Effects of colchicine and trimethoxybenzene on glucose oxidation in isolated brown fat cells from rats

Effects of colchicine and trimethoxybenzene on glucose oxidation in isolated brown fat cells from rats

Gen. Pharmac., Vol. 10. pp. 47 to 49. © Pergamon Press Ltd 1979, Printed in Great Britain 0306-3623/79/0101-0047502.00/0 E F F E C T S OF C O L C H ...

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Gen. Pharmac., Vol. 10. pp. 47 to 49. © Pergamon Press Ltd 1979, Printed in Great Britain

0306-3623/79/0101-0047502.00/0

E F F E C T S OF C O L C H I C I N E A N D TRIMETHOXYBENZENE ON GLUCOSE OXIDATION IN I S O L A T E D B R O W N FAT CELLS F R O M RATS JUDITH W. ROSENTHAL Biology Department, Kean College, Union, NJ 07083, U.S.A. (Received 19 June 1978)

Abstract--1. Glucose oxidation in isolated brown fat cells from rats is reduced in the presence of colchicine. 2. The effect of colchicine is seen in the absence or presence of insulin and with or without calcium in the incubation medium. 3. Trimethoxybenzene is as effective as colchicine in reducing glucose oxidation in control and insulin treated fat cells. 4. The ability of colchicine to reduce glucose oxidation in fat cells most likely is independent of the cytoplasmic microtubular system. After 1 hr of digestion at 37°C in a shaking water bath the cells were filtered through nylon chiffon and washed Colchicine, an anti-mitotic agent, has been shown to twice in 4~o albumin buffer. The cells were incubated for reduce glucose oxidation in mouse epididymal adi- 3 hr at 37°C in a shaking water bath in plastic culture pose tissue (Winand et al., 1973) and in isolated white tubes containing 1.5 ml of 4% albumin buffer with 2.3 mM fat cells from rats (Soifer et al., 1971; Loten & Jean- uniformly labelled [14C]glucose to which test agents were added. All tubes were gassed with 100~o oxygen and renaud, 1974); this effect is seen in both the absence capped. Carbon dioxide was collected on rolled filter and presence of insulin. ~ o l c h i c i n e not only interferes papers in disposable plastic center wells (Kontes Glass Co, with microtubule formation (Soifer et al., 1971) but Vineland, N.J.) attached to rubber caps. At the end of the also binds to the labile microtubular subunits called incubation period 0.2 ml phenethylamine was added to the tubulins which are found in adipocytes (Schimmel, filter paper and 0.25 ml of 1 N H2SO,~ was added to the 1975). The effects of colchicine on glucose oxidation medium. The filter paper, and wells were removed after appear, however, to be dissociable from its inhibitory 1 hr, added to 10 ml of toluene containing 0.4% Omnifluor action on cytoplasmic microtubule formation. For (New England Nuclear, Boston, MA) and counted in a Beckman LS-100 scintillation counter. Aliquots of the example, colchicine can inhibit glucose uptake by fat medium were counted in Bray's scintillation cocktail (Bray, cell ghosts (Cheng & Katsoyannis, 1975). 