Download Hormonal and Nutritional Substrate Control of Cardiac Lysosomal

CARDIAC LYSOSOMAL ENZYMES/Wildenihal
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Hamilton, P Dow. Washington, D C , American Physiological Society,
1963, pp 1160-1161
Jones AW Altered ion transport in vascular smooth muscle from
spontaneously hypertensive rats: influences of akiosterone, norepinephrine, and angiotensin. Circ Res 33: 563-572, 1973
Friedman SM: An ion-exchange approach to the problem of inlracellular
sodium in the hypertensive process. Circ Res 34 (suppl I): 123-128, 1974
Jones AW, Hart RG: Altered ion transport in aortic smooth muscle
during dcoxycorticosteronc acetate hypertension in the rat. Circ Res 37:
333-341, 1975
Friedman SM, Nakashima M, Friedman CL: Cell Na and K in the rat
tail artery during the development of hypertension induced by desoxycorticosterone acetate Proc Soc Exp Biol Med 150: 171-176, 1975
Friedman SM: Lithium substitution and the distribution of sodium in the
rat tail artery. Circ Res 34: 168-175, 1974
Friedman SM, Mar M, Nakashima M: Lithium substitution analysis of
N» and K phases in a small artery. Blood Vessels 11: 55-64, 1974
Friedman SM: H + and cation analysis of biological fluids in the intact
animal. In Glass Electrodes for Hydrogen and Other Cations, edited by
G Eiscnman. New York, Marcel Dekkcr, 1967, pp 442-463
Friedman SM: Comparison of net fluxes of Li and Na in vascular smooth
muscle. Blood Vessels 12: 219-235, 1975
Ronve G: Ionic composition of the rat aorta after incubation in saline
Life Sciences (I) 8: 919-927, 1969
Palate V, Guslafson B, Friedman SM: Maintenance of the ionic
composition of the the incubated artery. Can J Physiol Pharmacol
49: 106-112, 1971
Friedman SM, Nakashima M, Palaty V: Glass electrode measurement of
ion transfers dunng rewarming of the single rat tail artery. Can J Physiol
Pharmacol 47: 863-869, 1969
Kleinzeller A- Cellular transport of water. In Metabolic Transport,
*d 3, vol 6, edited by WE Hokin New-York, Academic Press, 1972,
pp 91-131
Friedman SM, Nakashima M, Palaty V, Walters BK: Vascular resistance and Na + -K + gradients in the perfused rat-tail artery. Can J Physiol
Pharmacol 51: 410-417, 1973
Guignard J-P, Friedman SM: Intraluminal pressure and ionic distribution in the tail artery of rats Circ Res 27: 505-512, 1970
Jones AW: Altered transport in large and small arteries from spontaneously hypertensive rats and the influence of calcium Circ Res 34 (suppl
I): 117-122, 1974
441
17. Guignard J-P, Friedman SM: Vascular ionic effects of angiotensin II in
the rat Proc Soc Exp Biol Med 137: 147-150, 1971
18. Keatinge WR: Ionic requirements for arterial action potential. J Physiol
(Lond) 194: 169-182, 1968
19. Somlyo AP, Somlyo AV: Vascular smooth muscle. I. Normal structure,
pathology, biochemistry, and biophysics. Pharmacol Rev 20: 197-272,
1968
20. Morns DJ, Davis RP: AWosterone; current concepts. Metabolism 23:
473-494, 1974
21. Matthes KJ, Junge-HUlsing G, Schmitt G, Wagner H, Oberwitler W,
Hauss WH: (Jber die Beziehung zwischen gesttJrtem Mesenchymstoffwechsel und VcrBnderungen der Lipidkonzentration in der Gefasswand
bei arterieller Hypertension. J Atherosclerosis Res 9: 305-318, 1969
22. Villamil MF, Nachev P, Kleeman CR' Effect of prolonged infusion of
angiotensin II on ionic composition of the arterial wall. Am J Physiol
218: 1281-1286, 1970
23. Hollander W, Kramsch DM, Farmelant M, MadofT IM- Arterial wall
metabolism in experimental hypertension of coarctation of the aorta of
short duration. J Clin Invest 47: 1221-1229, 1968
24. Todd ME, Friedman SM" The ultrastructure of peripheral arteries during
the development of DOCA hypertension in the rat. Z Zellforsco Mikrosk
Anat 128: 538-554, 1972
25. Palaty V, Gustafson B, Friedman SM: Sodium binding in the arterial
wall. Can J Physiol Pharmacol 47: 763-770, 1969
26. Palaty V, Gustafson B, Friedman SM: The role of protein-polysaccharides in hydration of the arterial wall. Can J Physiol Pharmacol 48:
54-60, 1970
27. Edelman IS, Fimognari GM On the biochemical mechanism of action of
aldostcrone. Recent Prog Horm Res 24: 1-44, 1968
28. Knox WH, Sen AK. Mechanism of action of aldosterone with particular
reference to (Na + K)-ATPase. Ann NY Acad S;i 242: 471-488, 1974
29. Friedman SM, Scott GH, Nokasfiima M L Vascular morphology in
hypertensive states in the rat. Anat Rec 171: 529-544, 1971
30. Friedman SM, Nakashima M, Mar M: Morphological assessment of
vasoconstriction and vascular hypertrophy in sustained hypertension in
the rat. Microvasc Res 3: 416-425, 1971
31. Folkow B, HallbSck M, Lundgren Y, Sivertsson R, Weiss L: Importance
of adaptive changes in vascular design for establishment of primary
hypertension, studied in man and in spontaneously hypertensive rats.
