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Elevated plasma levels of pancreastatin (PST) in patients with non-insulin-dependent diabetes mellitus (NIDDM)
Regulatory Peptides, 30 (1990) 159-164
159
Elsevier REGPEP 00951
Elevated plasma levels of pancreastatin (PST) in patients with non-insulin-dependent diabetes mellitus (NIDDM) Akihiro Funakoshi 1, Kayoko Tateishi 2, Hirotsugu Shinozaki 3, Masahiro Matsumoto 4 and Hideyuki Wakasugi ~ I National Kyushu Cancer Center, 2First Department of Biochemistry, Fukuoka University, 3 Sanshin-kai Hara Hospital, Fukuoka and 4Municipal Kitakyushu Kokura Hospital, Kitakyushu (Japan)
(Received 26 March 1990; revised version received and accepted 17 May 1990) K e y words: Pancreastatin; N I D D M
Summary Pancreastatin (PST) is known as the peptide which inhibits first phase of glucosestimulated insulin secretion. Fasting plasma PST levels and responses of PST after oral glucose ingestion in patients with non-insulin-dependent diabetes meUitus (NIDDM) were studied with human PST-specific radioimmunoassay. Fasting plasma PST in N I D D M patients was not different from healthy controls, although a slightly higher level of PST was observed in patients treated with sulfonylurea among N I D D M patients. No significant increase in plasma PST was observed after a glucose ingestion in healthy controls, In contrast, plasma PST levels in N I D D M patients rose significantly after glucose ingestion. These results suggest a possible pathophysiologlcal role for PST in NIDDM.
Introduction Pancreastatin (PST) isolated recently from porcine pancreas [ 1] is known to inhibit the first phase of glucose-induced insulin secretion and augment glucagon secretion stimulated by arginine in the isolated perfused pancreas [2]. We have reported that PST
Correspondence: A. Funakoshi, National Kyushu Cancer Center, Notame 3-1-1, Minami-ku, Fukuoka
815, Japan. 0167-0115/90/$03.50 © 1990 Elsevier Science Publishers B.V. (BiomedicalDivision)
160
elevated blood glucose levels after glucose stimulation in conscious rats [3,4]. Plasma PST levels in patients with non-insulin-dependent diabetes mellitus (NIDDM), thus, were interesting for us. We, therefore, investigated fasting plasma PST and changes of PST after oral glucose ingestion in patients with N I D D M using a recently developed radioimmunoassay (RIA) for human PST [5,6]. Materials and Methods
Subjects The groups studied included 59 patients with N I D D M and 26 healthy control subjects having no family history of diabetes mellitus or obesity for fasting plasma PST determination. N I D D M patients did not have significant diabetic complications, hepatic cirrhosis, or pancreatic exocrine dysfunction. The patients were treated with three different regimens: diet restriction alone (diet group, n = 22), oral hypoglycemic drug (sulfonylurea): SU group, n = 22) and insulin (insulin group, n = 15). A 75 g oral glucose tolerance test (OGTT) was performed in seven healthy subjects and 12 patients with N I D D M who consist of diet group (n = 22) and SU group (n = 6). After an overnight fast, an indwelling cannula was placed in an antecubital vein and blood was drawn. Blood samples were taken 0, 30, 60, 90, 120 and 180 min after ingestion of a 75 g glucose solution. The blood samples for measurement of glucose, insulin and PST were immediately placed in chilled tubes containing trasylol and 1.2 mg/ml EDTA and centrifuged. The plasma was separated into aliquots. All aliquots were stored at - 40 °C and were thawed only once, just before use. Informed consent for the studies was obtained from all subjects.
