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Hexose metabolism in pancreatic islets. Effect of (−)-hydroxycitrate upon fatty acid synthesis and insulin release in glucose-stimulated islets
Biochimie (1991) 73, 1287-1290 © Soci6t6 fran~aise de biochimie et biologie mol6culaire / Elsevier, Paris
1287
Hexose metabolism in pancreatic islets. Effect of (-)-hydroxycitrate upon fatty acid synthesis and insulin release in glucose-stimulated islets A Sener, WJ Malaisse* Laboratoly of Experimental Medicine, Brussels Free University, 115, Boulevard de Waterloo, B-I O00 Brussels, Belgium (Received 21 May 1991; accepted 8 August 1991)
Summary - - Anaplerotic reactions leading to the de novo synthesis of fatty acids were recently proposed to participate in the coupling of metabolic to secretory events in the process of glucose-stimulated insulin release. In an attempt to validate such a proposal, the effect of (-)-hydroxycitrate upon fatty acid synthesis and insulin release was investigated in glucose-stimulated rat pancreatic islets. The inhibitor of ATP citrate-lyase, when tested in the 1.0-2.0 mM range, failed to affect glucose-stimulated insulin release, but also failed to inhibit the incorporation of 14C-labelled acetyl residues derived from L-[U-14C]leucine into islet lipids. A partial inhibition of fatty acid labelling by 3H20 was only observed in islets incubated for 120 rain in the presence of 5.0 mM (-)-hydroxycitrate and absence of CaCI 2. These findings suggest that (-)-hydroxycitrate is not, under the present experimental conditions, a useful tool to abolish fatty acid synthesis in intact pancreatic islets.
pancreatic islets / insulin release / (-)-hydroxycitrate / fatty acid synthesis
Introduction T h e s t i m u l a t i o n o f i n s u l i n r e l e a s e b y D-glucose is c a u s a l l y l i n k e d to its m e t a b o l i s m in islet cells [ 1 ]. T h e c o u p l i n g o f m e t a b o l i c to m o r e d i s t a l e v e n t s in t h e secretory sequence may represent a mu!tifactorial p r o c e s s [2] a n d w a s r e c e n t l y p r o p o s e d to i n v o l v e a n a p l e r o t i c r e a c t i o n s l e a d i n g to the de novo s y n t h e s i s o f f a t t y a c i d s [3, 4]. In the l i g h t o f s u c h a p r o p o s a l , t h e p r e s e n t s t u d y a i m e d at e x p l o r i n g the e f f e c t o f ( - ) hydroxycitrate, a potent inhibitor of ATP citrate-lyase [5], u p o n f a t t y a c i d s y n t h e s i s a n d i n s u l i n s e c r e t i o n in g l u c o s e - s t i m u l a t e d islets.
Materials a n d m e t h o d s (-)-Hydroxycitric acid was either obtained from Roche (Basel, Switzerland) or donated by Dr H Brunengraber (Mount Sinai Medical Center, Cleveland, OH). In the latter case, the lactone derivative was incubated for 60 min at 80°C in 1.0 M NaOH and aliquots of a stock (}.2 M solution, adjusted at pH 7.4, were kept at - 20°C. All experiments were conducted in islets prepared by the collagenase method [6] for fed female albino rats.
*Correspondence and reprints
The methods used to measure insulin release [6] and the oxidation of 14C-labelled nutrients [7] were identical to those reported in the cited references. For measuring the incorporation of 3H from 3H20 or 14C fl ! 14Pl.h..-.--o~ ;~ -- *h~ ¢.. ...... 1 --~:~,.. ~ l t , , , , s~ I~.-]~IUII~VaI~, IlIIv Idlil.~ II~I.ILI? ~ 1 . ~ 1 l i l V I ~ ' L Y
~'r,--..-.-, ~
IlUlIi
~ae :-!~.
Ol
lbll~l
I:_:A~
IIi.,IIU~ ,
groups of 40 islets each were washed twice, after 120 min incubation, with a non-radioactive medium [6], placed in 0.2 ml of a solution of KCI (100 mM), EDTA (5 mM) and HCI (50 mM), frozen in liquid N 2 and disrupted by mechanical vibration [8]. The islet homogenate was heated for 10 min at 70°C, mixed with 3.0 ml of CHCIJCH3OH/I-ICI (200/100/1, v/v/v) and 20 lal of rat serum, kept overnight at 4°C and centrifuged for 10 rain at 1000 g and 4°C. After removal of the upper aqueous phase, the lower layer was washed 5 times with CH3OH/H20 (l/q, v/v). The lipid extract was removed, dried in vacuum, incubated for 60 min at 60°C in i.0 ml of a solution of KOH (0.5 M) in CH3CH2OH, mixed with 1.0 ml H20 and 2.0 ml CHCI 3 and again centrifuged for 10 min at 4°C. The upper aqueous and lower organic phases were then examined for their radioactive content by liquid scintillation. After correction for the blank value found, in the absence of islets in 1.0 lal of incubation medium, the results were taken as representative of the labelling of the glycerol moiety (aqueous phase) and fatty acyl moiety (organic phase) of islet lipids. A comparable approach, except for the hydrolysis of lipids, was used to measure the incorporation of 14C from L-[UlaC]leucine into islet lipids. All results are expressed as the mean (+ SEM), together with the number of individual observations (n) or degree of freedom
(dj~.
