Mechanism of hormone action. The role of butterfat in gluconeogenesis

Mechanism of hormone action. The role of butterfat in gluconeogenesis

Life Sciences Vol. 8, Part II, pp . 435-443, 1969 Printed in Great Britain. MECHANISM OF HORMONE ACTION . Pergamon Press TAE ROLE OF BUTTERFAT IN G...

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Life Sciences Vol. 8, Part II, pp . 435-443, 1969 Printed in Great Britain.

MECHANISM OF HORMONE ACTION .

Pergamon Press

TAE ROLE OF BUTTERFAT IN GLUCONEOGENESIS N. R. Sarkar

Animal Research Institute, Research Branch Canada Department of Agriculture, Ottawa, Canada

(Received 10 December 1968 ; in final form 29 January 1969) While studying the interrelationship between the rate of gluconeogenesis and activities of certain key enzymes involved in the process,

it became apparent

that the blood glucose levels in 48-hr fasting rats maintained exclusively with butterfat for 48 hr were not significantly different from those observed in rata after 48 hr fasting and lower than those found in rats after 96 hr fasting .

The

glucose production in butterfat fed rata after the administration of triamcinolone diacetate was also found to be considerably lower than had been previously found in fasting rats under similar conditions (1, 2) .

Aerrera et al .

(3) found

an increased production of glucose from alanine in the presence of linoleic acid in their liver perfusion experiments .

Similar results were also reported by

Williamson et al . (4) who used oleic acid instead of linoleic acid in their experiments .

Since synthesis of blood glucose was greatly auvpresaed in the exner-

invents reported in this Paper,

a detailed investigation of the changes in the

activities of asnartate and alanine aminotranaferases and PEPR in the livers of butterfat fed rate was undertaken in order to ascertain as to what extent these changes, if . there are any, can be related to the observed rate of gluconeogenesis in these animals . ?~aterials atul Methods ?gale albino rats weighing 140 to 150 g were purchased from Canadian BreedAbbreviations : PEP - ohosphoenolpyruvate ; PEPR - phoaphoenolpyruvate carboxykinase : PC - pyruvate carboxylase ; ATP - adenosine triphosphate ; NAD - nicotinamide-adenine dinucleotide .

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ing Compsnq, St . Constants la Prairie, Quebec . Five groups of rate initially fasted for 48 hr were maintained with butterfat diet ad libitum (5) and sacrificed at the end of 96 hr .

One group received

triamcinolone diacetate (10 mg/100 g body weighr_) 16 hr prior to sacrifice, the second and third groups received alanine and aspartate (1 mg/g body weight) respectively, 4 hr prior to sacrifice while the fourth group received 2 .5 ml (3x) of a 20x suspension of casein hydrolysate at 4 hr intervals commencing 16 hr prior to sacrifice .

The fifth group received no special treatment .

Ten rats were fasted for 48 hr and maintained with butterfat diet for an additional 48 hr and then sacrificed at the end of 112 hr .

For the last 16 hr

five of these rats were given a standard laboratory diet and the other five received hormone treatment . Ten rata were fasted for 96 hr, five of them received hormone 16 hr prior to sacrifice and the other five served as controls for this set of experiments . All animals were sacrificed at the end of 96 hr .

Five rata, maintained with the

laboratory diet during the entire period of experiment, served as controls . Five other rate were fasted for 48 hr and sacrificed at the end of this period .

The

hormone and amino acids were administered intraperitoneally and casein hydrolysate by stomach tube ae previously described (, 2) . The procedures for the preparation of homogenate and high speed supernatant fraction (cytosol), determinations of blood glucose, hepatic glycogen, and prorein contents, and the activity of phoaphorylase a were the same ae previously described (,1) .

The activities of aspartste and alanine aminoatransferases were

determined according to the procedures described in Sigma Technical Bulletin No . 505, based on the methods described by Reitman and Prankel (6), and Flroblewski and LaDue (7) .

The PEPR was assayed in the supernatant fraction by the method

described by Nordlie and Lardy (8) . Results The blood glucose levels were lowered from a control level of 120 mg ner 100 ml of blood to 66 and 78 mg per 100 ml of blood when the rats were fasted

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for 48 and 96 hr respectively .

The blood glucose level in butterfat fed rat

was 64 mg and elevated to 80, 88, 84 and 92 mg respectively after the administration of triamcinolone diacetate, alanine, aepartate, and casein hydrolysate, and to 102 mg when triamcinolone diacetate was administered after the removal of butterfat .

The blood glucose levels were elevated from 78 mg to 106 mg in 96-

hr fasting rate after the administration of the hormone . shown in Table I .

These reaulte are

The reaulte in Table I also demonstrate a lose in body weight

and liver weight of 20X and 29X respectively after 48-hr fasting and 30X and 42X respectively after 96-hr fasting .

No further significant changes in body

and liver weights were observed during the period when the 48-hr fasting rata were maintained with butterfat .

