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First published online July 20, 2006
Journal of Experimental Biology 209, 2929-2938 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02329
Metabolic organization and effects of feeding on enzyme activities of the dogfish shark (Squalus acanthias) rectal gland
1 Rosenstiel School of Marine and Atmospheric Science, University of Miami,
4600 Rickenbacker Causeway, Miami, FL 33149, USA
2 Bamfield Marine Sciences Centre, Bamfield, British Columbia,
Canada
3 Department of Biology, McMaster University, 1280 Main Street West,
Hamilton, Ontario L8S 4K1, Canada
4 Department of Biology, University of Victoria, Victoria, British Columbia,
Canada
* Author for correspondence at address 1 (e-mail: pwalsh{at}uottawa.ca)
Accepted 15 May 2006
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Summary |
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Key words: elasmobranch, shark, ketone body, ß-hydroxybutyrate, lipid metabolism
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)
demonstrated that little skate [Raja (now Leucoraja)
erinacea] hearts performed better when oxidizing ketone bodies as
fuels over fatty acids or glucose. Using isolated hepatocytes from the same
species, Mommsen and Moon demonstrated that whereas overall hepatic rates of
long chain fatty oxidation were generally low, substrates such as oleate (and
leucine for that matter) could support a net export of ketone bodies from the
liver (Mommsen and Moon,
1987). Likewise, isolated liver mitochondria of dogfish shark
(Squalus acanthias) had a great capacity to produce ketone bodies
from acetate, palmitoyl-CoA, pyruvate and alanine
(Anderson, 1990). In studies
using intact and cannulated dogfish, deRoos and colleagues demonstrated that
even in tissues such as brain, which in mammals is viewed as a highly
glucose-dependent tissue, ketone bodies are an important metabolic fuel in
elasmobranchs (deRoos, 1994).
Indeed, ketone-body-based metabolism appears to extend also to closely related
holocephalans (Speers-Roesch et al.,
2006a).
Whereas the very low concentration of serum albumin in elasmobranchs, and
thus the low capacity for plasma fatty acid binding and transport, had been
reported earlier (Fellows et al.,
1980; Fellows and Hird,
1981; Ballantyne et al.,
1993), an important connection was later made in an elegant review
article (Ballantyne, 1997).
Ballantyne reasoned that the strategy of using high concentrations of plasma
urea (several hundred millimolar) for osmotic balance (the ureosmotic
strategy) would likely disrupt serum albumin structure and function. Thus,
this ureosmotic strategy ultimately required a decreased reliance on fatty
acids, and an increased reliance on ketone bodies, as circulating metabolic
fuels (Ballantyne, 1997).
Clearly, elasmobranchs and holocephalans have an interesting overall
metabolic profile compared to teleosts and mammals. In addition, several
observations suggest that both circulating glucose and ketone body
concentrations are dynamic and can span a large range, depending upon feeding,
season, exercise, etc (e.g. Patent,
1973; Zammit and Newsholme, 1979;
deRoos et al., 1985; Gutierrez
et al., 1988; deRoos and deRoos,
1992; Richards et al.,
2003). It is especially the event of feeding, and the large NaCl
load that it generates which must be excreted, that is thought to activate the
gland through hormonal, acid-base, and other cues
(MacKenzie et al., 2002).
Indeed, the rectal gland is a highly aerobic organ that has served as a model
system for the principles of active NaCl co-transport for almost four decades
(for reviews, see Shuttleworth,
1988; Riordan et al.,
1994; Silva et al.,
1990; Silva et al.,
1996; Hazon et al.,
2003). However, surprisingly, in the many studies of transport and
salt secretion by elasmobranch rectal glands, scant data are available on the
substrate(s) that this important gland uses to fuel salt excretion. In most
studies of perfused rectal glands glucose is the typical substrate, and in
some cases supraphysiological concentrations have been used (e.g. T. J.
Shuttleworth, J. Thompson, R. S. Munger and C.M.W., manuscript submitted for
publication), but really no systematic published data support this choice of
in vitro fuel.