! 288
A Sener, WJ Malaisse
Results
Labelling of islet lipids by 3H20 and D-[U-14C] glucose Over 120 min incubation in the presence of either 3H20 or D-[U-~4C]glucose, a graded increase in the labelling of the fatty acyl moiety of islet lipids was observed at increasing concentrations of D-glucose (table I). At a high concentration of the hexose (16.7 raM), about one third of the newly formed fatty acids were derived from exogenous D-glucose. The labelling of the glycerol moiety of islet lipids was also measured in these experiments. When expressed as glycerol equivalent, it averaged in the presence of 16.7 mM D-glucose 0.58 -+ 0.08 pmol/ 120 min per islet in the case of o-[U-14C]glucose and, as such, was commensurate with the glucose-induced paired increment in the labelling of the same glycerol moiety by 3H20, ie 0.62_+0.14 pmol/120 min per islet. The latter value takes into account two correction factors. First, when expressed as 3H equivalent, the labelling of islet lipids represents twice that calculated by reference to the specific radioactivity of 3H20. Second, close to five 3H atoms are incorporated into L-glycerol-3-phosphate for each molecule of Dglucose converted to the latter metabolite. The tritiation of D-glucose metabolites indeed takes place in the reactions catalyzed by phosphogluco-isomerase on the C~ of D-fructose 6-phosphate, by triose phosphate isomerase on the C~ of glycerone-3-phosphate, and by NAD-linked glycerophosphate dehydrogenase on the C.z_ of L-glycerol-3-phosphate [9, 10]. The glycerol moiety of lipids may be labelled in a cycling process including both lipolysis and re-esterification of fatty ,,at~itt~ lt~iA~--t-~ll.. --1 . . . . . • _'.__,Jc' ___ u~au~, ul~lU~lltCtltly, ~lyg~lUl may tD e r e - incorpolt~i¢ll rated in lipids, since islet homogenates display glycerol kinase activity [ 11].
Insulin release When groups of 8 islets each were pre-incubated for 30 min in the presence of 2.8 mM D-glucose and 4.0 mM CaC12 and then incubated for 120 min in the
presence of 16.7 mM D-glucose at the same high CaCI2 concentration, the release of insulin averaged 210.9 + 7.2 l.tU/120 min per islet (table II). The latter output represented 150.4 + 9.2% (df = 78) of that recorded when both the pre-incubation and incubation were conducted at normal CaC12 concentration (1.0 mM). At the high CaCI2 concentration, the presence of (-)-hydroxycitrate (2.0 mM) in both the pre-incubation and incubation media failed to affect significantly insulin release, the hormonal output during incubation averaging 91.4 + 5.1% of the mean control value recorded within the same experiments in the absence of the drug (df= 78; P > 0.09).
Oxidative data As shown in table HI, (-)-hydroxycitrate (2.0 mM) failed to affect significantly the oxidation of D-[UlaC]glucose (16.7 mM) in the presence of a high concentration of CaCI2 (4.0 mM). In these experiments the oxidation of the hexose was not significantly different in the presence of 1.0 mM and 4.0 mM CaC12, averaging respectively 61.5 + 4.4 and 67.1 + 5.2 pmol/120 min per islet (n = 8 in both cases). In the presence of D-glucose, (-)-hydroxycitrate (1.0 to 2.0 mM) also failed to affect significantly the oxidation of L-[U-laC]leucine, [1-14C]acetate or [2-14C]acetate. In the absence and presence of (-)-hydroxycitrate, respectively, the oxidation of [2-14C]acetate only represented 45.5 + 5.0% (df = 30; P < 0.001) and 43.3 + 7.3% (df= 32; P < 0.001) of that of [1-14C]acetate. As judged from data collected in separate experiments, the oxidation of 14C-labelled acetate appeared lower in the absence of CaCI2 than at a high CaC12 concentration (4.0 mM).
Effect of (-)-hydroxycitrate upon the synthesis of fatty acids In the presence of 7.0 mM D-glucose, (-)hydroxycitrat~ failed to affect significantly the labelling of islet lipids by L-[U-14C]leucine (0.5 mM), whether in the absence of CaCI2 or at normal CaCI2 concentration (table IV).
Table I. Labelling of islet lipids by 3H20 and D-[U-14C]glucoseat increasing concentrations of D-giucose.