Circ Res 32 (suppl I): 2-13, 1973
Hormonal and Nutritional Substrate Control of
Cardiac Lysosomal Enzyme Activities
KERN WILDENTHAL, M.D.,
SUMMARY
Prolonged starvation is known to induce significant
alterations in several cardiac lysosomal enzymes, particularly the
acid proteinase cathepsin D. To determine what specific factors
might mediate these changes, fetal mouse hearts in organ culture
were maintained in media designed to simulate selected hormonal or
nutritional substrate changes that accompany starvation. Reduced
concentrations of glucose caused an increase in the activity of
/9-acetylglucosaminidase but had no effect on cathepsin D or acid
phosphatase activities (i.e., effects opposite from those of starvation).
Also, high concentrations of free fatty acid, acetoacetate, and /S-OHbutyrate induced an increase in cathepsin D ( + 18%) and a simulta-
PH.D.
neous decrease in glucosaminidase ( - 1 9 % ) , with little change in add
phosphatase. Furthermore, glucagon had no effect on any of the enzymes, whereas growth hormone caused a small (6%) increase in
cathepsin D activity. In addition, insulin deprivation caused significant increases (7-25%) in the activities of all three enzymes. Insulin deprivation and excess ketones, but not tbe other interventions,
increased the proportion of enzyme activity which was nonsedimentable. These results suggest the possibility that lysosomal alterations
during starvation may be related in part to prolonged insulin deficiency and exposure to high concentrations of ketones and free fatty
acids.
PROLONGED starvation is accompanied by characteristic
alterations in the activities and distribution of several
cardiac lysosomal enzymes.'- 2 The specific activity of
cathepsin D, the major acid proteinase in the heart, is
increased by 20-40% after 3-6 days of food deprivation in
mice, rats, and rabbits, and the proportion of activity that is
present in the nonsedimentable (i.e., non-particulate-bound)
fraction of the tissue homogenate becomes much greater
From the Pauline and Adolph Weinberger Laboratory for Cardiopulmonary Research, Departments of Physiology and Internal Medicine, University
of Texas Health Science Center at Dallas, Dallas, Texas.
Supported by grants from the Moss Heart Fund and the National Heart
and Lung Institute (HL 14706), and performed while Dr. Wildenihal held a
U S . Public Health Service Research Career Development Award (HL
70125).
Received March 23, 1976: accepted for publication May 24. 1976.
442
CIRCULATION RESEARCH
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than in hearts of fed animals. Immunohistochemical localization studies have revealed the changes to be confined
specifically to cardiac myocytes rather than interstitial or
endothelial cells. The myocytes display not only increased
amounts of the enzyme but also alterations in its subcellular
distribution that suggest an increased presence of cathepsin
D outside secondary lysosomes.1 Some other enzymes of
lysosomal origin are affected differently by starvation: acid
phosphatase activity is increased minimally, whereas the
activity of /S-acetylglucosaminidase remains constant or
declines.1-2
Prolonged starvation is accompanied by a number of
chronic metabolic changes that might play a role in causing
the alterations in cardiac lysosomal function. Concentrations of fatty acids and ketones become elevated, while
glucose is reduced. Levels of growth hormone and glucagon
rise, and insulin falls. The present study was undertaken to
test under controlled conditions which of these factors might
possess the potential to play a role in mediating the effects of
prolonged starvation on the heart. As a practical matter it
would be impossible to test the chronic effects of these
agents using conventional experimental models, in which the
period of relative stability is limited to a few hours.
