Download Oxidative metabolism in thermogenic tissues of the swordfish and

THE JOURNAL OF EXPERIMENTAL ZOOLOGY 261:llO-114 (1992)
RAPID COMMUNICATION
Oxidative Metabolism in Thermogenic Tissues of the
Swordfish and Mako Shark
J.S. BALLANTYNE, M.E. CHAMBERLIN, AND T.D. SINGER
Department of Zoology, University of Guelph, Guelph, Ontario, Canada NIG
2 W1 (J.S.B., T.D.S.); Department of Zoological and Biomedical Sciences and
College of Osteopathic Medicine, Ohio University, Athens, Ohio 45701
(M.E.C.)
Mitochondria isolated from the heater organ of the swordfish Xiphias gladius and
ABSTRACT
the warm lateral red muscle of the mako shark Isurus oxyrinchus display respiratory control, indicating that uncoupling of oxidative phosphorylation is not important in heat production. The mitochondria of the swordfish heater organ resemble those of mammalian brown adipose tissue in their
substrate preferences, oxidizing pyruvate plus malate, palmitoyl carnitine, and a-glycerophosphate
at high rates. The high hexokinase levels in the heater organ indicate exogenous glucose is a n important energy source. The high activity of cytosolic a-glycerophosphate dehydrogenase and the high
rates of mitochondrial oxidation of a-glycerophosphate indicate that cytosolic redox is balanced via
the a-glycerophosphate cycle. Calcium stimulates mitochondrial a-glycerophosphate oxidation, and
it is proposed that calcium released from the sarcoplasmic reticulum may act as the signal coupling
enhanced rates of substrate oxidation with calcium transport. Based on mitochondrial respiration
rates, heat generation by the intact heater organ was calculated. Complete oxidation of glucose produces heat at a greater rate than oxidation of lipids. Nevertheless, oxidation of either substrate could
produce sufficient heat to maintain tissue temperature 10°C above ambient a t 20°C.
The mitochondria from the red muscle of the mako shark oxidize pyruvate plus malate, glutamine, glutamate, proline, and P-hydroxybutyrate a t high rates. Palmitoyl carnitine was not oxidized.
The respiration rates are similar to those described for mitochondria isolated from “cold” muscle of
other elasmobranchs. Therefore, it does not appear that mako shark red muscle mitochondria are
specialized for thermogenesis.
Several large pelagic fishes maintain some tissues
at temperatures above those of the surrounding
water (Carey and Teal, ’69;Carey et al., ’81;Carey,
’82; Block, ’85).In swordfish (family Xiphiidae), one
of the eye muscles, the superior rectus, has become
modified specifically for heat production to warm
the eye and brain (Carey, ’82).Ultrastructural studies of billfish (family Istiophoridae) indicate that
a portion of the superior rectus muscle (“brainheater
organ”; Block, ’86) is modified for heat production
and contains abundant mitochondria, sarcoplasmic
reticula (SR), and T tubules. The contractile elements are largely absent.
Whereas the billfish heater organ appears to be
specially modified for thennogenesis, other fish muscles retain their contractile function and conserve
the heat by countercurrent heat exchangers. In
lamnid sharks, heat generated by skeletal red muscle contraction and associated metabolism is retained by countercurrent exchangers (Carey et
al., ’81).
The metabolic characteristics of mitochondria
found in these two types of “warmer-than-ambient”
0 1992 WILEY-LISS,INC.
fish tissues have not been described. Such studies
require freshly caught fish and depend on biochemical facilities being available at sea. Recently, we
had the opportunity to establish facilities at sea for
the biochemical analyses of fresh tissues of the
swordfish Xiphias gladius and the shortfin mako
shark Isurus oxyrinchus. Our goal was to characterize the metabolism of the swordfish heater organ
and mako shark warm red muscle using isolated
mitochondria and enzyme measurements.
MATERIALS AND METHODS
Animals
As part of a Canadian Department of Fisheries
and Oceans survey, swordfish and mako sharks were
obtained by long-lining off Corsair Canyon near
Georges Bank (66“W 41.5”N)between September
6, 1990, and September 11, 1990. Lines were set
Received March 25,1991;revision accepted July 2,1991.
THERMOGENIC TISSUES OF SWORDFISH AND MAKO SHARK
at night and retrieved the following morning. Only
live fish were used for the reported studies.
