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CARBOHYDRATE METABOLISM IN LIZARDS 1531

Studies on Carbohydrate Metabolism in Lizards H . S . ALI A T H A R , S . NAZRUL HASNAIN, a n d M . ZAIN-UL-ABEDIN *

Department of Biochemistry, Univeristy of Karachi, Karachi-32, Pakistan

(Z . Naturforsch. 27 b , 1531—1535 [1972] ; received July 7, 1972)

Carbohydrate Metabolism, Lizards, Saccharoid Fraction 1. Initial glycogen levels in the liver were found to be significantly higher than in the kidneys

of the two species studied. The glycogen levels in kidney were however considerably higher than in some mammalian species.

2. Total reducing capacity, true glucose, saccharoid fraction and titratable acidity increase gradually with concomitant decrease in the glycogen levels when liver and kidney homogenates of uromastix and liver homogenate of varanus were incubated for a period of 4 hours.

3. Total reducing capacity, true glucose and the titratable acidity increase gradually, while the glycogen and the saccharoid fraction remain unchanged when the kidney homogenates of varanus are incubated for 4 hours.

4. Total reducing capacity, true glucose and saccharoid fraction in the blood of uromastix are higher than in varanus. The blood glucose levels in both the species are higher than found in the mammals.

Carbohydrates are required by the living orga-nisms to provide energy for work and other meta-bolic activities. Metabolism of carbohydrates mainly deals with the metabolism of glucose and closely related substances. The principal route of carbo-hydrate metabolism is through glycolysis and citric acid cycle. Many workers have however contributed in working out the intricate mechanisms of pentose phosphate pathway1 - 4 and glucuronic acid cyc le 5 - 7 . The extent to which any one or more of these pathways is used by any organism depends upon several factors. Changes in the nature of environmental conditions of the organism are as-sociated with innumerable modifications in the pat-tern of life. Many adaptive changes of structure and behaviour are related to biochemical modifications which have been associated with the environmental changes. Reptiles occupy a pivotal position in the animal kingdom because they serve as a link between the poikilotherms and the homeotherms. Most of the studies that have been conducted on carbo-hydrate metabolism in reptiles are related to seasonal variations in blood sugar levels 8' 9, blood lactic acid9 and glycogen content of the tissues 10. The present study is concerned with the carbo-hydrate metabolism 'in vitro' with particular re-ference to blood sugar levels, saccharoid fraction of

Requests for reprints should be sent to H. S. ALI ATHAR, Department of Biochemistry, University of Karachi, Karachi-32, Pakistan.

* Present Address: Prof. M. ZAIN-UL-ABEDIN, Max-Planck-Institut für experimentelle Medizin, D-3400 Göttingen, W.-Germany.

blood, glycogen content of liver and kidney and time course of changes in the above parameters and titratable acidity of liver and kidney homogenates of two reptilian species.

Materials and Methods

1. Animals: The lizards of local species of Uro-mastix hardwickii and Varanus monitor were collected from fields near Karachi. Animals of both sexes were kept in wooden boxes without food for several days before the experiments. The animals were collected during the period May to October which is the active period of these animals.

2. Preparation of tissue homogenates: Livers of three animals were pooled and a 10% homogenate was prepared in 0.02 M Tris-HCl buffer pH 7.36 + 0.1 M NaCl, 0.0039 M KCl and 0.002 M CaCl2. In the case of varanus kidneys of three animals and in case of Uromastix kidneys of six to eight animals were homo-genised in the above buffer to give a 5% homogenate.

3. Studies with tissue homogenates: Samples of liver and kidney homogenates were drawn immediately at zero time and analysed for initial glycogen, glucose, total reducing capacity and titratable acidity. The rest of homogenates were incubated at 37 °C in a water bath with frequent shaking. Samples were drawn after 30, 90, 180 and 240 min and analysed for above para-meters.

a) Glycogen in the homogenates was precipitated with ethanol11 and then determined according to the method of F A L E S 1 2 .

b) True glucose was determined by N E L S O N - S O M O -GYI method 13.

c) Total reducing capacity of the homogenates at various time intervals was determined by F O L I N - W U method 14.