Laboratory methods Blood glucose was measured using a glucose analyzer (Beckman, Berkley, CA). Plasma Immunoreactive insulin (IRI) was determined using an Insulin RIA Bead kit (Dainabot Co., Tokyo, Japan). Plasma PST was measured by the previously described RIA method [5,6]. Briefly, [125I]Tyr-human PST-29 as the tracer, anti-human PST-29 antiserum (R711) and human PST-52 as the standard were used for RIA. The antiserum (R711) recognized the C-terminal portion and C-terminal glycine amide of human P ST [7]. The antiserum reacted 100~o with human PST-52 and human PST-48, 84~o with human PST-29 and 30~o with human PST-10, but less than 0.001 ~o with other pancreatic and gut hormones such as insulin, glucagon, somatostatin, pancreatic polypeptide, cholecystokinin, arginine vasopressin, and human gastrin-I. The sensitivity of the assay was 4.25 fmol/ml. Plasma samples (1 ml) were extracted with Sep-pak C18 cartridges and the extracts were dried and reconstituted in 1 ml of RIA buffer. 0.4 ml of the reconstituent was usually used in duplicate for RIA. Intraassay coefficients of variation for plasma samples containing PST at normal range and high level were 7.7 and 7.8 ~o, respectively. Interassay coefficients of variation for these samples were 10.4 and 8.2 ~o, respectively. The recovery (mean + SEM) of human PST-52 added to pooled plasma in a range of 9.7 to 38.7 fmol/ml was 79.1 + 1.8Yo. All samples from any one subjects in OGTT were run in the same assay.
161
[ p
Ir p,%.o~
1
20
-6
e= lo
0-
Control
Diet
SU
Insulin
NIDDM
Fig. 1. Fasting plasma PST levels in 26 healthy controls and 59 NIDDM patients. Vertical bars represent standard errors. Asterisks indicate significantdifferencefrom sulfonylurea(SU) groups.
Analysis of data Values were expressed as the mean + S.E. Results were analyzed by multiple analysis of variance (MANOVA) with repeated measures with respect to the treatment and time [8]. Student's unpaired test was employed for statistical analysis of fasting plasma PST. A value of P < 0.05 was considered significant.
Results
Fasting plasma PST in patients with NIDDM Fasting plasma PST levels were 13.2 + 0.55 pmol/1 in healthy controls and 11.2 + 0.38, 15.3 + 0.94 and 11.6 + 0.51 pmol/1 in diet, SU and insulin groups of N I D D M , respectively. The fasting plasma PST levels in the SU group were elevated significantly from the other two groups of N I D D M (Fig. 1), but not significant from controls.
Blood glucose and plasma insulin levels after 75 g oral glucose ingestion After ingestion of glucose, the mean glucose and insulin levels in the control subjects rose gradually from the basal levels and then declined to nearly basal levels within 180min (F value with respect to time was 4.27; P < 0 . 0 2 for glucose and 23.17; P < 0.001 for insulin) (Fig. 2). Ingestion of glucose significantly increased glucose level in both diet and SU groups, but did not increase insulin level in SU group (F value with respect to time was 11.2; P < 0.001 in diet group and 21.0; P < 0.001 in SU group for glucose and 9.96; P < 0.001 in diet group and 0.83; P = 0.55 in SU group for insulin) (Fig. 2). There were significant differences in glucose and insulin responses between control, diet group and SU group (F(2,16) -- 34.36; P < 0.001 for glucose and 4.55; P < 0.05 for insulin) (Fig. 2).
162 500
4O0
~300 8 ~ 200 m
iooi C
i
n
10
t
~0
r
if0
o
1~0
l
1~0 min
50
Diet group
40 •_=- 30
--= 20
/ ~
SU group Control
o o
3'0
I~o
90
12o
18o min
Time
Fig. 2. Responses of blood glucose (upper panel) and plasma insulin (lower panel) 7 in healthy controls, 6 diet groups and 6 SU groups in N I D D M to an oral glucose ingestion. There were significant differences in blood glucose and insulin responses between control, diet group and SU group (F(2,16)= 34.36; P < 0.001 for blood glucose and 4.55; P < 0.05 for insulin) by MANOVA with repeated measures.
20
"6 E
~
SUgroup Control Dietgroup
10
o_
G
~o
8'o
9'o
~~o
~~o ~in
Time Fig. 3. Response of plasma PST in 7 healthy controls, 6 diet groups and 6 SU groups in N I D D M to an oral glucose ingestion. There were significant differences in PST response between control and N I D D M (F(2,16) = 5.8; P < 0.02) by MANOVA with repeated measures.