Therefore, in the present study, one goal was to focus particularly on
patterns of fuel usage in the rectal gland of the dogfish shark Squalus
acanthias and how they might be affected by feeding and changes in
circulating substrate levels. Furthermore, in order to better characterize the
physiological variability of available substrates, and the metabolic scope of
the gland, we examined the effects of natural feeding on circulating
levels of ketone bodies and glucose and the activities of several enzymes of
ketone body and general metabolism. This paper is a companion to two others
recently published (Wood et al.,
2005; Kajimura et al.,
2006) that examine the effects of feeding on acid-base balance and
nitrogen metabolism and excretion, respectively, in the same species.
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) and pH (7.90±0.15). We found that
dogfish would not feed when held in smaller tanks or when isolated, but when
held in a large group (approximately 100 fish) in this very large tank, a few
dogfish would start feeding after about a week in captivity, and thereafter,
the others would quickly learn to do so. Twice weekly, the animals were fed a
ration equivalent to 2% of estimated total biomass of all the dogfish in the
circular tank, most of which was quickly devoured (fed treatment). Food
consisted of whole dead trawl fish (mainly flatfish, herring and eelpout).
Samples of food were taken, minced with a food processor and frozen at
-20°C for later content analysis, as reported
(Wood et al., 2005).
Experimental design
Effects of feeding on plasma metabolites and rectal gland enzyme activities
Some fish (starved treatment) were removed from the circular tank to a
separate 1500 l tank 1 week before the start of an experiment, were not fed
during this separation period, and then each dogfish was transferred to an
individual 40 l polyurethane-coated wooden box (seawater flow=1 l
min-1) as described (Wood et
al., 1995; Wood et al.,
2005). For the fed fish treatment, 1 h after the start of feeding
in the large 200,000 l circular tank as described above, fed fish were
transferred to these same 40 l boxes. These fish are the same individuals as
described (Kajimura et al.,
2006); therefore, data on nitrogen excretion, nitrogen
metabolizing enzymes, blood nitrogen metabolite levels, etc. are directly
comparable to the data of the present study.
This series examined the effect of feeding on blood chemistry and key enzymes of metabolism in the tissues. Dogfish were fed and then transferred to the 40 l boxes as described above. Separate groups were killed at `6 h' (4-8 h, N=15), `20 h' (19-22 h, N=13), `30 h' (29-31 h, N=8) and `48 h' (N=10) after feeding. A 1-week starved group (N=14) was also examined. At sacrifice fish were anaesthetized with MS-222, weighed, placed on an operating table and blood samples (by caudal puncture with a heparinized 10 ml syringe) and tissue samples were taken. Ten tissues were dissected; liver, muscle, gills, intestine, rectal gland, esophagus, anterior stomach (stomach 1), posterior stomach (stomach 2), brain and kidney (note that kidney samples were taken only from starved fish and at 48 h post-feeding). Plasma samples, obtained by centrifugation at 9000 g for 2 min, were stored frozen at -20°C. Tissue samples were frozen immediately with liquid nitrogen, and stored at -80°C for later enzymatic analysis. At sacrifice, the amount of food remaining in the stomach was collected by making a small incision at the distal end of the stomach and gently squeezing the contents into a tared beaker and weighing. Some fish had empty stomachs at sacrifice, and were excluded from the analysis, as we could not be certain that they had fed at 0 h. These fish constituted 4 out of 15 fish at 6 h, 2 out of 13 fish at 20 h, 1 out of 8 fish at 30 h and 1 out of 10 fish at 48 h after feeding. In addition, any solid food that was regurgitated into the boxes during the experiments was collected and weighed.
Perfused rectal gland studies
Dogfish that had starved for 7-10 days were anesthetized with MS-222 (0.2 g
l-1 seawater) and this dose was maintained by irrigation of the
gills with seawater via a pump. Mass, length and sex were noted.