Accordingly, an organ culture system was employed, in
which intact hearts from fetal mice can be maintained in a
reasonably stable functional state for several days under
precisely controlled conditions in vitro. 3 The use of any in
vitro model involves, of course, the dilemma of knowing to
what extent it is legitimate to extrapolate results to related
situations in vivo, and hearts in organ culture are no less
subject to this problem than are traditional systems. The use
of fetal hearts may compound the problem when attempts
are made to relate results to adult hearts. Nevertheless,
results in vitro may provide valuable hints about the
potential ability of specific interventions to elicit analogous
responses in vivo, so long as caution is used in drawing
extrapolations.
Methods
Intact hearts of 18- to 20-day fetal mice were maintained
in organ culture as described in detail previously.3 Briefly,
the hearts, which weighed 2-4 mg and measured < 1 mm in
thickness, were isolated under sterile conditions and explanted on stainless steel grids at the liquid-air interphase
of shallow culture chambers. They were maintained at
37.5°C and exposed to an atmosphere of 95% O a -I- 5%
COj. The basic nutritive solution was medium 199 or
Earle's salt solution (Grand Island Biologicals); additions
to or deletions from the basic medium were employed as required by the experimental protocol. Glucose was used in
concentrations ranging from 50 to 400 mg/100 ml of solution. Other substrates added to the basic medium included
sodium octanoate (2 mM), sodium acetoacetate (5 mM), and
sodium /5-OH-butyrate (10 mM). Studies on effects of variations in glucose concentration were made with medium 199
supplemented with 2 mM octanoate to ensure that changes
induced by low glucose could not be due to an inadequate
energy supply per se. For tests of hormonal effects, crystalline bovine insulin (50 fig/m\; Sigma), glucagon (0.5-50
jig/ml; Lilly), and growth hormone (20-100 //g/ml;
VOL. 39, No. 3, SEPTEMBER
1976
Miles) were added to the medium. For studies of effects of
long chain fatty acid, hearts were cultured in medium 199
supplemented with 35% fetal calf serum (Colorado Serum
Co.); oleic acid (final concentration = 1.7-2.0 mM) was
saponified with NaOH and then combined slowly at 45°C
with undiluted serum that had previously been chilled to
4°C, after which the serum was added to the chemically
defined medium.
After cultivation for 3 days, hearts were assayed for
protein and for activities of creatine phosphokinase (a
nonlysosomal enzyme which served as a general marker of
cellular viability) and cathepsin D, /3-acetylglucosaminidase,
and acid phosphatase (all of which are localized at least
partially in lysosomes in heart). Preliminary experiments
revealed that specific activities of the lysosomal enzymes
rose gradually during the 3-day period of cultivation; thus,
an agent which caused an increase in activity over matched
controls would do so by stimulating enzyme activity over
and above the normal effects of cultivation per se.
For measurements of total enzyme activities, the hearts
were homogenized vigorously in a 0.1% solution of Triton
X-100 with a Polytron homogenizer to disrupt cells and
organelles maximally. Cellular debris was sedimented at 350
g for 5 minutes, and assays were performed on the
supernatant fluid. Protein was measured by the technique of
Lowry et al.4 and creatine phosphokinase (CPK) by the
technique of Szasz et al. s The lysosomal enzymes were
assayed by modifications of the methods of Barrett 9 as
described previously.1 Briefly, cathepsin D activity was
determined from formation of Folin-reactive products from
purified hemoglobin at pH 3.2 and 45°C (Barrett's method
II); glucosaminidase, from cleavage of nitrophenol from
p-nitrophenyl-/3-acetyl-D-glucosaminide at pH 4.3 and
37°C; and acid phosphatase, from cleavage of nitrophenol
from /7-nitrophenyl phosphate at pH 4.5 and 37°C after
inhibition of nonlysosomal phosphatases with sodium acetate.6
For measurements of sedimentable vs. nonsedimentable
enzyme activity, the tissue was homogenized gently for 20
seconds in a Potter-Elvehjem homogenizer, using a buffered
solution (pH 7.4) of 0.25 M KC1, to retain intact lysosomes
insofar as possible.7 After initial centrifugation at 350 g to
remove undisrupted cells, nuclei, and debris, the supernatant
fluid was recentrifuged at 40,000 g for 20 minutes. The
supernatant fluid from this second centrifugation was assayed
for enzyme activity (termed "nonsedimentable" activity);
the pellet was rehomogenized vigorously in 0.1% Triton
X-100 and also assayed for enzyme activity (termed "sedimentable" activity). The ratio of nonsedimentable to total
(i.e., "sedimentable" plus "nonsedimentable") activity was
calculated as an index of lysosomal fragility or of the
presence of enzyme outside lysosomes, or both.
