**2.2.2 Results**

112 Steroids – Basic Science

The guinea pig utilizes a similar mechanism of adrenal steroidogenesis to that of humans. In a guinea pig model in which impaired glucose tolerance is induced by streptozotocin (STZ) treatment, we measured serum levels of DHEA, DHEA-S and c-peptide to determine if these

All experiments were performed using Hartley male guinea pigs with a body weight of 500- 600 g. Experimental protocols followed the Principals of Laboratory Animal Care and were approved by the Ethics Committee of Toho University School of Medicine. Until experiments began, guinea pigs were housed in groups of three in metabolism cages in a temperature-controlled room with a 12h light/dark cycle. They had free access to tap water

Under intra-abdominal anaesthesia (pentobarbital sodium 30mg/Kg), streptozotocin (STZ) was administrated to 12 guinea pigs intra-abdominally. After 4 weeks, a glucose tolerance test (50% glucose, 1g/Kg, intra-abdominal route) was performed. Impaired glucose tolerance (IGT) was defined as a blood glucose level of more than 300 mg/dl after 3 hrs. Six

**[Control]**

0 4 8 12

**[IGT]**

0 4 8 12

(W)

(W)

**2.2 Basic study**

were related to serum glucose levels.

control guinea pigs had intra-abdominal saline only.

3.5

3.0

DHEA

2.5

0

3.5

3.0

DHEA

2.5

0

Fig. 2. Changes in Concentrations of Serum DHEA

4.0

(ng/mL)

4.0

(ng/mL)

**2.2.1 Materials and methods**

and guinea pig chow.

Concentrations of serum DHEA showed no significant change during observation in the control group, however there was a tendency towards decrease in the IGT group (Fig. 2). Concentrations of serum DHEA-S also had no significant change in the control group. However, in the IGT group, concentrations of serum DHEA-S decreased significantly from 39.0±4.2 μg/dl (before STZ administration) to 27.5±5.0 μg/dl (after 8 weeks)(p<0.05)(Fig. 3).

Fig. 3. Changes in Concentrations of Serum DHEA-S

DHEA and Impaired Glucose Tolerance Clinical and Basic Study 115

Serum c-peptide levels showed no significant change during observation in the control group. However in the IGT group, these levels decreased significantly from 1.280±0.144 ng/ml (before) to 0.965±0.272 ng/ml (after 12 weeks)(Fig. 5 ). Serum c-peptide levels after DHEA-S administration were not significantly different between guinea pigs with DHEA-S and those without DHEA-S in both the control group and the IGT group. C-peptide levels continued to be significantly lower in the IGT group than in the control group (P<0.05) (Fig. 6).

**[Control]**

0 4 8 12

**[IGT]**

0 4 8 12

Coleman et al.(1982). first reported that DHEA had an effect on lowering blood glucose in animal experiments. Since this report, there have been many reports that DHEA and DHEA-S are related to insulin or blood glucose levels. However, their exact role has not been determined (Gansler et al.,1985; Farah et al.,1992; Barrett-Connor,1992; Yamaguchi et al.,1998). Some of these reports described the use of rats and mice in animal experiments, but few studies used guinea pigs which have a similar mechanism of adrenal steroidogenesis to that of humans (Strott et al.,1981; Hyatt et al.,1983) In our guinea pig models in which impaired glucose tolerance is induced by STZ treatment , serum levels of DHEA and DHEA-S were decreased. We measured serum c-peptide instead of serum

\* \* \*

(W)

(W)

Control DHEA-S + Control DHEA-S -

IGT DHEA-S + IGT DHEA-S - \* : p<0.05 vs control

1.75 2.00

(ng/mL)

1.50 1.25

0.75 1.00

0.50 0.25

1.75 2.00

(ng/mL)

1.50 1.25

0.75 1.00

0.50 0.25

CPR

**2.2.3 Discussion**

0

Fig. 6. Changes in serum c-peptide after DHEA-S administration

CPR

0

Blood glucose levels three hours after DHEA-S administration showed no significant change between guinea pigs with DHEA-S and those without DHEA-S in the control group. In the IGT group, three hour blood glucose levels had improved from 333.7±24.5 mg/dl (before) to 190.7±89.8 mg/dl (after 4 weeks) (Fig. 4). However FPG showed no significant change between the control group and the IGT group. The result was similar after DHEA-S administration.

Fig. 4. Changes in 3 hour blood glucose level

Fig. 5. Changes in serum C-peptide after STZ administration

Blood glucose levels three hours after DHEA-S administration showed no significant change between guinea pigs with DHEA-S and those without DHEA-S in the control group. In the IGT group, three hour blood glucose levels had improved from 333.7±24.5 mg/dl (before) to 190.7±89.8 mg/dl (after 4 weeks) (Fig. 4). However FPG showed no significant change between the control group and the IGT group. The result was similar after DHEA-S administration.

\*

Control DHEA-S+ Control DHEA-S-IGT DHEA-S+ IGT DHEA-S-

\* : p<0.05 vs 0W

Control IGT \* : p<0.05 vs 0W

0 4 8 12

0 4 8 12

Fig. 5. Changes in serum C-peptide after STZ administration

\*

(W)

(W)

\*

\*

450 (mg/dL)

400

350

300

250

180min plasma glucose

200

150

100

50

0

2.00 (ng/mL)

1.75

1.50

1.25

1.00

CPR

0.75

0.50

0.25

0

Fig. 4. Changes in 3 hour blood glucose level

Serum c-peptide levels showed no significant change during observation in the control group. However in the IGT group, these levels decreased significantly from 1.280±0.144 ng/ml (before) to 0.965±0.272 ng/ml (after 12 weeks)(Fig. 5 ). Serum c-peptide levels after DHEA-S administration were not significantly different between guinea pigs with DHEA-S and those without DHEA-S in both the control group and the IGT group. C-peptide levels continued to be significantly lower in the IGT group than in the control group (P<0.05) (Fig. 6).