1532 H. S. ALI ATHAR, S. N. HASNAIN, AND M. ZAIN-UL-ABEDIN

d) Titratable acidity: 5 ml of the homogenates at each time intervals was transferred to a centrifuge tube and immediately immersed in a boiling water bath for 5 min, cooled and centrifuged. A 2 ml aliquot was titrated against 0.02 M NaOH, using 2 drops of thymol blue as indicator.

4. Initial glycogen content of the tissues: Liver and kidney of the animals were removed immediately after sacrifice and rinsed in chilled saline. A piece of each tissue was transferred to a tube containing warm KOH. Glycogen was precipitated with ethanol and estimated by anthrone method.

The difference between the values of total reducing capacity and true glucose was taken as saccharoid fraction. Total reducing capacity and true glucose were also determined in blood of the two species.

Results

The initial glycogen levels in the liver and kid-ney of uromastix and varanus are shown in Table I. The initial glycogen levels were found to be 3.96

Table I. Glycogen content of liver and kidney of uromastix and varanus. The values are expressed as mg glucose per 100

mg wet wt. tissue.

Animal Liver Kidney

Uromastix 3.96 4- 0.48 * 0.33 ± 0.01 (8) (10)

Varanus 4.62 ± 0.47 0.23 ± 0.01 (11) (12)

* Numbers in parentheses indicate the number of observations. Each value represents a mean + S.E.

and 0.33 mg-% in the liver and kidney of uromastix respectively, whereas the liver and kidney of varanus were found to contain 4.62 and 0.23 mg-% glycogen respectively. Thus the glycogen concentration in the liver of uromastix was 10 times that of its kidney while in varanus the glycogen content in liver was 20 times more than in kidney.

Table II shows the time course of changes in carbohydrate content and titratable acidity in the liver homogenate of uromastix. The total reducing capacity, true glucose, saccharoid fraction and titratable acidity increased gradually whereas glyco-gen content decreased. The total reducing capacity and true glucose levels increased from 0.38 to 1.23 mg-% and 0.19 to 0.66 mg-% respectively in 4 hours. The saccharoid fraction was increased 2§ times, from 0.2 to 0.57 mg-%. The titratable aci-dity increased from 13.9 to 17.0 //eq/100 mg tissue. The glycogen levels were however decreased from 1.97 to 0.5 mg-%.

The time course of changes in carbohydrate content and titratable acidity in the kidney homo-genate of uromastix is shown in Table III. The true glucose and total reducing capacity are more than doubled in 4 hours. The total reducing capacity was increased from 0.39 to 0.82 mg-%, whereas true glucose increased from 0.18 to 0.45 mg-%. The sac-charoid fraction was increased from 0.21 to 0.40 mg-%, similarly titratable acidity increased from 12 .75 to 14.2 /ceq/100 mg tissue. During this period of time the glycogen level fell from 0.17 to 0.06 mg-% hence it was reduced to about J in 4 hours.

The time course of changes in carbohydrate con-tent and titratable acidity in the liver homogenate of varanus is shown in Table IV. The total reducing capacity and true glucose was increased 3 to 4 folds in 4 hours. The total reducing capacity was in-creased from 0.67 to 1.87 mg-% whereas true glu-cose level was increased from 0.28 to 1.17 mg-%. The saccharoid fraction and the titratable acidity also increased from 0.18 to 0.45 mg-%. The saccharoid fraction was increased from 0.39 to 0.70 mg-%. The titratable acidity was increased from 12.1 to 16.0/<eq/l00 mg tissue, hence an in-crease of 32% in 4 hours. The glycogen level registered a sharp decline in the first 90 min (from

Table II. Time course of changes in carbohydrate content and titratable acidity in the liver homogenate of uromastix.