Samples were maintained at 4°C at all times until assay.
Three to eight hearts from a single litter were pooled to
provide enough material for the assays. In all studies, results
from hearts exposed to the experimental interventions were
compared with results from control hearts from matched
littermates that had been cultured and assayed at the same
times under similar conditions. Statistical comparisons were
made using Student's /-test for paired observations. Values
CARDIAC LYSOSOMAL ENZYMES/Wildenthal
443
TABLE 1 Effect of Alterations in Hormones and Metabolic Substrates on Activities of Lysosomal Enzymes and Creatine
Phosphokinase in Fetal Mouse Hearts Maintained in Organ Culture for 3 Days
Cathepsin
D activity
Acid phosphatasc
activity (%)
0-Acetylglucosaminidasc
activity (%)
A. Effect of reduced glucose (50
mg/100 ml)
B. Effect of ketones (5 mM acetoacetate + 10 mM /3-OH butyrate)
C. Effect of fatty acids (2 mM
octanoate)
D. Effect of glucagon (5 jig/ml)
E. Effect of growth hormone (20
103
99 ± 2
106 ± 2*
100 ± 1
98 ± 2
98 ± 3
101 ± 3
F. Effect of insulin deprivation
125 ± 4 *
107 ± 2*
125 ±2*
2
101
±
1
115 ± 3*
108 ± 3*
101
104 ± 2
±
3
Crca.line
phosphokinase
activity (%)
112 ± 2 *
94 ± 4
101 ±2
106 ± 3
99±2
93 ±2*
104 ± 3
95 ± 3
85 ±2*
Each value represents the mean ± 1 SEM of 8-12 samples (five hearts per sample), expressed as a percentage of values from matched littermate controls The
control medium for comparison A contained glucose at a concentration of 200 mg/100 ml, the control medium for comparison F contained insulin at a
concentration of 50 /ig/m\; all other control and experimental media contained glucose at 100 mg/100 ml and lacked insulin.
• P < 0.05 compared to control media, by Student's l-test for paired observations.
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are expressed throughout the paper as the mean ± 1 SEM of
each group of measurements.
Results
Spontaneous and rhythmic beating persisted throughout
the 3-day culture periods in all hearts in all media. Except
for hearts exposed to glucagon, which causes tachycardia at
the higher concentrations used,8 beating rates were similar
in all media.
Variations in substrate composition failed to alter the
specific activity of CPK but did exert selective effects on the
three lysosomal enzymes that were assayed. Summary
results are given in Table 1.
Reductions in the concentration of glucose over a physiological range had no effect on cathepsin D or acid phosphatase activities, but produced a significant increase in the activity of j9-acetylglucosaminidase. This effect, which was
progressive over a wide range of glucose concentrations
(Table 2), was opposite to that which would have been expected if hypoglycemia participated in starvation-induced
alterations in cardiac glucosaminidase activity in vivo.2
Addition of high concentrations of ketones to the culture
medium produced effects which partially mimicked the net
effect of starvation. Acetoacetate plus /S-OH-butyrate at
levels of 5 mM and 10 mM, respectively (which simulate their
relative concentrations during prolonged starvation), caused
a 15% increase in the specific activity of cathepsin D (P <
0.01) and an 8% increase in acid phosphatase (P < 0.05).
The activity of glucosaminidase was unaltered in the same
Jiearts. Additional experiments jn whicfi the two ketones
were added separately to the culture medium revealed that
either alone was able to produce the observed increase in
catheptic activity.
