Makoto Otsuki

Effect of Gymnema sylvestre, R.Br. on glucose homeostasis in rats

Effect of Gymnema sylvestre, R.Br. (G. sylvestre; GS4) on glucose homeostasis was studied in rats. In the first set of experiments, the acute effect of GS4 was examined in both non-diabetic and streptozocin (30 mg/kg)-induced mildly diabetic rats. Administration of 1 g/kg body weight of GS4 to 18-h fasted non-diabetic rats significantly attenuated the serum glucose response to oral administration of 1 g/kg glucose. The immunoreactive insulin (IRI) response in GS4-administered rats was lower, but not significantly, than that in control rats. In mildly diabetic rats, a 60 min increment in serum glucose concentrations was significantly reduced by GS4 administration. No IRI response was observed in these diabetic rats irrespective of GS4 administration. In the second set of experiments, the chronic effect of GS4 was examined in mildly diabetic rats. Two weeks after the induction of diabetes, the rats were divided into two groups that had similar impairment of glucose tolerance assessed by an oral glucose loading test. The rats were fed for 32-35 days with either a control diet or a diet supplemented with GS4. After 4 weeks, GS4 showed a tendency to reduce the serum glucose concentrations in the fed state and to improve the glucose tolerance. Gain in body weight, food intake, pancreas weight and the pancreatic contents of IRI, protein, amylase and trypsinogen were unaltered in the GS4-treated group compared with the control. These results suggest the usefulness of G. sylvestre in the treatment of certain classes of non-insulin-dependent diabetes mellitus.

Green tea polyphenol (–)-epigallocatechin-3-gallate inhibits ethanol-induced activation of pancreatic stellate cells

Backgroundu2002 Activated pancreatic stellate cells (PSCs) play a central role in the pathogenesis of pancreatic fibrogenesis and inflammation. Ethanol, a major cause of chronic pancreatitis, directly induces PSC activation and oxidative stress. Inhibition of PSC activation or stimulation to PSC might be an effective therapeutic strategy for the prevention of pancreatic fibrosis, and (–)‐epigallocatechin‐3‐gallate (EGCG), a major component of green tea extracts, is a potent antioxidant of polyphenols. Therefore, we examined the mechanisms through which ethanol induces oxidative stress on PSCs and evaluated the effect of EGCG on activation and cell functions of ethanol‐stimulated PSCs.

Role of endogenous bile on basal and postprandial CCK release in humans

The role of intraduodenal bile in regulation of plasma cholecystokinin (CCK) levels were investigated in patients with obstructive jaundice under external bile diversion and under physiological bile flow into the duodenum by internal bile drainage. Basal plasma CCK levels determined by a specific and sensitive bioassay in patients under external bile drainage (2.2±0.2 pmol/liter; mean±se) were significantly higher than those in control subjects (1.0±0.3 pmol/liter). In control subjects, the peak CCK response (6.2±0.7 pmol/liter) to a test meal was seen at 45 min, whereas that in patients under external bile drainage, it was seen at 20 min after a test meal (17.6±3.2 pmol/liter;P<0.01 vs controls). After peak response, plasma CCK levels in controls gradually decreased, but remained significantly elevated during a 3-hr observation period. In patients under bile diversion, the test meal caused a prompt plasma CCK peak, with a transient fall followed by a continuous rise until 180 min postprandially. In six patients, external bile diversion was changed to internal biliary drainage with a stent tube within two weeks to maintain physiological bile flow into the duodenum. Internal bile drainage normalized basal (0.9±0.2 pmol/liter) as well as meal-stimulated CCK release (peak value: 5.0±0.8 pmol/liter). These results demonstrate that endogenous bile exerts tonic inhibition on basal and postprandial plasma CCK levels in humans.

High glucose activates rat pancreatic stellate cells through protein kinase C and p38 mitogen-activated protein kinase pathway.

Objective: Hyperglycemia is implicated in fibrosis in many organs. Exocrine and endocrine pancreas are closely linked both anatomically and physiologically, and pathological conditions in the exocrine gland can cause impairment of endocrine function and vice versa. Chronic pancreatitis causes pancreatic fibrosis and sometimes results in diabetes mellitus. Pancreatic stellate cells (PSCs) play a pivotal role in pancreatic fibrogenesis. However, the effects of high glucose concentrations on PSC activation have not been fully elucidated. Methods: Cultured PSCs were incubated in the presence of various concentrations of glucose. Pancreatic stellate cell proliferation, &agr;-smooth muscle actin (&agr;-SMA) expression, and collagen production were determined by colorimetric conversion assay, Western blot analysis, and Sirius red dye binding assay, respectively. Results: High glucose concentrations significantly increased PSC proliferation, &agr;-SMA expression, and collagen type I production in PSCs. High glucose concentrations activated protein kinase C (PKC) in PSCs, and PKC inhibitor GF109203X inhibited glucose-stimulated PSC proliferation, &agr;-SMA expression, and collagen secretion. High glucose also activated p38 mitogen-activated protein kinase (MAPK) in PSCs, and p38 MAPK inhibitor SB203580 inhibited glucose-stimulated collagen secretion. Conclusions: Our results indicate that high glucose concentrations stimulate PSC activation via PKC-p38 MAP kinase pathway and suggest that high glucose may aggravate pancreatic fibrosis.