D-glucose
Fatty acid moiety
(mt~l)
3H20a
Nil 8.3 16.7
0.44 _+ 0.10 (12) 1.02 _+ 0.18 (11) 1.45 _+ 0.22 (13)
o_lU_14C]glucosea 0.25 _ 0.03 (11) 0.45 _+ 0.07 (13)
Glycerol moiety 3H20b
o-[U-14C]glucoseC
2.29 + 0.63 (20) 3.28 + 0.64 (25) 3.07 + 0.66 (26)
028 + 0.06 (11) 0 58 + 0.08 (13)
aExpressed as pmol of acetyl equivalent/120 min per islet; bExpressed as pmol of 3H20 equivalent/120 min per islet; CExpressed as pmol of glycerol equivalent/120 min per islet.
(-)-hydroxycitrate and pancreatic islets Table II. Effect of (-)-hydroxycitrate upon insulin release evoked by D-glucose (16.7 mM) after 30 min pre-incubation. CaC12 (-)OH-citrate (mM) (mM) 1.0 4.0 4.0
Insulin output (lxU/120 min per islet)
Nil Nil 2.0
140.2 + 10.8 (40) 210.9 + 7.2 (40) 192.8 + 8.0 (40)
Over 120 min incubation, the incorporation of ~4C from L-[U-14C]leucine (0.2 raM) into islet lipids was also unaffected by (-)-hydroxycitrate (2.0 mM) at higher concentrations of both D-glucose (16.7 m M ) and CaCl 2 (4.0 mM), with a :nean ratio between experimental and control values of 85.2 _+ 13.9% ( d f = 22). In the presence of 7.0 m M D-glucose and 1.0 m M CaCI 2, (-)-hydroxycitrate even failed to decrease the labelling of islet lipids when the inhibitor of ATP citrate-lyase was present in the medium in high concentration (6.6 raM) during both a 60-min preincubation conducted in the presence of unlabelled Lleucine (0.5 mM) and a 120-min incubation performed at the same concentration of the amino acid mixed with a tracer amount of L-[U-t4C]leucine (data not shown). In the last set of experiments, groups of 50 islets each were incubated for 120 min in the presence of Dglucose (16.7 mM) and a tracer amount of 3H20 (0.25 Ci/ml) and then extracted for measurement of tritium incorporation into the fatty acyl moiety of islet lipids. At normal CaCl 2 concentration (1.0 mM) and in the absence of CaCl2, respectively, such an incorporation averaged 0.78 _+ 0.09 and 0.78 _+ 0.13 pmol of 3H20 equivalent per 120 min and islet (n = 14 in both cases). When (-)-hydroxycitrate was present at a 5.0 m M concentration in the CaZ+-deprived medium, the labelling of fatty acids still represented 61.8 _+ 17.5% ( d f = 26; P < 0.05) of the mean corresponding control value.
1289
Discussion
The present data confirm that ~4C-labelled acetyl residues generated from exogenous D-[u-laC]glucose are incorporated into the fatty acyl moiety of islet lipids [12]. Such an incorporation is more than two orders of magnitude lower than the rate of D-[U-~aC]glucose oxidation. It only accounts for about one-third of the total rate of fatty acid synthesis, as judged from the incorporation of 3H20 into the fatty acyl moiety of islet lipids. The latter difference is consistent with the knowledge that exogenous D-glucose only exerts a modest sparing action upon the catabolism of endogenous nutrients [13]. The entry into the Krebs cycle of unlabelled metabolites such as 2-ketoglutarate, succinyl CoA and oxalacetate derived from these endogenous nutrients and the compensatory exit from the Krebs cycle of partially ~4C-labelled metabolites, such as malate and citrate may accot:nt for the difference in l'~CO2 production from [1-14C]acetate and [214C]acetate, as already observed in a recent study [14]. The present work primarily aimed at investigating the effect of (-)-hydroxycitrate upon glucose-stimulated insulin release, in order to further explore the validity of the anaplerotic hypothesis for nutrientinduced insulin secretion [3, 4]. In our experiments, Table IV. Effect of (-)-hydroxycitrate upon the labelling of islet lipids by L-[U-14C]leucine (0.5 mM) in islets exposed to 7.0 mM D-glucose.
CaCI2 (raM)
(-)OH-citrate (mM)
Nil Nil 1.0 1.0 1.0
Nil 1.0 Nil 1.0 6.6
I4C incorporation a (fmol/120 rain per islet) i83 225 171 189 195
+ + + + +
i5 15 12 17 21
(i0) (10) (20) (10) (10)
aExpressed as acetyl equivalent.
Table III. Effect of (-)-hydroxycitrate upon [14C]nutrient oxidation.