163
Plasma P S T after 75 g oral glucose ingestion After glucose ingestion, plasma PST levels in the control subjects did not change within 180 min (F value with respect to time was 0.83; P = 0.55) (Fig. 3). In contrast, ingestion of glucose significantly increased plasma PST in patients with NIDDM (F value with respect to time were 8.25; P < 0.02 in diet group and 7.03; P < 0.05 in SU group) (Fig. 3). There were significant differences in PST response between control and NIDDM (F(2,16) = 5.8; P < 0.02) (Fig. 3).
Discussion
In the present study, we have measured the plasma PST in NIDDM patients treated with different regimens by a highly sensitive human PST RIA. We found that the fasting plasma PST levels in SU group were slightly higher than other two groups of NIDDM, but not significant different from healthy controls. Interestingly, the PST levels after oral glucose ingestion were significantly elevated in two groups (diet and SU) of NIDDM patients when analyzed by MANOVA with repeated measures with respect to time, although the PST response was not significantly observed in control group. We have not done the glucose ingestion test in insulin treated group because of high plasma glucose levels. We have reported in the previous study [9] that plasma PST increased in healthy volunteers after intrajejunal infusion of liquid meal containing 16 g protein, 56.4 g carbohydrate, and 12.5 g fat in 400 kcal/400 ml. Thus, protein or fat may be responsible for the triggering of initial PST release but glucose may not be in healthy controls. The small significant rise of plasma PST after glucose ingestion in NIDDM patients may suggest the hypersensitivity of PST-producing cells to glucose in the pancreas of these patients. Therefore, these results suggest that the hypersecretion of PST in NIDDM patients may work on inhibition of glucose-stimulated insulin secretion and induce hyperglycemia [3,4,10].
References 1 Tatemoto, K, Efendic, S., Mutt, V., Makk, G., Feistner, G.J. and Barchas, J. D., Isolation and structure of pancreastatin, a novel pancreatic peptide that inhibits insulin secretion, Nature (Lond.), 324 (1986) 476-478. 2 Efendic, S., Mutt, V., Tatemoto, K., Quan, C., Chang, D. and Ostenson, C.G., Pancreastatin and islet hormone release, Proc. Natl. Acad. Sci., USA 84 (1987) 7257-7260. 3 Funakoshi, A., Miyasaka, K., Kitani, K. and Tatemoto, K., Effect of pancreastatin on pancreatic endocrine function in the conscious rat, Regnl. Pept., 24 (1989) 225-231. 4 Miyasaka, K., Funakoshi, A., Yasunami, Y., Nakamura, R., Kitani, K., Tamamura, H., Funakoshi, S. and Fujii, N., Rat pancreastatin inhibits both pancreatic exocrine and endocrine secretions in rats, Regul. Pept., 28 (1990) 189-198. 5 Tateishi, K., Funakoshi, A., Jimi, A., Funakoshi, S., Tamamura, H., Yajima, H. and Matsuoka, Y., High plasma pancreastatin-like immunoreactivityin a patient with malignant insulinoma, Gastroenterology, 97 (1989) 1313-1319. 6 Tateishi, K., Funakoshi, A., Wakasugi, H., Iguchi, H., Shinozaki, H., Abe, M., Funakoshi, S., Tamamura, H., Yajima, H. and Matsuoka Y., Plasma pancreastatin-like immunoreactivity in various diseases, J. Clin. Endocrinol. Metab., 69 (1989) 1305-1308.
164 7 Funakoshi, S., Tamamura, H., Ohta, M., Yoshizawa, K., Funakoshi, A., Miyasaka, K., Tateishi, K., Tatemoto, K., Nakano, I., Yajima, H. and Fujii, N., Isolation and characterization of a tumor-derived human pancreastatin-related protein, Biochem. Biophys. Res. Commun., 164 (1989) 141-148. 8 Wallenstein, S., Zucker, C.L. and Fleiss, J.L., Some statistical methods useful in circulation research, Circ. Res., 47 (1980) 1-9. 9 Funakoshi, A., Tateishi, K., Shinozaki, H., Miyasaka, K., Ito, T. and Wakasugi, H., Plasma pancreastatin responses after intrajejunal infusion of liquid meal in patients with chronic pancreatitis, Dig. Dis. Sci., 1990, in press. 10 Ahren, B., Lindskog, S., Tatemoto, K. and Efendic, S., Pancreastatin inhibits insulin secretion and stimulates glucagon secretion in mice, Diabetes, 37 (1988) 281-285.
