Increased Insulin-Insensitive Glucose Transport in Polymorphonuclear Leukocytes from Non-InsulinDependent Diabetic Patients
Summary We studied the transport rate of a non-metabolizable hexose analogue, 3-O-methyl-D-glucose, in polymorphonuclear leukocytes (insulin-insensitive cells) from patients with untreated non-insulin-dependent diabetes mellitus. The mean glucose transport rate was significantly elevated in the diabetic patients compared with healthy controls (13.3 + 3.7 vs 10.4 ± 2 . 5 fl/cell.sec, m e a n ± S D , p < 0.01). In the diabetic subjects, glucose transport rates were positively correlated with HbA1c levels (r = 0.563, p < 0.01) but had no relations with ambient plasma glucose concentrations. Short-term incubation with 20 mM D-glucose had no effect on glucose transport in those cells. When glucose transport rates, HbA1c and fasting plasma glucose levels were simultaneously measured at weekly intervals over a four-week period in three diabetic subjects, the alterations in transport rates generally paralleled the changes observed in HbA1c levels rather than plasma glucose concentrations. It can be concluded that unlike insulin-sensitive cells such as adipocytes and muscle, glucose transport in human polymorphonuclear leukocytes, which are insulin insensitive cells, is increased in patients with non-insulin-dependent diabetes mellitus. Long-term, not shortterm, derangement of glucose metabolism seems to be associated with increased glucose transport rate found in those patients.
Rosen 1987) and differentiation (Kasahara, Hinkle, Ikawa and Amanuma 1986). Recent cDNA cloning has demonstrated that the facilitative glucose transporters comprise a family of structurally related proteins with different tissue distributions (Bell, Kayano, Buse, Burant, Takeda, Lin, Fukumoto and Seino 1990). However, little is known about pathophysiological changes in glucose transport induced by physiological stimuli or in various metabolic or endocrine disorders in humans. Although previous reports have suggested that insulin-stimulated glucose transport rate in adipocytes (Ciaraldi, Kolterman, Scarlett, Kao and Olefsky 1982; Kashiwagi, Verso, Andrews, Vasquez, Reaven and Foley 1983; Sinha, Taylor, Pories, Flickinger, Meelheim, Atkinson, Sehgal and Cam 1987) or muscle (Dohm, Tapscott, Pories, Dabbs, Flickinger, Meelheim, Fushiki, Atkinson, Elton and Caw 1988) from patients with non-insulin-dependent diabetes mellitus (NIDDM) are lower than that in healthy subjects, there is a paucity of literature relating to the glucose transport in insulininsensitive cells from those patients. Since human polymorphonuclear leukocytes (PMNLs) are insulin-insensitive cells with respect to glucose transport (Okuno and Gliemann 1988), we assayed the glucose transport rate in PMNLs of patients with N I D D M to examine whether or not insulin-insensitive glucose transport is affected by abnormalities in glucose metabolism. Materials and Methods Subjects
Key words Glucose Transport - Polymorphonuclear Leukocytes — Insulin Insensitive Cells — Diabetes Mellitus
Introduction Transport of hexoses is mediated by a facilitated diffusion process in mammalian cells. Changes in glucose transport rate in these cells are induced by treatment with hormones (Okuno and Gliemann 1987; Filetti, Damante and Foti 1987; Carter-Su, Rozsa, Wang and Stubbart 1988) or in association with cellular transformation (Hatanaka 1974; Kita-
gawa, Nishino and Iwashima 1985; Bimbaum, Haspel and
Horm.metab.Res. 23(1991)387-391 © Georg Thieme Verlag Stuttgart-New York
Two groups of subjects participated in Study A. The control group consisted of 29 healthy subjects (24 men and 5 women), 23—63 yrs of age (mean age, 43.3 ± 14.2 yrs). The study group consisted of 23 patients with untreated NIDDM (14 men and 9 women), 30—65 yrs of age (mean age, 55.4 ± 7.7 yrs). None had abnormal renal or liver function by routine tests. All patients were classified as NIDDM according to National Diabetes Data Group (1979) criteria. All subjects were consuming a weight-maintaining diet, which contained at least 200 g/day of carbohydrate, for at least 7 days before the study. Each subject was studied after an overnight fast. Table 1 represents some of the clinical characteristics of the subjects. In study B, glucose transport rates were measured once a week during a four weeks' period in three diabetic patients treated with diet alone, insulin or sulfonylurea and changes in transport rate were analyzed in reference to those in plasma glucose and HbAlc levels. The clinical data on the individual cases are described in the legend to Figure 4.
Received: 9 Oct. 1990
Accepted: 4 Febr. 1991
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Y. Okuno, Y. Nishizawa and H. Morii The Second Department of Internal Medicine, Osaka City University Medical School, Osaka, Japan
Horm. metab. Res. 23 (1991)
Y. Okuno, Y. Nishizawa and H. Morii
Tabe 1 Clinical characteristics of the study group. Control
NIDDM
Number
29
23
Sex Man Woman
24 5
14 9
Age (yr) BMI (kg/m2) Fasting plasma glucose (mM) HbA1c (%) Fasting plasma insulin (pM)
43.3 ±14.2 (23-63)
55.4 ±7.7 (30-65)
20.3 ±1.8
21.8 ±3.2
a
5.1 ±0.23
9.6 ±2.9
a
5.2 ±0.3
10.8 ±2.9
a
83.3 ±12.2
96.2 ±25.1
All data are expressed as mean ± SD. a p < 0.001, significantly different from control subjects. Fig. 1 0.05 mM 30MG transport rates in 29 healthy subjects ( • ) and 23 patients with NIDDM ( • ) .
Chemicals 3-0-[14C]-methyl-D-glucose (53 Ci/mol) was purchased from Amersham, U. K., unlabeled 30MG and phloretin from Sigma, U. S. A., high-molecular-weight dextran (Mr 180,000) from Nakarai Chemicals, Japan, and Mono-Poly resolving medium from Frow Laboratories, Inc., Australia. The other reagents were of analytical grade.