Addition to the medium of a soluble free fatty acid (FFA),
octanoate, produced no change in cathepsin D or acid
phosphatase activity. However, it did induce a small,
consistent decrease (7%) in glucosaminidase activity similar
to that observed in vivo after starvation or high fat diets.2 A
medium chain fatty acid such as octanoate is especially
useful in studies of fetal hearts, which in many species,
including mice, have not yet developed the capacity to utilize
long chain fatty acids, presumably because of inadequate
cardiac levels of carnitine and acylcarnitine transferase.*""
Despite their inability to be transported into mitochondria
and oxidized by fetal heart cells, however, long chain fatty
acids can be taken up and incorporated into triglycerides.
Accordingly, to establish whether the effect of fatty acids was
dependent on their oxidation or whether their uptake alone
would suffice to produce the changes, studies were repeated
using media containing oleate-enriched serum. The activity
of glucosaminidase in hearts maintained for 3 days in media
containing unenriched serum (FFA = 260 n») was 784 ± 55
TABLE 2 Effect of Glucose Concentration on the Activity of fi-Acetylglucosaminidase in Fetal
Mouse Hearts Maintained in Organ Culture for 3 Days
Glucose concentration
/3-Acetylglucosaminidase activity
(nmol nitrophenyl/hr x mg protein)
% of activity at 400 mg/100 ml
4O0mg/100ml
200mg/100ml
lOOmg/lOOml
50mg/l00ml
668 ±35
752±29*t
821±33*t
838 ± 36*
113%
123%
125%
Hearts of littcrmatcs were compared by Student's (-test at four concentrations of glucose. Each value represents the
mean ± 1 SEM of five matched samples (15 hearts).
• P < 0.05, compared to value at 400 mg/100 ml.
t P < 0.05, compared to preceding concentration.
CIRCULATION RESEARCH
444
Katonu and Octanotc Acid Atnort
140 -
Kctonn and Ocfanoic Add Pitisnt
120
g
100
J«
80
o
•6
Ivlty
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nmol nitrophenol/hour x mg protein, and the activity in
hearts of matched littermates exposed to increased oleate
(FFA = 1840 MM) was 769 ± 39 (-2% ± 4%, P > 0.30),
indicating that long chain fatty acids could not mimic the
effect of octanoate in fetal mouse hearts.
The effect of combined addition to the medium of fatty
acids and ketones is shown in Figure 1. The individual
effects of each alone were additive, and together they
produced an 18 ± 6% increase in cathepsin D activity (from
88 ^g tyrosine/hour x mg protein in controls to 104 in
hearts receiving the fatty substrates) and a simultaneous 19
± 3% decrease in glucosaminidase activity (from 765 nmol
nitrophenol/hour x mg protein to 619), while acid phosphatase was increased only minimally—i.e., changes analogous
to those induced in vivo by starvation.
Hormonal influences on cardiac lysosomal enzymes are
also shown in Table 1. Glucagon in concentrations of 0.5-50
fig/m] produced no changes in cathepsin D, acid phosphatase, or glucosaminidase. Growth hormone had no effect on
the latter two enzymes, but caused a small (6%) increase in
the specific activity of cathepsin D. In contrast, insulin
deprivation produced changes in the activities of all
enzymes. Cathepsin D and glucosaminidase were affected
similarly (+25% for both, P < 0.01), while acid phosphatase activity was increased less markedly (Fig. 2).
In addition to its effects on total enzyme activities, each
agent was tested for its effect on the distribution of enzyme
activity between nonsedimentable and sedimentable fractions of the tissue homogenate. The proportion of activity
which was nonsedimentable was unaltered by changes in
glucose concentration or by the addition to the medium of
glucagon or growth hormone. Oleic acid had no effect, but
octanoate caused small decreases in the nonsedimentable
60
u
4
I
40
UJ
20
Cattwptln D
Add PhosphatoM
£-oe«tylgluco«amlnldOM
FIGURE I Combined effects of ketones (5 mM acetoacetate + 10
/MM fi-OH-butyrate) and fatty acids (2 /MM octanoale) on specific
activities of lysosomal enzymes in fetal mouse hearts maintained in
organ culture for 3 days. Comparisons were made between hearts of
matched littermates, and results are expressed as a percentage of
control values. The data are derived from assays on 12 matched
samples (60 heans in each group). Variance bars represent ± / SEM.
• - P < 0.05, compared to values obtained in hearts cultured in the
absence of ketones and fatty acids. Glucose concentration was 100
mg/IOO ml in control and experimental media.