159
Elsevier REGPEP 00951
Elevated plasma levels of pancreastatin (PST) in patients with non-insulin-dependent diabetes mellitus (NIDDM) Akihiro Funakoshi 1, Kayoko Tateishi 2, Hirotsugu Shinozaki 3, Masahiro Matsumoto 4 and Hideyuki Wakasugi ~ I National Kyushu Cancer Center, 2First Department of Biochemistry, Fukuoka University, 3 Sanshin-kai Hara Hospital, Fukuoka and 4Municipal Kitakyushu Kokura Hospital, Kitakyushu (Japan)
(Received 26 March 1990; revised version received and accepted 17 May 1990) K e y words: Pancreastatin; N I D D M
Summary Pancreastatin (PST) is known as the peptide which inhibits first phase of glucosestimulated insulin secretion. Fasting plasma PST levels and responses of PST after oral glucose ingestion in patients with non-insulin-dependent diabetes meUitus (NIDDM) were studied with human PST-specific radioimmunoassay. Fasting plasma PST in N I D D M patients was not different from healthy controls, although a slightly higher level of PST was observed in patients treated with sulfonylurea among N I D D M patients. No significant increase in plasma PST was observed after a glucose ingestion in healthy controls, In contrast, plasma PST levels in N I D D M patients rose significantly after glucose ingestion. These results suggest a possible pathophysiologlcal role for PST in NIDDM.
Introduction Pancreastatin (PST) isolated recently from porcine pancreas [ 1] is known to inhibit the first phase of glucose-induced insulin secretion and augment glucagon secretion stimulated by arginine in the isolated perfused pancreas [2]. We have reported that PST
Correspondence: A. Funakoshi, National Kyushu Cancer Center, Notame 3-1-1, Minami-ku, Fukuoka
815, Japan. 0167-0115/90/$03.50 © 1990 Elsevier Science Publishers B.V. (BiomedicalDivision)
160
elevated blood glucose levels after glucose stimulation in conscious rats [3,4]. Plasma PST levels in patients with non-insulin-dependent diabetes mellitus (NIDDM), thus, were interesting for us. We, therefore, investigated fasting plasma PST and changes of PST after oral glucose ingestion in patients with N I D D M using a recently developed radioimmunoassay (RIA) for human PST [5,6]. Materials and Methods
Subjects The groups studied included 59 patients with N I D D M and 26 healthy control subjects having no family history of diabetes mellitus or obesity for fasting plasma PST determination. N I D D M patients did not have significant diabetic complications, hepatic cirrhosis, or pancreatic exocrine dysfunction. The patients were treated with three different regimens: diet restriction alone (diet group, n = 22), oral hypoglycemic drug (sulfonylurea): SU group, n = 22) and insulin (insulin group, n = 15). A 75 g oral glucose tolerance test (OGTT) was performed in seven healthy subjects and 12 patients with N I D D M who consist of diet group (n = 22) and SU group (n = 6). After an overnight fast, an indwelling cannula was placed in an antecubital vein and blood was drawn. Blood samples were taken 0, 30, 60, 90, 120 and 180 min after ingestion of a 75 g glucose solution. The blood samples for measurement of glucose, insulin and PST were immediately placed in chilled tubes containing trasylol and 1.2 mg/ml EDTA and centrifuged. The plasma was separated into aliquots. All aliquots were stored at - 40 °C and were thawed only once, just before use. Informed consent for the studies was obtained from all subjects.
