brain
of the trout. However, it is clear from this study that GLUT-2 mRNA is expressed in specific
brain regions. GLUT-2 relative expression increased only in the hindbrain, not the
hypothalamus unlike changes noted for GK activity and expression that occurred in both
regions. The absence of GLUT-2 expression changes by circulating glucose levels in the
hypothalamus is similar to that observed in mammals where transport of glucose into
hypothalamus through GLUT-2 is considered not to be limiting (27). In contrast, transport of
glucose into trout BB and hindbrain was induced by glycemia in the present experiment
suggesting a significant difference between tissues with respect to the control of glucose
uptake by GLUT-2.
Levels of glycogen in the hypothalamus and hindbrain responded directly to changes
in plasma glucose levels whereas in the other brain regions only marginal changes in
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glycogen were observed. These changes occurred in the same direction as those observed for
GK activity and expression in agreement with the known effect of increased GK activity in
mammals stimulating GSase activity leading ultimately to increased glycogen synthesis (33).
Changes observed in glycogen levels in the present experiment can be also attributed to
changes in GSase activity. The parallel change observed in GK activity/expression and
glycogen levels was previously reported in fed, fasted and re-fed rainbow trout where we
postulated that plasma glycemia was modulating the glucose sensing mechanisms within the
brain (36); our present data support this postulate. Moreover, changes noted in tissue levels of
glucose and glucose 6-phosphate agree in general with those for glycogen levels. In addition
to changes in glucose and glycogen levels, changes were also noted in levels of
glyceraldehyde 3-phosphate that increased in both the hypothalamus and hindbrain of fish
under hyperglycemia and decreased under hypoglycemia. Therefore, levels of this glycolytic
intermediate might be strongly associated with increased GK activity and with the finding that
increased glycolytic potential also occurred in the hypothalamus and hindbrain under
hyperglycemic conditions whereas a decrease was noted under hypoglycemic conditions. This
is in contrast to the mammalian situation where hypoglycemia may lead to enhanced
glycolysis in the hypothalamus (43) but is in agreement with a previous study in which PK
activity in the same brain regions decreased in fasted fish and recovered to fed values in refed fish (36). These results demonstrate that the hypothalamus and hindbrain of the trout brain
increase both glycogen production and glycolysis possibly due to low activities of GSase and
thus limited capacity for glycogen production.
In addition to glucose, glucosensing neurons in mammals respond to a variety of
metabolites including lactate, ketone bodies and fatty acids (11). Moreover, the electrical
activity of glucose-responsive hypothalamic neurons in mammals may be regulated by
glucose through enhanced production of lactate by glial cells and its oxidation by
neighbouring neurons (1). In the present study, changes observed in plasma levels of lactate
and amino acids generally paralleled those of plasma glucose. Interestingly, levels of amino
acids increase in hypothalamus (but not in other brain regions) after some hyperglycemic
treatments suggesting that in this specific brain region a connection may exist between
glucose sensitivity and the capacity for detecting and using other fuels by the fish brain (34).
The results as a group support our previous hypothesis (36) that proposed the
existence of glucosensors in the rainbow trout hypothalamus and hindbrain. These
glucosensors respond to changes in plasma glucose with similar changes in GK activity and
mRNA expression and glycogen levels but with a different response regarding GLUT-2. This
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metabolic response may be involved in the orexigenic or anorexigenic responses observed in
rainbow trout subjected to hypoglycemic or hyperglycemic conditions (35).