Rectal glands were cannulated in situ until all three ports had been
fitted with PE 50 tubing. The artery was first, followed by collecting duct
and vein. The artery and vein were flushed with dogfish saline (mmol
l-1) (NaCl, 257; Na2SO4, 7; MgSO4,
3; KCl, 4; CaCl2, 2; Na2HPO4, 0.1; urea 400;
trimethylamine oxide (TMAO), 80; NaHCO3 6; solutions were gassed
with 0.275% CO2 and balance oxygen, yielding a pH of 7.9 to 8.0)
with 50 i.u. heparin, while the duct was flushed with 500 mmol l-1
NaCl (we also found that heparin was useful in minimizing clots and mucous
plugs). All saline solutions were processed through a 0.45 µm filter prior
to use. The gland was then removed and connected to a PE-50 line in series
with a thermostatted saline reservoir, peristaltic pump, thermostatted
windkessel/bubble trap and temperature equilibration coil, similar to the
set-up described by Shuttleworth et al. (T. J. Shuttleworth, J. Thompson, R.
S. Munger and C.M.W., manuscript submitted for publication). The gland rested
on a thermostatted Plexiglass platform (12±1.0°C). Perfusion began
at approximately 0.25 ml min-1 until the gland was cleared of blood
and it was then secured to the platform to prevent movement (by taping the
catheters). Input flow was then increased to about 1.0 ml min-1 and
collection of duct secretion commenced. Typically, a period of 30 min with 5
mmol l-1 glucose and no forskolin stimulation preceded substrate
tests to allow the gland to equilibrate. Forskolin was then added (from a
stock of 25 mg ml-1 DMSO to a final concentration of 5 µmol
l-1, i.e. 4.1 µl DMSO per 50 ml perfusate) to stimulate gland
secretion via activation of cAMP-mediated pathways
(Morgan and Valentich, 1991).
Each substrate/substrate combination test period was 60 min (substrates were
added to perfusate as 100-fold concentrated stocks, neutralized when
necessary), and duct secretions were collected and venous flow rates were
monitored (gravimetrically) every 30 min. Typically venous outflow rates were
60% of inflow rates due to the previously described venous bypass
(Kent and Olson, 1982).
Experiments typically lasted 5.5 h, allowing five substrate comparisons per
gland. Since secretion rates invariably declined slightly over time for a
given gland, the order of substrate addition was randomized to prevent bias.
Chloride in the duct secretions was determined with a Radiometer CMT 10
Chloride Titrator (Copenhagen, Denmark). Urea was assayed colorimetrically
(Rahmatullah and Boyde, 1980).
Chloride secretion rates were calculated from the volume of duct secretion,
the chloride concentration of the duct secretion, flux time and gland mass to
yield mmoles Cl- h-1 g-1.
Analytical techniques and statistics
Plasma glucose levels were measured using an infinity glucose hexokinase
liquid stable reagent (Thermo-Trace, Noble Park, Victoria, Australia). Plasma
ß-hydroxybutyrate levels were measured using a ß-hydroxybutyrate
LiquiColor test (Stanbio Laboratory, Boerne, TX, USA). Plasma acetoacetate
levels were measured according to published methods
(Williamson et al., 1962).
For analysis of enzyme activity, tissues were homogenized on ice in 5-10
volumes of homogenization buffer (20 mmol l-1
K2HPO4, 10 mmol l-1 Hepes, 0.5 mmol
l-1 EDTA, 1 mmol l-1 dithiothreitol, 50% glycerol,
adjusted with NaOH to pH 7.5 at 24°C) using a Brinkman Polytron.
Homogenates were centrifuged at 8000 g for 20 min at 4°C.
The supernatant, or a 1:9 or 1:99 dilution in homogenization buffer was used
directly for assaying the activity of ß-hydroxybutyrate dehydrogenase (EC
3.1.1.30; BHBDH, ketone body metabolism), malic enzyme (EC 1.1.140; ME, NADPH
generation), 6-phosphogluconate dehydrogenase (EC 1.1.1.44; 6-PGDH, pentose
shunt pathway), lactate dehydrogenase (EC 1.1.1.27; LDH, anaerobic
glycolysis), NADP-isocitrate dehydrogenase (EC 1.1.1.41; ICDH, oxidative
metabolism) and citrate synthase (EC 2.3.3.1; CS, mitochondrial abundance) by
previously described methods (Mommsen et
al., 1980). To assess nitrogen metabolism, glutamate dehydrogenase
(EC 1.4.1.3; GDH), alanine amino transferase (EC 2.6.1.2; AAT) and aspartate
amino transferase (EC 2.6.1.1; DAT, formerly ASP AT) were assayed by the
methods of Walsh and Henry (Walsh and
Henry, 1990) and glutamine synthetase (transferase units, EC; GS)
by the methods of Webb and Brown (Webb and
Brown, 1980). Not all tissues were assayed for all enzymes. The
protocol used for the determination of Na+/K+ ATPase
activity followed that of McCormick
(McCormick, 1993). In all
cases assay temperature was 22°C.
