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Plasma and testicular testosterone in experimental diabetic rats
Diabetes Research and Clinieal Practice, 3 (1987) 81-84 Elsevier
81
DRC 00108
Plasma and testicular testosterone in experimental diabetic rats Hisako Fushimi ~, Toru Inoue 1, Akihito Ohtsuka ~, Keisuke K a n a o 2, Shizumori Ishihara 2, Takahiro Tsujimura 3, H a r u o N u n o t a n i 3, Takeshi Minami 4 and Y u k o Okazaki 4 ~Department of Medichle, ZDepartment of Radioisotope, and 3Department of Pathology, Sumitomo Hospital, and 4Department of Pharmacology, Kinki University, Osaka, Japan (Received 30 May 1986, revised received 31 August 1986, accepted 31 August 1986)
Key words: Streptozotocin diabetic rats; Testosterone; (Plasma); Testicular testosterone; LH; Prolactin
Summary Testicular and plasma testosterone levels were found to be decreased markedly in streptozotocin diabetic rats compared with those of controls. Treatment with 6 units NPH insulin daily for one week almost normalized plasma testosterone levels parallel to the increase in body and liver weights in diabetic rats, while testosterone levels in testicles were not significantly changed. Plasma prolactin and LH levels were unchanged among control, diabetic and diabetic insulin-treated rats. Thus, testosterone reduction in the testis might play a role in diabetic impotence.
Introduction Although the existence of impotence in diabetics has been widely known for years [1,2], its etiology is unclear. It has been suggested to be secondary to diabetic neuropathy [3,4] or diabetic vascular disturbances [5], psychologic factors [6] or endocrine disorders, but this has not been verified. Among endocrine disorders, plasma gonadotropin and testosterone have been studied in diabetics [1,2,7]. However, because some studies reported low urinary 17-ketosteroid excretion [1] and low levels of plasma testosterone [7], but others reported normal
Address for correspondence: Dr. H. Fushimi, Department of Medicine, Sumitomo Hospital, 5-2-2, Nakanoshima, Kita-ku, Osaka, 530, Japan.
plasma testosterone levels [3,8,9], normal gonadotropin levels [10] or normal gonadotropin response [11,12], the role of endocrine disorders in diabetic importence is now rarely considered. On the other hand, researchers have recently reported decreased fertility with reduced serum testosterone levels and testicular morphoiogic changes in B B rats [13,14], and also decreased steroid biosynthesis in isolated Leydig cells from testes of streptozotocin (stz) diabetic rats [15,16]. Moreover, Berchtold et al. [7] reported low plasma testosterone levels in juvenile diabetes (mean age: 28 + 2 years), while others studied older age groups. Younger groups may show more sensitive reflective changes in hormonal levels in the diabetic stage. We therefore studied young diabetic rats and in this paper describe the marked decrease in testicular and plasma testosterone levels in stz diabetic rats.
0168-8227/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
82 TABLE I BLOOD G L U C O S E LEVELS, BODY A N D O R G A N WEIGHTS O F CONTROL, DIABETIC A N D DIABETIC INSULINT R E A T E D RATS No. of animals: 6 per group. Weights of testis and kidney were for a pair. Values are mean 4- SE. Insulin treatment with 6 U of NPH insulin subcutaneously, once daily, was commenced 7 days prior to, and continued until, sacrifice. FBG
Body weight (g) 7 days prior
Testis (g)
Liver (g)
Sacrifice
ont o, Diabetic
Kidney (g)
1 540 4- 19
a
195 4- 7
189 -k 9
2.67 4- 0.10
9.75 4-
2.51 4- 0.17
2.90 4- 0.08
14.97 4-
2.45 4- 0.07
b Diabetic insulin-treated
478 4-
192 4- 10
222 4- 7
a p < 0.001; b p < 0.0I.