Time Carbohydrates mg glucose/100 mg tissue wet weight Titratable Acidity [min] Total Reducing True Glucose Saccharoid Glycogen (/<eq of lactate/100 m<

Capacity Fraction tissue)

0 0.38 ± 0.02 * 0.19 ± 0.03 0.20 ± 0.02 1.97 ± 0.55 13.9 ± 0.89 30 0.68 ± 0 . 1 0 0.32 ± 0 . 0 8 0.36 ± 0.02 1.39 ± 0.46 15.4 ± 0.87 90 0.91 ± 0 . 1 3 0.54 ± 0.13 0.37 ± 0.03 0.91 ± 0.32 16.4 ± 0.90

180 1.07 ± 0 . 1 3 0.59 ± 0.08 0.48 ± 0 . 1 1 0.69 ± 0.20 16.4 ± 0.65 240 1.23 ± 0 . 1 5 0.66 ± 0 . 0 1 0.57 ± 0.15 0.50 ± 0.15 17.0 ± 0.49

* Mean + S.E. Each value is a mean of 4 observations.

CARBOHYDRATE METABOLISM IN LIZARDS 1 5 3 3

Table III. Time course of changes in carbohydrate content and titratable acidity in the kidney homogenate of uromastix.

Time Carbohydrates mg glucose/100 mg tissue wet weight Titratable Acidity [min] Total Reducing True Glucose Saccharoid Glycogen (/ /eq of lactate/100 mg

Capacity Fraction tissue)

0 0.39 ± 0.01 * 0.18 ± 0 . 0 1 0.21 ± 0.02 0.17 ± 0.01 12.75 ± 1.65 30 0.55 ± 0.02 0.25 ± 0.01 0.30 ± 0.005 0 . 1 0 ± 0 . 0 1 13.15 ± 1.52 90 0.68 ± 0.01 0.35 ± 0.02 0.33 ± 0.08 0.08 ± 0.02 13.50 ± 1.57

180 0.78 ± 0.01 0.39 ± 0.03 0.39 ± 0.08 0.08 ± 0.01 13.85 ± 1.57 240 0.82 ± 0.01 0.45 ± 0.05 0.40 ± 0.06 0.06 4- 0.01 14.20 ± 1.46

* Mean + S.E. Each value is a mean of 4 observations.

Table IV. Time course of changes in carbohydrate content and titratable acidity in the liver homogenate of varanus.

Time Carbohydrate mg glucose/100 mg tissue wet weight Titratable Acidity [min] Total Reducing True Glucose Saccharoid Glycogen (//eq of lactate/100 mg

Capacity Fraction tissue)

0 0.67 ± 0.04 * 0.28 ± 0.01 0.39 ± 0.03 3.40 ± 0.50 12.10 ± 0.36 30 1.10 ± 0.06 0.58 ± 0.01 0.52 ± 0.05 2.40 ± 0.65 12.95 ± 0.28 90 1.43 ± 0 . 0 7 0.99 ± 0.06 0.51 ± 0.02 0.84 ± 0.23 14.75 ± 0.62

180 1.79 ± 0.12 1.12 ± 0.10 0.67 ± 0.07 0.45 ± 0.12 16.0 ± 0 . 4 4 240 1.87 ± 0.12 1.17 ± 0.08 0.70 ± 0.09 0.30 ± 0.02 16.0 ± 0 . 5

* Mean ± S.E. Each value is a mean of 4 observations.

Table V. Time course of changes in carbohydrate content and titratable acidity in the kidney homogenate of varanus.

Time Carbohydrate mg/100 mg tissue wet weight Titratable Acidity [min] Total Reducing True Glucose Saccharoid Glycogen (//eq lactate/100 mg

Capacity Fraction tissue)

0 0.43 ± 0.08 * 0 . 1 0 ± 0 . 0 1 0.33 ± 0.06 0.037 ± 0.01 10.30 ± 0.79 30 0.40 ± 0.03 0.14 ± 0.02 0.26 ± 0.04 0.035 ± 0.01 11.35 ± 0 . 7 2 90 0.52 ± 0.06 0.18 ± 0.03 0.34 ± 0.04 0.061 ± 0.01 12.15 ± 1.08

240 0.61 ± 0.06 0.25 ± 0.03 0.36 4 - 0.03 0.037 ± 0.01 12.30 ± 1.04

* Mean ± S.E. Each value is a mean of 4 observations.