1960). The present experiments were carried out to examLipids were extracted from isolated fat cell samples using ine the effects of colchicine on glucose oxidation in chloroform-methanol (2:1 v/v). The extracts were filtered isolated brown fat cells from rats. The e,ffects of col- through No. 1 Whatman paper and total lipids prepared chicine were studed in both the absence and presence by the method of Folch et al. (Folch et al., 1957) washing of insulin and/or calcium, since calcium has been the chloroform-methanol extracts once with 0.74% KCI and found to inhibit polymerization of microtubules three times with pure solvents upper phase containing (Weisenberg, 1972; Borisy et al., 1974). Finally, tri- 0.74% KC1. Total cell lipids were determined by evaporating an aliquot of lipid extract in tared vessels and weighing. methoxybenzene, which has no anti-mitotic activity [14C]Tripalmitin was used as a recovery standard. Each (Fitzgerald, 1976) but resembles the A-ring of the col- incubation flask contained approximately 10 mg of lipid. chicine molecule, (see Fig. 1) was tested for its ability Crystalline insulin was a gift of Eli Lilly and Company to alter glucose oxidation in isolated brown fat cells. (Indianapolis, IN). Colchicine and EDTA (ethylenediamine tetraacetic acid)were purchased from Sigma Chemical Co (St. Louis, MO); trimethoxybenzene was purchased from MATERIALS AND METHODS the Aldrich Chemical Co (Metuchen, N.J.). Colchicine and Fat cells were obtained by enzymatic digestion of the trimethoxybenzene were dissolved in 95°~, ethanol and brown dorsal interscapular adipose tissue of normal female equal volumes of ethanol were added to all other flasks. rats of Sprague-Dawley descent (Sunrise Laboratory AniEach experiment was carried out 2 or more times on mals, Whitehouse Station, N.J.). The rats (approximately separate days and all samples were prepared in duplicate. 200g) were fed laboratory chow ad libitum. Isolated fat The values are given as the Mean + S.E.M. and are corcells were obtained by digestion of minced adipose tissue rected for the zero time controls. The results are expressed with crude bacterial collagenase (Worthington Biochemical as/zmoles of glucose converted to carbon.dioxide per gram Corp, Freehold, N.J.), using a modification of the method lipid per 3 hr incubation. Statistical analyses were perof Rodbell (Rodbell, 1964). One mg of collagenase per ml formed using the Student t-test. buffer was used to digest the tissue which was pooled from several rats. The pH of the phosphate buffer was adjusted RESULTS to 7.4 after addition of bovine Fraction V albumin (Armour Pharmaceuticals, Berkeley Heights, N.J.). The As shown in Table 1, colchicine ( 1 0 - 2 M ) can phosphate buffer contained: NaC1, 128raM; CaCI2, 1.4raM: MgSO4, 1.4mM: KC1, 5.2mM and NazHPO,, reduce glucose oxidation in isolated brown fat cells both in the absence and in the presence of insulin. 10mM (Fain et al., 1973). INTRODUCTION