VOL. 39, No. 3, SEPTEMBER
140
1976
EJInuilin PrtMtit
tntulin AbMfrt
120
100
80
60
40
20
0
Cattwpiin D Add Photpnatat*
/9-octtytqluco»amintdoM
FIGURE 2 Effect of insulin deprivation on specific activities of
lysosomal enzymes in fetal mouse hearts maintained in organ
culture for 1 days. Comparisons were made between hearts of
matched littermates, and results are expressed as a percentage of
control values. The data are derived from assays on nine matched
samples (45 hearts in each group). Variance bars represent ±1 SEM.
* = P < 0.05, compared to values obtained in hearts cultured in
the presence of insulin (50 fig/ ml).
fraction (i.e., changes opposite to those induced by starvation). As shown in Table 3, both insulin deficiency and
excess ketones produced increases in the nonsedimentable
fraction. The changes were slightly more pronounced for
cathepsin D than for glucosaminidase (as is true for hearts of
starved animals), but the changes were small compared to
those encountered in vivo.1 2
Discussion
Starvation-induced alterations in lysosomal enzymes have
been demonstrated in heart,1- ' skeletal muscle, 11 and
liver. " • " Unlike liver, changes in heart and skeletal muscle
are not "pan-lysosomal" but rather are quite distinct for
different enzymes: 1 - 212 the specific activities of some
enzymes of lysosomal origin increase during prolonged
starvation (e.g., cathepsin D and, to a lesser extent, acid
phosphatase), while the activities of others may remain
constant or even decrease (e.g., /3-acetylglucosaminidase).
These alterations, along with simultaneous changes in the
lability of the enzymes, develop gradually, and significant
changes are not demonstrable for a day or more after
starvation has begun.1 2- l2 Interestingly, starvation-induced
increases in the degradation rates of cardiac and skeletal
muscle proteins also appear only after a lag period of a day
or more, despite earlier alterations in rates of protein
synthesis,1" which suggests the possibility that increases in
lysosomal proteolytic activity or enzyme "availability" or
both may be causally linked with increases in muscle protein
degradation during starvation, possibly in concert with
increased activities of nonlysosomal proteinases.11- I7 "' e
The metabolic basis for starvation-induced lysosomal
changes has been uncertain. Both insulin deprivation and
glucagon excess are known to alter lysosomal function and
protein degradation in perfused liver,20 and the hypoinsulinemia and nyperglucagonemia of starvation have therefore seemed likely candidates for mediating hepatic changes.
CARDIAC LYSOSOMAL ENZYMES/Wildenthal
445
TABLE 3 Effects of Insulin Deprivation and of Acetoacetate (5 /MM) and fi-OH-butyraie (10
mM) on the Proportion of Lysosomal Enzyme Activities Present in the Nonsedimentable
Fraction of Cardiac Homogenates
% nonsedimentable
calhepsin D activity
(100 x nonsedimentablc/
total activity)
% nonsedimentable
0-acetylglucosaminidase
activity (100 x
nonsedimentable/
total activity)
A.
Medium 199 (n = 8)
Medium 199 + ketones (n - 8 )
Difference
P
46 ± 1.9
49 ± 1.7
+ 3 ± 0.8
<0.01
50 ± 1.7
51 ± 1.6
+ 1 ±0.5
>0.05
B.
Medium 199 with insulin (n = 12)
Medium 199 without insulin (n - 12)
Difference
41 ± 1 . 6
45 ± 1.7
+4 ± 1.4
47 ± 1.3
50 ± 1.4
P
<0.0l
+ 3 ± 1.1
<0.05
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For each intervention, hearts of matched littermates (eight hearts per sample) were compared by Student's (-test for
paired observations Each value represents the mean ± I SEM
Lack of insulin has been shown in cultured hearts to increase
the activity of cathepsin D, particularly in the nonsedimentable fraction of the tissue homogenate,*1- 12 and this change
can be correlated with concomitant increases in the rate of
protein degradation.12 On the other hand, short-term studies
of perfused rat hearts 23 - 2t have suggested that insulindependent alterations in lysosomal function and protein
degradation develop rapidly, unlike the delayed appearance
of starvation-induced changes. Furthermore, in short-term
studies of perfused hearts glucosaminidase is affected in a
similar way and to a similar degree as cathepsin D.2<
The present experiments confirm that insulin deficiency in
vitro is a potent stimulator of lysosomal enzyme activity and
demonstrate that in cultured hearts, as in perfused hearts,
glucosaminidase is altered in the same way as cathepsin D,
even after several days. Thus, cardiac lysosomal alterations
produced by insulin deprivation in vitro do not mimic
exactly those observed after prolonged starvation. Therefore, although hypoinsulinemia may well contribute to some
of the delayed effects of starvation, it seems unlikely to be
the sole explanation for the complex lysosomal alterations
that are observed in vivo.