All data are reported as mean ± s.e.m. All data were normally distributed, and multiple comparisons for differences in metabolite concentrations and enzyme activities at different sampling times were evaluated by one-way ANOVA followed by Dunnett's paired multiple comparison test (Figs 2, 3, 4 and 6) and a LSD post-hoc test for all the other data. Significance was accepted at P<0.05. For rectal gland substrate preference experiments, because of inter-preparation variability, it was often convenient to express data as a percentage of the control period, and if so, arcsine-transformed values were used in statistical tests.
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Results |
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Oxidative metabolism and/or activity of hydrogen shuttles, as indicated by ICDH activity was similar in most tissues of starved dogfish, with the highest activity in anterior stomach, liver and posterior stomach (Fig. 4). ICDH activity incurred a massive (>10-fold) increase in rectal gland such that by 19-22 h, this enzyme clearly had the highest activity of tissues examined. This increase was followed by a rapid reduction to starved control levels by 29-31 h (Fig. 4). Notably, liver, anterior stomach and intestine also exhibited similar, yet less pronounced patterns of peak ICDH activity at 19-22 h (Fig. 4). Because of these high levels of ICDH induced by feeding in the rectal gland, we also measured a more direct indicator of oxidative metabolism in the rectal gland (only), mitochondrial citrate synthase (Fig. 5), for comparison. The values of CS measured were rather high in comparison to tissues for other species (see Discussion) and showed a significant increase beginning at 20 h post-feeding (Fig. 5).
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The pentose shunt enzyme 6-PGDH showed significant increases in liver,
gill, intestine, esophagus and anterior stomach with feeding, peaking at 19-22
h at 
1 U g-1 in gill (results not shown). Interestingly,
rectal gland, a tissue in which other enzymes showed plasticity had rather
constant levels of 6-GPDH at 0.6 U g-1 in all treatments
(results not shown).
The activation of secretory function of the rectal gland was reflected in the initial activation of Na+/K+ ATPase shortly after feeding (Fig. 7). This was followed by a return to fasting enzyme activity levels at 20 h and a subsequent slow increase of activity at 48 h (Fig. 7).
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Rectal gland metabolic substrate preferences
In general, the perfused rectal gland preparation in our hands showed good
viability, and secretion rates/activation by forskolin compared well with
previous studies. For all preparations, chloride concentration of rectal gland
secretions ranged from 454 to 582 mmol l-1, and urea concentration
from 7 to 28.8 mmol l-1.
In the first series of experiments, we wished to determine potential effects of glucose concentration and we compared the typically used (and indeed physiological) 5 mmol l-1 to the supraphysiological concentration of 30 mmol l-1 used in some studies. We found no effect of glucose concentration in this range (5 mmol l-1 glucose = 0.594±0.232 mmol Cl- h-1 g-1; 30 mmol l-1 glucose = 0.592± 0.094 mmol Cl- h-1 g-1).
In the next series of experiments, we wished to examine if the addition of other substrates to the baseline of 5 mmol l-1 glucose could alter chloride secretion. Among a variety of substrates (lactate, alanine, glutamate, acetoacetate), only ß-hydroxybutyrate (BHB) had the ability to increase secretion rate by 60-87%, without any discernable difference between the effects of 0.5 to 5 mmol l-1 BHB (Table 2). Notably, the augmentation of secretion rate by BHB had some residual effect in that when some glands were switched back to glucose only, elevated secretion rate was maintained for at least another hour (Table 2).