Materials and methods
concentration of 1 mM, cooled in ice and centrifuged at 1000 × g for 30 min to separate plasma. Plasma was frozen at - 8 0 ° C until use. Testicles were removed immediately. Blood was rinsed off, and the testicles were frozen on dry ice and kept at - 8 0 ° C until use. The testicles were homogenized by a Polytron homogenizer with 5 mM Tris buffer Ph 7.4 containing 1 m M E G T A , and the homogenates were centrifuged at 10,000 x g for 20 min to obtain supernatants for testosterone analysis. Testosterone
Experimental diabetes mellitus in Wistar male rats was induced by intravenous injection of stz (80 mg/kg body weight) via the tail vein at 7 weeks of age. The rats were kept on water and standard laboratory chow ad libitum for 6 weeks, with agematched normal rats employed as controls. The rats were then exsanguinated. The following experiments were carried out at 4°C (unless otherwise mentioned). Blood was mixed with E G T A at a final
TABLE 2 PLASMA A N D T E S T I C U L A R TESTOSTERONE C O N C E N T R A T I O N S IN CONTROL, DIABETIC A N D DIABETIC INSULIN-TREATED RATS No. of animals: 6 per group. Values are mean 4- SE (cf. Table I). Testosterone Plasma (ng/dl)
on rol Diabetic
LH Testis (,ug/100 g)
FSH
Plasma (ng/ml)
Prolactin Plasma (ng/ml)
940 '49 /a 91 4- 7
2.44 4- 0 . 2 4 )
Ha
3.81 + 0.05
0.1~
4.15 4- 0.20
4.05 4- 0.11
0.11,
4.53 4- 0.08
! Diabetic insulin-treated a p < 0.001; b p < 0.01.
223 + 40
2.35 + 0.27
J
83 concentrations in plasma and testicular supernatant were determined using the testosterone RIA kit Eiken. Plasma FSH and LH were also determined using the hFSH and hLSH RIA kit Eiken. Plasma prolactin was also measured by the prolactin RIA bead kit Dainabot. Blood glucose was measured using the glucose oxidase method. Values were shown as mean + SEM. The histology of a portion of each specimen was studied light-microscopically.
Results
As shown in Table 1, body and testicular weight were reduced significantly in diabetic rats, along with liver weight. Insulin treatment for 7 days increased body weight as well as liver weight significantly. Plasma and testicular testosterone concentrations were decreased significantly, but subsequent insulin treatment increased plasma testosterone levels significantly, while the testicular level remained unchanged (Table 2). Plasma LH and prolactin levels did not differ significantly among the three groups. Plasma FSH was not detectable with our method. Light-microscopic study did not detect any histologic changes.
mones (FSH and prolactin) were well detected. Hormone levels or their responses to synthetic LHRH are reported to be influenced by aging [17-19]. Therefore, the absence of an increase in our diabetic or insulin-treated diabetic rats may be due to the young age of our rats. Hyperprolactinemia also usually accompanies a low plasma testosterone level [20]. A longer period of hypotestosteronemia might be necessary to increase the prolactin concentration. In this study, insulin treatment increased plasma testosterone levels but the testicular concentration was unchanged, partially because control of blood glucose was insufficient. Further study is necessary on this point. Low plasma testosterone levels in spontaneously diabetic BB rats with reduced fertility [13], and also low plasma testosterone and reduced steroid synthesis in Leydig cells from testes of stz diabetic rats [15] with improvement by insulin treatment [16] have been reported previously. These data and our finding of a marked decrease in diabetic testicular testosterone concentration and plasma testosterone levels in young rats suggest that the possibility of a relation between endocrine testicular dysfunction and diabetic importence is not to be neglected. Further investigation is necessary as to whether this marked decrease takes place only during the young age period and is compensated later or persists throughout diabetic life.