Oxidation rate (pmol/120 rain per islet) Control (-)OH-citrate
D-glucose (mM)
CaCI2 (m~;)
(-)OH-citrate (raM)
D-[U-14C]glucose (16.7)
16.7
4.0
2.0
67.1 + 5.2 (8)
65.4 + 6.2 (8)
L-[U-14C]leucine (0.5) L-[U-14C]leucine (0.5)
7.0 7.0
Nil 1.0
1.0 1.0
11.8 + 0.8 (10) 12.4 + 0.5 (10)
13.3 + 1.0 (10) 13.8 + 0.8 (i0)
[14C]nutrient (mM)
[ 1 14C]acetate [2-14C]acetate
(1.0) (1.0)
16.7 16.7
Nil Nil
2.0 2.0
9.4 + 0.6 (7) 4.2 + 0.5 (8)
9.9 + 2.1 (8) 3.4 + 0.7 (8)
[ 1-14C]acetate
(1.0) (1.0)
16.7 16.7
4.0 4.0
1.5 1.5
18.7 __+ 2.3 (9) 9.7 __+ 1.0 (10)
20.3 +__ 2.8 (10) 10.3 __+ 1.5 (10)
-
[2-14C]acetate
! 290
A Sener, WJ Malaisse
the concentration of CaCI_~ was raised above its usual value (!.0 mM) in order to compensate for the chelation of Ca 2÷ by (-)-hydroxycitrate. No significant inhibitory action of (-)-hydroxycitrate upon glucoseinduced insulin secretion was observed, even when the islets were pre-incubated for 30 min in the presence of the drug. The significance of this negative observation is considerably obscured by the finding that (-)hydroxycitrate also failed to abolish the synthesis of fatty acids, as judged from results obtained with either L-[U-14C]leucine or even 3H20. The former amino acid was used as a radioactive tracer in order to avoid labelling of the glycerol moiety of islet lipids. It should be realized, however, that laC-labelled acetoacetate generated from L-[U-~4C]leucine could be incorporated directly into lipid without mediation of the citrate pathway [15]. The failure of (-)-hydroxycitrate to suppress the synthesis of fatty acids is probably attributable to its poor uptake by islet cells, since the drug was tested at a concentration several orders of magnitude higher than that required to inhibit ATP citrate-lyase in acellular systems [5]. In this respect, the situation in pancreatic islets appears similar to that characterized in liver cells, in which (-)-hydroxycitrate causes either no inhibition or only a partial decrease of lipid synthesis [16, 17]. As a matter of tact, a modest but significant inhibition of fatty acid synthesis in glucose-stimulated islets was only observed with 3H20 as tracer over 120 min incubation in the presence of a high concentration (5.0 mM) of (-)hydroxycitrate, the drug being tested in CaCLdeprived media. In conclusion, the present results suggest that (-)hydroxyci_trate may not represent a . ~ f i d tanl tr~ abolish fatty acid synthesis in pancreatic islets and, hence, to assess the possible participation of the latter process in the stimuius-secretion coupling of nutrientinduced insulin release.
Acknowledgments This study was supported by grants from the Belgian Foundation for Scientific Medical Research and Belgian Ministry of Scientific Policy. The authors are grateful to M Mahy for technical assistance and C Demesmaeker for secretarial help.
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6 7
8
9
10
11 12 13
14
15 16
References 17 Malaisse WJ, Sener A, Herchuelz A, Hutton JC (1979) Insulin release: the fuel hypothesis. Metabolism 28, 373-386
MalaisseWJ, Malaisse-Lagae F, Sener A (198.4) Coupling factors in nutrient-induced insulin release. ExpeHentia 40, 1035-1043 Matschinsky FM, Corkey BE, Prentki M, Meglasson MD, Erecinska M, Shimizu T, Ghosh A, Parker J (1989) Metabolic connectivity and signalling in pancreatic Bcells. Excetpta Medica ICS 800, 17-26 Corkey BE, Glennon MC, Chen KS, Deeney JT, Matschinsky FM, Prentki M (1989) A role for malonyI-CoA in glucose-stimulated insulin secretion from clonal pancreatic 13-cells. J Biol Chem 264, 21608-21612 Sullivan AC, Singh M, Srere PA, Glusker JP (1977) Reactivity and inhibitor potential of hydroxycitrate isomers with citrate synthase, citrate lyase, and ATP citrate lyase. J Biol Chem 252, 7583-7590 Malaisse-Lagae F, Malaisse WJ (1984) Insulin release by pancreatic islets. In: Methods in Diabetes Research (Lamer J, Pobl SL, eds) Wiley, NY, 147-152 Malaisse WJ, Sener A (1988) Hexose metabolism in pancreatic islets. Feedback control of D-glucose oxidation by functional events. Biochim Biophys Acta 971, 246254 Malaisse WJ, Hutton JC, Kawazu S, Sener A (1978) The stimulus-secretion coupling of glucose-induced insulin release. XXXI. Metabolic effects of menadione in isolated islets. Eur J Biochem 87, 121-130 Malaisse-Lagae F, Liemans V, Malaisse