Preparation of cells PMNLs were prepared without exposure to hypotonic medium. With a heparinized syringe, about 15 ml of venous blood was sampled from each subject. A part of the blood was used for measurement of plasma glucose, immunoreactive insulin (IRI) and HbA1c. One vol. of 3% dextran in 0.9% NaCl was added to 2 vol. of the blood to enhance rouleaux formation of erythrocytes. After sedimentation for 30 min at 24 °C, the supernatant containing mononuclear cells, platelets and some remaining erythrocytes was layered on top of Mono-Poly resolving medium (Ferrante and Thong 1980) and centrifuged for 30 min at 300 x g. The erythrocytes were stuck to the bottom of the tube after this procedure and the PMNLs were recovered from the band of the highest density near the bottom. They were then washed twice at 24 °C in the buffer containing 31 mM Mops and Krebs' salts as described elsewhere (Okuno, Plesner, Larsen and Gliemann 1986) and adjusted to a concentration of 2—4 x 10 cells/ml. The purity of the PMNLs was 96-99% with 1-4% lymphocytes. Samples contaminated by more than 1 % erythrocytes were not used. The total and differential counts of the PMNLs were normal in all subjects.
Transport assay This was carried out at 37 °C, pH 7.4, as previously described {Okuno et al. 1986). In brief, 15 ul buffer with 56 nCi (10* cpm) 14C-labelled and 0.05 mM unlabelled 30MG was placed in a 7.5 ml round-bottomed tube (Evergreen Scientific, U. S. A.). PMNLs were allowed to equilibrate with 0.05 mM unlabelled 30MG for 15 min at 37 °C (Okuno et al. 1986). At time zero, 45 ul of the PMNL suspension was squirted onto the 15 ul buffer containing labelled and unlabelled 30MG. Thus, the 30MG concentration was the same on both sides of the membrane and the equilibration of the extra-cellular [ C]30MG with its intracellular distribution space was measured (equilibrium exchange). Incubation was terminated at 5 sec by the addition
of 3.5 ml stopping solution (0.3 mM phloretin in Mops buffer containing 0.1 uM HgCl2). The cells were pelleted by centrifugation at about 4000 x g for 1 min and the supernatant was discarded. This procedure was repeated once. Finally 2.5 ml of scintillation fluid was added and the radioactivity was determined. Blank values, determined by the addition of stopping solution before adding the cells, were subtracted from all measurements. They contained about 20% of the counts present when the cells were allowed to equilibrate with labelled 30MG for 15 min. In an in vitro study, the short-term regulatory effects of glucose on glucose transport in normal PMNLs was examined. PMNLs from each of four healthy men (aged 37, 35, 29 and 26 yrs) were incubated with or without 20 mM D-glucose at 37 °C for 60 min. After the cells were washed with above-mentioned buffer, the glucose transport rate was assayed. The glucose transport rate was expressed as glucose clearance rate per cell (in fl/cell-sec). Plasma glucose was measured with an Auto Analyzer. The serum IRI concentration was determined by the double antibody method. HbA1c was measured with high pressure liquid chromatography (Cole, Soeldner, Dunn and Bunn 1978).
Analytical procedure Statistical analysis was performed with unpaired Student's t-test. Linear regression analysis was used to determine the correlation coefficients. Data are expressed as mean ± SD.
Results Study A Figure 1 shows the 30MG transport rates in the healthy subjects (n = 29) and patients with NIDDM (n = 23). The mean of transport rate was 13.3 ±3.7 fl/cell-sec in the diabetic patients which was significantly (p < 0.01) elevated than the control level (10.4 ± 2.5). In healthy subjects, neither fasting plasma glucose concentrations nor HbAlc levels was
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388
Glucose Transport in Polymorphonuclear Leukocytes
Horm. metab. Res. 23 (1991)
25 n
Fig. 3 Correlation between the transport rates and HbA1c levels measured simultaneously In the diabetic patients.
related to glucose transport rates (data not shown). In diabetic patients, there was no significant correlation between fasting plasma glucose concentrations and glucose transport rates (Fig. 2), but the latter was significantly correlated with the levels of HbAlc(Fig. 3). In vitro study The glucose transport rate of normal PMNLs was not affected by short-term (60 min) treatment with 20 mM D-glucose: the mean transport rate was 12.7 + 3.7 fl/cell-sec in the absence of glucose and 12.5+4.7 fl/cell-sec in the presence of glucose, without a significant difference between the two values (n = 4). Study B The results of study B are illustrated in Figure 4. In case 1 and case 2 in which either sulfonylurea or insulin was administered, glucose transport rates gradually decreased in parallel with HbAlc levels rather than plasma glucose concentrations. On the other hand, glucose transport rates in the patient (case 3) with stable levels of glucose and HbAlc remained unchanged over the same period. Discussion We have already demonstrated that exchange of 30MG between incubation medium and the intracellular water of PMNLs occurred via facilitated diffusion with a Km of about 4 mM {Okuno et al. 1986) and that insulin did not acutely stimulate glucose transport rate (Okuno and Gliemann 1988). In this study, the mean transport rate of PMNLs was significantly higher in the NIDDM group than in the healthy control group. Although there was a considerable difference in mean age between the two groups, the possibility can be ruled out that the increased transport rate in the diabetic subjects was due to the higher age (55.4 + 7.7 vs 43.3 ±14.2 years), because it is known that glucose transport in PMNLs is not altered by variance in age (23—63 years) (Okuno and Morii 1989).
Fig. 4 Changes in fasting plasma glucose concentration ( • • ) , HbAlc level ( O — O ) and transport rate (A • ) in three diabetic patients. Case 1 (left panel) a 46-year-old man with a six-year history of diabetes mellitus. An improvement in glycemic control was obtained through diet and sulfonylurea threapy after admission. He had retinal microaneurysms but was free of diabetic neuropathy or nephropathy. Case 2 (central panel) a 56-year-old man with a five-year history of diabetes mellitus. Insulin was started immediately after hospitalization, because he complained of severe thirst and general fatigue. He had no diabetic complications. Case 3 (right panel) a 61-year-old woman with a three-year history of diabetes mellitus. Although her glycemic control has been good and stable, she admitted to our hospital because of diabetic education. She suffered from mild peripheral neuropathy.
Previous studies have shown that chemotactic factors such as C5a enhance transport of glucose in PMNLs (Okuno and Gliemann 1988). It has also been shown that glucose transport is increased in PMNLs isolated from patients with acute bacterial infections (Bibi, DeChatelet and McCall 1977). Since our patients were free of either a viral or a bacterial infection and the total and differential counts of PMNLs were normal in all individuals, "inflammation" was unlikely to be responsible for the increase in glucose transport.
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Fig. 2 Correlation between the glucose transport rates and fasting plasma glucose concentrations measured simultaneously in the diabetic patients.
