Effects of branched-chain-enriched amino acid solution on insulin and glucagon secretion and blood glucose level in liver cirrhosis.

BACKGROUND: The aim of this study was to determine whether therapeutically used branched-chain amino acids (BCAAs) solution influences glucose metabolism in liver cirrhosis (LC). METHODS: BCAAs solution (200 ml) was infused in LC patients at different stages, and plasma concentrations of glucose and pancreatic hormones were determined. RESULTS: In patients with mild LC, BCAAs caused a significant increase in glucose level (maximal increment, 12.5+/-2.5 mg/dl) with a great increase in insulin (maximal increment, 39.5+/-8.3 microU/ml) and a small increase in glucagon secretion (maximal increment, 101.0+/-16.0 pg/ml). In patients with advanced LC, BCAAs caused a great increase in glucagon secretion (220.5+/-19.4 pg/ml) with only a slight increase in glucose levels (5.8+/-2.2 mg/dl). CONCLUSION: BCAAs solution causes hyperglycemia in mild LC due to insulin resistance, whereas it causes only a slight increase in severe LC due to hepatic glucagon resistance. Thus, there is a possibility that BCAAs solution may lead to hypoglycemia in advanced LC with hepatic glycogen depletion.

Defects of cholecystokinin (CCK)-A receptor gene expression and CCK-A receptor-mediated biological functions in Otsuka Long-Evans Tokushima Fatty (OLETF) rats.

Recent studies in genetically obese and diabetic Otsuka Long-Evans Tokushima Fatty (OLETF) rats suggest defects of cholecystokinin (CCK)-A receptor gene expression and CCK-A receptor-mediated biological functions such as pancreatic juice, protein, and gastric acid secretion. The present studies were undertaken to further examine CCK-A receptor gene expression and CCK-A receptor-mediated biological functions in the pancreas, stomach, and brain of OLETF rats. Expression of the CCK-A receptor gene could not be detected in the stomach, pancreas and brain by the reverse-transcription polymerase chain reaction (RT-PCR) method and Southern blotting of the PCR products. Southern blot analysis of genomic DNA from OLETF and control Long-Evans Tokushima Otsuka (LETO) rats with CCK-A receptor fragment as a probe revealed different restriction bands. Expression of the CCK-B receptor gene was observed in the stomach, pancreas, and brain in both OLETF and LETO rats by the RT-PCR method, with expression of the CCK-B receptor gene markedly enhanced in OLETF rats compared with that in LETO rats. Consistent with the defect of CCK-A receptor gene expression, CCK-A receptor-mediated biological functions were not observed in these organs. Perfused exocrine and endocrine pancreas of OLETF rats were insensitive to CCK stimulation but not to carbamylcholine stimulation. Basal gastric acid and pepsinogen secretions in OLETF rats were higher than in LETO rats. OLETF rats showed a significantly higher average daily food intake, gained body weight faster, and were heavier than LETO rats. The present study confirmed that OLETF rats have CCK-A receptor gene anomalies and demonstrated deficient CCK-A receptor-mediated biological function in the pancreas, stomach, and brain.

CCK-, secretin-, and cholinergic-independent pancreatic fluid hypersecretion in protease inhibitor-treated rats.