3.40 to 0.84 mg-%) and was then reduced gradually to 0.30 mg-% at the end of 4 hours incubation period. During the total incubation period the glyco-gen level was reduced to l / l 0 the initial levels.

Table V represents the time course of changes in carbohydrate content and titratable acidity in the kidney homogenate of var anus. The total reducing capacity and true glucose levels increased gradually from 0.43 to 0.61 mg-% and 0.10 to 0.25 mg-% respectively. The saccharoid fraction however did not increase significantly. The titratable acidity was increased from 10.3 to 12.3//eq/100 mg tissue. The glycogen level remained constant during 4 hour period of incubation.

The saccharoid fraction in blood of uromastix and var anus is shown in Table VI. The saccharoid fraction in blood of uromastix and varanus were approximately 35.74 and 31.0 mg-% respectively.

Table VI. Saccharoid fraction of blood of uromastix and varanus.

Animal Total True Saccharoid Reducing Glucose Fraction Capacity

m g - % m g - % m g - % m g - %

Uromastix 157.80 ± 3 . 1 0 * 122.11 ± 4.91 35.74 ± 3 . 1 2 (10) (10) (10)

Varanus 138.30 ± 9.65 107.30 ± 7.20 31.0 ± 3 . 6 (6) (6) (6)

* Mean + S.E. Numbers in parentheses indicates the number of observations.

Discussion

The two reptilian species used in the present stu-dies — namely uromastix and varanus are taxonomi-cally very closely related to each other. Both of them belong to the natural order Lacertilia and have

1534 H. S. ALI ATHAR, S. N. HASNAIN, AND M. ZAIN-UL-ABEDIN

adapted for the desert life and live deep in burrows in sandy areas. Inspite of this they do have anatomi-cal and physiological differences distinct enough to place them in two different genera.

The glycogen levels in the kidney and liver tis-sues (Table I) are very much similar in both the species. The livers of uromastix and varanus were found to contain 3.96 and 4.62 mg glycogen per 100 mg wet wt of tissue. These values are similar to those observed in other animal species. WEBER and CANTERO 15 reported 4 mg-% glycogen in rat liver while LYON and PORTER 16 reported 5 . 0 7 mg-% in mice liver. In human beings the liver glycogen content is 3.15 mg-% 17 while in a lizard Varanus greseus, it is 4.4 mg-% 10. The glycogen content in kidneys of both the reptiles is considerably less in comparison to that of their livers. This is not sur-prising because liver plays an important role in ac-cumulating and storing the excess sugar from the blood in the form of tissue glycogen. This reserve carbohydrate is released into the blood circulation when sugar level falls below a critical level in the blood. Such a regulatory mechanism by the liver has been known for a long time.

Although the levels of liver glycogen in various species are about the same, there is a considerable difference in the glycogen levels of the kidneys of different species. KREBS et al.18 have reported 0 . 0 1

mg-% glycogen in rat kidney but higher glycogen content in the kidneys of reptiles has been re-ported 19. Perhaps the high glycogen content in the kidneys of these reptiles indicates a higher meta-bolic activity in this tissue as compared to mammals.

During the course of incubation of the liver homogenate of uromastix (Table II) over a period of 4 hours there was a gradual drop in the glycogen content while the total reducing capacity, true glu-cose, saccharoid fraction and titratable acidity in-creased concomitantly. During the process of gly-colysis when glycogen breaks down, pyruvate and lactate will be accumulated, alongwith an increase in the free glucose and sugar phosphate levels. The total reducing capacity usually includes besides the

1 E. RACKER, Advances in Enzymol. 15, 141 [1954]. 2 E. RACKER, Harvey Lectures 51,143 [1955]. 3 B . L . HORECKER a n d A . H . MEHLER, A n n . R e v . B i o c h e m .

