Plasma amylin concentrations in fasted and fed rats quantified by a monoclonal immunoenzymometric assay.

Amylin is a 37-amino acid peptide co-secreted from the pancreatic beta-cell with insulin in response to nutrient stimuli. Plasma amylin concentrations in the rat are reported to vary widely. We have employed a recently-developed immunoenzymometric assay to quantify plasma amylin concentrations in fasted, fed and glucose-administered rats. Fasted amylin concentrations ranged between 1.02+/-0.09 and 1.63+/-0.15pM among three different common rat strains, and increased up to 7.70+/-0.80 pM after feeding. The differences among strains and between fasted and fed rats were all significant at P<0.01 or less. Intravenous glucose administration (5.2 mmol/kg) also significantly increased plasma amylin concentrations in fasted rats from 1.5+/-0.3pM to 3.4+/-0.5pM, and in fed rats from 4.6+/-1.1 pM to 9.1+/-1.7 pM. Plasma amylin/insulin molar ratios ranged between 2.3+/-0.2% and 3.6+/-0.5% (mean 3.0%), but did not differ among strains, or between the fasted vs fed state in any strain. In conclusion, a new sensitive immunoenzymometric assay revealed fasting plasma concentrations which are lower than previously reported, and which are significantly increased by stimulation with feeding or glucose administration.

Nephrectomy decreases amylin and pramlintide clearance in rats.

Amylin is a 37 amino acid hormone, co-secreted with insulin from the pancreatic beta-cell in response to nutrient stimuli. Because the human amylin analog, pramlintide, is being tested in patients with diabetes mellitus, a known risk factor for nephropathy, we examined the role of the kidney on amylin and pramlintide metabolism and action in functionally nephrectomized rats. Nephrectomy markedly altered amylin metabolism: it increased incremental area under the plasma amylin concentration curve 3.6-fold (P<0.001) and increased the elimination half-life from 17+/-1 to 26+/-2 minutes (P < 0.01) after subcutaneous injection of 100 microg amylin. Nephrectomy decreased plasma amylin clearance from 20.3+/-1.1 to 7.9+/-0.4 mL/min (P < 0.0001). Thus, at these doses in the rat, the kidney is important for metabolizing amylin and pramlintide.

Effects of rat amylin on renal function in the rat.

Amylin is a peptide secreted from the pancreatic beta-cell along with insulin in response to nutrient stimuli. Amylin has been reported to delay gastric emptying, inhibit glucagon secretion and gastric acid secretion, increase plasma lactate, plasma glucose and plasma renin activity, and decrease plasma calcium. Receptors for amylin have been found in the rat nucleus accumbens and the kidney. In the present experiments, amylin was administered to anesthetized rats by continuous intravenous infusions at varied rates. Amylin significantly increased urine flow at an infusion rate resulting in a plasma concentration of approximately 52 pM, and at a concentration of approximately 193 pM, it increased sodium excretion, glomerular filtration rate and renal plasma flow. Renal calcium and potassium excretion were significantly elevated at plasma amylin concentrations of approximately 52 pM and 193 pM, respectively. Higher concentrations of plasma amylin decreased plasma calcium and potassium and blunted urinary excretion of these electrolytes. Thus, of the renal responses tested, diuresis and natriuresis appeared to be the most sensitive to infused amylin. These renal effects occurred only at plasma concentrations above the normal range, but within the range of concentrations reported in insulin resistant rats.

Comparison of the in vitro and in vivo pharmacology of adrenomedullin, calcitonin gene-related peptide and amylin in rats.