A
111
D
C
Mitochondriaf isolation
Mitochondria were isolated and characterized on
board ship. Brain heater organs were dissected from
swordfish heads, and lateral red muscle was excised
from a region near the backbone of the mako shark.
The tissue was minced, placed in cold isolation
medium, and homogenized with three passes of a
loosely fitting pestle of a Potter-Elvehjem homogenizer. The medium for isolating mitochondria from
swordfish brain heater muscle consisted of 250 mM
sucrose, 1mM EGTA (ethylene glycol bis-(@-aminoethyl ether) N,N,N',N'-tetraacetic acid), 30 mM
HEPES (N-[2-hydroxyethyl]piperazine-Nr-[2-ethanesulfonic acid]),and 1%BSA (bovine serum albumin, Sigma A-7030, essentially fatty acid free).
Mako muscle mitochondria were isolated using a
medium composed of 500 mM sucrose, 150 mM KC1,
20 mM HEPES, 10 mM Na2 EDTA (ethylenediFig. 1. Typical traces of respiration of mitochondria isoaminetetraacetic acid), 5 mM MgC12,and 0.1% BSA. lated from the mako shark red muscle and swordfish heater
The pH of both solutions was adjusted to 7.2 at 20°C. organ. ADP (400 nM) was added prior to the addition of substrate (GLN, 12.5 mM glutamine; a-GP, 1 mM a-glycerophosThe homogenates were centrifuged at 918g for 10 phate).
A: Mako shark mitochondria (0.79 mg proteidml). B:
minutes. The resulting supernatants were removed Swordfish heater organ mitochondria (0.38 mg protein/ml). C:
and centrifuged at 9660g for 10 minutes. The pel- Swordfish heater organ mitochondria (0.31 mg protein/ml). D:
lets were removed and resuspended in isolation Swordfish heater organ mitochondria (0.31 mg proteiniml); 1
medium and again centrifuged at 9,660g for 10 min- mM CaCl, added prior to the addition of a-glycerophosphate.
utes. The final pellets were resuspended in isolaEnzyme measurements
tion medium t o yield a final protein concentration
of 3-17 mg/ml.
Maximal enzyme activities were measured in
samples of brain heater tissue that had been froMitochondria1 respiration
zen in liquid nitrogen and stored at - 80°C. These
Oxygen consumption was measured using a assays were performed at the University of Guelph
Clark-type electrode in temperature-controlled using a Varian DMS UV-visible spectrophotome(20°C) 1ml cells. One volume of mitochondrial sus- ter (Varian Canada Inc., Georgetown, Ontario).
pension was added t o 9 volumes of a reaction Reaction rates for all enzymes were determined at
medium consisting of 150 mM KCl, 30 mM HEPES, 20°C in 50 mM imidazole buffer, pH 7.2. Lactate
10 mM KH2P04,and 1%BSA (swordfish heater dehydrogenase and hexokinase were measured acorgan) or 400 mM urea, 200 mM TMAO (trimeth- cording to the method of Singer and Ballantyne
ylamine oxide), 150 mM KC1, 50 mM sucrose, 30 ('89). Glycerol kinase, a-glycerophosphate dehydromM HEPES, 1%BSA, and 10 mM KH2P04,pH 7.2 genase, and pyruvate kinase were assayed as des(mako red muscle). The pH of the reaction media cribed by Vijayan et al. ('91) except that 0.2 mM
was adjusted to 7.2 at 20°C. State 3 respiration NADH was used in the pyruvate kinase assay.
(defined by Chance and Williams, ,551,which occurs
RESULTS
in the presence of saturating ADP concentrations
(0.4mM), was used to assess maximal rates of subThe methods described above yield mitochondria
strate oxidation.
displaying tightly coupled respiration (see Fig.
lA,B). The highest respiratory control ratios (the
Protein determination
ratio of the respiratory rate in the presence of ADP
Protein contents of mitochondrial and tissue t o that after ADP has been phosphorylated) were
preparations were determined with a microassay of obtained when swordfish (9.76 3.03, n = 4) and
Bradford ('76) using appropriate blanks and BSA mako(ll.21 k 1.39,n = 3)wereoxidizingglutamine.
Heater organ mitochondria oxidize a wide variety
as a standard.