WJ (1989) Phosphoglucoisomerase-catalyzed interconversion of hexose-phosphates. Isotopic discrimination between hydrogen and tritium. Mol Cell Biochem 89, 5767 Maggetto C, Manuel y Keenoy B, Zahner D, Bodur H, Sener A, Malaisse WJ (1991) Interconversion of D-fructose 1,6-bisphosphates and triose phosphates in human erythrocytes. Biochim Biophys Acta (in press) Yilmaz MT, Sener A, Malaisse WJ (1987) Glycerol phosphorylation and oxidation in pancreatic islets. Mol Cell EndocHno152, 251-256 Berne C (1975) The metabolism of lipids in mouse pancreatic islets. I--he biosynthesis of triacyigiycerois and phospholipids. Biochem J 152, 667--673 Malaisse WJ, Best L, Kawazu S, Malaisse-Lagae F, Sener A ~1983) The stimulus-secretion coupling of glucose-induced insulin release. LX. Fuel metabolism in islets deprived of exogenous nutrient. Arch Biochem Biophys 224, 102-110 Malaisse WJ, Sener A (1991) Hexose metabolism in pancreatic islets. Unequal oxidation of the two carbons of glucose-derived acetyl residues. Arch Biochem Biophys 292, in press Robinson AM, Williamson DH (1980) Physiological roles of ketone bodies as substrates and signals in mammalian tissues. Physiol Rev 60, 143-187 Berkhout TA, Havekes LM, Pearce NJ, Groot PHE (1990) The effect of (-)-hydroxycitrate on the activity of the lowdensity-lipoprotein receptor and 3-hydroxy-3-methylglutaryl-CoA reductase levels in the human hepatoma cell line Hep G2. Biochem J 272, 181-186 Gibbons GF, Attwell Thomas CP, Pullinger CR (1986) The metabolic route by which oleate is converted into cholesterol in rat hepatocytes. Biochem J 235, 1924
1287
Hexose metabolism in pancreatic islets. Effect of (-)-hydroxycitrate upon fatty acid synthesis and insulin release in glucose-stimulated islets A Sener, WJ Malaisse* Laboratoly of Experimental Medicine, Brussels Free University, 115, Boulevard de Waterloo, B-I O00 Brussels, Belgium (Received 21 May 1991; accepted 8 August 1991)
Summary - - Anaplerotic reactions leading to the de novo synthesis of fatty acids were recently proposed to participate in the coupling of metabolic to secretory events in the process of glucose-stimulated insulin release. In an attempt to validate such a proposal, the effect of (-)-hydroxycitrate upon fatty acid synthesis and insulin release was investigated in glucose-stimulated rat pancreatic islets. The inhibitor of ATP citrate-lyase, when tested in the 1.0-2.0 mM range, failed to affect glucose-stimulated insulin release, but also failed to inhibit the incorporation of 14C-labelled acetyl residues derived from L-[U-14C]leucine into islet lipids. A partial inhibition of fatty acid labelling by 3H20 was only observed in islets incubated for 120 rain in the presence of 5.0 mM (-)-hydroxycitrate and absence of CaCI 2. These findings suggest that (-)-hydroxycitrate is not, under the present experimental conditions, a useful tool to abolish fatty acid synthesis in intact pancreatic islets.
pancreatic islets / insulin release / (-)-hydroxycitrate / fatty acid synthesis
Introduction T h e s t i m u l a t i o n o f i n s u l i n r e l e a s e b y D-glucose is c a u s a l l y l i n k e d to its m e t a b o l i s m in islet cells [ 1 ]. T h e c o u p l i n g o f m e t a b o l i c to m o r e d i s t a l e v e n t s in t h e secretory sequence may represent a mu!tifactorial p r o c e s s [2] a n d w a s r e c e n t l y p r o p o s e d to i n v o l v e a n a p l e r o t i c r e a c t i o n s l e a d i n g to the de novo s y n t h e s i s o f f a t t y a c i d s [3, 4]. In the l i g h t o f s u c h a p r o p o s a l , t h e p r e s e n t s t u d y a i m e d at e x p l o r i n g the e f f e c t o f ( - ) hydroxycitrate, a potent inhibitor of ATP citrate-lyase [5], u p o n f a t t y a c i d s y n t h e s i s a n d i n s u l i n s e c r e t i o n in g l u c o s e - s t i m u l a t e d islets.
Materials a n d m e t h o d s (-)-Hydroxycitric acid was either obtained from Roche (Basel, Switzerland) or donated by Dr H Brunengraber (Mount Sinai Medical Center, Cleveland, OH). In the latter case, the lactone derivative was incubated for 60 min at 80°C in 1.0 M NaOH and aliquots of a stock (}.2 M solution, adjusted at pH 7.4, were kept at - 20°C. All experiments were conducted in islets prepared by the collagenase method [6] for fed female albino rats.