Plasma cholecystokinin (CCK) levels in fed rats increased from 2.59 +/- 0.13 pmol/l to the peak of 27.6 +/- 4.1 pmol/l at 1 h after a single oral administration of synthetic protease inhibitor (PI; ethyl N-allyl-N-[(E)-2-methyl-3-[4-(4-amidino-phenoxycarbonyl)phenyl] propenoyl] amino acetate methansulfonate; 20 mg/kg body wt), but then returned to the preloading value at 12 h after administration. The pancreatic fluid secretion, rich in chloride but poor in bicarbonate, was significantly elevated at 6-12 h postfeeding (100.9 +/- 8.2 vs. 27.3 +/- 2.3 microliters/30 min in control rats, P < 0.01). Loxiglumide (50 mg.kg body wt-1.h-1), atropine (100 micrograms.kg body wt-1.h-1), or antisecretin serum (100 microliters/rat) at 12 h postfeeding did not modify the fluid hypersecretion. Loxiglumide, when given together with PI, completely abolished fluid hypersecretion, but it could not inhibit hypersecretion when applied 3 h after PI treatment. Labeling with 5-bromo-2'-deoxyuridine showed active proliferation of acinar cells at 3 h after PI treatment (3.56 +/- 0.29% vs. 0.46 +/- 0.08% in control, P < 0.001), but not in rats given loxiglumide together with PI. In rats that fasted from 12 h before to 12 h after PI feeding, neither pancreatic fluid hypersecretion nor active proliferation of acinar cells was observed. These results suggest that pancreatic fluid hypersecretion in fed rats at 6-12 h after PI treatment is caused not by CCK-, secretin-, or cholinergic-dependent mechanisms but probably by acinar cell proliferation.

Plasma cholecystokinin levels in acute pancreatitis.

Recent studies have shown that cholecystokinin (CCK) is involved in the induction and development of acute pancreatitis in experimental animals. In the present study we determined basal plasma CCK concentrations by a specific and sensitive radioimmunoassay using antiserum OAL656 in 17 patients with acute pancreatitis due to gallstone in the common bile duct (n = 7), alcoholic (n = 4), post endoscopic retrograde pancreatography (n = 1), and unknown causes (n = 4), and 37 patients with cholelithiasis (n = 18) and choledocholithiasis (n = 19). Plasma CCK concentrations in patients with gallstone pancreatitis on hospital day 1 (mean +/- SEM, 6.78 +/- 1.39 pM) were significantly higher than those in patients with other causes (1.33 +/- 0.16 pM) or in 20 healthy control subjects (1.55 +/- 0.11 pM). There was no relationship between plasma CCK and serum pancreatic enzyme levels, the severity of acute pancreatitis, or serum bilirubin concentrations. Plasma CCK levels in patients with acute symptomatic cholelithiasis (n = 7; 4.35 +/- 0.90 pM) and choledocholithiasis (n = 8; 4.52 +/- 1.17 pM) were significantly higher than those in patients without symptoms (cholelithiasis, n = 11, 1.40 +/- 0.17 pM; choledocholithiasis, n = 11, 1.88 +/- 0.49 pM) but tended to be lower than those in patients with gallstone pancreatitis. These present observations suggest that the increase in plasma CCK levels in gallstone pancreatitis appears not to be the cause but to be the result of gallstone pancreatitis probably due to a transient disturbance of bile flow into the duodenum by stones or edema of the bile duct. Our present results provide some evidence for the usefulness of CCK receptor antagonists for the treatment of biliary colics and acute pancreatitis.

Role of endogenous cholecystokinin and cholecystokinin-A receptors in the development of acute pancreatitis in rats.

Recent studies provide significant evidence that cholecystokinin (CCK) is involved in the induction and development of acute pancreatitis in experimental animals. However, the results obtained with specific CCK-A (peripheral) receptor antagonists are still controversial. The present studies were undertaken to evaluate the involvement of endogenous CCK and the CCK-A receptors in the development of severe acute pancreatitis induced in Otsuka Long-Evans Tokushima Fatty (OLETF) rats that have a selective defect in the CCK-A receptor. Three models of severe acute pancreatitis were induced by retrograde intraductal infusion of 4% sodium taurocholate, by the closed duodenal loop, or by a single intraperitoneal injection of 500 mg/100 g body weight of L-arginine in OLETF rats and control Long-Evans Tokushima Otsuka (LETO) rats. Plasma CCK levels rose up to 4- to 14-fold over the preloading values after the onset of acute pancreatitis in all three models in both groups of rats. However, histologic alterations as well as the magnitudes of increase in serum amylase and lipase activity and the pancreatic wet weight were significantly less in the OLETF rats than those in the LETO rats. In addition, 72 h after the onset of arginine pancreatitis, massive destruction of pancreatic parenchyma with a significant reduction in serum amylase and lipase activities and pancreatic wet weight was observed in the LETO rats, whereas these changes were not seen in OLETF rats. These results suggest that endogenous CCK and CCK-A receptors play a role in the development of severe acute pancreatitis in rats.