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48

JUDITH W. ROSENTHAL

COLCHICINE

DISCUSSION

H

CH30. T

OH30

CH30

TRIMETHOXYBENZINE

CH30CH30-

CH30 Fig. 1. Molecular structure of colchicine and trimethoxybenzene.

Glucose conversion to carbon dioxide was reduced to approximately 60% of the basal and 30% of the insulin stimulated state by this concentration of colchicine. Since calcium has been shown to be a potent inhibitor of microtubule polymerization (Weisenberg, 1972; Borisy et al., 1974) removal of calcium from the incubation medium should enhance polymerization, perhaps counteracting the effect of colchicine on glucose oxidation if this is a microtubule-dependent event. However, as shown in Table 2, the reduction of glucose oxidation by colchicine in both control and insulin stimulated cells was still observed even with the removal of calcium from the incubation medium. When 1 m M E D T A (a chelating agent which binds calcium) was present in the calcium free medium the effects of colchicine were enhanced. Finally, the effects of trimethoxybenzene were compared with those of colchicine (Table 3). Trimethoxybenzene which has been found to have no effect on tubulin polymerization (Fitzgerald, 1976) is more effective than colchicine in reducing glucose oxidation in control and insulin treated brown fat cells.

The ability of colchicine to depress glucose oxidation has previously been demonstrated in isolated white fat cells of rats (Soifer et al., 197l; Loten & Jeanrenaud, 1974) and mouse epididymal fat (Winand et al., 1973) and is now confirmed and documented for isolated brown fat cells of rats. Originally, the ability of insulin to stimulate microtubule formation in white fat cells (Soifer et al., 1971) and the reduction in glucose utilization in the presence of colchicine (Soifer et al., 1971; Loten & Jeanrenaud, 1974) suggested that these cytoplasmic structures might be involved in the action of insulin. However, additional studies with colchicine, a potent anti-mitotic agent, indicate that its effects on fat cell glucose metabolism appear to be related to the plasma membrane glucose transport system and not the cytoplasmic microtubular system (Cheng & Katsoyannis, 1975). For example, glucose uptake and conversion to carbon dioxide by fat cell ghosts is reduced by colchicine in both the absence and presence of insulin (Cheng & Katsoyannis, 1975). The results reported in this paper provide additional evidence that the reduction in glucose conversion to carbon dioxide by colchicine is not a microtubule-dependent event. Calcium, l mM, a potent inhibitor of microtubule polymerization in a variety of in vitro systems (Weisenberg, 1972; Borisy et al., 1974) did not alter the response of fat cells to colchicine or insulin. In addition, trimethoxybenzene, which has no demonstrable antimitotic activity (Fitzgerald, 1976) can mimic the effects of colchicine on glucose oxidation. The molecular features of the colchicine molecule which result in anti-mitotic activity and inhibition of tubulin polymerization remain unclear. It has been suggested by Wilson (Wilson et al., 1974) that the integrity of the 7-membered C ring is essential for these effects. However, Fitzgerald (Fitzgerald, 1976) has presented evidence that a bicyclic structure involving both the A and C rings of colchicine are essental for anti-mitotic activity. The trimethoxyphenyl component is according to Fitzgerald (Fitzgerald, 1976), only one of the two sites on the colchicine molecule involved in binding to tubulin. Thus, the effects of trimethoxybenzene on glucose oxidation in brown fat cells suggest that this agent is altering glucose transport independently of the microtubule

Table 1. Effects of colchicine on glucose oxidation in isolated brown fat cells in the absence or presence of insulin

Additions None Colchicine, 10 -6 M

Colchicine, 10 4 M Colchicine, 10 -2 M

~tmoles glucose converted to CO2/gm lipid/3 hr Increment due No insulin Plus insulin to insulin 3.06 3.24 2.89 1.78

+ 1.23 ± 1.40 +_ 1.19 4- 0.8l

10.22 _+ 4.17 9.76 +_ 3.24 9.32 _+ 2.97 2.98 4- 1.21

7.16 6.52 6.43 1.20

Isolated brown fat cells were incubated for 3 hr in the absence or presence of colchicine with or without insulin (0.16 mU/ml). The results are the average of 3 experiments carried out in duplicate and are expressed as pmoles of glucose converted to carbon dioxide per gram lipid per 3 hr + SEM.

Effects of colchicine and trimethoxybenzene on rats

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Table 2. Effects of calcium on isolated brown fat cell glucose oxidation, with and without insulin and colchicine pmoles glucose converted to CO2/gm lipid/3 hr _ C a 2+

Additions None Insulin, 0 . 1 6 m U / m l Colchicine, 5 x 10 -3 M Insulin + Colchicine

+ C a 2+

7.22 15.75 4.81 7.32

-Ca

± 0.72 _+ 4.15 ± 0.91 4- 1.59

3.83 8.21 2.69 4.05

+EDTA

2+

4- 0.23 + 0.07 _+ 0.08* 4- 0,13"

5.83 12.18 2.10 3.00

4- 0.41 +_ 1.53 +_ 0.17" 4- 0.20*

Isolated brown fat cells were incubated for 3 hr in normal buffer (+Ca2+), buffer containing no calcium ( - C a 2 + ) , and buffer with no calcium plus 1 m M EDTA ( - C a 2+, + E D T A ) . Insulin (0.16mU/ml) and/or colchicine (5 × 10 -3 M) were added to some of the incubation tubes. The results are the average of 2 experiments carried out in duplicate and are expressed as pmoles of glucose converted to carbon dioxide per gram lipid per 3 hr ± SEM. * P < 0.02 when compared to control values. Table 3. Effects of colchicine and trimethoxybenzene on glucose oxidation in isolated brown fat cells ~moles glucose converted to CO2/gm lipid/3 hr No Plus Increment due insulin insulin to insulin