*Correspondence and reprints
The methods used to measure insulin release [6] and the oxidation of 14C-labelled nutrients [7] were identical to those reported in the cited references. For measuring the incorporation of 3H from 3H20 or 14C fl ! 14Pl.h..-.--o~ ;~ -- *h~ ¢.. ...... 1 --~:~,.. ~ l t , , , , s~ I~.-]~IUII~VaI~, IlIIv Idlil.~ II~I.ILI? ~ 1 . ~ 1 l i l V I ~ ' L Y
~'r,--..-.-, ~
IlUlIi
~ae :-!~.
Ol
lbll~l
I:_:A~
IIi.,IIU~ ,
groups of 40 islets each were washed twice, after 120 min incubation, with a non-radioactive medium [6], placed in 0.2 ml of a solution of KCI (100 mM), EDTA (5 mM) and HCI (50 mM), frozen in liquid N 2 and disrupted by mechanical vibration [8]. The islet homogenate was heated for 10 min at 70°C, mixed with 3.0 ml of CHCIJCH3OH/I-ICI (200/100/1, v/v/v) and 20 lal of rat serum, kept overnight at 4°C and centrifuged for 10 rain at 1000 g and 4°C. After removal of the upper aqueous phase, the lower layer was washed 5 times with CH3OH/H20 (l/q, v/v). The lipid extract was removed, dried in vacuum, incubated for 60 min at 60°C in i.0 ml of a solution of KOH (0.5 M) in CH3CH2OH, mixed with 1.0 ml H20 and 2.0 ml CHCI 3 and again centrifuged for 10 min at 4°C. The upper aqueous and lower organic phases were then examined for their radioactive content by liquid scintillation. After correction for the blank value found, in the absence of islets in 1.0 lal of incubation medium, the results were taken as representative of the labelling of the glycerol moiety (aqueous phase) and fatty acyl moiety (organic phase) of islet lipids. A comparable approach, except for the hydrolysis of lipids, was used to measure the incorporation of 14C from L-[UlaC]leucine into islet lipids. All results are expressed as the mean (+ SEM), together with the number of individual observations (n) or degree of freedom
(dj~.
! 288
A Sener, WJ Malaisse
Results
Labelling of islet lipids by 3H20 and D-[U-14C] glucose Over 120 min incubation in the presence of either 3H20 or D-[U-~4C]glucose, a graded increase in the labelling of the fatty acyl moiety of islet lipids was observed at increasing concentrations of D-glucose (table I). At a high concentration of the hexose (16.7 raM), about one third of the newly formed fatty acids were derived from exogenous D-glucose. The labelling of the glycerol moiety of islet lipids was also measured in these experiments. When expressed as glycerol equivalent, it averaged in the presence of 16.7 mM D-glucose 0.58 -+ 0.08 pmol/ 120 min per islet in the case of o-[U-14C]glucose and, as such, was commensurate with the glucose-induced paired increment in the labelling of the same glycerol moiety by 3H20, ie 0.62_+0.14 pmol/120 min per islet. The latter value takes into account two correction factors. First, when expressed as 3H equivalent, the labelling of islet lipids represents twice that calculated by reference to the specific radioactivity of 3H20. Second, close to five 3H atoms are incorporated into L-glycerol-3-phosphate for each molecule of Dglucose converted to the latter metabolite. The tritiation of D-glucose metabolites indeed takes place in the reactions catalyzed by phosphogluco-isomerase on the C~ of D-fructose 6-phosphate, by triose phosphate isomerase on the C~ of glycerone-3-phosphate, and by NAD-linked glycerophosphate dehydrogenase on the C.z_ of L-glycerol-3-phosphate [9, 10]. The glycerol moiety of lipids may be labelled in a cycling process including both lipolysis and re-esterification of fatty ,,at~itt~ lt~iA~--t-~ll.. --1 . . . . . • _'.__,Jc' ___ u~au~, ul~lU~lltCtltly, ~lyg~lUl may tD e r e - incorpolt~i¢ll rated in lipids, since islet homogenates display glycerol kinase activity [ 11].
Insulin release When groups of 8 islets each were pre-incubated for 30 min in the presence of 2.8 mM D-glucose and 4.0 mM CaC12 and then incubated for 120 min in the
presence of 16.7 mM D-glucose at the same high CaCI2 concentration, the release of insulin averaged 210.9 + 7.2 l.tU/120 min per islet (table II). The latter output represented 150.4 + 9.2% (df = 78) of that recorded when both the pre-incubation and incubation were conducted at normal CaC12 concentration (1.0 mM). At the high CaCI2 concentration, the presence of (-)-hydroxycitrate (2.0 mM) in both the pre-incubation and incubation media failed to affect significantly insulin release, the hormonal output during incubation averaging 91.4 + 5.1% of the mean control value recorded within the same experiments in the absence of the drug (df= 78; P > 0.09).
