9 1992 by The Humana Press, Inc. All rights of any nature, whatsoever, reserved. 0163-4984/92/3201 - ~ 3 3 1 $02.00
Effect of Acute Zinc Deficiency on Insulin Receptor Binding in Rat Adipocytes MARIE J E A N N E GOMOT, 1 PATRICE FAURE, ~ ANNE-MARIE ROUSSEL *'2 CHARLES COUDRAY, ~ MIREILLE OSMAN, 2 AND ALAIN FAV1ER 1
ILaboratoire de Biochimie C, H6pital A. /Vlichallon, BP217 X, 38043 Grenoble C6dex, France; and 2Laboratoire de Biochirnie C, UFR de Pharmacie, 38700 La Tronche, France Received January 31, 1991; Accepted May 2, 1991
ABSTRACT Considerable data have been reported on the relationship between insulin resistance and zinc deficiency. In this study, insulin receptor binding was measured in isolated rat adipocytes. Two assays were carried out at 37~ (binding and internalization) and 16~ (binding) using 125I insulin 0.05-20 nM. A decreased insulin receptor binding was observed in zinc-deficient rat adipocytes, but we could not make any distinction between the specific zinc depletion effects and the effects of the caloric restriction induced by zinc deficiency. Index Entries: Insulin resistance; zinc deficiency; rat adipocytes;
insulin binding.
INTRODUCTION An insulin resistance related to zinc depletion has been observed by m a n y authors w h o reported a decreased glucose tolerance (1,2) and insulin secretion (3,4). Moreover, a mild zinc deficiency without any caloric restriction resulted in a lower glucose incorporation and oxidation in rat adipocytes (5). The cause of these abnormalities is yet unclear, but *Author to whom all correspondence and reprint requests should be addressed. Biological Trace Element Research
331
Vol. 32, 1992
332
Gomot et al.
could be related to the perturbation of insulin receptor binding. In the present article, we studied the insulin receptor binding in zinc-deficient rat adipocytes using various 125I insulin levels.
MATERIALS A N D METHODS Animals and Diets (Table 1) Twenty five male Wistar rats (initial wt 80-100 g) (IFFA CREDO 69210, L'Arbreles, France) were housed in acid-washed, stainless-steel cages and allowed free access to deionized distilled water delivered by a stainless-steel watering system. The absence of zinc released by these materials was checked. The rats were divided into three groups fed for 4 wk on the adequate diet. The ad libitum group (n = 5) was fed ad libitum on a zinc-adequate diet--100 ppm. The zinc-deficient group (n = 15) was fed on a zinc-deficient diet--< 0.2 ppm. The pair-fed group (n = 5) was fed on the zinc-adequate diet with the same quantities that had been consumed by the zinc-deficient group the day before, according to the pair-feeding method.
Trace Element Analysis Samples of diet, plasma, and bone were mineralized with nitric acid 25%. The amount of zinc was measured by flameless atomic absorption spectrophotometry (Perkin Elmer 460). Control value (Liver NIST, 1577A) was 128 mg/L. Laboratory value was 123 mg/L.
Isolated Adipocytes Obtention The epidydimal adipose tissues of each group were pooled and sliced. The adipocytes were isolated by the method described by Rodbell and Cushman (6,7). The size and number of fat cells were measured according to Lavau et al. (8). Cells were used within 4 h.
Insulin Receptor Binding Assays Fat cell suspension (0.3 mL) was added to 125Iinsulin (300 mCi/mg; CEA, Saday, France) and unlabeled insulin (Endopancrine| Choay, France) in final concentrations between 0.05-20 nM. Then the samples were shaken for 1 h into an agitating water bath. Two assays were carried out at 37~ to investigate binding and internalization, and 16~ to investigate insulin binding. Biological Trace Element Research
Vol, 32, 1992
Effects of Acute Zinc Deficiency
333
Table 1 Centesimal Diet Compositiona Egg albumin Corn starch Sucrose Corn oil Mineral mixture and amino acids b Vitaminsc
14.5 38.0 38.0 4.5 4.0 1.0
~The diets were purchased from INRA F-Clermond-Ferrand (Lamand). bSalt mixture, expressed in g/103 g: L-cystein, 2.356; L-tryptophan, 0.392; Ca pantothenate, 0.392; CaCO3, 9.42; KH2PO4, 10.68; NaC1, 7.85; NH 4 Fe citrate, 1.29; Mn2SO4, 0.03; CuSO4, 0.03; CaC12, 0.03; KI, 0.00023; MgSO4, 7.51. cVitamin mixture: Vitamin A, 1980 UI; D3, 2500 UI; BI, 2 mg; B2, 1.5 mg; B3, 7 mg; B12, 5 la,g; C, 80 mg; E, 17 mg; PP, 10 mg.