Effects of intravenous infusion of amino acids on cholecystokinin release and gallbladder contraction in humans.

We investigated the effect of intravenous infusions of the therapeutically available amino acid solutions Moripron and Morihepamin (Roussel Morishita, Osaka, Japan) on gallbladder contraction and cholecystokinin (CCK) release in healthy male volunteers. Plasma CCK levels were measured by radioimmunoassay, using the antibody OAL-656, which is specific for the aminoterminus of CCK-8 and thus recognizes biologically active forms of all CCKs. The volume of the gallbladder was calculated by ultrasonographic measurements. Intravenous infusion of Moripron at the rate of 3.33 ml/min for 60 min, caused gallbladder contraction, with a peak response of 31.3 +/- 8.6% of the fasting volume at 45-60 min, and a significant increase in plasma CCK concentration, from 1.8 +/- 0.2 pmol/l to a peak of 9.9 +/- 1.5 pmol/l, at 30-45 min. The maximum gallbladder contraction and the peak CCK release during the Moripron infusion were not significantly different from findings after a test meal. There was a close relationship between the peak plasma CCK concentration and the maximal gallbladder contraction during the administration of Moripron, and this agent, even when infused at the rate of 1.67 ml/min, significantly increased plasma CCK levels and gallbladder contraction. Intravenous infusion of Morihepamin had no significant influence on gallbladder volume or plasma CCK levels. The discrepancy in responses appeared to be related to differences in composition between Moripron and Morihepamin, and not to the total dose of amino acid. Intravenous infusions of amino acids appear to have different effects on gallbladder contraction and plasma CCK secretion depending on the amino acids composition. Our findings suggest that an intravenous infusion of Moripron could be used for the prophylaxis of acute acalculous cholecystitis and sludge formation due to reduced biliary motility in patients on total parenteral nutrition.

Pharmacological profile of TP-680, a new cholecystokininA receptor antagonist.

1. The pharmacological characteristics of a newly developed serine derivative (R)-1-[3-(3-carboxypyridine-2-yl) thio-2-(indol-2-yl)carbonylamino]propionyl-4-diphenylmethyl- piperazine (TP-680), a cholecystokinin type A (CCKA) receptor antagonist, were studied and compared with those of MK-329 and loxiglumide. 2. TP-680 showed approximately 2 and 22 times greater selectivity for peripheral CCKA receptors relative to brain CCK (CCKB) receptors than MK-329 and loxiglumide, respectively, when IC50 values for inhibition of [125I]-CCK-8 binding in isolated acini and cerebral cortex were compared. 3. TP-680 was approximately 17 times less potent than MK-329, but was 106 times more potent than loxiglumide in inhibiting 100 pM CCK-8-stimulated amylase release from rat pancreatic acini. The antagonism produced by TP-680 was specific for CCK in that the effects of other receptor secretagogues or agents bypassing receptors were not altered. 4. TP-680 caused a parallel rightward shift of the dose-response curve for CCK-8-stimulated amylase release as did MK-329 and loxiglumide. However, in contrast to MK-329 and loxiglumide, TP-680 suppressed the maximal responses of CCK-8-induced amylase release in a concentration-dependent fashion, indicating that TP-680 is an unsurmountable antagonist. 5. Repeated washing of acini after a 30 min treatment with TP-680 restored the responsiveness but not the sensitivity, causing a residual inhibition on the action of CCK-8. 6. The addition of loxiglumide prior to or together with application of TP-680 protected CCK receptors from unsurmountable and irreversible antagonism by TP-680. 7. Our results indicate that TP-680 is a potent and the most selective CCKA receptor antagonist for the pancreas reported to date.

High-fat hypercaloric diet induces obesity, glucose intolerance and hyperlipidemia in normal adult male Wistar rat.