*
J.S. BALLANTYNE ET AL.
112
TABLE 1. Oxidation of substrates by mitochondria isolated
from the heater organ of the swordfish Xiphias gladius and
warm red muscle of the mako shark Isurus oxyrinchus'
Substrate
Pyruvate (10 mM) +
malate (0.1 mM)
a-Glycerophosphate
(5mM) +
CaCl, (1mM)
Succinate (5 mM)
Glutamate (10 mM)
Glutamine (12.5 mM)
Proline (50 mM)
Octanoyl carnitine
(40pM) +
malate (0.1mM)
Palmitoyl carnitine
(40pM) +
malate (0.1 mM)
Acetoacetate (0.3 mM)
p-Hydroxybutyrate
(25 mM swordfish);
(10 mM mako)
Endogenous
State 3 respiration rate
(nmoles 0,iminlmg
mitochondrial motein)
Swordfish heater Mako red muscle
151.28 ? 61.80 (4) 34.34 t 12.53 (3)
167.03 t 91.16 (4)
ND
106.66 t 49.50 (3) 21.74 t 3.25 (3)
167.16 t 48.10 (4) 45.94 t 13.81 (3)
148.81 t- 50.39 (4) 49.84 t 2.21 (3)
37.48 t 16.56 (4) 38.43 - 41.16 (2)
116.33 47.61 (4)
ND
TABLE 2. Maximal activities of selected enzymes in the
brain heater organ of the swordfish'
Enzyme
Hexokinase
Pyruvate kinase
Lactate dehydrogenase
glycerol kinase
a-glycerophosphate dehydrogenase
Activity
2.34 2 1.76(4)
116.42 t 23.69(4)
104.96 t 15.37(4)
0.08 t 0.02 (4)
10.83 t 2.99(4)
'Values given are means ? SEM. Units are micromoles substrate convertedlminigm. Methods are as described in Materials and Methods.
Numbers of preparations are given in parentheses.
Brown fat mitochondria are poorly coupled due to
the presence of the regulated proton channel thermogenin. When the channel is activated, the pH
*
gradient across the inner mitochondrial membrane
is dissipated, and oxidative metabolism proceeds
at a high (uncoupled) rate resulting in substantial
143.70 t 49.80 (4)
ND
heat production (Nedergaard and Cannon, '90).
Block and Carey ('87) indicated that thermogenin
ND
21.00 - 26.94 (2)
60.32 t 27.34 (4) 46.53 t 3.92 (3) is not present in the heater organ of billfish and
proposed that the mitochondria in this tissue are
coupled and produce ATP. The findings of our study
18.41 t 8.97 (3) 3.72 ? 2.24(3) support their proposition, since mitochondria iso'Values given are means t SE. The number of mitochondrial prep- lated from the heater organ had high respiratory
arations are in parentheses, and ND indicates not detected. Substrates
control ratios and high rates of oxidation in vitro
not oxidized: sarcosine (5 mM), p-alanine (5 mM), citrate (20 mM), are dependent on ADP.
alanine (10 mM), glycine (10 mM), isocitrate (1 mM), betaine (10 mM),
According to the model of Block and Carey ('87),
aspartate (10 mM), citrulline ( 2 mM), and arginine (1 mM).
mitochondrial respiration in the intact heater organ
of substrates (Table11, and oxidationof a-glycerophos- is stimulated by cytosolic ADP generated by the
phate was stimulated fourfold by the addition of Ca2+-ATPasein the SR. Calcium continuously leaks
CaC12(Fig. lC,D). Although only octanoyl carnitine across the SR membrane, maintaining high rates
and palmitoyl carnitine are listed in Table 1, the of ATP hydrolysis and high rates of oxidative metabheater organ also oxidized the carnitine esters of olism. Block and Carey ('87) focused on the hydrolstearic, decanoic, and hexanoic acids. In contrast ysis of ATP in heat production. Currently accepted
to the heater organ mitochondria, the mitochondria estimates of the heat released upon hydrolysis of
isolated from the red muscle of the shark did not ATP to ADP and Pi, under physiological conditions
oxidize palmitoyl carnitine, although there was a of pH, Mg2+, and ionic strength, indicate that
very slight oxidation of hexanoyl carnitine. The pre- about 50% of the energy available is released as
ferred substrates for the shark mitochondria were heat (Alberty, '69). This is equivalent to about - 6.0
pyruvate plus malate, amino acids (glutamate, glu- kcal/mole. Far more heat is generated in the comtamine, and proline), and P-hydroxybutyrate (Table bustion of substrates for the synthesis of ATP. As1).The activities of hexokinase, a-glycerophosphate suming an enthalpy of combustion of - 2,398 kcall
dehydrogenase,pyruvate kinase, and glycerol kinase mole for palmitate (Weast,'70) and 23 0 2 consumed
and lactate dehydrogenase in the swordfish heater yielding 138ATP with - 6.0 kcal/moleconserved per
ATP, oxidation of palmitate with ATP synthesis reorgan are presented in Table 2.