RESULTS Clinical symptoms of zinc deficiency--loss of appetite, alopecia, skin lesions--were observed as soon as the second week of the diet, according to plasma and bone zinc levels, which were significantly lower in the zinc-deficient group (Table 2). The effect of the zinc depletion was to impair the growth of the rats (Table 2). There were 0.3-3.106 cell/mL in the adipose cell suspensions. The size of adipocytes in the zinc-deficient group was significantly lower than in the other groups (Table 3). The insulin receptor binding was significantly lower in the zincdeficient and in the pair-fed groups compared to the ad libitum group. However, we did not observe a significant difference between the zincdeficient and the pair-fed groups, at 16~ (Fig. 1) and 37~ (Fig. 2).
DISCUSSION Zinc is known to enhance the specific insulin receptor binding (9,10) and to have an insulin-like effect on isolated fat cells (11). Furthermore, glucose tolerance is impaired in zinc-deficient animals. In the present study, we observed in zinc-deficient rats a decreased insulin receptor binding. At least three hypotheses can explain this phenomenon: It could be related to the effects of the caloric restriction induced by zinc deficiency, since we could not observe any difference between the zinc-deficient and the pair-fed groups. Moreover, an impaired receptor synthesis could be one of the possible hypotheses, according to the impaired protein synthesis that occurs in the zinc-deficient
Biological Trace Element Research
Vol. 32, 1992
Gomot et al.
334
Table 2 Effect of Diets on Body Wt, Food Intake, and Trace Element Levels ~ ZD n=7 Bodywt, g Food intake, g/24 h/rat Zn plasma, ~mol/L Cu plasma, p,mol/L Zn femoral bone, p,g/g dry "Values are mean bp < 0.05 b e t w e e n < .05 b e t w e e n 0.05 b e t w e e n
~
PF n=7
113 + 12b'c 18c 6.1 + 2.1 b'c 19.6 _+ 1.5 c
AL n=7
171 + 5~ 18~ 24.9 _+ 0.5 16.4 + 0.7 a
112 + 5b'c
192 _ 20 30 25.6 + 0.7 21.5 + 2.1
243 + 9
252 + 13
-+ SE f o r n s h o w n . ZD/PF. ZD/AL. PF/AL.
Table 3 Weight and Volume of Isolated Adipocytes for Each Group/500 Cells
VoI, pL Weight, ng
200
CZ
PF
AL
43 _+ 8 39 + 3
73 _+ 9 67 + 9
85 + 5 78 + 8
~mo]/lO 6
150
100
.........
50
RL RATS
........ PF RATS ~ZD
0 O. 05
1
5
i0
RATS
20
Insulln,nM
Fig. 1.
Insulin binding to rat adipocytes at 16~
rats. (12,13) Recently, it has b e e n r e p o r t e d that modification of cell m e m b r a n e fatty acid c o m p o s i t i o n , as o b s e r v e d d u r i n g zinc deficiency, c o u l d affect insulin b i n d i n g to t h e s e r e c e p t o r s (14) b y d e c r e a s i n g cell m e m b r a n e fluidity a n d r e c e p t o r translocation into the cells. In conclusion, the relationship a m o n g zinc status, insulin secretion, a n d glucose m e t a b o l i s m r e m a i n s unclear. Isolated fat cells s e e m to b e a Biological Trace Element Research
Vol. 32, 1992
335
Effects o f A c u t e Zinc Deficiency {mo]/]O 6 150
100
50 AL RATS ........ PF RATS --ZD
0.05
1
3 Insulin,
Fig. 2.
