J. Embryol. exp. Morph. Vol. 40, pp. 115-124, 1977
115
Printed in Great Britain © Company of Biologists Limited 1977
Hormone control in regeneration:
effects of somatostatin on appendage regeneration,
blood glucose and liver glycogen in
Diemictylus viridescens
1
By S. V E T H A M A N Y - G L O B U S , M. G L O B U S ,
J. A. H A R T F O R D , I. F R A S E R A N D D . W E B E R
From the Department of Biology, University of Waterloo, Canada
SUMMARY
In the present communication, synthetic somatostatin, a hypothalamic factor which has
a known inhibitory effect o n the release of growth hormone, thyroid-stimulating hormone,
prolactin, insulin and glucagon in man and other mammals, was found to have an inhibitory
effect on limb and tail regeneration in adult Diemictylus viridescens, when the newts were
treated with a daily dose of 3-5 or 15 /^g/animal for a period of 34 days post-amputation. At
the higher dose, the animals exhibited total inhibition of appendage regeneration in a few
cases and the remainder showed a considerable delay compared to the controls; none of the
experimental animals reached the advanced four-digit stage achieved by the controls.
Furthermore, the blood glucose and liver glycogen values in the somatostatin-treated
animals were significantly lower than the control values. Mechanisms in the storage, mobili­
zation and utilization of glucose (involving hormones) are discussed in relation to appendage
regeneration in the newt and possible controls of regeneration at the level of the hypothalamus
are suggested.
INTRODUCTION
Previous work in appendage regeneration in the newt, Diemictylus viridescens
has implicated several hormones including, insulin, adrenocorticosteroids,
thyroxine, growth hormone and prolactin (Vethamany-Globus & Liversage,
1973a, b, c; and reviews by Schotte, 1961: Schmidt, 1968 and Thornton, 1968).
On the basis of a series of experiments, Schotte and his associates (1961)
postulated that the pituitary-adrenal synergism is a major controlling factor,
especially in the early stages of normal limb regeneration which include wound
healing and dedifferentiation. Wilkerson (1963) and Bromley & Thornton (1974)
showed that mammalian somatotrophin, injected into hypophysectomized
newts, also supported regeneration of the limb and other workers have demon­
strated that limb regeneration is thyroxine-dependent as well (Richardson,
1
Author's
Canada.
address:
Department of Biology, University of Waterloo, Waterloo, Ontario,
116
S. VETHAMANY-GLOBUS AND OTHERS
1940, 1945; Schotte & Washburn, 1954; Schmidt, 1968). Furthermore, it was
observed that prolactin alone increased survival and supported limb regeneration in hypophysectomized newts, but less effectively than the combination of
prolactin and thyroxine (Connelly, Tassava & Thornton, 1968; Tassava, 1969;
Bromley & Thornton, 1974) and on the basis of these findings Tassava (1969)
proposed that a prolactin-thyroxine synergistic control is operative in limb
regeneration. In a recent series of expei iments, Vethamany-Globus & Liveisage
(1973 a, b, c) demonstrated an insulin involvement in limb and tail regeneration
in adult Diemictylus viridescens. They showed (a) that insulin insufficiency
resulted in an interference with normal limb and tail regeneration, (b) that tail
blastemata in vitro require insulin for growth and differentiation and (c) that
a combination of hormones, namely, insulin, growth hormone, hydrocortisone
and thyroxine, gives optimum growth and cartilage differentiation. They
suggested that no single hormone has the unique power of promoting normal
regeneration and proposed a multiple-hormone control of limb and tail
regeneration in the newt.