Oxidative data As shown in table HI, (-)-hydroxycitrate (2.0 mM) failed to affect significantly the oxidation of D-[UlaC]glucose (16.7 mM) in the presence of a high concentration of CaCI2 (4.0 mM). In these experiments the oxidation of the hexose was not significantly different in the presence of 1.0 mM and 4.0 mM CaC12, averaging respectively 61.5 + 4.4 and 67.1 + 5.2 pmol/120 min per islet (n = 8 in both cases). In the presence of D-glucose, (-)-hydroxycitrate (1.0 to 2.0 mM) also failed to affect significantly the oxidation of L-[U-laC]leucine, [1-14C]acetate or [2-14C]acetate. In the absence and presence of (-)-hydroxycitrate, respectively, the oxidation of [2-14C]acetate only represented 45.5 + 5.0% (df = 30; P < 0.001) and 43.3 + 7.3% (df= 32; P < 0.001) of that of [1-14C]acetate. As judged from data collected in separate experiments, the oxidation of 14C-labelled acetate appeared lower in the absence of CaCI2 than at a high CaC12 concentration (4.0 mM).
Effect of (-)-hydroxycitrate upon the synthesis of fatty acids In the presence of 7.0 mM D-glucose, (-)hydroxycitrat~ failed to affect significantly the labelling of islet lipids by L-[U-14C]leucine (0.5 mM), whether in the absence of CaCI2 or at normal CaCI2 concentration (table IV).
Table I. Labelling of islet lipids by 3H20 and D-[U-14C]glucoseat increasing concentrations of D-giucose.
D-glucose
Fatty acid moiety
(mt~l)
3H20a
Nil 8.3 16.7
0.44 _+ 0.10 (12) 1.02 _+ 0.18 (11) 1.45 _+ 0.22 (13)
o_lU_14C]glucosea 0.25 _ 0.03 (11) 0.45 _+ 0.07 (13)
Glycerol moiety 3H20b
o-[U-14C]glucoseC
2.29 + 0.63 (20) 3.28 + 0.64 (25) 3.07 + 0.66 (26)
028 + 0.06 (11) 0 58 + 0.08 (13)
aExpressed as pmol of acetyl equivalent/120 min per islet; bExpressed as pmol of 3H20 equivalent/120 min per islet; CExpressed as pmol of glycerol equivalent/120 min per islet.
(-)-hydroxycitrate and pancreatic islets Table II. Effect of (-)-hydroxycitrate upon insulin release evoked by D-glucose (16.7 mM) after 30 min pre-incubation. CaC12 (-)OH-citrate (mM) (mM) 1.0 4.0 4.0
Insulin output (lxU/120 min per islet)
Nil Nil 2.0
140.2 + 10.8 (40) 210.9 + 7.2 (40) 192.8 + 8.0 (40)
Over 120 min incubation, the incorporation of ~4C from L-[U-14C]leucine (0.2 raM) into islet lipids was also unaffected by (-)-hydroxycitrate (2.0 mM) at higher concentrations of both D-glucose (16.7 m M ) and CaCl 2 (4.0 mM), with a :nean ratio between experimental and control values of 85.2 _+ 13.9% ( d f = 22). In the presence of 7.0 m M D-glucose and 1.0 m M CaCI 2, (-)-hydroxycitrate even failed to decrease the labelling of islet lipids when the inhibitor of ATP citrate-lyase was present in the medium in high concentration (6.6 raM) during both a 60-min preincubation conducted in the presence of unlabelled Lleucine (0.5 mM) and a 120-min incubation performed at the same concentration of the amino acid mixed with a tracer amount of L-[U-t4C]leucine (data not shown). In the last set of experiments, groups of 50 islets each were incubated for 120 min in the presence of Dglucose (16.7 mM) and a tracer amount of 3H20 (0.25 Ci/ml) and then extracted for measurement of tritium incorporation into the fatty acyl moiety of islet lipids. At normal CaCl 2 concentration (1.0 mM) and in the absence of CaCl2, respectively, such an incorporation averaged 0.78 _+ 0.09 and 0.78 _+ 0.13 pmol of 3H20 equivalent per 120 min and islet (n = 14 in both cases). When (-)-hydroxycitrate was present at a 5.0 m M concentration in the CaZ+-deprived medium, the labelling of fatty acids still represented 61.8 _+ 17.5% ( d f = 26; P < 0.05) of the mean corresponding control value.