Discussion
Insulin treatment with 6 units once-daily was found to be insufficient to reduce plasma levels significantly; however, body and liver weights were suitably increased by the treatment, and the plasma testosterone level was almost normalized, although the testicular testosterone concentration was not increased. The doses of insulin given might have not been sufficient. Insulin treatment with larger doses is currently being conducted in our laboratory. Testosterone, FSH, LH and prolactin were all determined by RIA methods using human hormone antibody and this may be one of the reasons why FSH levels were undetectable in rat plasma. In the diabetic rat group, LH was not elevated in spite of the low plasma level of testosterone. The other hor-
References 1 Sch6flting, K., Federlin, K., Ditschuneit, H. and Pfeiffer, E.F., Disorders of sexual function in male diabetics, Diabetes, 12: 519-527, 1963. 2 Robin, A. and Babbott, D., Impotence and diabetes mellitus, J. Am. Med. Assoc., 168: 498-500, 1958. 3 Kolodny, R.C., Kahn, C.B., Goldstein, H.H. and Barnett, D.M., Sexual dysfunction in diabetic men, Diabetes, 23: 306--309, 1974. 4 Melman, A., Henry, D.P., Felter, D.L. and O'Connor, B., Effect of diabetes upon penile sympathetic nerves in impotent patients, South. Med. J., 73: 307-309, 1980. 5 Jevtich, M.J., Edson, M., Jarman, W.D. and Herrera, H.H., Vascular factor in erectile failure among diabetics, Urology, 19: 163-168, 1982. 6 Hosking, D.J., Bennet, T., Hampton, J.R., Evans, D.F., Clark. A.J. and Robertson, G., Diabetic impotence: studies
84
7
8
9
10
11
12
13
of nocturnal erection during REM sleep, Br. Med. J., 2: 1394-1396, 1979. Berchtold, P., Berger, M., Cuppers, H.J. et al., Non-glucoregulatory hormones (T4, T3, yT3, TSH, testosterone) during physical exercise in juvenile type diabetes, Horm. Metab. Res., lfl: 269-297, 1978. Schiavi, R.C. and White, D., Androgens and male sexual function: a review of human studies, J. Sex Marital Tber., 2: 214-228, 1976. Faerman, I., Vilar, O., Rivarola, M.A. et al., Impotence and diabetes: studies of androgenic function in diabetic impotent males, Diabetes, 21: 23-30, 1972. Jensen, S.B., Hagen, C., Froland, A. and Pedersen, P.B., Sexual function and pituitary axis in insulin treated diabetic men, Acta Med. Scand., (Suppl.) 624: 65-68, 1979. Rastogi, G.K., Chakraborti, J. and Sinha, M.K., Serum gonadotropins (LH and FSH) and their response to synthetic LHRH in diabetic men with and without impotence, Horm. Metab. Res., 6: 335-336, 1974. Wright, A.D., London, D.R., Holder, G., Williams, J.W. and Rudd, B.F., Luteinizing release hormone tests in impotent diabetic males, Diabetes, 25: 975-977, 1976. Murray, F.T., Caomeron, D.F. and Orth, J.M., Gonadal dysfunction in the spontaneously diabetic BB rat, Metabolism, 32: 141-147, 1983.
14 Murray, F.T., Orth, J.M., Gunsalus, G. et al., The pituitary-testicular axis in the streptozotocin diabetic male rat: evidence for gonadotroph, Seritoli cell and Leydig cell dysfunction, Int. J. Androl., 4: 265-280, 1981. 15 Kfihn-Velten, N., Waldenburger, D. and Sta~b, W., Evaluation of steroid biosynthetic lesions in isolated Leydig cells from the testes of streptozotocin-diabetic rats, Diabetologia, 23: 529-533, 1982. 16 Calvo, J.C., Biella de Souza Valle, L., Barafiao, J.L., Tesone, M. and Charreau, E.H., NADPH generating enzymes in Leydig cells from diabetic rats, Horm. Metab. Res., 11: 161-164, 1979. 17 Isurigi, K., Fukutani, K., Takayasu, H., Wakabayashi, K. and Tamaoki, B., Age-related changes in serum luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels in normal men, J. Clin. Endocrinol. Metab., 39: 955957, 1974. 18 Johnson, L., Petty, C.S. and Neaves, W,B., Influence of age on sperm production in old age, J. Clin. Endocrinol. Metab., 39: 40-46, 1974. 19 Vermeulen, A., Rubens, R. and Verdonck, L., Testosterone secretion and metabolism in male senescence, J. Clin. Endoerinol., 34: 730-735, 1972. 20 Stegmayr, B. and Skogstr6m, K., Hyperprolactinaemia and testosterone production, Horm, Res., 21: 224-228, 1985.