leases 65.5%of the heat of combustion. This amounts
DISCUSSION
to 1,571kcal/mole of palmitate. In contrast, hydrolWhereas all animal tissues produce heat, only two, ysis of the 138 ATP produced yields only 828 kcal.
mammalian brown fat and fish brain heater organ, ATP hydrolysis, therefore, can only account for
appear to be specifically designed for heat produc- 34.5%of the total heat produced.
In the present study, calcium stimulated the oxition. Both tissues have abundant mitochondria
indicative of a high metabolic rate, yet maintenance dation of a-glycerophosphate in swordfish brain
of these high rates occurs by different mechanisms. heater organ. A similar phenomenon has been ob-
THERMOGENIC TISSUES OF SWORDFISH AND MAKO SHARK
served in another type of muscle, insect flight muscle (Sacktor, '76). Such activation of mitochondrial
respiration may play a role in coupling SR calcium
release to mitochondrial respiration. In swordfish
brain heater organ, calcium leaking from the SR
may stimulate mitochondrial respiration before
cytosolic levels of ADP rise. A similar phenomenon has been observed in several cell types (Balaban,
'90). The coupling between the Ca2+-ATPaseand
mitochondria oxidative phosphorylation may be
either ADP or cytosolic calcium. Elucidation of the
mechanism of respiratory control in intact heater
organ cells must await further experimentation.
Oxidation rates of swordfish heater organ mitochondria exceed those reported for other fish even
when differences in temperature are taken into
account (Moyes et al., '89, '90; Chamberlin et al.,
'91). If high rates of substrate oxidation are to be
used to warm the eye and brain, heat must be produced at a rate greater than it is lost to the environment. Using the mitochondrial respiration rates
we have obtained (Table 11,it is now possible to estimate the rates of heat production of an intact heater
organ. Swordfish heater organ contains 35 nmol
cytochrome c/g tissue (Carey, '82) and animal mitochondria typically contain 0.45 nmol cytochrome
c/mg mitochondrial protein (Tzagoloff,'82). Therefore, oxidation of palmitoyl carnitine in the isolated
mitochondria can be converted to a respiration rate
of 11,176.7 nmol 02/min/g tissue in the intact tissue. Since 23 O2 are consumed for every palmitate
oxidized, palmitate oxidation would yield 1.16 cal/g
tissue or 0.0806 Wlg tissue. A heater organ from a
large swordfish may weigh 150 g (Carey, '82) and
would produce 12.09 W. A similar calculation for
glucose oxidation, based on mitochondrial pyruvate
oxidation and the production of two pyruvate molecules for every glucose molecule, yields a heat production of 0.365 W/g or 54.7 W for 150 g tissue. The
value we have calculated for pyruvate oxidation
(0.365 Wlg) is similar t o rates of 0.5 W per gram
reported for brown adipose tissue (see Rothwell and
Stock, '85, for review). These calculated values include the heat released by ATP hydrolysis.
If it is assumed that the thermal conductivity
(Weast, '70) of the heater organ is the same as that
of water, a 150 g sphere of tissue with a n average
diffusion distance of 3.296 cm (radius of the sphere)
would lose 2.336 W when faced with a 10°C temperature gradient (Carey, '82, reports temperature
gradients of 10"-14"C for free ranging swordfish).
Clearly, at 20"C, the oxidation of palmitate or glucose can yield sufficient heat t o maintain a 10°C
temperature gradient between the environment and
113
a 150 g heater organ. It should be noted, however,
that our calculations of heat production are based
on state 3 rates of mitochondrial respiration, which
undoubtedly exceed the rate in the intact heater
organ. Although mitochondrial respiration rate in
the intact heater organ has not been measured,
mitochondria in aerobic mammalian cells operate
at 25% of their maximal capacity (Harris et al., '81).