Recently, several hypothalamic factors have been isolated, characterized and
synthesized which exert selective control over the release of pituitary hormones
(see review by Serially, Arimura & Kastin, 1973) and can selectively alter the
levels of pituitary hormones in vivo without the need for surgical removal of
the gland. One such hypothalamic factor which inhibits the release of growth
hormone from the adenohypophysis, was recently isolated from sheep hypothalami, characterized as a peptide and synthesized (Brazeau et al. 1973); it is
now referred to as somatostatin (SRIF-somatotrophin release inhibiting
factor). Infusion of somatostatin in man and other mammals was found to
inhibit the secretion of growth hormone, thyroid stimulating hormone (TSH)
and prolactin (Siler, Yen & Vale, 1974; Yen, Siler & DeVane, 1974), and also
reduces the blood levels of insulin and glucagon in baboons and man by acting
directly on both alpha and beta cells of the islets of Langerhans (Koerker,
Goodner & Ruch, 1974a; Koercker, Ruch & Chideckel, 1914b \ Smith, Woods &
Johnson, 1974; Yen et al. 1974). In view of these inhibitory actions of somatostatin on growth hormone, prolactin, TSH, insulin and glucagon, an
attempt was made, in the present experiments, to evaluate the effects of
somatostatin on limb and tail regeneration in the newt. Moreover, these
hormones are closely related to carbohydrate, protein, lipid and nucleic acid
metabolism (Tepperman, 1965). In this study of SRIF-induced metabolic
changes in the adult newt, we have attempted to monitor the hormone effects
on carbohydrate metabolism (blood glucose levels and liver glycogen content),
though probably the effects are not exclusive to it.
Hormone control in regeneration
117
MATERIALS AND METHODS
Adult newts, Diemictylus viridescens, from Nashville, Tennessee, U.S.A.,
were kept in deionized water at 20 ± 1 °C, and fed ground beef twice weekly.
After acclimatizing to laboratory conditions for at least 1 month prior to experimentation, medium sized animals of both sexes weighing 2-5 g were selected
for the experiments.
Right forelimb amputations, just proximal to the elbow, were performed
after the newts were anaesthetized in a 1:1000 solution of M.S. 222 (tricaine
methanesulphonate, Sandoz). Synthetic linear somatostatin, generously supplied
by Dr R. Deghenghi, Ayerst Research Laboratories, Montreal, Canada, was
kindly prepared and supplied to us by Dr P. Brazeau, University of Montreal,
Montreal, Canada, in the form of a somatostatin-protamine-zinc suspension
(PZ-SRIF). Each of the experimental animals received a daily intraperitoneal
injection of PZ-SRIF starting at 2 days prior to amputation and continuing
every 24 h thereafter for 36 days (i.e. 34 days post-amputation). Two different
doses of somatostatin, namely 3-5 and 15 ^g/0-03 ml/animal, were administered.
One set of control animals received daily injections of protamine zinc (PZ),
and a second set of control animals (Y) were allowed to regenerate without
treatment. During the regeneration period of 34 days urine-sugar analyses
were done periodically using Testape (Lilly and Co., Canada Ltd, Toronto).
Upon termination of the experiments (34 days post-amputation), the animals
were anaesthetized in M.S. 222 for approximately 30 min and blood samples
ranging from 50 to 100 /*1 were collected (by cardiac puncture) for blood-sugar
analysis using graduated lambda pipettes which had been previously flushed
with a heparin solution. Each tube of the measured aliquots of blood (also
containing heparin solution, 0-5 JJX) was quick-frozen in an ethanol-dry ice bath
and stored at - 20 °C for subsequent glucose analysis. The animals were fasted
for at least 48 h prior to the termination of the experiment. Blood glucose levels
in 10/4 samples (done in triplicate) were measured by a micromethod utilizing
an enzymatic (glucose oxidase) determination of glucose recommended by
Mattenheimer (1970). A correction factor obtained by use of a heparin blank,
was applied to the data.
The content of liver glycogen in PZ-SRIF-treated and control animals was
determined by an anthrone method modified by Carroll, Longley & Roe (1956).
For this purpose the livers were dissected, quick-frozen at - 55 °C and stored
at - 20 °C for subsequent glycogen analysis, the values of which are reported
as mg per gram of wet weight of liver.
Limb and tail blastemata from experimental and control animals were fixed
in Bouin's fluid, and decalcified in 8 % sulfosalicylic acid in 70 % alcohol for
3-4 weeks prior to embedding in paraffin wax. They were then sectioned at
8 /*m, stained with hematoxylin and counterstained with orange G-eosin.
118
S. VETHAMANY-GLOBUS AND OTHERS
RESULTS
A total of 62 adult salamanders were used in these experiments. An initial
series of 32 animals were given a daily dose of 3-5 jug of somatostatin (in
protamine-zinc suspension) per animal, administered over a period of 38 days.