81
DRC 00108
Plasma and testicular testosterone in experimental diabetic rats Hisako Fushimi ~, Toru Inoue 1, Akihito Ohtsuka ~, Keisuke K a n a o 2, Shizumori Ishihara 2, Takahiro Tsujimura 3, H a r u o N u n o t a n i 3, Takeshi Minami 4 and Y u k o Okazaki 4 ~Department of Medichle, ZDepartment of Radioisotope, and 3Department of Pathology, Sumitomo Hospital, and 4Department of Pharmacology, Kinki University, Osaka, Japan (Received 30 May 1986, revised received 31 August 1986, accepted 31 August 1986)
Key words: Streptozotocin diabetic rats; Testosterone; (Plasma); Testicular testosterone; LH; Prolactin
Summary Testicular and plasma testosterone levels were found to be decreased markedly in streptozotocin diabetic rats compared with those of controls. Treatment with 6 units NPH insulin daily for one week almost normalized plasma testosterone levels parallel to the increase in body and liver weights in diabetic rats, while testosterone levels in testicles were not significantly changed. Plasma prolactin and LH levels were unchanged among control, diabetic and diabetic insulin-treated rats. Thus, testosterone reduction in the testis might play a role in diabetic impotence.
Introduction Although the existence of impotence in diabetics has been widely known for years [1,2], its etiology is unclear. It has been suggested to be secondary to diabetic neuropathy [3,4] or diabetic vascular disturbances [5], psychologic factors [6] or endocrine disorders, but this has not been verified. Among endocrine disorders, plasma gonadotropin and testosterone have been studied in diabetics [1,2,7]. However, because some studies reported low urinary 17-ketosteroid excretion [1] and low levels of plasma testosterone [7], but others reported normal
Address for correspondence: Dr. H. Fushimi, Department of Medicine, Sumitomo Hospital, 5-2-2, Nakanoshima, Kita-ku, Osaka, 530, Japan.
plasma testosterone levels [3,8,9], normal gonadotropin levels [10] or normal gonadotropin response [11,12], the role of endocrine disorders in diabetic importence is now rarely considered. On the other hand, researchers have recently reported decreased fertility with reduced serum testosterone levels and testicular morphoiogic changes in B B rats [13,14], and also decreased steroid biosynthesis in isolated Leydig cells from testes of streptozotocin (stz) diabetic rats [15,16]. Moreover, Berchtold et al. [7] reported low plasma testosterone levels in juvenile diabetes (mean age: 28 + 2 years), while others studied older age groups. Younger groups may show more sensitive reflective changes in hormonal levels in the diabetic stage. We therefore studied young diabetic rats and in this paper describe the marked decrease in testicular and plasma testosterone levels in stz diabetic rats.
0168-8227/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
82 TABLE I BLOOD G L U C O S E LEVELS, BODY A N D O R G A N WEIGHTS O F CONTROL, DIABETIC A N D DIABETIC INSULINT R E A T E D RATS No. of animals: 6 per group. Weights of testis and kidney were for a pair. Values are mean 4- SE. Insulin treatment with 6 U of NPH insulin subcutaneously, once daily, was commenced 7 days prior to, and continued until, sacrifice. FBG
Body weight (g) 7 days prior
Testis (g)
Liver (g)
Sacrifice
ont o, Diabetic
Kidney (g)
1 540 4- 19
a
195 4- 7
189 -k 9
2.67 4- 0.10
9.75 4-
2.51 4- 0.17
2.90 4- 0.08
14.97 4-
2.45 4- 0.07
b Diabetic insulin-treated
478 4-
192 4- 10
222 4- 7
a p < 0.001; b p < 0.0I.