Horm. metab. Res. 23 (1991) Several investigators reported that insulinstimulated glucose transport in epitrochlearis muscle or adipocytes from streptozotocin (STZ)-induced diabetic rats was lower than that in normal control animals {Wallberg-Henriksson 1986; Wallberg-Henriksson, Zetan and Henriksson 1987; Wieringa and Krans 1978; Kobayashi and Olefsky 1979). Similar results were obtained with adipocytes or muscle from patients with N I D D M (Ciaraldi et al. 1982; Kashiwagi et al. 1983; Sinha et al. 1987; Dohm et al. 1988). Consistent with these results is the finding that patients with NIDDM are characterized by a marked impairment in in vivo insulin-stimulated glucose uptake {Ginsberg, Kimmerling, Olefsky and Reaven 1975; DeFronzo, Deibert, Hendler, Felig and Soman 1979; Rizza, Mandarino and Gerich 1981; Kolterman, Gray, Griffin, Bernstein, Insel, Scarlett and Olefsky 1981; Bogardus, Lillioja, Howard, Reaven and Mott 1984; Donner, Fraze, Chen and Reaven 1985). In contrast, Baron, Kolterman, Bell, Mandarino and Olefsky (1985) demonstrated that non-insulin dependent diabetic patients have basal rates of non-insulin mediated glucose uptake that are about 2 times that of healthy subjects. However few data are available concerning the possible effect of diabetic states on the glucose transport in insulin-insensitive cells. Oka, Asano, Shibasaki, Lin, Tsukuda, Akanuma and Takaku (1990) very recently reported that in the rat liver, in which, unlike in adipocyte and muscle tissue, insulin does not acutely activate glucose transport, glucosetransporter protein and mRNA levels are increased about twofold in streptozotocin-induced rats. In this study, we also found that the transport rate of PMNLs (insulin-insensitive cells) from patients with N I D D M is significantly elevated compared with the healthy subjects. Therefore, it seemed reasonable to assume that the glucose transport rate in insulin-insensitive cells and sensitive cells may be regulated independently in diabetic states. Glucose transport in PMNLs does not seem to be affected by short-term glycemic control, first because no significant correlation was found between transport rates and ambient plasma glucose concentrations and secondly because 60 min of incubation with 20 mM glucose did not exert any appreciable effect on glucose transport. On the other hand, a positive correlation was noted between transport rates and HbAlc levels in our patients. Moreover, when glucose transport rate, H b A l c and plasma glucose concentration were assayed simultaneously during 4 weeks of follow-up, changes of glucose transport were observed to generally parallel those of HbAlc, but not glucose levels. In healthy subjects, values of glucose transport rates assayed on 4 different days separated by 1-week interval were very stable (coefficient of variation = about 7%) {Okuno and Morii 1989). Thus, these changes observed in glucose transport do not seem to be ascribed to method variation. Since the HbA1c level is a reliable index of long-term blood glucose control (Koenig, Peterson, Jones, Saudek, Lehrman and Cerami 1976), these results suggest that long-term, not short-term, derangement of glucose metabolism is associated with increased glucose transport rate in PMNLs in N I D D M . PMNLs move through bessel walls and enter the tissues only half a day after they are released from the bone marrow {Nathan 1988). Therefore, the glucose transport system of the immature cells, myeloblasts, present in the bone marrow appears to be modified by the long-term blood glucose abnormalitiy.
Y. Okuno, Y. Nishizawa and H. Morii It has been proposed that glucose may regulate the rate of its own transport, although the precise mechanisms remain to be elucidated. Van Putten and Krans (1985) and Sasson and Cerasi (1986) reported that this regulatory mechanism was present in 3T3-L1 preadipocytes and rat skeletal muscle and that basal glucose transport in these cells appeared to decrease rather than increase upon prolonged exposure to high glucose concentrations. These results are discrepant from our data that long-term blood glucose abnormality may increase glucose transport rate in PMNLs. The reason for this difference may be one or more of the following possibilities. First, they used insulin-sensitive cells and we used insulin-insensitive cells. Second, increased glucose transport in PMNLs of diabetic subjects with long-term blood glucose abnormality may be influenced by other hormonal and metabolic factors as well as glucose itself. Third, the duration of abnormality in glucose metabolism of our patients was much longer than the incubation time (24—48 hrs) used in their experiments. In conclusion, the insulin-insensitive glucose transport of PMNLs, unlike insulin-sensitive glucose transport, is increased in patients with N I D D M and long-term, not short-term, derangement of glucose metabolism seems to be associated with the increased glucose transport found in PMNLs from those patients. Acknowledgements This study was supported in part by a research grant from the Ministry of Education, Culture and Science of Japan References Baron, A. D., O. G. Kolterman, J. Bell, L. J. Mandarino, J. M. Olefsky: Rates of noninsulin-mediated glucose uptake are elevated in type II diabetic subjects. J. Clin. Invest. 76:1782-1788 (1985) Bell, G. I., T. Kayano, J. B. Buse, C. F. Burant, J. Takeda, D. Lin, H. Fukumoto, S. Seino: Molecular biology of mammalian glucose transporters. Diabetes Care 13:198-208 (1990) Bibi, S. S., L. R. DeChatelet, C. E. McCall: Human toxic neutrophils. IV. Incorporation of amino acids and uptake of 2-deoxy-D-glucose J. Infect. Dis. 135:949-951(1977) Birnbaum, M. J., H. C. Haspel, O. M. Rosen: Transformation of rat fibroblasts by FSV rapidly increases glucose transporter gene transcription. Science 235:1495-1498(1987) Bogardus, C, S. Lillioja, B. V. Howard, G. Reaven, D. Mott: Relationship between insulin secretion, insulin action and fasting plasma glucose concentration in nondiabetic and non-insulin-dependent diabetic subjects. J. Clin. Invest. 74:1238-1246 (1984) Carter-Su, C, F. W. Rozsa, X. Wang, J. R. Stubbart: Rapid and transitory stimulation of 3-O-methylglucose transport by growth hormone. Am. J. Physiol. 255: E723-729 (1988) Ciaraldi, T. P., O. G. Kolterman, J. A. Scarlett, M. Kao, J. M. Olefsky: Role of glucose transport in the postreceptor defect of non-insulindependent diabetes mellitus. Diabetes 31:1016-1022 (1982) Cole, R. A., J. S. Soeldner, P. J. Dunn, H. F. Bunn: A rapid method for determination of glycosylated hemoglobin using high pressure liquid chromatography. Metabolism 27:289-301 (1978) DeFronzo, R. A., A. D. Deibert, R. Hendler, P. Felig, V. J. Soman: Insulin sensitivity and insulin binding to monocytes in maturityonset diabetes. J. Clin. Invest. 63:936-939 (1979) Dohm, G. L., E. B. Tapscott, W. J. Pories, D. J. Dabbs, E. G. Flickinger, D. Meelheim, T. Fushiki, S. M. Atkinson, C. W. Elton, J. F. Caro: An invitro human muscle preparation suitable for metabolic studies decreased insulin stimulation of glucose transport in muscle from morbidly obese and diabetic subjects. J. Clin. Invest. 82: 486-494(1988)
