A c t a Physiol Scand 1992, 146, 205-211

Effects of somatostatin on glucagon-stimulated glycogenolysis and gluconeogenesis in hepatocytes cultured in vitro J. ROSA, J. ROSA and V. F I S T E R Department of Physiology, Zagreb University School of Medicine, Zagreb, Croatia ROSA,J., ROSA,J. & FISTER,V. 1992. Effects of somatostatin on glucagon-stimulated glycogenolysis and gluconeogenesis in hepatocytes cultured in vitro. Acta Physiol Scand 146, 205-211. Received 26 November 1991, accepted 29 April 1992. ISSN 0001-6772. Department of Physiology, Zagreb University School of Medicine, Croatia. The effects of somatostatin (SS-14) on glycogenolysis and gluconeogenesis in rat hepatocytes cultured in vitro in a serum-free medium were investigated. Somatostatin (122 nmol 1-’) did not significantly change the basal glucose production with or without pyruvate (10 mrnol I-’). Glucagon strongly (over 100%) increased the glucose production in hepatocytes incubated in a medium supplemented with 10 mmol I-’ pyruvate. This increase in glucose production is the result of increased rates of gluconeogenesis and glycogenolysis. Somatostatin partially inhibited the glucagon stimulated increase in glucose production. Glucagon also significantly increased the glucose production in a glucose-free medium without pyruvate, which resulted from an increase of glycogenolysis. Somatostatin did not inhibit the increase in glucose production in these conditions. After a 4 h ‘fast’, glycogen in hepatocytes fell to a very low level. Glucose production was minimal. After the addition of pyruvate, there was a increase in gluconeogenesis and glucose production. Glucagon stimulated the rate of gluconeogenesis. Somatostatin completely inhibited this glucagon-stimulated increase in gluconeogenesis.

Key words: glucagon, gluconeogenesis, glycogenolysis, hepatocytes, somatostatin. Somatostatin has been shown to inhibit the secretion of a number of hormones, such as growth hormone, thyrotropin, insulin, glucagon and, under certain conditions, adrenocorticotropic hormone and prolactin (Oliver & Wagle 1975, Vale et al. 1975, Reichlin 1983a, Reichlin 1983b). Administration of somatostatin has been found to cause a change in blood sugar level, which is believed to reflect suppression of glucagon secretion (Altszuler et al. 1976. Dileepan & Wagle 1985). The other possibility is a direct action of somatostatin on liver metabolism. Somatostatin concentration in the hepatic portal vein is significantly higher than those obtained in peripheral veins (Berelowitz et al. 1978, Patel et al. 1980). Most of the somatostatin (preCorrespondence : Jagoda RoSa, Department of Physiology. Zagreb University School of Medicine, Salata 3, P.O. Box 978, 41000 Zagreb, Croatia.

dominantly SS-28) entering the portal vein originates from the intestinal tract (Brazeau 1986). There is some evidence that pancreatic somatostatin (predominantly SS-14) is released into islet capillaries (Aponte et al. 1985). In this study, direct effects of somatostatin (SS-14) on glycogenolysis and gluconeogenesis hepatocytes cultured in vitro were investigated and glucose production was measured in a glucose-free medium. W e found specific effects of somatostatin on glucose production, depending on experimental conditions and medium supplementation.

METHODS In all experiments, male adult Wistar rats, each weighing 150-250 g. were used. Waymouth MB 752/1 medium and Swim’s S-77 medium were purchased from Grand Island Biological Company (NY, USA)

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a

b

Fig. 1. Glucose production (pmol mg-' protein 2 h-') in cultured hepatocytes incubated in Hanks-Hepes medium without glucose with ( b ) or without (a) pyruvate (10 mmol I-'), treated with 0.1 pmol I-' glucagon 122 nmol 1-' somatostatin ( 0 )or with 0.1 pmol I-' glucagon and and in untreated controls (0). Each value is the mean S E M for 122 nmol 1-I somatostatin (0) four to five plates in two separate experiments