Additions None Colchicine, 1 x 1 0 - 3 M Colchicine, 5 x 10 -3 M Colchicine, 1 x 1 0 - 2 M Trimethoxybenzene, 1 x 10 -3 M Trimethoxybenzene, 5 x 1 0 - 3 M Trimethoxybenzene, 1 x 1 0 - 2 M

1.64 1.74 1.40 1.23 1.43 0.95 0.60

+ 0.40 4- 0.44 ± 0.31 _+ 0.24 + 0.33 4- 0.18 4- 0.10"

2.75 2.89 2.07 1.47 2.50 1.57 0.91

_+ 0.62 + 0.56 4- 0.25 + 0.12 4- 0.51 + 0.23 4- 0.11"

1.11 1.15 0.67 0.24 1.07 0.62 0.31

Isolated brown fat ceils were incubated for 3 hr in the absence or presence of colchicine or trimethoxybenzene with or without insulin (0.16 mU/ml). The results are the average of 5 experiments carried out in duplicate and are expressed as/~moles of glucose converted to carbon dioxide per gram lipid per 3 hr + S.E.M. * P < 0.02 when compared to control values.

s y s t e m , p e r h a p s by i n t e r a c t i n g w i t h s o m e c o m p o n e n t o f t h e g l u c o s e t r a n s p o r t s y s t e m of fat cells. P r e s u m a b l y this s y s t e m is t h e s a m e as t h a t affected by colchicine. Acknowledgements--This work was supported in part by a grant from the American Philosophical Society and by Kean College of N.J. The author would like to thank both Joyce Balboa and Dot Stabile for their assistance in the preparation of this manuscript.

REFERENCES BORISY G . G., OLMSTED J. B., MARCUM J. M . & ALLEN

C. (1974) Microtubule assembly in vitro. Fedn. Proc. 33, 167-174. BRAY G. A. (1960) A simple efficient liquid scintillator for counting aqueous solutions in a liquid scintillation counter. Analyt. Biochem. exp. Med. 1, 279-285. CHENG K. & KATSOYANNIS P. G. (1975) The inhibition of sugar transport and oxidation in fat cell ghosts by colchicine. Biochem. biophys. Res. Commun. 64, 1069-1075. FAIN J. N., JACOaS M. D. & CLEMENT-CORMIER~. C. (1973) Interrelationship of cyclic AMP, lipolysis, and respiration in brown fat cells. Am. J. Physiol. 244, 346351. G.P. 10/1--O

FITZGERALD T. J. (1976) Molecular features of colchicine associated with antimitotic activity and inhibition of tubulin polymerization. Biochem. Pharmac. 25, 1383-1387. FOLCH J., LEES M. & SLOANE-STANLEY G. H. (1957) A simple method for the isolation and purification of total lipids from animal tissue. J. biol. Chem. 226, 497-509. LOTEN E. G. & JEANRENAUD I . (1974) Effects of cytochalasin B, colchicine and vincristine on the metabolism of isolated fat cells. Biochem. J. 140, 185 192. RODBELL M. (1964) Metabolism of isolated fat cells. J. biol. Chem. 239, 375 380. SCmMMEL R. J. (1975) Characterization of a colchicine receptor protein in rat epididymal adipose tissue. Biochem. biophys. Acta 399, 181-190. SOIFER D., BRAUN T. & HECI-ITER O. (1971) Insulin and microtubules in rat adipocytes. Science 172, 269-271. WEISENBERG R. C. (1972) Microtubule formation in vitro in solutions containing low calcium concentrations. Science 177, 1104~1105. WILSON L., BAMBURG J. R., MIZEL S. B, GRISHAM L. M. & CRESWELL K. M. (1974) Interaction of drugs with microtubule proteins. Fedn. Proc. 33, 158 166. WINAND J., DEHAYE J. & CHRISTOPHE J. (1973) Effets de la colchicine, de l'eau lourde et de la cytochalasine B sur le metabolisme du tissu adipeux de la souris normale et de la souris obese-hyperglycemique. Archs int. Physiol. Bioehim. 81, 603.