24,207 [1955]. 4 H. G. WOOD, Physiol. Rev. 35, 841 [1955]. 5 S . HOLLMANN a n d 0 . TOUSTER, N o n - G l y c o l y t i c P a t h w a y s

of Metabolism of Glucose. Academic Press, New York 1964.

true glucose a number of other components in-cluding sugar phosphates 20. The difference between the total reducing capacity and true glucose is re-ferred to as saccharoid fraction. During the break-down of glycogen the increase in saccharoid fraction indicates the accumulation of sugar phosphates. Since the end product of glycolysis is the pyruvic and/or lactic acid, the increase in titratable acidity therefore is not surprising.

Similar pattern of changes in total reducing capa-city, true glucose, saccharoid fraction, titratable aci-dity and glycogen was observed in the liver homo-genate of varanus (Table IV).

The time course of changes in carbohydrate con-tent and titratable acidity in the kidney homogenate of uromastix (Table III) show that total reducing capacity, true glucose, saccharoid fraction and titratable acidity increased gradually with time whereas glycogen decreased. Time course of changes in total reducing capacity, true glucose, saccharoid fraction, titratable acidity and glycogen in the kid-ney homogenate of varanus is shown in Table V. A gradual increase in total reducing capacity, true glucose and titratable acidity was noted whereas the saccharoid fraction and glycogen content showed no significant change.

Kidney homogenates of uromastix and varanus showed a different pattern of changes. Increase in total reducing capacity, true glucose, and saccharoid fraction was more in case of uromastix than in varanus and specifically saccharoid fraction showed no signi cant change in the latter.

The total reducing capacity, true glucose and the saccharoid fraction were higher in the blood of uromastix than in varanus (Table VI) . These levels are however significantly higher than those found in mammals 21. The saccharoid fraction is related to the blood sugar level and varies with total reducing capacity. This is in agreement with the findings of K H A N and RAHMAN 22 who reported that the change in the value of saccharoid fraction was similar to that of sugar and the behaviour was similar in nor-mal as well as in diabetes.

6 J . J. BURNS, A m e r . J. M e d . 2 6 , 7 4 0 [ 1 9 5 9 ] . 7 J. L. STROMINGER. Physiol. Rev. 40, 55 [I960] . 8 M . ZAIN-UL-ABEDIN a n d M . H . QAZI , C a n a d . J . B i o c h e m .

43, 831 [1965]. 9 E . H U T T O N a n d J . GOODNIGHT, P h y s i o l . Z o o l . 3 0 , 1 9 8

[1957]. 1 0 G . H A G G A G . K . A . RAHIM, a n d F . KHALIL, C o m p . B i o c h e m .

Physiol. 16, 457 [1965],

SODIUM SUBSTITUTES AND RECEPTOR POTENTIAL 1535

11 HASSID and ABRAHAM, Chemical Procedures for Analysis of Polysaccharides, in: Methods of Enzymology, Vol . 3, Academic Press, New York 1957.

12 F. W. FALES, J. biol. Chemistry 193, 113 [1951] . 13 N. NELSON and M. SOMOGYI, as cited in Practical clinical

B i o c h e m i s t r y , H . V A R L E Y , 4 t h e d i t i o n , W . HEINEMANN, Medical Book Ltd., London 1967.

14 O. FOLIN and H. Wu, as cited by OSER, in Hawk's Practical Physiological Chemistry, 14th edition, McGraw-Hill, New York 1965.

1 5 G . W E B E R a n d A . C A N T E R O , S c i e n c e [ W a s h i n g t o n ] 1 2 0 , 851 [1954].

1 6 J . B . L Y O N a n d J . P O R T E R . J . b i o l . C h e m i s t r y 2 3 8 , 1 [1963].