(o),

\VOJBA2000 medium is \Vaymouth \IB 752/1 medium containing the following additions per litre : 2 g albumin; 36 mg alanine; 53 mg serine; 644 mg glutamine; and 50 mg garamycine. Glucagon and collagen (type 111 acid-soluble) were purchased from Sigma Chemical Company (St Louis, 110, US.1). Collagenase CLS I1 (131 C mg-') was purchased from Northington (NJ, US.4). Somatostatin was purchased from Ferring (Malmo, SLveden), and trypsin inhibitor from Calibiochem (La Jolla, C.1, USA). Male rats were deprived of food 12 h prior to liver perfusion. Hepatocytes were isolated b!- a modified collagenase perfusion technique of Berry & Friend (1969). Calcium-free Swim's S-77 medium containing collagenase (0.5 mg m1-I) and insulin 0.5 pg m1-l) was used for perfusion of the liver. Usually more than 800b of cells excluded trypan blue as a measure of viability. After two washings with the same medium without collagenase, the cells were suspended to a final concentration of 106 cells ml-' in WOb/Bh,,oo medium. Three ml of the cell suspension were placed in 60 mm Petri dishes which had previously been coated with collagen. T h e culture dishes were maintained at 37 O C in an atmosphere of j o b CO, and 959; air. Four h after inoculation of culture dishes. the medium was replaced with fresh WO,/BA,,,, medium and cultures were incubated for 20 h. After having been 24 h in culture, the medium was removed and cells were incubated in glucose-free Hanks-Hepes medium, containing 10 mmol I-' pyru-

vate, without hormones (control) or with the addition of glucagon (0.1 pmol l-l), somatostatin (122 nmol l-') or both hormones. The glucose released into the medium was determined enzymatically with glucose oxidase. T h e rate of glycogenolysis was measured b>- incubating the cultures in glucose-free Hanks-Hepes medium without the addition of pyruvate. T h e rate of gluconeogensis was measured in cultures first incubated 4 h in glucose-free Hanks-Hepes medium without pyruvate and then in glucose-free Hanks-Hepes medium with the addition of 10 mmol I-' pyruvate. T h e incubation medium was removed and hepatocytes were washed three times with cold saline and frozen immediately in liquid nitrogen. T h e cells were digested in 0.2 N NaOH and an aliquot was taken for the determination of glycogen and protein. T h e amount of glycogen was determined by the Roehring & Allred method (1974) and protein by the Lowry et al. method (1951).

RESULTS Somatostatin (SS-14) alone, i n a concentration

of 122 nmol I-' did not change the basic glucose production (Fig. l a ) . Glucagon alone, in a concentration of 0.1 pmol 1-' caused a significant (30°10) increase

of glucose production. In the

presence of somatostatin, glucagon also sig-

Somatostatin a.nd glucose production

0.91 o.a

a

b

Fig. 2. Glycogen concentration (glycosyl units pmol mg-' protein) in cultured hepatocytes incubated in Hanks-Hepes medium without glucose with (b) or without (a) pyruvate (10 mmol I-'), treated with 0.1 pmol I-' glucagon (Q) 122 nmol 1-' somatostatin or with 0.1 pmol glucagon and 122 nmol 1-' somatostatin (0) and in untreated controls (0).Each value is the mean$SEM for four to five plates in two separate experiments.

(m)

1.6

f

0

h

Ps

lo

i3

05

I

1

2

I

3

I

4

0

1

i

j

i

Hours Fig. 3. Glucose production (pmol mg-' protein) in cultured hepatocytes incubated in Hanks-Hepes medium without glucose, treated with 0.1 pmol 1-' glucagon (-e-) and in untreated controls (-O-), without somatostatin (a) or in presence of 122 nmol I-' somatostatin (b). Each value is the mean SEM for four to five plates in two separate experiments.