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Based on the above result of BHB augmentation of the effect of glucose, and on the high BHBDH values obtained for rectal gland (Fig. 2), in the next experimental series we wished to determine if BHB could support chloride secretion alone, or alternatively, if glucose was strictly required by the gland. Clearly, preparations were not viable when perfusion was begun with BHB only, as their chloride secretion rates were low in comparison to most preparations (Table 3). Even when augmented with 5 mmol l-1 glucose in the second flux period, secretion rates declined (Table 3). We also attempted starting the gland with 5 mmol l-1 glucose and then switching to BHB alone, or subsequently adding back glucose, but preparations so treated also exhibited decline (Table 3). In all cases in these preparations, we could detect an `on the spot' substantial decline in the volume secreted in the second 30-min portion of each hour-long flux when glands were presented with only BHB as substrate (results not shown).
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Plasma glucose concentration remained relatively stable following feeding
(Fig. 1C), although it
underwent a temporary decrease of about 30% during the 4-8 h post-feeding
period. A similar decrease in blood glucose was shown when dogfish were
exposed to exogenous insulin (deRoos et
al., 1985). It would be interesting to determine if feeding
release of insulin is responsible for this decrease in plasma glucose.
Although peptide sequences have been determined for shark insulins and sharks
respond to injection of homologous insulins with decreases in plasma glucose
[but no changes in plasma BHB or acetoactate
(Anderson et al., 2002)],
post-prandial responses in plasma insulin and subsequent metabolic changes are
yet to be described.
In terms of enzyme activity measurements, we noted increases in
Na+/K+ ATPase activity at 6 h and 48 h
(Fig. 7) that are consistent
with activation of the gland by feeding. Notably, the increase in activity is
biphasic, a point that we return to below. One of the most striking results to
emerge from this study are the overall enzyme activity values for the rectal
gland. It has been known for some time that the tissue is extremely
metabolically active when in secretion mode; for example, even unstimulated
glands have oxygen consumption rates of 0.1-0.3 µmol g-1
min-1, whereas stimulated glands can achieve rates of 1.4 µmol
g-1 min-1 (Silva et
al., 1980; T. J. Shuttleworth, J. Thompson, R. S. Munger and
C.M.W., manuscript submitted for publication). Our results demonstrate that
these rates are also reflected in enzyme activities. Notably, per mass, enzyme
activities for rectal gland are higher than all other tissues measured (for
BHBDH and ICDH, Figs 2,
4) or are equivalent to those
of the highest tissues measured (for ME and LDH, Figs
3,
6). The CS data
(Fig. 5) are also notable in
that the 20 to 30 U g-1 we report are similar to values for red
muscle in tuna (Guppy et al.,
1979)!
The LDH data are particularly interesting in light of prior measurements of
substantial lactate in the gland (7 mmol l-1)
(Silva et al., 1980), values
that are close to those in white muscle in this species
(Richards et al., 2003). The
inability of lactate to augment glucose-based secretion rates
(Table 2) indicates that the
high LDH activity seen in the rectal gland is probably not for use of
lactate from the circulation, but for production of lactate. Given the rather
high oxygen consumption rate of the gland, we speculate that there may be
brief periods when demand for chloride secretion outpaces the ability of the
circulation to deliver oxygen to fuel aerobic metabolism, and that a certain
fraction of the gland's secretion output is met periodically by anaerobic
glycolysis.
In addition to having generally high metabolic rates reflected in enzyme
activity, the metabolic poise of the rectal gland seems to be rather plastic
at the molecular level. Short-term (i.e. 4-8 h) increases in enzyme activity
were noted for BHBDH, ICDH, LDH and Na+/K+ ATPase. A
similar observation of short-term activation of Na+/K+
ATPase activity was made for European dogfish Scyliorhinus canicula
at 3-12 h post-feeding (MacKenzie et al.,
2002). These changes in both studies take place presumably during
a time course when mechanisms other than transcriptional or translational
control would predominate [and indeed MacKenzie et al. did not find an
increase in Na+/K+ ATPase message in the short term
(MacKenzie et al., 2002)]. It
would be interesting to examine the mechanisms of short-term enzyme activation
in this gland, especially considering that there is not precedent for typical
post-translational modifications (e.g. phosphorylation/dephosphorylation) for
many of the enzymes we examined.