This treatment did not alter the rate or the normal pattern of limb and tail
regeneration nor did it result in changes in the mean values of blood glucose
and liver glycogen levels. However, when the dose was increased from 3-5 to
15/tg/animal, statistically significant alterations were observed in the above
levels as well as in the rate of appendage regeneration. The results obtained
are summarized in Table 1 and graphically illustrated in Figs 1 and 2. In this
series of 30 animals, comprised of a control group (Y), a PZ group and a
PZ-SR1F group, the latter exhibited loss of appetite, they were less active than
the Y and PZ animals and during the experiment one PZ and one PZ-SRIF
animal died. The mortailty rate was very low and probably not directly
attributable to SRIF treatment.
Limb regenerates. After 34-36 days of regeneration, the majority of the Y
and PZ animals exhibited normal regenerates of an advanced four-digit stage,
whereas none of the PZ-SRIF animals had regenerated to that extent (see
Fig. 1). Two somatostatin-treated animals failed to regenerate a limb and the
remainder of them showed considerable retardation. Of these, 3 reached a
late-cone stage, with a central cartilage condensation, and 4 of them showed
the beginnings of digit formation. Two untreated control and 2 PZ animals
showed a similar delay, reaching only the late-cone stage; however, all 4 of these
had an epidermal blister on the blastema, which was presumably responsible for
the delay in limb regeneration. Epidermal blisters were not observed in the
PZ-SRIF animals.
Tail regenerates. The majority of the controls (8 of the 10 Y animals and 6
of the 9 PZ animals) had well advanced tail regenerates with 6-12 centra along
the segmental cartilage rod, ventral to the regenerating spinal cord. When tail
regenerates of the three groups were compared (Fig. 1), the somatostatintreated animals showed a pronounced delay in regeneration; 5 of the 10 PZSRIF tails exhibited mid-cone stage blastemata without cartilage whorls (i.e.
no centra). Moreover, when individual PZ-SRIF animals showed a delay in
limb regeneration, a corresponding delay in tail regeneration was frequently
observed. Although a daily dose of 15/tg of somatostatin did not completely
inhibit limb and tail regeneration in all of the treated animals, it did interfere
with the rate of regeneration, resulting in a considerable delay when compared
with PZ and Y control groups.
Blood glucose values. Blood samples, used in the determination of blood
glucose levels, were taken from experimental animals after a 48-h fast period
and anesthesia in MS-222 for 30 mins. Table 1 shows the mean blood glucose
values (+ standard deviation) for each group. A test for significance of the
Hormone control in regeneration
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oo
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AA
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119
k
DD
PZ
PZ-SRIF
PZ
. Y
Fig. 1. Stages in limb and tail regeneration achieved by the Y, PZ and PZ-SRIF
groups of animals. A and A, Uninjected controls for limb and tail respectively;
O and • , PZ injected controls for limb and tail respectively; • and • , PZ-SRIF
treated animals for limb and tail respectively. Each symbol represents a single
animal. Stages 1-6 in limb regeneration: 1 = no regeneration; 2 = early-cone
stage; 3 = mid-cone stage; 4 = late-cone stage with cartilage; 5 = beginning digit
stage; 6 = advanced digit stage; + = epidermal blister; s = spike, a-f stages in
tail regeneration: a = no regeneration; b = early cone stage; c = mid cone with
central cartilage; d = 4-5 centra in the cartilage rod; e = 6-8 centra in the
cartilage rod and f = 9-12 centra in the cartilage rod.
Table 1. Effects of somatostatin on limb and tail regeneration, blood
glucose and liver glycogen, in adult newt
Limb
regenerates
A
,
Group
A
B
C
*
D
Blood
Liver
Glucose
Glycogen
(Mean±s.D., (Mean±s.D.,
Tail
regenerates
E
,
F
*
G
*
H
mg/100 ml
of blood)
mg/g of
wet weight)
Y control
— 1/10 4/10 — 5/10 1/10 1/10 8/10 81-87 ± 19-55 32-51 ±14-14
group
PZ control — — 2/9 2/9 5/9
— 3/9 6/9 61-36 + 20-45 34-84 ±9-90
group
PZ-SRIF 2/9 — 3/9 4/9 — 5/9 3/9 1/9 43-72± 11-55 12-33 ±9-84
A = no regeneration; B = mid-cone stage; C = late-cone stage with cartilage; D =
beginning digit stage; E = advanced digit stage; F = mid-cone, tail regenerate with central
cartilage rod; G = young tail regenerate showing 4-5 centra; H = advanced tail regenerate
showing 6-12 centra.