Materials and methods
concentration of 1 mM, cooled in ice and centrifuged at 1000 × g for 30 min to separate plasma. Plasma was frozen at - 8 0 ° C until use. Testicles were removed immediately. Blood was rinsed off, and the testicles were frozen on dry ice and kept at - 8 0 ° C until use. The testicles were homogenized by a Polytron homogenizer with 5 mM Tris buffer Ph 7.4 containing 1 m M E G T A , and the homogenates were centrifuged at 10,000 x g for 20 min to obtain supernatants for testosterone analysis. Testosterone
Experimental diabetes mellitus in Wistar male rats was induced by intravenous injection of stz (80 mg/kg body weight) via the tail vein at 7 weeks of age. The rats were kept on water and standard laboratory chow ad libitum for 6 weeks, with agematched normal rats employed as controls. The rats were then exsanguinated. The following experiments were carried out at 4°C (unless otherwise mentioned). Blood was mixed with E G T A at a final
TABLE 2 PLASMA A N D T E S T I C U L A R TESTOSTERONE C O N C E N T R A T I O N S IN CONTROL, DIABETIC A N D DIABETIC INSULIN-TREATED RATS No. of animals: 6 per group. Values are mean 4- SE (cf. Table I). Testosterone Plasma (ng/dl)
on rol Diabetic
LH Testis (,ug/100 g)
FSH
Plasma (ng/ml)
Prolactin Plasma (ng/ml)
940 '49 /a 91 4- 7
2.44 4- 0 . 2 4 )
Ha
3.81 + 0.05
0.1~
4.15 4- 0.20
4.05 4- 0.11
0.11,
4.53 4- 0.08
! Diabetic insulin-treated a p < 0.001; b p < 0.01.
223 + 40
2.35 + 0.27
J
83 concentrations in plasma and testicular supernatant were determined using the testosterone RIA kit Eiken. Plasma FSH and LH were also determined using the hFSH and hLSH RIA kit Eiken. Plasma prolactin was also measured by the prolactin RIA bead kit Dainabot. Blood glucose was measured using the glucose oxidase method. Values were shown as mean + SEM. The histology of a portion of each specimen was studied light-microscopically.
Results
As shown in Table 1, body and testicular weight were reduced significantly in diabetic rats, along with liver weight. Insulin treatment for 7 days increased body weight as well as liver weight significantly. Plasma and testicular testosterone concentrations were decreased significantly, but subsequent insulin treatment increased plasma testosterone levels significantly, while the testicular level remained unchanged (Table 2). Plasma LH and prolactin levels did not differ significantly among the three groups. Plasma FSH was not detectable with our method. Light-microscopic study did not detect any histologic changes.
mones (FSH and prolactin) were well detected. Hormone levels or their responses to synthetic LHRH are reported to be influenced by aging [17-19]. Therefore, the absence of an increase in our diabetic or insulin-treated diabetic rats may be due to the young age of our rats. Hyperprolactinemia also usually accompanies a low plasma testosterone level [20]. A longer period of hypotestosteronemia might be necessary to increase the prolactin concentration. In this study, insulin treatment increased plasma testosterone levels but the testicular concentration was unchanged, partially because control of blood glucose was insufficient. Further study is necessary on this point. Low plasma testosterone levels in spontaneously diabetic BB rats with reduced fertility [13], and also low plasma testosterone and reduced steroid synthesis in Leydig cells from testes of stz diabetic rats [15] with improvement by insulin treatment [16] have been reported previously. These data and our finding of a marked decrease in diabetic testicular testosterone concentration and plasma testosterone levels in young rats suggest that the possibility of a relation between endocrine testicular dysfunction and diabetic importence is not to be neglected. Further investigation is necessary as to whether this marked decrease takes place only during the young age period and is compensated later or persists throughout diabetic life.