A small amount of glycogen (0 .56 mg/100 mg of

liver weight) was found in butterfat fed rata .

A loss in body weight of 15X, a

gain in liver weight of 18X, and an increase in glycogen content from 0 .56 to 2 .46 mg were noticed in butterfat fed rate after hormone treatment .

Aowever in

96-hr fasting rats an increase in liver weight by 31X and glycogen content from 0 to 3 .68 mg were noted after the administration of hormone .

An increase in

phosphorylase a activity from 48X to 70X and a decrease in body weight by 20X were also noticed in these hormone-treated butterfat fed rats . Activities of Hepatic Aapartate and Alanin e Aminotransferases in Butterfat Fed Rata Before and After Treatment The activities of aspartate and alanine aminotransferases were increased by 42X and 50X respectively after fasting for 48 hr and did not show any further change during next 48 hr, the period when these animals were maintained with butterfat (Figs . 1 and 2) .

The activities of the enzymes were increased by 38X

and 50X respectively in butterfat fed rats treated with hormone and 70X and 82X respectively if administered after the withdrawal of butterfat .

A 35X increase

in aspartate aminotranaferase activity and a 38X increase in alanine aminotransferase activity were also found in butterfat fed rats after the administration of alanine and aepartate respectively and 40X and 46X respectively (not shown) after the administration of casein hydrolysate .

The corresponding values in 96-

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The activities of aspartate aminotransferase (Fig . 1) and alanine aminotransfer . ase (Fig . 2) in the liver cytosols from rats under different metabolic states . The results are expressed in Sigma unite (Sigma Technical Bulletin No . 505) per ml of rat liver cytosol (1 g liver homogenized in 9 ml 0 .25 M sucrose, centrifuged at 105,000 x g for 2 hr) . The vertical lines represent the standard deviations . The number of animals in each experiment was 5 . a : Control, b : 4R-hr fasted rat, c : Butterfat fed rat, d : Butterfat fed rat plus laboratory diet (refeeding), e : Butterfat fed rat plus alanine, f : Butterfat fed rat plus aspartate, g : Butterfat fed rat plus hormone, h : Butterfat diet removed then hormone administered, and is 96-hr fasted rat . For details see Materials and Methods in the text . hr fasting rat were 180X and 2001 respectively of the control .

The refeeding of

the butterfat fed rats with a standard laboratory diet brought back the enzyme activities to control levels . T}ie results of 9'able 7T show that aspartate and alanine aminotransferases in the sera and muscle cytosols of animals were not affected after hormone

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treatments . TABLA, II The activities of aspartate and alanine aminotransferaees in the sera and cytosols prepared from muscles and livers of fasting rate before and after treatment with triamcinolone Treatment

Liver cytosol

Muscle cytosol

Serum

Aapartate

Alanine

Aspartate

Alanine

Aspartate

Alanine

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563

185

1066

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4 .0

0 .52

48-hr fasted

786

324

938

34 .6

3 .7

0 .49

1617

570

1135

34 .0

3 .4

0 .33

Hormonetreated, 48-hr fasted

For the conditions of the experiments see in the text . The results are expressed in Sigma unite /mg of protein . Each value represents the average of results from 5 different animals . Discussion The effect of fat on gluconeogenesis has been previously studied by Herrera et al . (1), Williamaon et al . (2), and Devis and Gibaon (9) .

The former imeat-

igators found increased glucose production from alanine by perfused livers in the presence of linoleic acid and oleic acid .

Davia and Gibaon (9) noted an in-

crease in PEP production from pyruvate by rabbit liver mitochondria in the presence of low concentration of oleate .

These authors also observed strong sup-

pression of PEP production at aupraoptimal concentration of oleate .

The results

presented in this paper, on the other hand, indicate that the gluconeogenic effect of hormone noted in fasting rats, is greatly suppressed when administered to butterfat fed rats .

The lack of adequate supply of amino acids Which are be-

lieved to be the major precursors of glucose synthesis in fasting and hormone treated rata, might be the cause of the observed lowered blood glucose levels . It is also of considerable interest to note that blood glucose levels in butterfat fed rats can be significantly elevated by administering such amino acids as

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alanine, aspartate or glutamate or a mixture of amino acids (casein hydrolysate), but not to the extents noted previously when administered to fasting rats . The suppression of PEP production noted by Davia and Gibaon (9) was attributed by them to insufficient availability of ATP, that is required for carboxylation of pyruvate to oxaloacetate . The results presented in this paper possibly suggest that the supply of amino acids to liver cells and hence their formation from extrahepatic tissue protein catabolism, is partially blocked in butterfat fed rats .

The possible

existence of a relationship between the rate of autolysis of muscle proteins and the release of amino acids in vivo has been suggested by Sarkar (10)~in order to explain the high rate of gluconeogenesis in hormone-treated rate .

It has also

been found that a 48-hr fasting rat can retain its body weight during the period (48 hr) when the animal was maintained with butterfat .