10
RATS
20
nM
Insulin binding to rat adipocytes at 37~
good experimental model for a n e w approach of the zinc-depletion effects. Nevertheless, a disturbing parameter for the interpretation of the results is the influence of the caloric restriction induced by zinc deficiency.
REFERENCES 1. J. Quaterman and E. Florence, Br. ]. Nutr. 28, 75-79 (1972). 2. D. G. Hendricks and A. W. Mahoney, ]. Nutr. 102, 1079-1084 (1972). 3. D. E. Brown, C. S. Penhos, L. Recant, and J. C. Smith, Proc. Soc. Exp. Biol. Med. 150, 557-560 (1975). 4. H. P. Roth and M. Kirchgegessner, Int. Z. Vit. Ern. Forsch 45, 201-208 (1975). 5. P. G. Reeves and B. L. O'Dell, Br. J. Nutr. 49, 441-452 (1983). 6. M. Rodbell, J. Biol. Chem. 239, 2, 375-380 (1964). 7. S. W. Cushman, J. Cell. Biol. 46, 326-341 (1970). 8. M. Lavau, C. Susini, J. Knittle, S. Blanche-Hirst, and M. R. C. Greenwood, Proc. Soc. Exp. Biol. Med. 156, 251-256 (1977). 9. E. R. Arquila, W. Packers, and S. Miyamoto, Endocrinology 103, 4, 1440-1449 (1978). 10. M. Fields, S. Reiser, and J. C. Smith, Nutr. Rep. Internat. 28, 1, 163-169 (1983). 11. J. M. May and C. S. Contoreggi, J. Biol. Chem. 257, 8, 4362-4368 (1981). 12. J. C. Wall-Work, G. J. Fosmire, and H. H. Sanstead, Br. ]. Nutr. 45, 127-136 (1981). 13. C. L. White, Biol. Tr. Elem. Res. 17, 175--187 (1988). 14. J. C. Field, J. Biol. Chem. 19, 1143-1150 (1990).
Biological Trace Element Research
Vol. 32, 1992
Effect of Acute Zinc Deficiency on Insulin Receptor Binding in Rat Adipocytes MARIE J E A N N E GOMOT, 1 PATRICE FAURE, ~ ANNE-MARIE ROUSSEL *'2 CHARLES COUDRAY, ~ MIREILLE OSMAN, 2 AND ALAIN FAV1ER 1
ILaboratoire de Biochimie C, H6pital A. /Vlichallon, BP217 X, 38043 Grenoble C6dex, France; and 2Laboratoire de Biochirnie C, UFR de Pharmacie, 38700 La Tronche, France Received January 31, 1991; Accepted May 2, 1991
ABSTRACT Considerable data have been reported on the relationship between insulin resistance and zinc deficiency. In this study, insulin receptor binding was measured in isolated rat adipocytes. Two assays were carried out at 37~ (binding and internalization) and 16~ (binding) using 125I insulin 0.05-20 nM. A decreased insulin receptor binding was observed in zinc-deficient rat adipocytes, but we could not make any distinction between the specific zinc depletion effects and the effects of the caloric restriction induced by zinc deficiency. Index Entries: Insulin resistance; zinc deficiency; rat adipocytes;
insulin binding.
INTRODUCTION An insulin resistance related to zinc depletion has been observed by m a n y authors w h o reported a decreased glucose tolerance (1,2) and insulin secretion (3,4). Moreover, a mild zinc deficiency without any caloric restriction resulted in a lower glucose incorporation and oxidation in rat adipocytes (5). The cause of these abnormalities is yet unclear, but *Author to whom all correspondence and reprint requests should be addressed. Biological Trace Element Research
331
Vol. 32, 1992
332
Gomot et al.
could be related to the perturbation of insulin receptor binding. In the present article, we studied the insulin receptor binding in zinc-deficient rat adipocytes using various 125I insulin levels.