If a similar situation occurs in heater organs, our
calculations indicate that a 150 g heater organ
could maintain a 10°C gradient when oxidizing
either glucose or fats. The larger surface area to
volume ratios of the heater organs of small fish
would have greater difficulty in maintaining a 10°C
gradient, especially if they are oxidizing lipids.
The high rates of heat production from glucose
oxidation may help to explain the high activities
of glycolytic enzymes in this tissue. The very high
hexokinase levels detected in this tissue indicate
that exogenous glucose supplies much of the carbohydrate oxidized in this tissue. This is supported
by ultrastructural studies that indicate that there
are no stores of glycogen or lipid in billfish heater
organs (Block,'86). High rates of glycolysis require
rapid regeneration of cytosolic NAD. Lactate dehydrogenase activity is relatively low in the heater
organ and may have a minor role in balancing
cytosolic redox. In contrast, a-glycerophosphate is
rapidly oxidized by the mitochondria and cytosolic
NAD-dependenta-glycerophosphatedehydrogenase
is found in high activity. The a-glycerophosphate
cycle balances redox in insect flight muscle (Sacktor,
'76) and squid mantle muscle (Storeyand Hochachka,
'76) and may play a similar role in the heater organ.
In mammalian liver, the a-glycerophosphatecycle
(Wernette et al., '81) is responsive to changing cytoplasmic calcium mediated by catecholamines. Catecholamine stimulation of brain heater function has
been proposed (Block and Carey, '87); perhaps it
affects the a-glycerophosphate cycle in this tissue
as well.
Although swordfish heater organ mitochondria
are tightly coupled, they do resemble brown adipose tissue mitochondria with respect to their substrate preferences. Mitochondria from both tissues
oxidize pyruvate plus malate, palmitoyl carnitine,
and a-glycerophosphate at high rates (Nicholls et
al., '72). Other metabolic similarities lie in the high
activities of hexokinase and a-glycerophosphate
dehydrogenase in both tissues (Flatmark and Pedersen,'7 5 ) .Whereas the role of a-glycerophosphate
dehydrogenase in redox balance is discussed above,
another role for this enzyme may involve metabolism
of glycerol, a by-product of triglyceride metabolism.
J.S. BALLANTYNE ET AL.
114
There is no evidence to suggest high levels of glycerol
kinase in brown adipose tissue; indeed, glycerol is
released by brown adipose tissue during thermogenesis (Hull and Hardman, '70). The swordfish heater
organ also resembles brown adipose tissue in that it
lacks significant levels of glycerol kinase.
An unusual finding of the present study is the
oxidation of ketone bodies in swordfish heater organ
mitochondria. It is generally assumed that ketone
body oxidation does not occur in teleost fishes
(Zammit and Newsholme, '79; Beis et al., '80). Nevertheless, this enzyme has been measured in only
a few teleost species. Swordfish are the most advanced
teleosts examined to date for this enzyme. Unless
this enzyme arose independently in the Xiphiidae,
it seems likely that ketone body oxidation exists
in more primitive teleosts. Assays for this enzyme
in other teleost families seems warranted.
The warm red muscle of the mako shark displays
no special characteristics for thermogenesis. The
mitochondrial substrate preferences as well as respiration rates are similar to those described for the
red muscle of the dogfish (Moyeset al., '90; Chamberlin and Ballantyne, unpublished data), an elasmobranch with "cold' red muscles. The mitochondria are well-coupled,indicating that ADP produced
during muscle activity stimulates oxidative metabolism and heat production.
ACKNOWLEDGMENTS
This research was funded through a Natural Sciences and Engineering Research Council of Canada (NSERC) operating grant to J.S.B. and by a
NSERCIDepartment of Fisheries and Oceans joint
subventions grant to J.S.B. Ship time was generously provided by the Department of Fisheries and
Oceans. We gratefully acknowledge the enthusiastic assistance of the captain and crew of the E.E.
Prince in obtaining fish. In addition, we also thank
Jaime Alvarado for his assistance, Martin Gerrits
for shore support, and the inventor of duct tape for
equipment security in stormy seas.
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