Beneficial effects of the synthetic trypsin inhibitor camostate in cerulein-induced acute pancreatitis in rats

The therapeutic effect and the mechanism of action of the synthetic trypsin inhibitor camostate were studied in a rat model of acute interstitial pancreatitis induced by four subcutaneous injections of 20 μg/kg body weight of cerulein at hourly intervals. Rats with acute pancreatitis were given either 100 mg/kg body weight camostate or volume- and pH-adjusted water via an orogastric tube 30 min after the last cerulein injection. The elevation of serum amylase activity was significantly reduced by camostate treatment and the peak value was seen 1 hr earlier than that observed in the rats that did not receive camostate. Camostate also inhibited the reduction in pancreatic content of lipase and amylase seen during experimental pancreatitis. These effects were accompanied by alleviation of the histologic signs of acute pancreatitis such as cellular infiltration and acinar cell vacuolization. After oral administration, camostate and its metabolite were absorbed from the intestine and were detectable in plasma for more than 6 hr in concentrations high enough to have antiprotease activity. In addition, camostate in the duodenum was able to increase pancreatic juice flow and protein output and to stimulate endogenous secretin release. These results suggest that oral administration of camostate reduces the severity of cerulein-induced acute pancreatitis by releasing endogenous secretin and by its antiprotease activity.

Persistent destruction of the basement membrane of the pancreatic duct contributes to progressive acinar atrophy in rats with experimentally-induced pancreatitis

Background and Aims: The imbalance between synthesis and degradation of extracellular matrix (ECM) proteins is a characteristic feature in chronic pancreatitis. We evaluated the relationship between type IV collagen structure in the basement membrane (BM) and the development of acinar atrophy or the regeneration from acinar injury. Methods: Three different models of pancreatitis were induced in rats by repetitive intraperitoneal injections of 500 mg/100 g body weight of arginine (Arg) or 20 μg/kg body weight of caerulein (Cn) or a single retrograde intraductal infusion of 40 μL/100 g body weight of 3% sodium taurocholate (NaTc). We examined the changes in type IV collagen structure by immunostaining, and the serial changes in the gelatinolytic activity of pro- and active matrix metalloproteinase-2 by zymography in these models of pancreatitis. Results: The pancreas appeared to be histologically normal on day 35 after the first intraperitoneal Cn injection and on day 42 after intraductal infusion of NaTc, whereas 85% to 90% of acinar tissue was replaced by fatty tissue and dilated pancreatic ducts on day 54 after the first intraperitoneal Arg injection. Immunoreactivity for type IV collagen appeared as a discontinuous line along the BM of ducts, vessels, tubular complexes, and acinar cells on day 40 in Arg-induced pancreatitis, whereas it was detected as a continuous line along the BM on day 35 in Cn-induced pancreatitis and on day 42 in NaTc-induced pancreatitits. Gelatinolytic activity of active MMP-2 increased significantly from day 13 to day 40 after the first intraperitoneal Arg injection, whereas it decreased to the baseline level on day 35 after the first intraperitoneal Cn injection and on day 42 after intraductal infusion of NaTc. Conclusions: Our findings indicate that a long-term increase in gelatinolytic activity of active MMP-2 in Arg-induced pancreatitis causes continuous disorganization of type IV collagen in the BM and progressive acinar atrophy, whereas a transient increase in gelatinolytic activity of active MMP-2 is involved in the regeneration of type IV collagen structure in the BM and recovery from acinar injury.

Oral glucose ingestion stimulates cholecystokinin release in normal subjects and patients with non-insulin-dependent diabetes mellitus

The role of glucose in the regulation of plasma cholecystokinin (CCK) level was investigated in healthy control subjects and patients with non-insulin-dependent diabetes mellitus (NIDDM). Plasma CCK concentration was determined by a specific and sensitive bioassay and by a highly sensitive and reliable double-antibody radioimmunoassay using OAL-656 as an antiserum. In control subjects, ingestion of Trelan G-75 (1,200 mOsm/L,225 mL), which is equivalent to 75 g glucose as metabolic products, caused a rapid and significant increase in plasma CCK bioactivity from 1.3 +/- 0.2 to a peak of 5.8 +/- 0.6 pmol/L and immunoreactive CCK concentration from 1.2 +/- 0.1 to 4.6 +/- 0.6 pmol/L. Ingestion of 75 g glucose in 225 mL water (33.3% solution) increased plasma CCK bioactivity to a similar degree to that observed following Trelan G-75 (peak response, 4.5 +/- 0.4 pmol/L). The same volume of 0.9% NaCl solution or water failed to increase plasma CCK concentration. A smaller dose of glucose (50 b/150 mL water) increased plasma CCK concentration, although the peak level (3.0 +/- 0.5 pmol/L) was less than that observed following 75 g glucose. In patients with NIDDM, Trelan G-75 ingestion increased CCK concentration, but the peak level was lower, albeit insignificantly, than that of normal subjects. When the maximal increment of plasma CCK above the basal value was compared between control and NIDDM subjects, the differences were statistically significant (NIDDM, 3.6 +/- 0.1 pmol/L; control, 5.0 +/- 0.4; P < .01). However, integrated CCK responses to Trelan G-75 in NIDDM (165.8 +/- 15.5 pmol/120 min) were not significantly different from those in control subjects (189.8 +/- 15.9 pmol/120 min). Peak CCK bioactivity occurred within 10 to 30 minutes of ingestion, preceding the increase in glucose and insulin. These results suggest a possible effect of CCK on insulin release in humans, and that the CCK secretory response to glucose in well-controlled diabetic patients is not significantly altered.