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I

c

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30

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do -

Minutes Fig. 4. Glucose production (jtmol mg-' protein) in cultured hepatocytes after 4 h fasting incubated in Hanks-Hepes medium without glucose in presence 10 mmol I-' pyruvate and treated nith 122 nmol I-' somatostatin (-O-) or 0.1 pmol I-' glucagon and 122 nmol I-' somatostatin (-@-) (b), or with 0.1 pmol I-' glucagon (-A-) and in untreated controls with (-O-) or without (-@-) pyrurate (a). Each point is the meanfSEM for four to five plates in two separate experiments

nificantly ( 3 2 O,,) increased the basal glucose production. In both cases, the increase in glucose production was the result of increased glycogenolysis caused by glucagon (Fig. 2a). T h e addition of pyruvate (10 mmol 1-l) did not significantly change the basal glucose production, in either the presence or absence of somatostatin (Fig. 1 b). However, glucagon greatly increased ( 103°,,) the glucose production in culture:; supplemented with pyruvate. This was only partially the result of an increase in glycogenolysis and presumably primarily the result of an increase in gluconeogensis. In the presence of somatostatin glucagon significantly ( 1900) increased the glucose production (Fig. l b ) and decreased the concentration of glycogen by 40 (Fig. 2b). Somatostatin did not significantly change either the basal glucose production or the glucagon-stimulated glucose production in hepatocytes incubated in glucose-free Hanks-Hepes medium without the addition of pyruvate during a 4 h period (Fig. 3 b).

If the cultures were pre-incubated in glucosefree Hanks-Hepes medium for 4 h and then incubated in same medium during a 90min period, the basal glucose production was very low (Fig. 4a). The addition of pyruvate very strongly ( < 700",) increased the glucose production in cultures preincubated in glucose-free HanksHepes medium. There was an almost linear increase in glucose production throughout the 90-min period. Glucagon increased the glucose production during the first 30 min by more than 5Oo0. This increase was somewhat smaller during the next 60 min. The addition of somatostatin, however did not change the basal glucose production. In the presence of somatostatin, glucagon did not increase glucose production during a 90 min period (Fig. 4b).

DISCUSSION The concentration of glycogen in hepatocytes cultured for 24 h in serum-free WO,/BA,,,,

Somatostatin and glucose production medium was approximately equal to the glycogen content in rat livers of animals fed ad libitum. The basal glucose production after incubation of the cells in a glucose-free medium was 0.71 pmol 2 h-'. Presumably the major part of glucose produced in this period was delivered by quick activation of glycogenolysis. Somatostatin did not change the basal glucose production (Fig. 1). This is in agreement with all previous studies since such an effect of somatostatin on the basal glucose output has not been observed by any group of authors (Cherrington et al. 1977, 1983, Oliver & Wagle 1975. Sacks et al. 1977. Sacca et al. 1979, Stevenson et al. 1984). However, the addition of glucagon increased the basal gltlcose production in a medium without pyruvate by 30% and in a medium supplemented with 10 mmol 1-' of pyruvate by more than 100%. This increase presumably resulted from the activation gluconeogenesis but as well as from increased glycogenolysis (Fig. 2). In the presence of somatostatin, the increase of glucose production caused by glucagon was very small (1 9 %) in a medium supplemented with pyruvate, which may have resulted from the inhibition of glycogenolysis as well as of gluconeogenesis. Several previous in vitro studies were controversial concerning the direct effect of somatostatin on glucagon - stimulated glucose production in the liver. Oliver & Wagle (1975), working on hepatocyte suspension, showed that somatostatin inhibited glucose production in cells treated with glucagon, Sacks et al. (1977) made the same conclusion working on the liver perfused in vitro. This is in agreement with our results showing an inhibition of glucagonstimulated glucose production in cultured hepatocytes. On the other hand, some investigations on liver slices, liver perfusion and suspension of liver cells did not show any inhibitory action of somatostatin on either basal or glucagon-stimulated glucose production (Chideckel et al. 1975, Gerich et al. 1975, Cherrington et al. 1977, Jacobus & Wittman 1978). The reasons for discrepancies among these findings could be the use of different experimental models and different experimental conditions. We used a stationary hepatocyte culture, which has several advantages over perfused liver, liver slices or freshly prepared cell suspensions (RoSa & Rubin 1980). Sen0 et al. (1988) have demonstrated that glucagon-induced glucose output is lower with glucose than without