MATERIALS A N D METHODS Animals and Diets (Table 1) Twenty five male Wistar rats (initial wt 80-100 g) (IFFA CREDO 69210, L'Arbreles, France) were housed in acid-washed, stainless-steel cages and allowed free access to deionized distilled water delivered by a stainless-steel watering system. The absence of zinc released by these materials was checked. The rats were divided into three groups fed for 4 wk on the adequate diet. The ad libitum group (n = 5) was fed ad libitum on a zinc-adequate diet--100 ppm. The zinc-deficient group (n = 15) was fed on a zinc-deficient diet--< 0.2 ppm. The pair-fed group (n = 5) was fed on the zinc-adequate diet with the same quantities that had been consumed by the zinc-deficient group the day before, according to the pair-feeding method.
Trace Element Analysis Samples of diet, plasma, and bone were mineralized with nitric acid 25%. The amount of zinc was measured by flameless atomic absorption spectrophotometry (Perkin Elmer 460). Control value (Liver NIST, 1577A) was 128 mg/L. Laboratory value was 123 mg/L.
Isolated Adipocytes Obtention The epidydimal adipose tissues of each group were pooled and sliced. The adipocytes were isolated by the method described by Rodbell and Cushman (6,7). The size and number of fat cells were measured according to Lavau et al. (8). Cells were used within 4 h.
Insulin Receptor Binding Assays Fat cell suspension (0.3 mL) was added to 125Iinsulin (300 mCi/mg; CEA, Saday, France) and unlabeled insulin (Endopancrine| Choay, France) in final concentrations between 0.05-20 nM. Then the samples were shaken for 1 h into an agitating water bath. Two assays were carried out at 37~ to investigate binding and internalization, and 16~ to investigate insulin binding. Biological Trace Element Research
Vol, 32, 1992
Effects of Acute Zinc Deficiency
333
Table 1 Centesimal Diet Compositiona Egg albumin Corn starch Sucrose Corn oil Mineral mixture and amino acids b Vitaminsc
14.5 38.0 38.0 4.5 4.0 1.0
~The diets were purchased from INRA F-Clermond-Ferrand (Lamand). bSalt mixture, expressed in g/103 g: L-cystein, 2.356; L-tryptophan, 0.392; Ca pantothenate, 0.392; CaCO3, 9.42; KH2PO4, 10.68; NaC1, 7.85; NH 4 Fe citrate, 1.29; Mn2SO4, 0.03; CuSO4, 0.03; CaC12, 0.03; KI, 0.00023; MgSO4, 7.51. cVitamin mixture: Vitamin A, 1980 UI; D3, 2500 UI; BI, 2 mg; B2, 1.5 mg; B3, 7 mg; B12, 5 la,g; C, 80 mg; E, 17 mg; PP, 10 mg.
RESULTS Clinical symptoms of zinc deficiency--loss of appetite, alopecia, skin lesions--were observed as soon as the second week of the diet, according to plasma and bone zinc levels, which were significantly lower in the zinc-deficient group (Table 2). The effect of the zinc depletion was to impair the growth of the rats (Table 2). There were 0.3-3.106 cell/mL in the adipose cell suspensions. The size of adipocytes in the zinc-deficient group was significantly lower than in the other groups (Table 3). The insulin receptor binding was significantly lower in the zincdeficient and in the pair-fed groups compared to the ad libitum group. However, we did not observe a significant difference between the zincdeficient and the pair-fed groups, at 16~ (Fig. 1) and 37~ (Fig. 2).
DISCUSSION Zinc is known to enhance the specific insulin receptor binding (9,10) and to have an insulin-like effect on isolated fat cells (11). Furthermore, glucose tolerance is impaired in zinc-deficient animals. In the present study, we observed in zinc-deficient rats a decreased insulin receptor binding. At least three hypotheses can explain this phenomenon: It could be related to the effects of the caloric restriction induced by zinc deficiency, since we could not observe any difference between the zinc-deficient and the pair-fed groups. Moreover, an impaired receptor synthesis could be one of the possible hypotheses, according to the impaired protein synthesis that occurs in the zinc-deficient
Biological Trace Element Research
Vol. 32, 1992
Gomot et al.