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Donner, C. C , E. Fraze, Y. D. I. Chen, G. M. Reaven: Quantitation of insulin-stimulated glucose disposal in patients with non-insulindependent diabetes mellitus. Diabetes 34: 831-835 (1985) Ferrante, A., Y. H. Thong: Optimal conditions for simultaneous purification of mononuclear and polymorphonuclear leukocytes from peripheral blood by the Hypaque-Ficoll method. J. Immunol. Methods36:109-117(1980) Filetti, S., G. Damante, D. Foti: Thyrotropin stimulates glucose transport in cultured rat thyroid cells. Endocrinology 120: 2576—2581 (1987) Ginsberg, H., G. Kimmerling, J. M. Olefsky, G. M. Reaven: Demonstration of insulin resistance in untreated adult-onset diabetic subjects with fasting hyperglycemia. J. Clin. Invest. 55: 454—461 (1975) Hatanaka, M.: Transport of sugars in tumor cell membranes. Biochim. Biophys. Acta 355:77-104 (1974) Kasahara, M., P. C. Hinkle, Y. Ikawa, H. Amanuma: Decrease in glucose transport activity of Friend erythroleukemia cell caused by dimethylsulfoxide, a differentiation-inducing reagent. Biochim. Biophys. Acta 856:615-623 (1986) Kashiwagi, A., M. A. Verso, J. Andrews, B. Vasquez, G. Reaven, J. E. Foley: In vitro insulin resistance of human adipocytes isolated from subjects with non-insulin-dependent diabetes mellitus. J. Clin. Invest. 72:1246-1254 (1983) Kitagawa, K., H. Nishino, A. Iwashima: Analysis of hexose transport in untransformed and sarcoma virus-transformed mouse 3T3 cells by photoaffinity binding of cytochalasin B. Biochim. Biophys. Acta821:63-66(1985) Kobayashi, M., J. M. Olefsky: Effects of streptozotocin-induced diabetes on insulin binding, glucose transport and intracellular glucose metabolism in isolated rat adipocytes. Diabetes 28: 87—95 (1979) Koenig, R. J., C. M. Peterson, R. L, Jones, C. Saudek, M. Lehrman, A. Cerami: Correlation of glucose regulation and hemoglobin A l e in diabetes mellitus. N. Eng. J. Med. 295:417-420 (1976) Kolterman, O, G., R. S. Gray, J. Griffin, P. Bernstein, J. Insel, J. A. Scarlett, J. M. Olefsky: Receptor and post receptor defects contribute to the insulin resistance in non-insulin-dependent diabetes mellitus. J. Clin. Invest. 68:957-969 (1981) Nathan, D. G.: Hematologic diseases. In: Wyngaarden, J. B., L. H. Smith (eds): Cecil Textbook of Medicine, 18th. W. B. Saunders Com., Philadelphia London Toronto Tokyo (1988), pp. 873 - 1 0 8 1 National Diabetes Data Group: Classification and diagnosis of diabetes mellitus and other categories of glucose intolerance. Diabetes 28:1039-1057(1979) Oka, Y., T. Asano, Y. Shibasaki, J. L. Lin, K. Tsukuda, Y. Akanuma, F. Takaku: Increased liver glucose-transporter protein and mRNA in streptozotocin-induced diabetic rats. Diabetes 39:441—446 (1990)
Horm. metab. Res. 23 (1991) Okuno, Y, L. Plesner, T. R. Larsen, J. Gliemann: Facilitated transport of 3-O-methyl-D-glucose in human polymorphonuclear leukocytes. FEBS Lett. 195:303-308(1986) Okuno, Y., J. Gliemann: Enhancement of glucose transport by insulin at 37 °C in rat adipocytes is accounted for by increased Vmax. Diabetologia 30:426-430 (1987) Okuno, Y, J. Gliemann: Effect of chemotactic factors on hexose transport in polymorphonuclear leukocytes. Biochim. Biophys. Acta 941:157-164(1988) Okuno, Y, H. Morii: Clinical application of measurement of glucose transport in human polymorphonuclear leukocytes. Diabetes Res. Clin. Pract. (Supplement 1 ) 7 : 5 - 9 (1989) Rizza, R. A., L. J. Mandarino, J. E. Gerich: Mechanism and significance of insulin resistance in non-insulin-dependent diabetes mellitus. Diabetes 30:990-995(1981) Sasson, S., E. Cerasi: Substrate regulation of the glucose transport system in rat skeletal muscle. J. Biol. Chem. 261:16827-16833 (1986) Sinha, M. K, L. G. Taylor, W. J. Pories, E. G. Flickinger, D. Meelheim, S. Atkinson, N. S. Sehgal, J. F. Cam: Long-term effect of insulin on glucose transport and insulin binding in cultured adipocytes from normal and obese humans with and without non-insulin-dependent diabetes. J. Clin. Invest. 80: 1073-1081 (1987) van Putten, J. P. M., H. M. J. Krans: Glucose as a regulator of insulinsensitive hexose uptake in 3T3 adipocytes. J. Biol. Chem. 260: 7996-8001(1985) Wallberg-Henriksson, H.: Repeated exercise regulates glucose transport capacity in skeletal muscle. Acta Physiol. Scand. 127: 39—43 (1986) Wallberg-Henriksson, H., N. Zetan, J. Henriksson: Reversibility of decreased insulin-stimulated glucose transport capacity in diabetic muscle with in vitro incubation. Insulin is not required. J. Biol. Chem. 262:7665-7671 (1987) Wieringa, T., H. M. J. Krans: Reduced glucose transport and increased binding of insulin in adipocytes from diabetic and fasted rats. Biochim. Biophys. Acta 538:563-570 (1978)
Requests for reprints should be addressed to: Yasuhisa Okuno, M. D. Second Department of Internal Medicine Osaka City University Medical School 1-5-7, Asahi-machi, Abeno-ku Osaka 545 (Japan)
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Glucose Transport in Polymorphonuclear Leukocytes
Summary We studied the transport rate of a non-metabolizable hexose analogue, 3-O-methyl-D-glucose, in polymorphonuclear leukocytes (insulin-insensitive cells) from patients with untreated non-insulin-dependent diabetes mellitus. The mean glucose transport rate was significantly elevated in the diabetic patients compared with healthy controls (13.3 + 3.7 vs 10.4 ± 2 . 5 fl/cell.sec, m e a n ± S D , p < 0.01). In the diabetic subjects, glucose transport rates were positively correlated with HbA1c levels (r = 0.563, p < 0.01) but had no relations with ambient plasma glucose concentrations. Short-term incubation with 20 mM D-glucose had no effect on glucose transport in those cells. When glucose transport rates, HbA1c and fasting plasma glucose levels were simultaneously measured at weekly intervals over a four-week period in three diabetic subjects, the alterations in transport rates generally paralleled the changes observed in HbA1c levels rather than plasma glucose concentrations. It can be concluded that unlike insulin-sensitive cells such as adipocytes and muscle, glucose transport in human polymorphonuclear leukocytes, which are insulin insensitive cells, is increased in patients with non-insulin-dependent diabetes mellitus. Long-term, not shortterm, derangement of glucose metabolism seems to be associated with increased glucose transport rate found in those patients.