Laboratory methods Blood glucose was measured using a glucose analyzer (Beckman, Berkley, CA). Plasma Immunoreactive insulin (IRI) was determined using an Insulin RIA Bead kit (Dainabot Co., Tokyo, Japan). Plasma PST was measured by the previously described RIA method [5,6]. Briefly, [125I]Tyr-human PST-29 as the tracer, anti-human PST-29 antiserum (R711) and human PST-52 as the standard were used for RIA. The antiserum (R711) recognized the C-terminal portion and C-terminal glycine amide of human P ST [7]. The antiserum reacted 100~o with human PST-52 and human PST-48, 84~o with human PST-29 and 30~o with human PST-10, but less than 0.001 ~o with other pancreatic and gut hormones such as insulin, glucagon, somatostatin, pancreatic polypeptide, cholecystokinin, arginine vasopressin, and human gastrin-I. The sensitivity of the assay was 4.25 fmol/ml. Plasma samples (1 ml) were extracted with Sep-pak C18 cartridges and the extracts were dried and reconstituted in 1 ml of RIA buffer. 0.4 ml of the reconstituent was usually used in duplicate for RIA. Intraassay coefficients of variation for plasma samples containing PST at normal range and high level were 7.7 and 7.8 ~o, respectively. Interassay coefficients of variation for these samples were 10.4 and 8.2 ~o, respectively. The recovery (mean + SEM) of human PST-52 added to pooled plasma in a range of 9.7 to 38.7 fmol/ml was 79.1 + 1.8Yo. All samples from any one subjects in OGTT were run in the same assay.
161
[ p
Ir p,%.o~
1
20
-6
e= lo
0-
Control
Diet
SU
Insulin
NIDDM
Fig. 1. Fasting plasma PST levels in 26 healthy controls and 59 NIDDM patients. Vertical bars represent standard errors. Asterisks indicate significantdifferencefrom sulfonylurea(SU) groups.
Analysis of data Values were expressed as the mean + S.E. Results were analyzed by multiple analysis of variance (MANOVA) with repeated measures with respect to the treatment and time [8]. Student's unpaired test was employed for statistical analysis of fasting plasma PST. A value of P < 0.05 was considered significant.
Results
Fasting plasma PST in patients with NIDDM Fasting plasma PST levels were 13.2 + 0.55 pmol/1 in healthy controls and 11.2 + 0.38, 15.3 + 0.94 and 11.6 + 0.51 pmol/1 in diet, SU and insulin groups of N I D D M , respectively. The fasting plasma PST levels in the SU group were elevated significantly from the other two groups of N I D D M (Fig. 1), but not significant from controls.
Blood glucose and plasma insulin levels after 75 g oral glucose ingestion After ingestion of glucose, the mean glucose and insulin levels in the control subjects rose gradually from the basal levels and then declined to nearly basal levels within 180min (F value with respect to time was 4.27; P < 0 . 0 2 for glucose and 23.17; P < 0.001 for insulin) (Fig. 2). Ingestion of glucose significantly increased glucose level in both diet and SU groups, but did not increase insulin level in SU group (F value with respect to time was 11.2; P < 0.001 in diet group and 21.0; P < 0.001 in SU group for glucose and 9.96; P < 0.001 in diet group and 0.83; P = 0.55 in SU group for insulin) (Fig. 2). There were significant differences in glucose and insulin responses between control, diet group and SU group (F(2,16) -- 34.36; P < 0.001 for glucose and 4.55; P < 0.05 for insulin) (Fig. 2).
162 500
4O0
~300 8 ~ 200 m
iooi C
i
n
10
t
~0
r
if0
o
1~0
l
1~0 min
50
Diet group
40 •_=- 30
--= 20
/ ~
SU group Control
o o
3'0
I~o
90
12o
18o min
Time
Fig. 2. Responses of blood glucose (upper panel) and plasma insulin (lower panel) 7 in healthy controls, 6 diet groups and 6 SU groups in N I D D M to an oral glucose ingestion. There were significant differences in blood glucose and insulin responses between control, diet group and SU group (F(2,16)= 34.36; P < 0.001 for blood glucose and 4.55; P < 0.05 for insulin) by MANOVA with repeated measures.
20
"6 E
~
SUgroup Control Dietgroup
10
o_
G
~o
8'o
9'o
~~o
~~o ~in
Time Fig. 3. Response of plasma PST in 7 healthy controls, 6 diet groups and 6 SU groups in N I D D M to an oral glucose ingestion. There were significant differences in PST response between control and N I D D M (F(2,16) = 5.8; P < 0.02) by MANOVA with repeated measures.
163
Plasma P S T after 75 g oral glucose ingestion After glucose ingestion, plasma PST levels in the control subjects did not change within 180 min (F value with respect to time was 0.83; P = 0.55) (Fig. 3). In contrast, ingestion of glucose significantly increased plasma PST in patients with NIDDM (F value with respect to time were 8.25; P < 0.02 in diet group and 7.03; P < 0.05 in SU group) (Fig. 3). There were significant differences in PST response between control and NIDDM (F(2,16) = 5.8; P < 0.02) (Fig. 3).