We also observed changes in enzyme activities after a longer time course
post-feeding (for ICDH, CS, and Na+/K+ ATPase) where it
is more likely that translational and transcriptional control could be
involved. Control by transcription for Na+/K+ ATPase was
examined in European dogfish Scyliorhinus canicula
(MacKenzie et al., 2002), and
changes in mRNA were only observed 1 day after feeding at a time when enzyme
activities were not elevated. It will be interesting to determine if a similar
lack of transcript elevation is seen for the three genes showing late enzyme
activation in the present study, namely CS, ICDH and
Na+/K+ ATPase.
Given the above results on rapid clearance of BHB, and the high enzyme activities of the gland (particularly for BHBDH), we wished to determine substrate preferences of the gland in an in vitro perfused system. Indeed, several parameters of gland function (responsiveness of chloride transport to forskolin stimulation, secretory flow rate, chloride secretion rate, ability to exclude urea from the secretion, etc.) indicated that our preparation was viable for several hours after isolation, in accordance with previous studies (e.g. T. J. Shuttleworth, J. Thompson, R. S. Munger and C.M.W., manuscript submitted for publication). Prior to testing of several substrates, we examined the use of a more realistic glucose concentration as a baseline substrate, and indeed found that a typical plasma glucose value of 5 mmol l-1 could support gland function as effectively as 30 mmol l-1 (see Results); therefore subsequent experiments were conducted at this concentration.
Several substrates representing carbohydrate (lactate), amino acid (alanine, glutamate) and ketone body (acetoacetate, ß-hydroxybutyrate) metabolism were examined for their ability to augment the chloride secretion rate supported by 5 mmol l-1 glucose. Indeed, only BHB was able to further stimulate the gland (Table 2), and it did so equally at concentrations typical of fed (0.5 mmol l-1) or starved (5 mmol l-1) dogfish plasma.
However, BHB alone was unable to sustain the chloride transport function of
the gland. Even in different combinations/arrangements (glucose first,
followed by BHB, etc), the viability of the gland was compromised after only a
short period when the sole fuel source provided was BHB. Thus we conclude that
glucose is one of the likely primary substrates for the gland in
vivo, but that BHB can supplement the glucose-based metabolism. This
arrangement makes sense from more than one perspective. First, at the
biochemical level, the reactions of glycolysis using glucose as a substrate
prior to entry of acetyl CoA into the mitochondria will provide two additional
ATP molecules per mole of substrate, as well as two NADH equivalents per mole
at the glyceraldehyde-3-phosphate dehydrogenase step, and two NADH equivalents
at the pyruvate dehydrogenase step (all four of which can be transferred to
the electron transport system). One mole of BHB will provide only one NADH
equivalent (via BHBDH) en route to acetyl CoA. Thus, on a
per mole basis, glucose is a more efficient substrate than BHB. Another
advantage of using primarily glucose as a fuel is that, if
NADH/NAD+ ratios skew unfavorably, or if oxygen demand simply
outpaces supply, some of the pyruvate could be shunted towards lactate
production by LDH to replenish cytoplasmic NAD+ and maintain redox
balance. Interestingly, the ability of BHB to augment glucose-fueled chloride
secretion would imply that, at least under our experimental conditions, the
utilization of acetyl-CoA by rectal gland mitochondria did not reach its
maximum. It is likely that exogenous delivery of BHB can increase the rate of
chloride transport, linked to rapid delivery of intramitochondrial electrons
through the BHBDH reaction and perhaps minor adjustments in intramitochondrial
redox. An analogous situation would be the rapid, partial, utilization of
-glycerophosphate by insect flight muscle mitochondria. However, it
appears that the rectal gland mitochondria will only respond if already primed
with glucose. At this point, and using analogous mammalian models, we can
assume that the subsequent steps in BHB metabolism are relatively slow
delivering acetyl-CoA to citrate synthase via a CoA transferase,
using mitochondrial succinyl-CoA as CoA donor, and thiolase. By the same
token, BHB alone should have provided at least a short oxidative surge, but
the time course of our experiments was not designed to detect such short-term
effects.