120
S. VETHAMANY-GLOBUS AND OTHERS
100
1000
•fb
60 =
60
1
T3
O
O
20
PZ
i
20
PZ-SRIF
Fig. 2. Histogram showing the mean blood glucose values ± S.D., expressed in mg per
100 ml of blood (D), and the mean values ± S.D. of liver glycogen ( • ) , expressed in
mg per g of wet weight for Y, PZ and PZ-SRIF groups of animals. Y = uninjected
controls; PZ = protamine-zinc-injected controls; PZ-SRIF = somatostatin-treated
animals.
difference between the means was performed using the standard T-test with
N-l degrees of freedom. A comparison between somatostatin-treated animals
and controls (Y and PZ series) showed a significant decrease in the mean blood
glucose concentration of PZ-SRIF animals (P < 0-001 and P < 0-05 respectively). In all groups, considerable variability in blood glucose values was
recorded (62-101 mg% for the Y group, 41-82 mg% for the PZ group and
32-55mg% for the PZ-SRIF group); however, similar variations in blood
glucose levels have previously been reported in both larval and adult Ambystoma
tigrinum (Bartell & Frye, 1974). Also the basal blood glucose concentration in
adult Diemictylus viridescens, as measured under our experimental conditions,
was considerably higher than levels reported for Ambystoma tigrinum (Bartell &
Frye, 1974) and for Taricha torosa (Wurster & Miller, 1960). In a subsequent
study (Vethamany-Globus, Globus, Fraser & Weber, 1977) we have attributed
this elevation of blood glucose to the MS-222 anesthesia, and in particular to
the time of anesthesia. Despite the MS-222-induced elevation in the blood
glucose levels, the mean value of PZ-SRIF animals, as noted above, was significantly lower than that of the control animals, suggesting a decreased ability,
on the part of the former, to rapidly mobilize glucose into the blood. During
the course of the experiments, glucose excretion into the urine was monitored;
however, none of the animals (PZ-SRIF, PZ or Y) exhibited glucosuria.
Hormone control in regeneration
121
Liver glycogen. The mean values ( ± standard deviation) of liver glycogen
content in Y, PZ and PZ-SRIF groups, presented in Table 1 and Fig. 2, are
expressed as milligrams of glycogen per gram of wet weight of liver. The PZSRIF group exhibited a mean value (12-331 ±9-84mg glycogen) which was
significantly lower than that of the PZ (34-84 ± 9-9 mg) and Y (32-511 ± 14-135
mg) control groups (P < 0-001 and P < 0-01 respectively). The difference
between the means of the PZ and Y series was not statistically significant.
Presumably, the somatostatin treatment interfered with glycogenesis and/or
glycogenolysis, resulting in low values for liver glycogen as well as blood
glucose.
DISCUSSION
Previous studies have shown that adrenocorticosteroids, growth hormone,
thyroxine and prolactin are of prime importance in limb regeneration in adult
Diemictylus viridescens (see reviews by Schotte, 1961; Schmidt, 1968; Thornton,
1968). In addition, Vethamany-Globus & Liversage (1973 a, b, c) have established
that insulin is essential in the promotion of growth and differentiation of the
regeneration blastema, and have proposed a multiple hormone control of limb
and tail regeneration. If we assume that an SRIF-mediated inhibition of growth
hormone, TSH, insulin and glucagon release, similar to that found in mammals
does exist in salamanders, then regeneration would be seriously affected by the
SRIF-induced depressed metabolic state. Thus, consideration of hypothalamic
factors in regeneration is appropriate. It has been shown, for example, that
removal of the pituitary gland from its hypothalamic connections, followed by
autoplastic, heterotopic implantation of the gland, can restore regeneration in
hypophysectomized animals (Schotte & Tallon, 1960; Dent, 1970; Liversage &
Liivamagi, 1971). Although an intact hypothalamic-hypophyseal tract does not
appear to be necessary for regeneration to occur, the involvement of hypothalamic factors, such as SRIF, transported via the systemic route, cannot be
ruled out. Indeed, the present results support that contention.