1289
Discussion
The present data confirm that ~4C-labelled acetyl residues generated from exogenous D-[u-laC]glucose are incorporated into the fatty acyl moiety of islet lipids [12]. Such an incorporation is more than two orders of magnitude lower than the rate of D-[U-~aC]glucose oxidation. It only accounts for about one-third of the total rate of fatty acid synthesis, as judged from the incorporation of 3H20 into the fatty acyl moiety of islet lipids. The latter difference is consistent with the knowledge that exogenous D-glucose only exerts a modest sparing action upon the catabolism of endogenous nutrients [13]. The entry into the Krebs cycle of unlabelled metabolites such as 2-ketoglutarate, succinyl CoA and oxalacetate derived from these endogenous nutrients and the compensatory exit from the Krebs cycle of partially ~4C-labelled metabolites, such as malate and citrate may accot:nt for the difference in l'~CO2 production from [1-14C]acetate and [214C]acetate, as already observed in a recent study [14]. The present work primarily aimed at investigating the effect of (-)-hydroxycitrate upon glucose-stimulated insulin release, in order to further explore the validity of the anaplerotic hypothesis for nutrientinduced insulin secretion [3, 4]. In our experiments, Table IV. Effect of (-)-hydroxycitrate upon the labelling of islet lipids by L-[U-14C]leucine (0.5 mM) in islets exposed to 7.0 mM D-glucose.
CaCI2 (raM)
(-)OH-citrate (mM)
Nil Nil 1.0 1.0 1.0
Nil 1.0 Nil 1.0 6.6
I4C incorporation a (fmol/120 rain per islet) i83 225 171 189 195
+ + + + +
i5 15 12 17 21
(i0) (10) (20) (10) (10)
aExpressed as acetyl equivalent.
Table III. Effect of (-)-hydroxycitrate upon [14C]nutrient oxidation.
Oxidation rate (pmol/120 rain per islet) Control (-)OH-citrate
D-glucose (mM)
CaCI2 (m~;)
(-)OH-citrate (raM)
D-[U-14C]glucose (16.7)
16.7
4.0
2.0
67.1 + 5.2 (8)
65.4 + 6.2 (8)
L-[U-14C]leucine (0.5) L-[U-14C]leucine (0.5)
7.0 7.0
Nil 1.0
1.0 1.0
11.8 + 0.8 (10) 12.4 + 0.5 (10)
13.3 + 1.0 (10) 13.8 + 0.8 (i0)
[14C]nutrient (mM)
[ 1 14C]acetate [2-14C]acetate
(1.0) (1.0)
16.7 16.7
Nil Nil
2.0 2.0
9.4 + 0.6 (7) 4.2 + 0.5 (8)
9.9 + 2.1 (8) 3.4 + 0.7 (8)
[ 1-14C]acetate
(1.0) (1.0)
16.7 16.7
4.0 4.0
1.5 1.5
18.7 __+ 2.3 (9) 9.7 __+ 1.0 (10)
20.3 +__ 2.8 (10) 10.3 __+ 1.5 (10)
-
[2-14C]acetate
! 290
A Sener, WJ Malaisse
the concentration of CaCI_~ was raised above its usual value (!.0 mM) in order to compensate for the chelation of Ca 2÷ by (-)-hydroxycitrate. No significant inhibitory action of (-)-hydroxycitrate upon glucoseinduced insulin secretion was observed, even when the islets were pre-incubated for 30 min in the presence of the drug. The significance of this negative observation is considerably obscured by the finding that (-)hydroxycitrate also failed to abolish the synthesis of fatty acids, as judged from results obtained with either L-[U-14C]leucine or even 3H20. The former amino acid was used as a radioactive tracer in order to avoid labelling of the glycerol moiety of islet lipids. It should be realized, however, that laC-labelled acetoacetate generated from L-[U-~4C]leucine could be incorporated directly into lipid without mediation of the citrate pathway [15]. The failure of (-)-hydroxycitrate to suppress the synthesis of fatty acids is probably attributable to its poor uptake by islet cells, since the drug was tested at a concentration several orders of magnitude higher than that required to inhibit ATP citrate-lyase in acellular systems [5]. In this respect, the situation in pancreatic islets appears similar to that characterized in liver cells, in which (-)-hydroxycitrate causes either no inhibition or only a partial decrease of lipid synthesis [16, 17]. As a matter of tact, a modest but significant inhibition of fatty acid synthesis in glucose-stimulated islets was only observed with 3H20 as tracer over 120 min incubation in the presence of a high concentration (5.0 mM) of (-)hydroxycitrate, the drug being tested in CaCLdeprived media. In conclusion, the present results suggest that (-)hydroxyci_trate may not represent a . ~ f i d tanl tr~ abolish fatty acid synthesis in pancreatic islets and, hence, to assess the possible participation of the latter process in the stimuius-secretion coupling of nutrientinduced insulin release.
Acknowledgments This study was supported by grants from the Belgian Foundation for Scientific Medical Research and Belgian Ministry of Scientific Policy. The authors are grateful to M Mahy for technical assistance and C Demesmaeker for secretarial help.
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