Rosen 1987) and differentiation (Kasahara, Hinkle, Ikawa and Amanuma 1986). Recent cDNA cloning has demonstrated that the facilitative glucose transporters comprise a family of structurally related proteins with different tissue distributions (Bell, Kayano, Buse, Burant, Takeda, Lin, Fukumoto and Seino 1990). However, little is known about pathophysiological changes in glucose transport induced by physiological stimuli or in various metabolic or endocrine disorders in humans. Although previous reports have suggested that insulin-stimulated glucose transport rate in adipocytes (Ciaraldi, Kolterman, Scarlett, Kao and Olefsky 1982; Kashiwagi, Verso, Andrews, Vasquez, Reaven and Foley 1983; Sinha, Taylor, Pories, Flickinger, Meelheim, Atkinson, Sehgal and Cam 1987) or muscle (Dohm, Tapscott, Pories, Dabbs, Flickinger, Meelheim, Fushiki, Atkinson, Elton and Caw 1988) from patients with non-insulin-dependent diabetes mellitus (NIDDM) are lower than that in healthy subjects, there is a paucity of literature relating to the glucose transport in insulininsensitive cells from those patients. Since human polymorphonuclear leukocytes (PMNLs) are insulin-insensitive cells with respect to glucose transport (Okuno and Gliemann 1988), we assayed the glucose transport rate in PMNLs of patients with N I D D M to examine whether or not insulin-insensitive glucose transport is affected by abnormalities in glucose metabolism. Materials and Methods Subjects
Key words Glucose Transport - Polymorphonuclear Leukocytes — Insulin Insensitive Cells — Diabetes Mellitus
Introduction Transport of hexoses is mediated by a facilitated diffusion process in mammalian cells. Changes in glucose transport rate in these cells are induced by treatment with hormones (Okuno and Gliemann 1987; Filetti, Damante and Foti 1987; Carter-Su, Rozsa, Wang and Stubbart 1988) or in association with cellular transformation (Hatanaka 1974; Kita-
gawa, Nishino and Iwashima 1985; Bimbaum, Haspel and
Horm.metab.Res. 23(1991)387-391 © Georg Thieme Verlag Stuttgart-New York
Two groups of subjects participated in Study A. The control group consisted of 29 healthy subjects (24 men and 5 women), 23—63 yrs of age (mean age, 43.3 ± 14.2 yrs). The study group consisted of 23 patients with untreated NIDDM (14 men and 9 women), 30—65 yrs of age (mean age, 55.4 ± 7.7 yrs). None had abnormal renal or liver function by routine tests. All patients were classified as NIDDM according to National Diabetes Data Group (1979) criteria. All subjects were consuming a weight-maintaining diet, which contained at least 200 g/day of carbohydrate, for at least 7 days before the study. Each subject was studied after an overnight fast. Table 1 represents some of the clinical characteristics of the subjects. In study B, glucose transport rates were measured once a week during a four weeks' period in three diabetic patients treated with diet alone, insulin or sulfonylurea and changes in transport rate were analyzed in reference to those in plasma glucose and HbAlc levels. The clinical data on the individual cases are described in the legend to Figure 4.
Received: 9 Oct. 1990
Accepted: 4 Febr. 1991
Downloaded by: National University of Singapore. Copyrighted material.
Y. Okuno, Y. Nishizawa and H. Morii The Second Department of Internal Medicine, Osaka City University Medical School, Osaka, Japan
Horm. metab. Res. 23 (1991)
Y. Okuno, Y. Nishizawa and H. Morii
Tabe 1 Clinical characteristics of the study group. Control
NIDDM
Number
29
23
Sex Man Woman
24 5
14 9
Age (yr) BMI (kg/m2) Fasting plasma glucose (mM) HbA1c (%) Fasting plasma insulin (pM)
43.3 ±14.2 (23-63)
55.4 ±7.7 (30-65)
20.3 ±1.8
21.8 ±3.2
a
5.1 ±0.23
9.6 ±2.9
a
5.2 ±0.3
10.8 ±2.9
a
83.3 ±12.2
96.2 ±25.1
All data are expressed as mean ± SD. a p < 0.001, significantly different from control subjects. Fig. 1 0.05 mM 30MG transport rates in 29 healthy subjects ( • ) and 23 patients with NIDDM ( • ) .
Chemicals 3-0-[14C]-methyl-D-glucose (53 Ci/mol) was purchased from Amersham, U. K., unlabeled 30MG and phloretin from Sigma, U. S. A., high-molecular-weight dextran (Mr 180,000) from Nakarai Chemicals, Japan, and Mono-Poly resolving medium from Frow Laboratories, Inc., Australia. The other reagents were of analytical grade.