Discussion
Insulin treatment with 6 units once-daily was found to be insufficient to reduce plasma levels significantly; however, body and liver weights were suitably increased by the treatment, and the plasma testosterone level was almost normalized, although the testicular testosterone concentration was not increased. The doses of insulin given might have not been sufficient. Insulin treatment with larger doses is currently being conducted in our laboratory. Testosterone, FSH, LH and prolactin were all determined by RIA methods using human hormone antibody and this may be one of the reasons why FSH levels were undetectable in rat plasma. In the diabetic rat group, LH was not elevated in spite of the low plasma level of testosterone. The other hor-
References 1 Sch6flting, K., Federlin, K., Ditschuneit, H. and Pfeiffer, E.F., Disorders of sexual function in male diabetics, Diabetes, 12: 519-527, 1963. 2 Robin, A. and Babbott, D., Impotence and diabetes mellitus, J. Am. Med. Assoc., 168: 498-500, 1958. 3 Kolodny, R.C., Kahn, C.B., Goldstein, H.H. and Barnett, D.M., Sexual dysfunction in diabetic men, Diabetes, 23: 306--309, 1974. 4 Melman, A., Henry, D.P., Felter, D.L. and O'Connor, B., Effect of diabetes upon penile sympathetic nerves in impotent patients, South. Med. J., 73: 307-309, 1980. 5 Jevtich, M.J., Edson, M., Jarman, W.D. and Herrera, H.H., Vascular factor in erectile failure among diabetics, Urology, 19: 163-168, 1982. 6 Hosking, D.J., Bennet, T., Hampton, J.R., Evans, D.F., Clark. A.J. and Robertson, G., Diabetic impotence: studies
84
7
8
9
10
11
12
13
of nocturnal erection during REM sleep, Br. Med. J., 2: 1394-1396, 1979. Berchtold, P., Berger, M., Cuppers, H.J. et al., Non-glucoregulatory hormones (T4, T3, yT3, TSH, testosterone) during physical exercise in juvenile type diabetes, Horm. Metab. Res., lfl: 269-297, 1978. Schiavi, R.C. and White, D., Androgens and male sexual function: a review of human studies, J. Sex Marital Tber., 2: 214-228, 1976. Faerman, I., Vilar, O., Rivarola, M.A. et al., Impotence and diabetes: studies of androgenic function in diabetic impotent males, Diabetes, 21: 23-30, 1972. Jensen, S.B., Hagen, C., Froland, A. and Pedersen, P.B., Sexual function and pituitary axis in insulin treated diabetic men, Acta Med. Scand., (Suppl.) 624: 65-68, 1979. Rastogi, G.K., Chakraborti, J. and Sinha, M.K., Serum gonadotropins (LH and FSH) and their response to synthetic LHRH in diabetic men with and without impotence, Horm. Metab. Res., 6: 335-336, 1974. Wright, A.D., London, D.R., Holder, G., Williams, J.W. and Rudd, B.F., Luteinizing release hormone tests in impotent diabetic males, Diabetes, 25: 975-977, 1976. Murray, F.T., Caomeron, D.F. and Orth, J.M., Gonadal dysfunction in the spontaneously diabetic BB rat, Metabolism, 32: 141-147, 1983.
14 Murray, F.T., Orth, J.M., Gunsalus, G. et al., The pituitary-testicular axis in the streptozotocin diabetic male rat: evidence for gonadotroph, Seritoli cell and Leydig cell dysfunction, Int. J. Androl., 4: 265-280, 1981. 15 Kfihn-Velten, N., Waldenburger, D. and Sta~b, W., Evaluation of steroid biosynthetic lesions in isolated Leydig cells from the testes of streptozotocin-diabetic rats, Diabetologia, 23: 529-533, 1982. 16 Calvo, J.C., Biella de Souza Valle, L., Barafiao, J.L., Tesone, M. and Charreau, E.H., NADPH generating enzymes in Leydig cells from diabetic rats, Horm. Metab. Res., 11: 161-164, 1979. 17 Isurigi, K., Fukutani, K., Takayasu, H., Wakabayashi, K. and Tamaoki, B., Age-related changes in serum luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels in normal men, J. Clin. Endocrinol. Metab., 39: 955957, 1974. 18 Johnson, L., Petty, C.S. and Neaves, W,B., Influence of age on sperm production in old age, J. Clin. Endocrinol. Metab., 39: 40-46, 1974. 19 Vermeulen, A., Rubens, R. and Verdonck, L., Testosterone secretion and metabolism in male senescence, J. Clin. Endoerinol., 34: 730-735, 1972. 20 Stegmayr, B. and Skogstr6m, K., Hyperprolactinaemia and testosterone production, Horm, Res., 21: 224-228, 1985.