Perspectives
After experimentally induced hyperglycemia and hypoglycemia in rainbow trout, we
demonstrated in both the Brockmann bodies and in the hypothalamus and hindbrain a
metabolic response that was distinct from that noted in other brain areas (midbrain,
telencephalon) and the liver (data not reported). The metabolic responses of the
hypothalamus, hindbrain and BB to changes in plasma glucose were characterized by parallel
changes in relative GLUT-2 expression, GK activity and expression, glycolytic potential, and
levels of glycogen. These changes are consistent with those reported in mammalian -cells
and glucose-excited (GE) neurons providing support for the presence of glucosensors in
rainbow trout responsive to hyperglycemia and hypoglycemia. Further studies are needed to
elucidate whether the mechanisms involved in glucose sensing post-GK induction are also
similar to those previously reported in mammals (11,31). Furthermore, considering that in fish
amino acids (in particular, arginine and lysine) are considered to be stronger insulin
secretogogues than glucose (15), whether expression profiling of -cells exposed to altered
glucose would reveal differential expression of genes involved in amino acid turnover and
nitrogen disposal (T.P. Mommsen, personal communication) would be important to
determine. Also, since glucose constitutes a small portion of the normal fish diet (40), it is
possible that the fish pancreas developed the capacity to produce and secrete insulin
independent of glucose, or at least in a nutrient milieu containing low glucose. Therefore, we
can hypothesize additional roles for glucosensors being involved in insulin release produced
not only by glucose but also by other secretagogues including amino acids (BB), or in the
detection of alternative fuels (BB, hypothalamus and hindbrain) including amino acids and
lactate.
Acknowledgements
This study was supported by research grants from Ministerio de Educación y Ciencia
and FEDER (AGL2004-08137-c04-03/ACU), and Xunta de Galicia
(PGIDT05PXIC31202PN). SP was recipient of a predoctoral fellowship from the
Universidade de Vigo. The authors wish to thank Dr. Paloma Morán (Universidade de Vigo)
for assisting in sequencing of genes.
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16
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Figure legends
Fig. 1. Glucose, lactate, -amino acid, protein, acetoacetate, and cortisol levels in plasma of
rainbow trout sampled 6 h after intraperitoneal injection of 5 ml·kg-1 of Cortland saline alone
(control, C) or saline containing bovine glucagon (GLN, 100 µg·kg-1), bovine insulin (INS, 4
mg·kg-1), 2-deoxy-D-glucose (2-DG, 100 mg·kg-1) or D-glucose (GLU, 500 mg·kg-1). Each
value is the mean + SEM of n = 8 fish per group. *, significantly different from control group
(P<0.05). When necessaary values were log transformed prior to statistical analysis. Different
letters indicate significant differences among treated groups (One-way ANOVA, P<0.05).
Fig. 2. Relative abundance of hexokinase-IV or glucokinase (GK) and GLUT-2 mRNA in
hypothalamus, hindbrain and Brockmann bodies of rainbow trout sampled 6 h after
intraperitoneal injection of 5 ml·kg-1 of Cortland saline alone (control, C) or saline containing
bovine glucagon (GLN, 100 µg·kg-1), bovine insulin (INS, 4 mg·kg-1), 2-deoxy-D-glucose (2DG, 100 mg·kg-1) or D-glucose (GLU, 500 mg·kg-1). Relative abundance of RT-PCR products
is presented in arbitrary units as mean + SEM (n = 4) between GK and GLUT-2 and -actin
expression. Further details as in legend to Fig. 1.
Fig. 3. Hexokinase IV or glucokinase (GK) and low Km hexokinase (HK) activities in
hypothalamus, hindbrain, midbrain, telencephalon, and Brockmann bodies of rainbow trout
sampled 6 h after intraperitoneal injection of 5 ml·kg-1 of Cortland saline alone (control, C) or
saline containing bovine glucagon (GLN, 100 µg·kg-1), bovine insulin (INS, 4 mg·kg-1), 2deoxy-D-glucose (2-DG, 100 mg·kg-1) or D-glucose (GLU, 500 mg·kg-1). Further details as in
legend to Fig. 1.