Preparation of cells PMNLs were prepared without exposure to hypotonic medium. With a heparinized syringe, about 15 ml of venous blood was sampled from each subject. A part of the blood was used for measurement of plasma glucose, immunoreactive insulin (IRI) and HbA1c. One vol. of 3% dextran in 0.9% NaCl was added to 2 vol. of the blood to enhance rouleaux formation of erythrocytes. After sedimentation for 30 min at 24 °C, the supernatant containing mononuclear cells, platelets and some remaining erythrocytes was layered on top of Mono-Poly resolving medium (Ferrante and Thong 1980) and centrifuged for 30 min at 300 x g. The erythrocytes were stuck to the bottom of the tube after this procedure and the PMNLs were recovered from the band of the highest density near the bottom. They were then washed twice at 24 °C in the buffer containing 31 mM Mops and Krebs' salts as described elsewhere (Okuno, Plesner, Larsen and Gliemann 1986) and adjusted to a concentration of 2—4 x 10 cells/ml. The purity of the PMNLs was 96-99% with 1-4% lymphocytes. Samples contaminated by more than 1 % erythrocytes were not used. The total and differential counts of the PMNLs were normal in all subjects.
Transport assay This was carried out at 37 °C, pH 7.4, as previously described {Okuno et al. 1986). In brief, 15 ul buffer with 56 nCi (10* cpm) 14C-labelled and 0.05 mM unlabelled 30MG was placed in a 7.5 ml round-bottomed tube (Evergreen Scientific, U. S. A.). PMNLs were allowed to equilibrate with 0.05 mM unlabelled 30MG for 15 min at 37 °C (Okuno et al. 1986). At time zero, 45 ul of the PMNL suspension was squirted onto the 15 ul buffer containing labelled and unlabelled 30MG. Thus, the 30MG concentration was the same on both sides of the membrane and the equilibration of the extra-cellular [ C]30MG with its intracellular distribution space was measured (equilibrium exchange). Incubation was terminated at 5 sec by the addition
of 3.5 ml stopping solution (0.3 mM phloretin in Mops buffer containing 0.1 uM HgCl2). The cells were pelleted by centrifugation at about 4000 x g for 1 min and the supernatant was discarded. This procedure was repeated once. Finally 2.5 ml of scintillation fluid was added and the radioactivity was determined. Blank values, determined by the addition of stopping solution before adding the cells, were subtracted from all measurements. They contained about 20% of the counts present when the cells were allowed to equilibrate with labelled 30MG for 15 min. In an in vitro study, the short-term regulatory effects of glucose on glucose transport in normal PMNLs was examined. PMNLs from each of four healthy men (aged 37, 35, 29 and 26 yrs) were incubated with or without 20 mM D-glucose at 37 °C for 60 min. After the cells were washed with above-mentioned buffer, the glucose transport rate was assayed. The glucose transport rate was expressed as glucose clearance rate per cell (in fl/cell-sec). Plasma glucose was measured with an Auto Analyzer. The serum IRI concentration was determined by the double antibody method. HbA1c was measured with high pressure liquid chromatography (Cole, Soeldner, Dunn and Bunn 1978).
Analytical procedure Statistical analysis was performed with unpaired Student's t-test. Linear regression analysis was used to determine the correlation coefficients. Data are expressed as mean ± SD.
Results Study A Figure 1 shows the 30MG transport rates in the healthy subjects (n = 29) and patients with NIDDM (n = 23). The mean of transport rate was 13.3 ±3.7 fl/cell-sec in the diabetic patients which was significantly (p < 0.01) elevated than the control level (10.4 ± 2.5). In healthy subjects, neither fasting plasma glucose concentrations nor HbAlc levels was
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Horm. metab. Res. 23 (1991)
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Fig. 3 Correlation between the transport rates and HbA1c levels measured simultaneously In the diabetic patients.
related to glucose transport rates (data not shown). In diabetic patients, there was no significant correlation between fasting plasma glucose concentrations and glucose transport rates (Fig. 2), but the latter was significantly correlated with the levels of HbAlc(Fig. 3). In vitro study The glucose transport rate of normal PMNLs was not affected by short-term (60 min) treatment with 20 mM D-glucose: the mean transport rate was 12.7 + 3.7 fl/cell-sec in the absence of glucose and 12.5+4.7 fl/cell-sec in the presence of glucose, without a significant difference between the two values (n = 4). Study B The results of study B are illustrated in Figure 4. In case 1 and case 2 in which either sulfonylurea or insulin was administered, glucose transport rates gradually decreased in parallel with HbAlc levels rather than plasma glucose concentrations. On the other hand, glucose transport rates in the patient (case 3) with stable levels of glucose and HbAlc remained unchanged over the same period. Discussion We have already demonstrated that exchange of 30MG between incubation medium and the intracellular water of PMNLs occurred via facilitated diffusion with a Km of about 4 mM {Okuno et al. 1986) and that insulin did not acutely stimulate glucose transport rate (Okuno and Gliemann 1988). In this study, the mean transport rate of PMNLs was significantly higher in the NIDDM group than in the healthy control group. Although there was a considerable difference in mean age between the two groups, the possibility can be ruled out that the increased transport rate in the diabetic subjects was due to the higher age (55.4 + 7.7 vs 43.3 ±14.2 years), because it is known that glucose transport in PMNLs is not altered by variance in age (23—63 years) (Okuno and Morii 1989).
Fig. 4 Changes in fasting plasma glucose concentration ( • • ) , HbAlc level ( O — O ) and transport rate (A • ) in three diabetic patients. Case 1 (left panel) a 46-year-old man with a six-year history of diabetes mellitus. An improvement in glycemic control was obtained through diet and sulfonylurea threapy after admission. He had retinal microaneurysms but was free of diabetic neuropathy or nephropathy. Case 2 (central panel) a 56-year-old man with a five-year history of diabetes mellitus. Insulin was started immediately after hospitalization, because he complained of severe thirst and general fatigue. He had no diabetic complications. Case 3 (right panel) a 61-year-old woman with a three-year history of diabetes mellitus. Although her glycemic control has been good and stable, she admitted to our hospital because of diabetic education. She suffered from mild peripheral neuropathy.
Previous studies have shown that chemotactic factors such as C5a enhance transport of glucose in PMNLs (Okuno and Gliemann 1988). It has also been shown that glucose transport is increased in PMNLs isolated from patients with acute bacterial infections (Bibi, DeChatelet and McCall 1977). Since our patients were free of either a viral or a bacterial infection and the total and differential counts of PMNLs were normal in all individuals, "inflammation" was unlikely to be responsible for the increase in glucose transport.
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Fig. 2 Correlation between the glucose transport rates and fasting plasma glucose concentrations measured simultaneously in the diabetic patients.