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glucose at every glucose concentration. Glucose output is inhibited by somatostatin (SS-14) when glucose is not added. We measured the glucose production in a glucose-free medium and demonstrated the inhibition of glucagoninduced glucose production in cultures supplemented with pyruvate. Hepatocytes were cultured for 24 h in serum-free WO,/BA,,,, medium with a high glycogen content, and after incubation in glucose-free medium without pyruvate, there was a quick and strong degradation of glycogen and this process was stimulated by glucagon, however, there was no effect of somatostatin on glucose production in these conditions. On the other hand, in hepatocytes with very low glycogen and in a glucose-free medium supplemented with 10 mmol l-1 pyruvate, there was an activation of gluconeogenesis stimulated by glucagon. Somatostatin completely inhibited this glucagon-induced increase in gluconeogenesis. The presence of glucose in the medium and conditions where there was only activation of glycogenolysis, may have been responsible for most negative somatostatin effects in previous experiments. The possible damage of somatostatin receptors could not be excluded in some experiments, since it is known that somatostatin inhibition of insulin release from isolated pancreatic islets was only demonstrated if the islets were insulated under very mild conditions (Okamoto et al. 1975). Most commercially available collagenase preparations are contaminated with trypsin which can damage protein receptors on the plasma membrane. We added a trypsin inhibitor to collagenase solution to prevent this deleterious effect. T h e mechanism of action of somatostatin is presently unknown. Specific somatostatin receptors on hepatocytes have not yet been demonstrated (Leitner et al. 1980). There is a structural relationship between somatostatin and glucagon, both of which have the same four amino acids occurring in the same sequence. Somatostatin is behaving as a partial agonist acting on the same receptors (Oliver et al. 1976). It is impossible to explain our results by this concept and we believe that the site of somatostatin interference with glucagon action must be far beyond membrane receptors. Somatostatin has been shown to inhibit cyclic AMP accumulation but there are contradicting reports regarding the involvement of cyclic AMP in the mechanism of action of somatostatin (Dileepan

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& Wagle 1983). I t is possible that somatostatm acts b!- decreasing intracellular calcium in hepatocytes. Somatostatin reduces m e m b r a n e permeability t o calcium i n the pancreatic x-cell (Reichlin 1983). If the s a m e mechanism is operative i n hepatocytes it would b e very easy 1 0 explain the inhibition of glucagon-stimulatt d gluconegenesis in hepatocytes only. T h e r e is a report stating that calcium deficient!. does not reduce accumulation of cyclic A\lP in response to glucagon b u t diminishes stimulation of gluconeogenesis bj- exterior cyclic AAiP. Ci1uc;igon has a rapid stimulator? effect on the flus of calcium from m e d i u m to tissue (Pilkis r t ill. 1972). K n e e r et a / . 1979). F u r t h e r studies are required t o elucidate the mechanism of somatostatin action i n heparoc!-tes.