Fig. 6. Changes in serum c-peptide after DHEA-S administration

## **2.2.3 Discussion**

Coleman et al.(1982). first reported that DHEA had an effect on lowering blood glucose in animal experiments. Since this report, there have been many reports that DHEA and DHEA-S are related to insulin or blood glucose levels. However, their exact role has not been determined (Gansler et al.,1985; Farah et al.,1992; Barrett-Connor,1992; Yamaguchi et al.,1998). Some of these reports described the use of rats and mice in animal experiments, but few studies used guinea pigs which have a similar mechanism of adrenal steroidogenesis to that of humans (Strott et al.,1981; Hyatt et al.,1983) In our guinea pig models in which impaired glucose tolerance is induced by STZ treatment , serum levels of DHEA and DHEA-S were decreased. We measured serum c-peptide instead of serum

DHEA and Impaired Glucose Tolerance Clinical and Basic Study 117

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Gracia-Webb, P. Bottomly, S. Bonser, AM. (1983). Instability of C-peptide reactivity in plasma and serum stored at -20℃. *Clinica Chimica Acta*, 129, 103-106. Hashimoto, K. Nishioka, T. Takao, T. Numata, Y. (1993). Low plasma corticotropin-releasing

Ho RS et al.(1988). In-vIvo and in-vitro glucose metabolism in a low-dose streptozotocin rat

Hubert, GD. Schriock, ED. Givens, JR. Buster, JE. (1991). Supression of

Massey, DE. Smyth, DG. (1975).  *Journal of Biological Chemistry,* 250,

McIntosh, MK. Berdanier, CD. (1991). Antiobesity effects of dehydroepiandrosterone are

Nakashima, N. Haji, M. Sakai, Y et al. (1995). Effect of dehydroepiandrosterone on glucose

Nesler, JE. Usiskin, KS. Barlascini, CO. Welty, DF. Clore, JN. Blackard, WG. (1989).

evaluation of possible mechanisms. *J Clin Endocrinol Metab,* 69 , 1040-1046. Nestler, JE. McClanahan, MA. Clore, JN. Blackard, WG. (1992). Insulin inhibits adrenal 17,

Nestler, JE. Beer, NA. Jakubowicz, DJ. Beer, RM. (1994). Effects of a reduction in circulating

Nestler, JE. (1995). Regulation of human dehydroepiandrosterone metabolism by insulin.

Parker, LN. Levin, ER. Lifrak, ET. (1985). Evidence for adaptation to severe illness. *Journal of*

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*Clinical Endocrinology and Metabolism,* 60, 947-952.

*Ann N Y Acad Sci,* 774, 73-81.

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of guinea pigs differs markedly from the insulin in their pancreas and plasma.

insulin because there were no reports of serum insulin measurements in guinea pigs (Massey&Smyth,1975; Rosenzweig et al.,1980; Gracia-Webb et al.,1983; Schlosser et al.,1987). Guinea pigs in the IGT group showed a significant decrease in serum c-peptide levels and it was speculated that this was not hyper-insulinemia. In IGT group guinea pigs, blood glucose levels improved after DHEA-S administration, however serum c-peptide levels were still significantly decreased. There was no correlation between serum c-peptide levels and DHEA or DHEA-S levels. In the STZ-induced model of diabetes, adult rats ranged from mild type 2 diabetes to type 1 diabetes depending upon STZ dose (Ho RS et al.,1988). In this experiment, fasting blood glucose levels in STZ-administered guinea pigs were not significantly different from those in control group. However, serum c-peptide levels were decreased and this state was thought to be approaching type 1 diabetes.

Similar to clinical data, it was thought that hyperglycemia itself suppressed DHEA and DHEA-S after prolonged hyperglycemia independent of serum insulin levels in the absence of hyperinsulinemia. In IGT group guinea pigs, serum c-peptide was still decreased after DHEA-S administration, however blood glucose levels improved significantly. It was thought that DHEA-S itself was involved in this improvement of blood glucose levels. In the hyperglycemic state in humans, the mechanism of decrease of DHEA and DHEA-S levels is not still clear. It has been reported that DHEA levels are low in situations of life-threatening stress(Parker et al., 1985; Wade et al.,1988). Long duration hyperglycemia in this experiment is a form of excessive stress. It was speculated that histological changes in the adrenal gland may occur. The zona fasciculata which secretes cortisol necessary to maintain life may become enlarged and the zona reticularis which secretes DHEA and DHEA-S may shrink. In addition to reports of the mechanism of the improvement of impaired glucose tolerance by DHEA and DHEA-S, further studies reported a number of other effects. These included acceleration of glucose uptake in cells, increasing sensitivity in insulin sensitive tissue and suppressing the activities of G6Pase and FBPase, the enzymes of glyconeogenesis in the liver(McIntosh & Berdanier,1991; Nakashima et al.,1995) However, many points remained unclear.