Often the activity of citrate synthase (our choice here) or cytochrome c
oxidase are used as a proxy for mitochondrial abundance. Considering that CS
in insect flight muscle is between 40 and 120 U g-1 of thorax
(scaling effects!) at 25°C (Heinrich
and Mommsen, 1985), the 25-40 U in rectal gland also compare
favorably with other highly aerobic tissues, such as bluefin tuna heart [77-95
U at 25°C (Blank et al.,
2004)]. Although these highly aerobic tissues are usually
characterized by relatively modest anaerobic capacities, as indicated by the
activity of LDH, it appears that the dogfish rectal gland has the best of both
worlds: a high aerobic capacity to drive transport processes, backed up by
substantial anaerobic capacity. Considering the relative inefficiency of
anaerobic glycolysis, the rectal gland seems to follow the motto: transport at
any cost! Of course, taking into consideration the small relative size of the
rectal gland (at 0.05% of body mass), it is unlikely that consumption rates of
glucose or BHB, or production rates of lactate, will make major contributions
to the metabolic turnover of the whole dogfish. It would be interesting to
repeat these experiments under conditions where rectal gland secretion was
maximal to determine if the mitochondria could be saturated, and to examine if
significant amounts of lactate were produced. As noted above, significant
levels of lactate have been reported previously in rectal gland
(Silva et al., 1980).
The choice of glucose as one apparent primary fuel for the rectal gland,
with the ability to supplement with BHB, also makes sense in the context of
the whole animal. When the gland is stimulated by neuroendocrine signals and
the alkaline tide (Wood et al.,
2005; C.M.W., R. S. Munger, J. Thompson, T. J. Shuttleworth,
manuscript submitted for publication) following feeding, glucose
concentrations are relatively stable, whereas BHB concentrations are rapidly
changing. Thus, the rectal gland would have a constant substrate supply and
not necessarily be competing with other tissues for BHB to perform its vital
physiological functions. A final caveat to our analysis is that lipid
metabolism was not examined. Indeed, recent enzymatic evidence suggests that a
few selected peripheral tissues in elasmobranches, including rectal gland, may
in fact be able to oxidize lipids as indicated by activities of enzymes such
as carnitine palmitoyl transferase
(Speers-Roesch et al., 2006b).
The perfused rectal gland preparation would be ideal to examine the ability of
substrates such as palmitate to support chloride secretion.
For decades, the elasmobranch rectal gland has proved to be an exciting physiological system for the study of transport processes. Further studies of its metabolic physiology and biochemistry in this context will surely yield similarly exciting insights into the mechanisms of rapid upregulation of metabolism.
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Acknowledgments |
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Footnotes |
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References |
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Anderson, P. M. (1990). Ketone body and phosphoenolpyruvate formation by isolated hepatic mitochondria from Squalus acanthias (spiny dogfish). J. Exp. Zool. 254,144 -154.[CrossRef]
Anderson, W. G., Ali, M. F., Einarsdottir, I. E., Schäffer, L., Hazon, N. and Conlon, J. M. (2002). Purification, characterization, and biological activity of insulins from the spotted dogfish, Scyliorhinus canicula, and the hammerhead shark, Sphyrna lewini. Gen. Comp. Endocrinol. 126,113 -122.[CrossRef][Medline]
Ballantyne, J. S. (1997). Jaws: the inside story. The metabolism of elasmobranch fishes. Comp. Biochem. Physiol. 118B,703 -742.[CrossRef]
Ballantyne, J. S., Glemet, H. C., Chamberlin, M. E. and Singer, T. D. (1993). Plasma nonesterified fatty acids of marine teleost and elasmobranch fishes. Mar. Biol. 116, 47-52.[CrossRef]
Blank, J. M., Morrissette, J. M., Landeira-Fernandez, A. M.,
Blackwell, S. B., Williams, T. D. and Block, B. A. (2004).
In situ cardiac performance of Pacific bluefin tuna hearts in
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deRoos, R. (1994). Plasma ketone, glucose, lactate and alanine levels in the vascular supply to and from the brain of the spiny dogfish shark (Squalus acanthias). J. Exp. Zool. 268,354 -363.[CrossRef]
deRoos, R. and deRoos, C. C. (1992). Effects of mammalian ACTH on potential fuels and gluconeogenic substrates in the plasma of the spiny dogfish shark (Squalus acanthias). Gen. Comp. Endocrinol. 87,149 -158.[CrossRef][Medline]
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