1 7 D . S . M A C I N T Y R E , S . PEDERSEN, a n d W . G . M A D D O C K , Proc. Soc. exp. Biol. Med. 47, 354 [1941].

1 8 H . A . KREBS, D . A . H . B E N N E T T , P . D E G A S Q U E T , T . GASCOYNE, a n d T . Y O S H I D A , B i o c h e m . J . 8 6 , 2 2 [ 1 9 6 3 ] .

1 9 M . ZAIN-UL-ABEDIN a n d B . K A T O R S K I , B i o c h e m . J . 1 0 2 , 189 [1967].

2 0 C . N . G R A Y M O R E a n d M . J . T O W L S O N , N a t u r e [ L o n d o n ] 195, 76 [1962].

21 J. S. ANINO, Clinical Chemistry, Principles and Procedures, 3rd ed. Boston, Little-Brown 1954.

2 2 I . A . K H A N a n d M . A . R A H M A N , N a t u r e [ L o n d o n ] 2 1 5 , 979 [1967].

The Effect of some Sodium Substitutes on the Receptor Potential of the Crayfish Photoreceptor Cell H . STIEVE, H . GAUBE, a n d T . MALINOWSKA

Institut für Neurobiologie der KFA Jülich

(Z. Naturforsch. 27 b, 1535—1546 [1972] ; received September 14, 1972)

Receptor potential, Na+-substitutes, ion dependence, photoreceptor cell, Crustacea Isolated crayfish retinas were perfused with four solutions in which Li+, NH4+, Tris H+ and

glucose were substituted for the sodium ions in the physiological salt solution. The changes of the extracellularly recorded receptor potential (ReP) evoked by short or long

stimuli were measured. The changes in the shape of ReP by test solutions were different for each Na-substitute.

For lithium ions as a Na-substitute (Tab. I and Fig. 3) the plateau value he was considerably decreased (to ~ 2 0 % ) contrary to the peak-amplitude /imax which even slightly increased.

Ammonium ions show quite a different effect than all the other substitutes. The ReP is de-creased strongly and irreversibly (Tab. II and Fig. 4 ) .

When Tris (hydroxymethyl-ammoniummethane-hydrochloride) is substituted for Na, Amax de-creased to about 60 per cent and the plateau is even more reduced (to 20 per cent; Tab. III) . Only the recovery-value for he (50% smaller) is markedly different contrary to our former experiments where choline was used as Na-substitute (decrease 20%).

Glucose as a substitute for sodium chloride caused strongly decreased peak-amplitude Amax 26% (Tab. IV and Fig. 9 ) . Increased osmotic pressure due to excess glucose causes irreversible damage of the ReP (Tab. V ) . All the changes except those produced by NH4+ were reasonably reversible.

The results can be explained by the following assumptions: a) the maximum of the ReP is caused mainly by an increase in the permeability of the cell mem-

brane for sodium. Ca- and Mg-ions also contribute to it to a certain degree. b) The plateau value of the ReP to long light stimuli is determined:

1. by the sodium concentration gradient, 2. by active transport processes, 3. by the Ca++- and Mg^-gradients, 4. the chloride gradient may perhaps contribute to this value.

It is generally accepted that the receptor potential of the invertebrate retina is caused by ion currents across the visual cell membrane. In this process positive ions permeating from the external medium into the cell play a decisive role.

In earlier publications we have reported ion sub-stitution experiments in the crustacean retina

Requests for reprints should be sent to Prof. Dr. H. STIEVE, Institut für Neurobiologie d. KFA Jülich GmbH., D-5170 Jülich 1, Postfach 365.

(STIEVE1'2). When external sodium was replaced by choline, we observed effects which were at least in part a specific result of the substitute ion. The present paper deals with a series of experiments per-formed with different sodium substitute ions in order to determine more exactly the specific role of sodium in the process leading to the receptor potential.

One should keep in mind that sodium ions play a different role in the visual system of invertebrates as compared to vertebrates; and that they possibly per-