Although a lose in body

weight was observed in these animals after hormone treatment, it was less marked than that previously found with fasting or fed rats .

A 32X loss in body weight

in rats upon hydrocortisone administration was also reported by Nordlie et al . (11) . The increased production of blood glucose observed in butterfat fed rats after the administration of a single amino acid or a mixture of amino acids can also be ascribed to the changes in the activities of aminotransferases induced by them .

Waldorf et al . (12) also reported increases in the activities of as-

partate and alanine aminotranaferases in the livers from rats maintained with high casein content (80X) diet .

These authors did not, on the other hand, notice

any change in the activities of these enzymes by feeding low carbohydrate at high fat diet .

Barnabei and Sereni (13) did not find any increase in tyrosine

aminotranaferase activity in perfused liver unless an adequate level of amino acids was present in the perfusate .

The administration of hormone causes an

elevation of the blood glucose level, and possibly does so by supplying amino acids to liver cells where they are comierted to gluCOSe via gluconeogenic pathway and by increasing the activities of the enzymes associated with the process .

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Whether or not the gluconeogenic effect of an individual amino acid (or a mixture of amino acide) ie due to activation or induction of aminotransferase (and poasibly other gluconeogenic enzymes), is now under investigation . The PEPK activity which was increased by 100X after 48 hr fasting, did not show any further change when these rats were maintained with butterfat .

ftowever

its activity was increased after the administration of hormone, but was less marked than that previously noted in hormone-treated fasting rats .

PEPK, accord-

ing to Rrebs (14), is the moat important enzyme in the control of gluconeogeneais, nonetheless the importance of aminotranaferases cannot be ignored since not only are the activities of these enzymes more markedly affected after fasting or hormone treatment, but also a close varallelism between the increased rate of gluconeogeneais and increased activities of the aminotranaferases could be found under all experimental conditions where marked increases in rates of gluconeogeneais are observed .

Furthermore, neither the activities of these en-

zymes in muscle nor those in serum are altered after the administration of hormone, and it is interesting to note that muscle is not a gluconeogenic tissue . Summary The study of Che effect of dietary fat on gluconeogeneais revealed no significant changes in body weight, liver weight (per 100 g body weight), blood glucose level, and activities of phoenhorylase a, aspartate and alanine amino tranaferases, and PEPK during the period when the 48-hr fasting rat was maintwined exclusively with butterfat for 48 hr .

The gluconeogenic effect (as meas-

ured from blood glucose levels) and the induction of aminotranaferases and PEPR by triamcinolone diacetate were less marked in butterfat fed rate than in fasting rata .

Increases in the activities of aminotranaferases were also observed

in butterfat fed rats after the administration of a single amino acid viz . aspartate, alanine and glutamate or a mixture of amino acids .

The results sug-

gested that the availability of amino acids for gluconeogeneais in the livers of butterfat fed rata is limited and can be possibly considered as one of the causes for lowered blood glucose levels in these animals .

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Acknowledgement I am thankful to Mrs . Eileen Cook and Mr . John Rusheleau for their skillful technical assistance . References 1.

N. R . SARRAR, Life Sciences 6,

2597 (1967) .

2.

N. R. BARRAR , Life Sciences 7, 481 (1968) .

3.

M. G . HERRERA, D . RAMM, N . RUDERMAN and G . F . CAAILL, JR ., in Adv . in Enzyme Regulation , Vol . IV, p . 225 (1966) .

4.

J . R. WILLIAMSON, R . A. KREISBERG and W . P . FELTS, Proc . Nat . Acad . Sci . U .S .A . 56, 247 (1966) .

5.

F . SAVER and J . D . ERFLE, J. Biol . Chem . 241,

30 (1966) .

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S . REITMAN and S . FRANKEL, Am . J. Clin . Path . 28, 56

7.

F . WROBLEWSRI and J. S . LaDUE, Proc . Soc . Expt . Biol . Med . 91, 569 (1957) .

8.

R. C . NORDLIE and H. A . LARRY, J. Biol . Chem . 238, 2259 (1963) .

9.

E . J . RAVIS and D . M . GIBSON, Biochem. Biophys . Res . Commun . 29, 815 (1967) .

(1957) .

10, N . K. SARRAR, European J . of Biochemistry - LETTERS, 2, 97 (1968) 11 . R . C . NORDLIE, F, E. VARRICCHIO and D . D . HOLTEN, Biochem. Biophys . Acta 97, 214 (1965) . 12 . ?~1, A . WALDORF, M . C . KIRK, H . LINKSWILPR and A . E . HARPER, Proc . Soc . Exptl . Biol . Med . 112,

764 (1963) .

13 . O . BARNABEI and F . SERENI, Biochem. Biophys . Res . Commun . 9, 188 (1962) . 14 . H . A . KREBS, in Adv . in Enzyme Regulation , Vol . IV, p . 339 (1966) .