Fig. 4. Glucose, glycogen, and glucose 6-phosphate levels in hypothalamus, hindbrain,
midbrain, and Brockmann bodies of rainbow trout sampled 6 h after intraperitoneal injection
of 5 ml·kg-1 of Cortland saline alone (control, C) or saline containing bovine glucagon (GLN,
100 µg·kg-1), bovine insulin (INS, 4 mg·kg-1), 2-deoxy-D-glucose (2-DG, 100 mg·kg-1) or Dglucose (GLU, 500 mg·kg-1). Further details as in legend to Fig. 1.
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Table 1. Glycogen synthase (GSase) and pyruvate kinase (PK) activities, -amino acid and
glyceraldehyde-3-phosphate (GAP) levels in hypothalamus of rainbow trout sampled 6 h
after intraperitoneal injection of 5 ml.kg-1 of Cortland saline alone (Control) or saline
containing bovine glucagon (100 µg.kg-1), bovine insulin (4 mg.kg-1), 2-deoxy-D-glucose
(100 mg.kg-1) or D-glucose (500 mg.kg-1). Each value is the mean ± SEM of n = 8 fish per
group. *, significantly different from control group (P<0.05). Different letters indicate
significant differences among treated groups (One way ANOVA, P<0.05).
Parameter
GSase activity
(mU·mg-1 protein)
PK activity
Optimal activity
(U·mg–1 protein)
Activity ratio (%)
-amino acid levels
(µmol.g-1 wet wt)
GAP levels
(nmol.g-1 wet wt)
Control
107.1 ± 9.2
Glucagon
145.3 ± 8.1*a
Treatment Group
Insulin
2-Deoxyglucose
81.9 ± 8.27*b
148.9 ± 10.4*a
8.90 ± 0.51
8.07 ± 0.39a
4.53 ± 0.15*b
8.32 ± 0.27a
7.32 ± 0.32a
24.9 ± 2.41
12.2 ± 1.43
24.5 ± 2.64a
16.1 ± 2.07a
25.5 ± 2.27a
12.1 ± 1.25a
38.0 ± 2.17*b
24.1 ± 2.6*b
38.8 ± 2.82*b
21.6 ± 1.04*b
23.5 ± 1.18
32.4 ± 1.54*a
11.5 ± 0.45*b
36.3 ± 1.23*b
33.5 ± 2.02*b
D-glucose
165.4 ± 19.6*a
Table 2. Glycogen synthase (GSase) and pyruvate kinase (PK) activities, -amino acid and
glyceraldehyde-3-phosphate (GAP) levels in hindbrain of rainbow trout sampled 6 h after
intraperitoneal injection of 5 ml.kg-1 of Cortland saline alone (Control) or saline containing
bovine glucagon (100 µg.kg-1), bovine insulin (4 mg.kg-1), 2-deoxy-D-glucose (100 mg.kg-1)
or D-glucose (500 mg.kg-1). Each value is the mean ± SEM of n = 8 fish per group. *,
significantly different from control group (P<0.05). Different letters indicate significant
differences among treated groups (One way ANOVA, P<0.05).
Parameter
GSase activity
(mU·mg-1 protein)
PK activity
Optimal activity
(U·mg–1 protein)
Activity ratio (%)
-amino acid levels
(µmol.g-1 wet wt)
GAP levels
(nmol.g-1 wet wt)
Control
102.5 ± 16.1
Glucagon
182.5 ± 12.9*a
Treatment
Insulin
73.6 ± 8.40*b
2-Deoxyglucose
192.7 ± 19.1*a
D-glucose
190.3 ± 16.3*a
11.86 ± 0.28
11.43 ± 0.45a
8.48 ± 0.23*b
11.7 ± 0.49b
11.3 ± 0.36b
17.1 ± 2.14
15.7 ± 1.30
17.6 ± 1.32a
16.2 ± 1.18
11.6 ± 1.52a
15.8 ± 1.51
25.1 ± 141*b
17.1 ± 1.04
27.7 ± 1.70*b
17.8 ± 0.96
21.7 ± 3.45
47.4 ± 2.82*a
12.2 ± 1.12*b
46.2 ± 5.19*a
47.9 ± 4.17*a
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