334
Table 2 Effect of Diets on Body Wt, Food Intake, and Trace Element Levels ~ ZD n=7 Bodywt, g Food intake, g/24 h/rat Zn plasma, ~mol/L Cu plasma, p,mol/L Zn femoral bone, p,g/g dry "Values are mean bp < 0.05 b e t w e e n < .05 b e t w e e n 0.05 b e t w e e n
~
PF n=7
113 + 12b'c 18c 6.1 + 2.1 b'c 19.6 _+ 1.5 c
AL n=7
171 + 5~ 18~ 24.9 _+ 0.5 16.4 + 0.7 a
112 + 5b'c
192 _ 20 30 25.6 + 0.7 21.5 + 2.1
243 + 9
252 + 13
-+ SE f o r n s h o w n . ZD/PF. ZD/AL. PF/AL.
Table 3 Weight and Volume of Isolated Adipocytes for Each Group/500 Cells
VoI, pL Weight, ng
200
CZ
PF
AL
43 _+ 8 39 + 3
73 _+ 9 67 + 9
85 + 5 78 + 8
~mo]/lO 6
150
100
.........
50
RL RATS
........ PF RATS ~ZD
0 O. 05
1
5
i0
RATS
20
Insulln,nM
Fig. 1.
Insulin binding to rat adipocytes at 16~
rats. (12,13) Recently, it has b e e n r e p o r t e d that modification of cell m e m b r a n e fatty acid c o m p o s i t i o n , as o b s e r v e d d u r i n g zinc deficiency, c o u l d affect insulin b i n d i n g to t h e s e r e c e p t o r s (14) b y d e c r e a s i n g cell m e m b r a n e fluidity a n d r e c e p t o r translocation into the cells. In conclusion, the relationship a m o n g zinc status, insulin secretion, a n d glucose m e t a b o l i s m r e m a i n s unclear. Isolated fat cells s e e m to b e a Biological Trace Element Research
Vol. 32, 1992
335
Effects o f A c u t e Zinc Deficiency {mo]/]O 6 150
100
50 AL RATS ........ PF RATS --ZD
0.05
1
3 Insulin,
Fig. 2.
10
RATS
20
nM
Insulin binding to rat adipocytes at 37~
good experimental model for a n e w approach of the zinc-depletion effects. Nevertheless, a disturbing parameter for the interpretation of the results is the influence of the caloric restriction induced by zinc deficiency.
REFERENCES 1. J. Quaterman and E. Florence, Br. ]. Nutr. 28, 75-79 (1972). 2. D. G. Hendricks and A. W. Mahoney, ]. Nutr. 102, 1079-1084 (1972). 3. D. E. Brown, C. S. Penhos, L. Recant, and J. C. Smith, Proc. Soc. Exp. Biol. Med. 150, 557-560 (1975). 4. H. P. Roth and M. Kirchgegessner, Int. Z. Vit. Ern. Forsch 45, 201-208 (1975). 5. P. G. Reeves and B. L. O'Dell, Br. J. Nutr. 49, 441-452 (1983). 6. M. Rodbell, J. Biol. Chem. 239, 2, 375-380 (1964). 7. S. W. Cushman, J. Cell. Biol. 46, 326-341 (1970). 8. M. Lavau, C. Susini, J. Knittle, S. Blanche-Hirst, and M. R. C. Greenwood, Proc. Soc. Exp. Biol. Med. 156, 251-256 (1977). 9. E. R. Arquila, W. Packers, and S. Miyamoto, Endocrinology 103, 4, 1440-1449 (1978). 10. M. Fields, S. Reiser, and J. C. Smith, Nutr. Rep. Internat. 28, 1, 163-169 (1983). 11. J. M. May and C. S. Contoreggi, J. Biol. Chem. 257, 8, 4362-4368 (1981). 12. J. C. Wall-Work, G. J. Fosmire, and H. H. Sanstead, Br. ]. Nutr. 45, 127-136 (1981). 13. C. L. White, Biol. Tr. Elem. Res. 17, 175--187 (1988). 14. J. C. Field, J. Biol. Chem. 19, 1143-1150 (1990).
Biological Trace Element Research
Vol. 32, 1992