There is strong evidence that genetic factors contribute to the development of obesity in humans as well as laboratory animals. Another important factor leading to obesity is an increase in energy intake. However, it is difficult to make normal rats obese by controlling daily food intake. There is no report of normal adult male Wistar rats becoming obese and diabetic on a high-fat diet. The aim of the present study was, therefore, to make normal adult Wistar rats obese by infusing high fat and hypercaloric diet through the cannula without disturbing the free movement and to investigate the influence of an increase in the caloric intake on body weight and glucose metabolism. High-fat hypercaloric diet (360 kcal/kg body wt./day; H group) or control diet (180 kcal/kg body wt./day; C group) was continuously infused into the stomach of normal adult male Wistar rats weighing approximately 300 g through gastric cannulas for 27 days. On day 28 after a 24-h fasting, serum concentrations of aspartate aminotransferase, alanine aminotransferase, total cholesterol, triglyceride, phospholipid, and free fatty acids (FFA) were determined, and intragastric glucose loading test (2 g/kg body wt.) was performed. The average weekly body weight gain in the H group was twice as much as that of the C group (40.0 +/- 2.4 vs. 19.4 +/- 1.9 g/week, P < 0.001). Serum levels of triglyceride, phospholipid, total cholesterol, and FFA were significantly elevated in the H group compared to those in the C group. Liver weight in the H group was significantly higher than that in the C group and showed steatosis. Pancreas weight (-13%) as well as protein (-12%), amylase (-53%) and trypsin content (-26%) were all reduced, whereas pancreatic DNA content was significantly increased in the H group compared to those in the C group. Serum glucose and insulin concentrations before and after glucose loading in the H group were significantly higher than those in the C group. Moreover, the insulin response relative to glucose response in the H group was significantly high compared to that in the C group, indicating the presence of insulin resistance. These results indicate that feeding of high-fat hypercaloric diet makes normal Wistar male adult rat obese associated with hyperlipidemia, hyperinsulinemia, and glucose intolerance.

Effects of MCI-727 on pancreatic exocrine secretion and acute pancreatitis in two experimental rat models.

The effects of a newly developed compound having antiulcer action, (Z)-2-(4-methylpiperazin-1-yl)-1-[4-(2-phenyl-ethyl)phenyl]-eth anone oxime hydrochloride monohydrate (MCI-727), on pancreatic exocrine secretion were studied in anesthetized rats and evaluated its preventive and therapeutic effects on acute pancreatitis in two experimental rat models. Intraduodenal administration of MCI-727 [25, 50, or 100 mg/kg body weight (wt)] stimulated a dose-dependent increase in pancreatic juice and bicarbonate output without increasing the protein output or plasma cholecystokinin concentration. MCI-727-stimulated pancreatic exocrine secretion was completely abolished by antisecretin serum but not by the cholecystokinin receptor antagonist loxiglumide (50 mg/kg body wt/h) or cholinergic receptor antagonist atropine (100 mu g/kg body wt/h). In rats with acute pancreatitis induced by four subcutaneous injections of 20 mu g/kg body wt cerulein at hourly intervals over 3 h, MCI-727 administered orally at a dose of 100 mg/kg body wt 30 min before the first cerulein injection significantly reduced the increases in serum amylase and lipase activity and pancreatic wet weight and induced improvements in the results of histologic examination. Moreover, when given 30 min before and 90 min after the first cerulein injection, MCI-727 had even more dramatic protective effects on all these parameters. In addition, even when administered immediately after the last cerulein injection, MCI-727 effectively ameliorated all these alterations of acute pancreatitis. However, MCI-727 had no apparent beneficial effects on the biochemical and histologic alterations of acute pancreatitis in the severe form induced by retrograde intraductal injection of 1.0 ml/kg body wt of 4% sodium taurocholate. These findings suggest that oral administration of MCI-727 stimulates pancreatic exocrine secretion by endogenous secretin release and that it has therapeutic as well as preventive effects on mild forms of acute pancreatitis in rats.

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.

Enlarging splenic vein aneurysm associated with stagnation of splenic venous blood flow.