Horm. metab. Res. 23 (1991) Several investigators reported that insulinstimulated glucose transport in epitrochlearis muscle or adipocytes from streptozotocin (STZ)-induced diabetic rats was lower than that in normal control animals {Wallberg-Henriksson 1986; Wallberg-Henriksson, Zetan and Henriksson 1987; Wieringa and Krans 1978; Kobayashi and Olefsky 1979). Similar results were obtained with adipocytes or muscle from patients with N I D D M (Ciaraldi et al. 1982; Kashiwagi et al. 1983; Sinha et al. 1987; Dohm et al. 1988). Consistent with these results is the finding that patients with NIDDM are characterized by a marked impairment in in vivo insulin-stimulated glucose uptake {Ginsberg, Kimmerling, Olefsky and Reaven 1975; DeFronzo, Deibert, Hendler, Felig and Soman 1979; Rizza, Mandarino and Gerich 1981; Kolterman, Gray, Griffin, Bernstein, Insel, Scarlett and Olefsky 1981; Bogardus, Lillioja, Howard, Reaven and Mott 1984; Donner, Fraze, Chen and Reaven 1985). In contrast, Baron, Kolterman, Bell, Mandarino and Olefsky (1985) demonstrated that non-insulin dependent diabetic patients have basal rates of non-insulin mediated glucose uptake that are about 2 times that of healthy subjects. However few data are available concerning the possible effect of diabetic states on the glucose transport in insulin-insensitive cells. Oka, Asano, Shibasaki, Lin, Tsukuda, Akanuma and Takaku (1990) very recently reported that in the rat liver, in which, unlike in adipocyte and muscle tissue, insulin does not acutely activate glucose transport, glucosetransporter protein and mRNA levels are increased about twofold in streptozotocin-induced rats. In this study, we also found that the transport rate of PMNLs (insulin-insensitive cells) from patients with N I D D M is significantly elevated compared with the healthy subjects. Therefore, it seemed reasonable to assume that the glucose transport rate in insulin-insensitive cells and sensitive cells may be regulated independently in diabetic states. Glucose transport in PMNLs does not seem to be affected by short-term glycemic control, first because no significant correlation was found between transport rates and ambient plasma glucose concentrations and secondly because 60 min of incubation with 20 mM glucose did not exert any appreciable effect on glucose transport. On the other hand, a positive correlation was noted between transport rates and HbAlc levels in our patients. Moreover, when glucose transport rate, H b A l c and plasma glucose concentration were assayed simultaneously during 4 weeks of follow-up, changes of glucose transport were observed to generally parallel those of HbAlc, but not glucose levels. In healthy subjects, values of glucose transport rates assayed on 4 different days separated by 1-week interval were very stable (coefficient of variation = about 7%) {Okuno and Morii 1989). Thus, these changes observed in glucose transport do not seem to be ascribed to method variation. Since the HbA1c level is a reliable index of long-term blood glucose control (Koenig, Peterson, Jones, Saudek, Lehrman and Cerami 1976), these results suggest that long-term, not short-term, derangement of glucose metabolism is associated with increased glucose transport rate in PMNLs in N I D D M . PMNLs move through bessel walls and enter the tissues only half a day after they are released from the bone marrow {Nathan 1988). Therefore, the glucose transport system of the immature cells, myeloblasts, present in the bone marrow appears to be modified by the long-term blood glucose abnormalitiy.
Y. Okuno, Y. Nishizawa and H. Morii It has been proposed that glucose may regulate the rate of its own transport, although the precise mechanisms remain to be elucidated. Van Putten and Krans (1985) and Sasson and Cerasi (1986) reported that this regulatory mechanism was present in 3T3-L1 preadipocytes and rat skeletal muscle and that basal glucose transport in these cells appeared to decrease rather than increase upon prolonged exposure to high glucose concentrations. These results are discrepant from our data that long-term blood glucose abnormality may increase glucose transport rate in PMNLs. The reason for this difference may be one or more of the following possibilities. First, they used insulin-sensitive cells and we used insulin-insensitive cells. Second, increased glucose transport in PMNLs of diabetic subjects with long-term blood glucose abnormality may be influenced by other hormonal and metabolic factors as well as glucose itself. Third, the duration of abnormality in glucose metabolism of our patients was much longer than the incubation time (24—48 hrs) used in their experiments. In conclusion, the insulin-insensitive glucose transport of PMNLs, unlike insulin-sensitive glucose transport, is increased in patients with N I D D M and long-term, not short-term, derangement of glucose metabolism seems to be associated with the increased glucose transport found in PMNLs from those patients. Acknowledgements This study was supported in part by a research grant from the Ministry of Education, Culture and Science of Japan References Baron, A. D., O. G. Kolterman, J. Bell, L. J. Mandarino, J. M. Olefsky: Rates of noninsulin-mediated glucose uptake are elevated in type II diabetic subjects. J. Clin. Invest. 76:1782-1788 (1985) Bell, G. I., T. Kayano, J. B. Buse, C. F. Burant, J. Takeda, D. Lin, H. Fukumoto, S. Seino: Molecular biology of mammalian glucose transporters. Diabetes Care 13:198-208 (1990) Bibi, S. S., L. R. DeChatelet, C. E. McCall: Human toxic neutrophils. IV. Incorporation of amino acids and uptake of 2-deoxy-D-glucose J. Infect. Dis. 135:949-951(1977) Birnbaum, M. J., H. C. Haspel, O. M. Rosen: Transformation of rat fibroblasts by FSV rapidly increases glucose transporter gene transcription. Science 235:1495-1498(1987) Bogardus, C, S. Lillioja, B. V. Howard, G. Reaven, D. Mott: Relationship between insulin secretion, insulin action and fasting plasma glucose concentration in nondiabetic and non-insulin-dependent diabetic subjects. J. Clin. Invest. 