In the current study, the long-term effects of somatostatin (SRIF) on the
adult newt were manifested in a considerable delay in limb and tail regeneration,
and in a significant decrease in both circulating blood glucose levels and liver
glycogen content, the latter two suggesting a hormone-induced derangement
in the carbohydrate metabolism. This is not surprising when one considers the
inhibitory action of SRIF on many diverse hormones, some of which are
closely linked to regeneration. Somatostatin has been found to inhibit secretion
of growth hormone (Brazeau et al. 1973), TSH (Siler et al. 1974) and prolactin
(Yen et al. 1974), and also has a direct effect on the beta cells of the pancreas
(Smith et al. 1974) inducing hypoglycemia and reducing the blood levels of
insulin in man (Alberti et al. 1973; DeVane, Silver & Yen, 1974; Mortimer
et al. 1974; Yen et al. 1974). Our data show that the mean liver glycogen
content in PZ-SRIF animals was significantly lower than the values obtained
122
S. VETHAMANY-GLOBUS AND OTHERS
for Y and PZ controls. Since both growth hormone and insulin are known to
influence the metabolism of liver glycogen, a decrease in the latter would be
expected if growth hormone and insulin levels were suppressed by somatostatin.
In this regard, growth hormone may activate glycogenesis by increasing the
transport of amino acids across the hepatic cell membrane, while insulin, on
the other hand, leads to an activation of hepatic glucokinase, tends to increase
the deposition of glycogen and decrease the output of glucose from the liver
(reviewed by Harper, 1973). Our data also show that the mean blood glucose
level of SRIF-treated animals was significantly lower than control animals,
which is consistent with the findings of DeVane et al. (1974) and Koerker et al.
(1974c) in mammals. Somatostatin has also been found to inhibit both glucagoninduced glycogenolysis and gluconeogenesis in isolated hepatic tissue (Oliver &
Wagle, 1975) and similar inhibitions could account for the decrease in circulating
blood glucose levels in SRIF-treated newts.
A total inhibition of all of the above-mentioned hormones was probably not
achieved in the SRIF-treated animals, as they survived and regenerated their
limbs and tails, albeit slowly. This is possibly due to the duration of the biological activity of the SRIF preparation which may not have extended over the
entire 24 h period between injections. Hence, the inhibition may have been
partly lifted prior to the subsequent injection, accounting for a partial rather
than a total deficiency of the hormones concerned and resulting in less drastic
metabolic derangements. Considering the spectrum of hormones involved in
SRIF inhibition, it is reasonable to suggest that SRIF-induced changes are not
exclusive to carbohydrate metabolism and may extend to protein, lipid and
nucleic acid metabolism as well; this remains to be determined.
As mentioned earlier in the results, the mean values of blood glucose for all
groups of animals were higher than the values reported in the literature for
other urodele species, namely, Taricha torosa (Wurster & Miller, 1960) and
Ambystoma tigrinum (Bartell & Frye, 1974). This elevation in blood sugar values
was found to be a response to stress, due to MS-222 anesthesia prior to termination of the experiments (Vethamany-Globus et al. 1977). Glycogenolysis is
normally stimulated by both epinephrine and glucagon. SRIF has no known
effect on epinephrine-induced glycogenolysis (Oliver & Wagle, 1975); nevertheless, the blood sugar levels of SRIF-treated animals failed to reach that of the
controls, even under the stress induced by anesthesia. These animals showed
instead an inability to mobilize hepatic glucose into the blood, as rapidly as the
controls. In this regard, an SRIF-induced deficiency of insulin and growth
hormone would be expected to lead to reduced deposition of liver glycogen.
The limited stores of liver glycogen we reported here, coupled with an SR1Finhibition of glucagon-promoted glycogenolysis was probably responsible for
the apparent inability, on the part of SRIF animals, to raise the blood sugar up
to the levels of controls. Work is currently in progress to assess SRIF-induced
histological changes in the pancreatic islets and the anterior pituitary gland.
Hormone control on regeneration
123
This work was supported by National Research Council of Canada Grant No. A9753 to
S. Vethamany-Globus and No. A6933 to M. Globus.
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WILKERSON,
(Received 17 November 1976, revised 7 February 1977)