We report a case of splenic vein aneurysm (SVA) that was enlarged during a 17-month follow-up. A 62-yr-old female with liver cirrhosis was followed up in our hospital. Real-time ultrasonography initially detected aechoic space at the middle part of the splenic vein, confirmed as SVA by contrast-enhanced computed tomography, magnetic resonance imaging and angiography. Magnetic resonance imaging showed the stagnation of blood flow in SVA, which suggests the presence of hypertension in the portal venous system. Moreover, SVA enlarged in parallel with the development of esophageal varices. These observations suggest that the persistent stagnation of blood flow in the portal venous system may have played a major role in the increase in size of the SVA in this case.

Effects of tetraprenylacetone on pancreatic exocrine secretion and acute pancreatitis in two experimental models in rats.

The effects of tetraprenylacetone (TPN), an acyclic polyisoprenoid with antiulcer actions, on pancreatic exocrine secretion, and its preventive and therapeutic effects on acute pancreatitis in two experimental models were studied in rats. Intraduodenal administration of TPN (0, 100, 200, and 400 mg/kg/h) caused dose-dependent increases in pancreatic juice and bicarbonate output without increasing protein output and plasma cholecystokinin (CCK) concentrations. TPN-stimulated pancreatic exocrine secretion was completely abolished by antisecretin serum but it was not by CCK receptor antagonist loxiglumide (50 mg/kg/h). In acute pancreatitis induced by four subcutaneous injections of 20 micrograms/kg cerulein at hourly intervals over, 3 h, TPN (400 mg/kg) given by an oral route either 1 h before the first cerulein injection or immediately after the last injection significantly reduced the increases in serum amylase and lipase activities and pancreatic wet wt. Pretreatment with TPN caused histologic improvements, whereas posttreatment failed to ameliorate histologic alterations. In severe type of acute pancreatitis induced by retrograde intraductal injection of 1.0 mL/kg of 4% sodium taurocholate, TPN exerted no apparent beneficial effects on biochemical and histologic alterations of acute pancreatitis. It is concluded that TPN given by an oral route stimulates pancreatic exocrine secretion through an increase in endogenous secretin release and causes beneficial effects on the experimental model of mild acute pancreatitis in rats.

Loss of sensitivity to cholecystokinin stimulation of isolated pancreatic acini from genetically diabetic rats.

Pancreatic exocrine function of a new inbred strain Otsuka Long-Evans Tokushima Fatty (OLETF) rat that develops spontaneous persistent hyperglycemia was evaluated in in vitro isolated pancreatic acini and compared with that in the control Long-Evans Tokushima Otsuka (LETO) rat. Serum glucose and insulin concentrations in the OLETF rats were significantly high (glucose: 270 +/- 12 vs. 208 +/- 10 mg/100 ml, P < 0.01; insulin: 12.4 +/- 1.7 vs. 4.9 +/- 0.6 ng/ml, P) < 0.01), whereas pancreatic wet weight was significantly low (803 +/- 20 vs. 1,138 +/- 17 mg, P < 0.01) compared with those in the LETO rat. Pancreatic acini isolated from the OLETF rat were totally insensitive to cholecystokinin (CCK)-8 stimulation at concentrations of up to 100 nM. However, neither the responsiveness nor the sensitivity to carbamylcholine, bombesin, and secretin of the acini from the OLETF rat was altered or even increased, probably due to the larger amylase content in the OLETF rat acini compared with those of the LETO rat acini (31.5 +/- 2.0 vs. 13.0 +/- 1.1 Somogyi units/micrograms DNA, P < 0.01). The responsiveness to fluoride, a direct activator of guanine nucleotide-binding protein, in the OLETF rat acini was similar to that in the LETO rat, suggesting that the transmembrane signaling and effectors and subsequent intracellular signal transduction molecules in the OLETF rat acini are normal. Moreover, 125I-CCK binding to the acini prepared from the OLETF rat was totally absent. These present results indicate that the OLETF rat has a selective defect in the binding of CCK to its receptors on the acinar cell surface.