On the basis of the present experiments, glucagon seems
unlikely to play a vital role in lysosomal function in fetal
mouse hearts, in contrast to its probable importance for
lysosomal function in liver. It is impossible from these data
alone to know whether this is a universal distinction between
cardiac and hepatic responses or whether it is species-specific. Like guinea pig hearts" but unlike liver and unlike
hearts of many other species, mouse hearts apparently lack
the capacity to respond to glucagon with increases in adenylate cyclase activity and cyclic AMP. 1 ' " Thus, if glucagoninduced lysosomal alterations are mediated through the
adenylate cyclase-cyclic AMP system, it might be expected
that mouse hearts (and, presumably, guinea pig hearts as
well) would fail to be affected, even though hearts of other,
responsive species could react the way liver does. Nevertheless, since starvation alters cardiac lysosomes similarly in
the mouse as in other species, 1 ' it seems doubtful that
glucagon is of primary importance in causing cardiac
changes in any species during starvation.
Growth liormone in very targe concentrations causes only
a minimal increase in cardiac cathepsin D activity, with no
change in the distribution of the enzyme. Thus, on the basis
of its effects in fetal hearts in vitro, its role in mediating
lysosomal changes in vivo therefore seems likely to be
negligible.
Substrate-dependent lysosomal changes are more complex. The effects of high concentrations of ketones and free
fatty acids result in directional changes in the activities of
cathepsin D, acid phosphatase, and glucosaminidase that
resemble those during starvation. Increases in cathepsin D
and, to a lesser extent, acid phosphatase are induced by
ketones, whereas metabolizable fatty acids produce a
decrease in glucosaminidase. Since these substrates cause a
reduction in the uptake and utilization of glucose and, since
reciprocal decreases in circulating levels of glucose often
occur simultaneously with increases in fatty acids and
ketones in vivo, it might have been expected that reduced
concentrations of glucose in vitro would cause effects similar
to those of ketones or fatty acids, or both. Paradoxically,
this was not the case, and decreased glucose availability was
accompanied by increased glucosaminidase activity over a
wide range of concentrations; thus, hypoglycemia seems
unlikely to play a direct role in causing the lysosomal
alterations that accompany starvation.
The demonstration that several of the interventions can
influence individual lysosomal enzymes in different ways
confirms in vitro the observation in several tissues in vivo,
including heart, that lysosomal enzyme respond to some
stimuli in a heterogeneous m a n n e r . ' 1 1 ' " In at least some
instances in vivo this could be due to an influx of migratory
cells and a resultant alteration in the cellular origin of the
lysosomes.11 " Such an influx is clearly impossible in an organ
culture system. One possible explanation is that there are
separate effects on the several subsets of distinct lysosomal
populations that are known to exist in heart."" 3 ' Alternatively, it remains possible that the synthesis or degradation
446
CIRCULATION RESEARCH
of certain enzymes within a given lysosomal population may
be altered independently from other enzymes in the same
lysosomes.
Finally, it should be reiterated that changes observed in a
relatively unphysiological system such as organ culture
should be extrapolated to hearts in vivo only with caution;
factors important in this highly catabolic model may be
different in some regards from those important in adult
hearts in vivo.22 and confirmatory studies in intact animals
would be valuable. It also should be noted that the relatively
small changes in hydrolytic enzyme activities and distribution noted here do not necessarily imply that concomitant
alterations in cellular catabolism must occur in all instances,
and actual measurements of the rate of proteolysis are
required before postulations about the ultimate metabolic
effect of the observed lysosomal changes should be entertained.22-30-"
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Acknowledgments
I am grateful to J R Wakeland, P.C. Morton, S C. Jasinski, A Vasil, and
M. Kennedy for invaluable technical assistance.
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Hormonal and nutritional substrate control of cardiac lysosomal enzyme activities.
K Wildenthal
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Circ Res. 1976;39:441-446
doi: 10.1161/01.RES.39.3.441
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