Potentiating effect of insulin on exocrine secretory function in isolated rat pancreatic acini

BACKGROUND/AIMSnInsulin is shown to exert various regulatory effects on the exocrine pancreatic function. We investigated the direct effect of insulin on exocrine pancreatic secretion.nnnMETHODSnThe effects of insulin on amylase release, 125I-secretin binding and Na(+)- and K(+)-activated adenosine triphosphate phosphohydrolase (Na+,K(+)-ATPase) activity were measured using the isolated rat pancreatic acini.nnnRESULTSnInsulin potentiated the amylase release elicited by secretin plus cholecystokinin (CCK), but not by either secretin or CCK alone. The potentiating effect of insulin was dependent on the concentration and preincubation time. Insulin had no effect on 125I-secretin binding. Ouabain, a specific Na+,K(+)-ATPase inhibitor, caused a concentration-dependent inhibition of the potentiated secretion by insulin without affecting the secretory response to secretin plus CCK. In membranes prepared from acini treated with insulin, Na+,K(+)-ATPase activity was significantly increased. Similar results were obtained when acini were treated with insulin in combination with secretin plus CCK.nnnCONCLUSIONSnInsulin exerts a direct effect on pancreatic acinar cells and potentiates exocrine secretion elicited by secretin in combination with CCK, in part, by increasing Na+,K(+)-ATPase activity.

Effect of islet hormones on secretin-stimulated exocrine secretion in isolated perfused rat pancreas

To clarify the effect of islet hormones on pancreatic ductular cell function, we measured the exocrine secretion elicited by 10 pM secretin in the presence or absence of islet hormones using an isolated perfused rat pancreas model. Insulin significantly increased secretin-stimulated pancreatic juice secretion, but not protein secretion. The potentiating effect of insulin on pancreatic juice secretion was concentration-dependent, and the maximal effect was observed with 1 μM insulin. Ouabain, a specific Na+,K+-ATPase inhibitor, caused concentration-dependent inhibition of the potentiating effect of insulin without affecting secretin action. Glucagon (100 nM) significantly inhibited secretin-stimulated pancreatic juice secretion and also tended to inhibit protein secretion. A somatostatin analog, SMS 201-995 (10 nM) significantly inhibited both the pancreatic juice and protein secretion stimulated by secretin. The inhibitory effect of SMS 201-995 was concentration-dependent and was maximal at 1–10 nM. These results demonstrate that insulin potentiates the secretory response to secretin, at least partly by increasing Na+,K+-ATPase activity, whereas glucagon and somatostatin inhibit this response. Thus, pancreatic islet hormones regulate the secretory function of pancreatic ductular and centroacinar cells.

Fasting prevents acute pancreatitis induced by cerulein in rats

We examined the effect of fasting on the course of experimental acute pancreatitis induced in rats by four subcutaneous injections of 20 μg/kg body weight of cerulein at hourly intervals. Rats were either fasted from 24 hr before to 9 hr after the first cerulein injection or fed ad libitumthroughout the experiment. Twenty-four hours of fasting reduced cerulein-induced increases in serum levels of amylase and anionic trypsin(ogen) to 50 and 70% of those in fed rats, respectively. Increases in pancreatic wet weight after cerulein injections were also less in fasted rats than in fed rats. Pancreatic content of trypsin was significantly decreased after a 24-hr fast, and no further changes were induced by cerulein injections. The histological signs of acute pancreatitis were greatly alleviated by fasting. However, 24 hr of fasting did not alter the sensitivity and responsiveness of the exocrine pancreas to cerulein in both in vivoand in vitro.Plasma CCK bioactivity and immunoreactive secretin concentration in 24-hr-fasted rats were significantly lower than those in fed rats. Administration of CCK receptor antagonist, loxiglumide, 12 hr prior to the induction of acute pancreatitis reduced the increase in serum amylase activity in fed rats to nearly the same levels as that in fasted rats and alleviated histological signs of pancreatitis to some extent. These present observations suggest that fasting lessens the severity of cerulein-induced acute pancreatitis by reducing endogenous CCK release.