of the endocrine pancreas and consequence of this

blockade on glucose homeostasis. 3 Clin Incest 5.5, 754-762. DILEEP.IU, K.N. & ~VAGLE, S.R. 1985. Somatostatin: a metabolic regulator. Life Sci 37. 2335-2343. GERICH, J.E., BIER,D., HA.AS, R., ~VOOD, C., BYRNE, R. 8; PENHOS, J.C. 1973. In vitro and in vioo effects of somatostatin on glucose, alanine and ketone metabolism in the rat (Abstract). Endocrinology Siippl 96, 128. J.KOBLS, K.E. & ~VITTMAN,J.S. 1978. T h e effect of somatostatin on gluconeogenesis and glycogenolysis in isolated rat hepatocytes (Abstract) Fed Pvoc 37, 339. KSEER,%,ll.,IVAGSER, M.J. & IARDY,H..4. 1979. Regulation b!- calcium of hormonal effects on gluconeogenesis. 3 Biol Chern 254, 12160-12168. LEITNER, J.K’., RIFKIN, R.M.,MAMAN, A. & SUSSMAN, K.E. 1980. The relationship between somatostatin binding and cyclic AMP-stimulated protein kinase inhibition. i21etabolism 29, 106.i-1074. ‘1his \\orb was supported b! a grant from the Croatian N.J., FARR,-4.L. & Research Fund U e thank 5fr Boris Tomasei IC for his LOWRY,O.H., ROSENBROUGH, RASDALL,R.J. 19.51. Protein measurement with the expert technical awstance Folin phenol reagent. f B d Chenz 193, 265--271. ( h . \ I O T O , H., S O T O , y.,h%IAYAMO10, s.,MABUCHJ, H. S. T.4mD.4, R. 1975. Inhibition by soniatostatin REFERENCES of insulin release from isolated pancreatic islets. FEBS Lett 54, 103-105. .-\LTSZULER, X., GOTTLIEB, B. & HAMPSHIRE,J. 1970. OLIVER, J R., LONG,K., WAGLE,S.R. & AL.LEN,D.O. Interaction of simatostatin, glucagon and insulin 1976. Somatostatin inhibition of glucagon-stimuon hepatic glucose output in the normal dox. lated adenosine 3’-5’-monophosphate accumulation Diabetes 25 (2), 116-121. in isolated hepatocytes (39547). Proc Soc Exp Biol APONTE,G . . GROSS,D. & y.4LlAD.A, T. (1985). .\led 153, 367-369. Capillar!- orientation of rat pancreatic D-cell OLIVER, J.R. & tV.AGLE, S.R. 1975. Studies on the processes : evidence for endocrine release of somatciinhibition of insulin release, glycogen01 statin. Am 3Phjlsiol249, (Gustrointest Lirer Ph),sto1 gluconeogenesis b!- somatostatin in the rat islets of 12), 6599-6606. langerhans and isolated hepatocytes. Biockenz BioBERELOWITZ, M.,KRONHEIXI, S., PIUSTONE, B. ik phys Res Cortim 62 (3), 772-777. SHIPIRO,B. 1978. Somatostatin-like immuno- PATEL, Y.C., n’HE4TLEY, T., FITZPATRICK, D. & reactivity in rat b100d.J. Clin Incest 61, 1410-1414. BROCK, G. 1980. .4 sensitive radioimmunoassay for BERRY,5I.N. 8; FRIEND,D.S. 1969. High-yield imrnunoreactive somatostatin in extracted plasma: preparation of isolated rat liver parenchymal cells : lleasurement and characterization of portal and a biochemical and fine structural study. J Cell B i d peripheral plasma in the rat. Endocrinology 107, 43, 50lt520. 306-313. BR.AZEAC, P. 1986. Somastatin: a peptide with PILKIS, S. CLAUS, T., JOHNSON, R,.L & PARK,C.R. unexpected physiologic activities. .4m f AJed 81 1972. Hormonal control of cyclic 3‘: 5’-AMP levels (SUPPI6B). 8-13. and gluconeogenesis in isolated hepatocytes from CHERRINGTOS, A.D., CALD\+’ELL,M.D., DIETZ, 51.R., fed rats. 3 Bid Chenr 250, 6328-6336. EXTON, J.H. & CROFFORD, O.B. 1977. T h e effect of REICHLIN, S. 1983a. Somatostatin. (First of two somatostatin on glucose uptake and production b! parts). *\-eD EtzglJ Med 309 (24), 1495--1501. rat tissues in ritro. Diabetes 26 (8), 740-748. REICHLIS, S. 1983b. Somatostatin. (Second of two CHERRISGTON, AD., LACY,K’.\V., ~VILLIAXIS, P.E. tLparts). S e w E t i g l 3 itled 309 ( 2 5 4 1556-1563. STEISER.K.E. 1983. Failure of somatostatin to ROEHRISG, K.L. & ALLRED,J.B. 1974. Direct enmodify effect of glucagon on carbohydrate metaq m a t i c procedure for determination of liver bolism in the dog. Am -7 P h p o l 244, E596E602. glycogen. -4nal Biorhem 58, 414-421. EL, E., p.4I.\tER, J., KEORKER, D.H., ESSISCK, ROSA,J. 8i RUBIN,E. 1980. Effects of‘ ethanol on J., DAVIDSON, l1.B. & GOODNER,C.J. 197:. ~ 43, amino acid uptake by rat liver cells. L NInacsr Somatostatin blockade of acute and chronic stimuli 366-372. J.?

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Effects of somatostatin on glucagon-stimulated glycogenolysis and gluconeogenesis in hepatocytes cultured in vitro.

The effects of somatostatin (SS-14) on glycogenolysis and gluconeogenesis in rat hepatocytes cultured in vitro in a serum-free medium were investigate...
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