Discussion
In the present study, we have measured the plasma PST in NIDDM patients treated with different regimens by a highly sensitive human PST RIA. We found that the fasting plasma PST levels in SU group were slightly higher than other two groups of NIDDM, but not significant different from healthy controls. Interestingly, the PST levels after oral glucose ingestion were significantly elevated in two groups (diet and SU) of NIDDM patients when analyzed by MANOVA with repeated measures with respect to time, although the PST response was not significantly observed in control group. We have not done the glucose ingestion test in insulin treated group because of high plasma glucose levels. We have reported in the previous study [9] that plasma PST increased in healthy volunteers after intrajejunal infusion of liquid meal containing 16 g protein, 56.4 g carbohydrate, and 12.5 g fat in 400 kcal/400 ml. Thus, protein or fat may be responsible for the triggering of initial PST release but glucose may not be in healthy controls. The small significant rise of plasma PST after glucose ingestion in NIDDM patients may suggest the hypersensitivity of PST-producing cells to glucose in the pancreas of these patients. Therefore, these results suggest that the hypersecretion of PST in NIDDM patients may work on inhibition of glucose-stimulated insulin secretion and induce hyperglycemia [3,4,10].
References 1 Tatemoto, K, Efendic, S., Mutt, V., Makk, G., Feistner, G.J. and Barchas, J. D., Isolation and structure of pancreastatin, a novel pancreatic peptide that inhibits insulin secretion, Nature (Lond.), 324 (1986) 476-478. 2 Efendic, S., Mutt, V., Tatemoto, K., Quan, C., Chang, D. and Ostenson, C.G., Pancreastatin and islet hormone release, Proc. Natl. Acad. Sci., USA 84 (1987) 7257-7260. 3 Funakoshi, A., Miyasaka, K., Kitani, K. and Tatemoto, K., Effect of pancreastatin on pancreatic endocrine function in the conscious rat, Regnl. Pept., 24 (1989) 225-231. 4 Miyasaka, K., Funakoshi, A., Yasunami, Y., Nakamura, R., Kitani, K., Tamamura, H., Funakoshi, S. and Fujii, N., Rat pancreastatin inhibits both pancreatic exocrine and endocrine secretions in rats, Regul. Pept., 28 (1990) 189-198. 5 Tateishi, K., Funakoshi, A., Jimi, A., Funakoshi, S., Tamamura, H., Yajima, H. and Matsuoka, Y., High plasma pancreastatin-like immunoreactivityin a patient with malignant insulinoma, Gastroenterology, 97 (1989) 1313-1319. 6 Tateishi, K., Funakoshi, A., Wakasugi, H., Iguchi, H., Shinozaki, H., Abe, M., Funakoshi, S., Tamamura, H., Yajima, H. and Matsuoka Y., Plasma pancreastatin-like immunoreactivity in various diseases, J. Clin. Endocrinol. Metab., 69 (1989) 1305-1308.
164 7 Funakoshi, S., Tamamura, H., Ohta, M., Yoshizawa, K., Funakoshi, A., Miyasaka, K., Tateishi, K., Tatemoto, K., Nakano, I., Yajima, H. and Fujii, N., Isolation and characterization of a tumor-derived human pancreastatin-related protein, Biochem. Biophys. Res. Commun., 164 (1989) 141-148. 8 Wallenstein, S., Zucker, C.L. and Fleiss, J.L., Some statistical methods useful in circulation research, Circ. Res., 47 (1980) 1-9. 9 Funakoshi, A., Tateishi, K., Shinozaki, H., Miyasaka, K., Ito, T. and Wakasugi, H., Plasma pancreastatin responses after intrajejunal infusion of liquid meal in patients with chronic pancreatitis, Dig. Dis. Sci., 1990, in press. 10 Ahren, B., Lindskog, S., Tatemoto, K. and Efendic, S., Pancreastatin inhibits insulin secretion and stimulates glucagon secretion in mice, Diabetes, 37 (1988) 281-285.