74:1238-1246 (1984) Carter-Su, C, F. W. Rozsa, X. Wang, J. R. Stubbart: Rapid and transitory stimulation of 3-O-methylglucose transport by growth hormone. Am. J. Physiol. 255: E723-729 (1988) Ciaraldi, T. P., O. G. Kolterman, J. A. Scarlett, M. Kao, J. M. Olefsky: Role of glucose transport in the postreceptor defect of non-insulindependent diabetes mellitus. Diabetes 31:1016-1022 (1982) Cole, R. A., J. S. Soeldner, P. J. Dunn, H. F. Bunn: A rapid method for determination of glycosylated hemoglobin using high pressure liquid chromatography. Metabolism 27:289-301 (1978) DeFronzo, R. A., A. D. Deibert, R. Hendler, P. Felig, V. J. Soman: Insulin sensitivity and insulin binding to monocytes in maturityonset diabetes. J. Clin. Invest. 63:936-939 (1979) Dohm, G. L., E. B. Tapscott, W. J. Pories, D. J. Dabbs, E. G. Flickinger, D. Meelheim, T. Fushiki, S. M. Atkinson, C. W. Elton, J. F. Caro: An invitro human muscle preparation suitable for metabolic studies decreased insulin stimulation of glucose transport in muscle from morbidly obese and diabetic subjects. J. Clin. Invest. 82: 486-494(1988)
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Donner, C. C , E. Fraze, Y. D. I. Chen, G. M. Reaven: Quantitation of insulin-stimulated glucose disposal in patients with non-insulindependent diabetes mellitus. Diabetes 34: 831-835 (1985) Ferrante, A., Y. H. Thong: Optimal conditions for simultaneous purification of mononuclear and polymorphonuclear leukocytes from peripheral blood by the Hypaque-Ficoll method. J. Immunol. Methods36:109-117(1980) Filetti, S., G. Damante, D. Foti: Thyrotropin stimulates glucose transport in cultured rat thyroid cells. Endocrinology 120: 2576—2581 (1987) Ginsberg, H., G. Kimmerling, J. M. Olefsky, G. M. Reaven: Demonstration of insulin resistance in untreated adult-onset diabetic subjects with fasting hyperglycemia. J. Clin. Invest. 55: 454—461 (1975) Hatanaka, M.: Transport of sugars in tumor cell membranes. Biochim. Biophys. Acta 355:77-104 (1974) Kasahara, M., P. C. Hinkle, Y. Ikawa, H. Amanuma: Decrease in glucose transport activity of Friend erythroleukemia cell caused by dimethylsulfoxide, a differentiation-inducing reagent. Biochim. Biophys. Acta 856:615-623 (1986) Kashiwagi, A., M. A. Verso, J. Andrews, B. Vasquez, G. Reaven, J. E. Foley: In vitro insulin resistance of human adipocytes isolated from subjects with non-insulin-dependent diabetes mellitus. J. Clin. Invest. 72:1246-1254 (1983) Kitagawa, K., H. Nishino, A. Iwashima: Analysis of hexose transport in untransformed and sarcoma virus-transformed mouse 3T3 cells by photoaffinity binding of cytochalasin B. Biochim. Biophys. Acta821:63-66(1985) Kobayashi, M., J. M. Olefsky: Effects of streptozotocin-induced diabetes on insulin binding, glucose transport and intracellular glucose metabolism in isolated rat adipocytes. Diabetes 28: 87—95 (1979) Koenig, R. J., C. M. Peterson, R. L, Jones, C. Saudek, M. Lehrman, A. Cerami: Correlation of glucose regulation and hemoglobin A l e in diabetes mellitus. N. Eng. J. Med. 295:417-420 (1976) Kolterman, O, G., R. S. Gray, J. Griffin, P. Bernstein, J. Insel, J. A. Scarlett, J. M. Olefsky: Receptor and post receptor defects contribute to the insulin resistance in non-insulin-dependent diabetes mellitus. J. Clin. Invest. 68:957-969 (1981) Nathan, D. G.: Hematologic diseases. In: Wyngaarden, J. B., L. H. Smith (eds): Cecil Textbook of Medicine, 18th. W. B. Saunders Com., Philadelphia London Toronto Tokyo (1988), pp. 873 - 1 0 8 1 National Diabetes Data Group: Classification and diagnosis of diabetes mellitus and other categories of glucose intolerance. Diabetes 28:1039-1057(1979) Oka, Y., T. Asano, Y. Shibasaki, J. L. Lin, K. Tsukuda, Y. Akanuma, F. Takaku: Increased liver glucose-transporter protein and mRNA in streptozotocin-induced diabetic rats. Diabetes 39:441—446 (1990)
Horm. metab. Res. 23 (1991) Okuno, Y, L. Plesner, T. R. Larsen, J. Gliemann: Facilitated transport of 3-O-methyl-D-glucose in human polymorphonuclear leukocytes. FEBS Lett. 195:303-308(1986) Okuno, Y., J. Gliemann: Enhancement of glucose transport by insulin at 37 °C in rat adipocytes is accounted for by increased Vmax. Diabetologia 30:426-430 (1987) Okuno, Y, J. Gliemann: Effect of chemotactic factors on hexose transport in polymorphonuclear leukocytes. Biochim. Biophys. Acta 941:157-164(1988) Okuno, Y, H. Morii: Clinical application of measurement of glucose transport in human polymorphonuclear leukocytes. Diabetes Res. Clin. Pract. (Supplement 1 ) 7 : 5 - 9 (1989) Rizza, R. A., L. J. Mandarino, J. E. Gerich: Mechanism and significance of insulin resistance in non-insulin-dependent diabetes mellitus. Diabetes 30:990-995(1981) Sasson, S., E. Cerasi: Substrate regulation of the glucose transport system in rat skeletal muscle. J. Biol. Chem. 261:16827-16833 (1986) Sinha, M. K, L. G. Taylor, W. J. Pories, E. G. Flickinger, D. Meelheim, S. Atkinson, N. S. Sehgal, J. F. Cam: Long-term effect of insulin on glucose transport and insulin binding in cultured adipocytes from normal and obese humans with and without non-insulin-dependent diabetes. J. Clin. Invest. 80: 1073-1081 (1987) van Putten, J. P. M., H. M. J. Krans: Glucose as a regulator of insulinsensitive hexose uptake in 3T3 adipocytes. J. Biol. Chem. 260: 7996-8001(1985) Wallberg-Henriksson, H.: Repeated exercise regulates glucose transport capacity in skeletal muscle. Acta Physiol. Scand. 127: 39—43 (1986) Wallberg-Henriksson, H., N. Zetan, J. Henriksson: Reversibility of decreased insulin-stimulated glucose transport capacity in diabetic muscle with in vitro incubation. Insulin is not required. J. Biol. Chem. 262:7665-7671 (1987) Wieringa, T., H. M. J. Krans: Reduced glucose transport and increased binding of insulin in adipocytes from diabetic and fasted rats. Biochim. Biophys. Acta 538:563-570 (1978)
Requests for reprints should be addressed to: Yasuhisa Okuno, M. D. Second Department of Internal Medicine Osaka City University Medical School 1-5-7, Asahi-machi, Abeno-ku Osaka 545 (Japan)
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Glucose Transport in Polymorphonuclear Leukocytes