Adrenomedullin has been reported to be structurally similar to a group of peptides that includes amylin, calcitonin and calcitonin gene-related peptide (CGRP). Human and rat adrenomedullin displaced [125I]CGRP from membranes of SK-N-MC cells (CGRP receptors) with affinities intermediate between those of rat amylin and rat CGRP alpha (Ki values 0.12 +/- 0.06, 0.017 +/- 0.007, 3.83 +/- 1.14 and 0.007 +/- 0.001 nM, respectively). In contrast Ki values for displacement of [125I]rat amylin from accumbens membranes (amylin receptors), and [125I]salmon calcitonin from T47D cells (calcitonin receptors) were lower than with rat amylin or rat CGRP alpha in these preparations (51 +/- 5, 34 +/- 2, 0.024 +/- 0.002, 0.31 +/- 0.07 nM, respectively, at amylin receptors; 33 +/- 5, 69 +/- 29, 2.7 +/- 1.5 and 13 +/- 3 nM, respectively, at calcitonin receptors). In anesthetized rats, the hypotensive potency of adrenomedullin was between that of amylin and CGRP alpha. In contrast, for amylin or calcitonin agonist actions (inhibition of [14C]glycogen formation in soleus muscle, hyperlactemia, hypocalcemia and inhibition of gastric emptying), human adrenomedullin was without measurable effect. Thus, in its binding behaviour and in its biological actions, adrenomedullin appeared to behave as a potent CGRP agonist, but as a poor amylin or calcitonin agonist.

Lactate production from the rat hindlimb is increased after glucose administration and is suppressed by a selective amylin antagonist: evidence for action of endogenous amylin in skeletal muscle.

By serially measuring blood flow and venous-arterial lactate differences across the hindlimb of the fasted anesthetized rat, we examined (1) whether exogenous amylin increased muscle lactate production in vivo, (2) whether glucose administration increased muscle lactate production, and (3), by using the selective amylin antagonist AC187 to block endogenous peptide, whether amylin secreted in response to glucose could mediate muscle lactate production. Abdominal aortic flow was unchanged by any treatment. Hindlimb lactate production was increased by both 100 micrograms s.c. amylin (4.0 +/- 0.4 cf 2.6 +/- 0.3 mumol/min after saline, P < 0.05) and by infusion of 2mmol D-glucose (3.0 +/- 0.2 cf 2.3 +/- 0.2 mumole/hr after saline, P < 0.03). The increase in hindlimb lactate production was prevented by infusion of AC187 (mean post-treatment venoarterial delta-lactate 140 +/- 11 microM; n.s. vs saline-treated delta-lactate 154 +/- 10 microM; P < 0.05 vs glucose-treated delta-lactate 201 +/- 14 microM). These findings are consistent with endogenous amylin secreted in response to a glucose challenge having acted at skeletal muscle to release lactate.

Gastric emptying is accelerated in diabetic BB rats and is slowed by subcutaneous injections of amylin.

Gastric emptying was measured in normal and insulin-treated spontaneously diabetic BB rats using the retention of an acaloric methylcellulose gel containing phenol red delivered by gavage. Dye content in stomachs removed after killing 20 min later was determined spectroscopically, and was compared to that in rats killed immediately after gavage to assess emptying. Diabetic rats had a markedly greater gastric emptying (90.3 +/- 1.7% passed) compared to normal Harlan Sprague Dawley rats (49.1 +/- 4.7% passed; p < 0.001) and non-diabetic BB rats (61.1 +/- 9.2% passed; p < 0.001). The pancreatic beta-cell peptide, amylin, which is deficient in insulin-dependent diabetes mellitus, dose-dependently inhibited gastric emptying in both normal and diabetic rats. The ED50 of the response in normal rats measured by phenol red and novel [3-3H]glucose gavage techniques was approximately 0.4 microgram. This dose was estimated to increase plasma amylin concentration by a mean of approximately 20 pmol/l to concentrations within the range observed in vivo. It is proposed that amylin could participate in the physiological control of nutrient entry into the duodenum and that the accelerated gastric emptying seen in BB rats could be related to their lack of amylin secretion.

Amylin regulation of carbohydrate metabolism.

This review describes how amylin may work in the control of carbohydrate metabolism by actions on gastric emptying and on muscle glycogen metabolism. Amylin, which is co-secreted with insulin from pancreatic beta-cells in response to nutrient stimuli, affects both carbohydrate absorption and carbohydrate disposal. Amylin appears to regulate carbohydrate metabolism as a partner to insulin. Defending fuel stores tends to be hierarchical; plasma glucose is defended first, then muscle glycogen, then liver glycogen, then fat. Fuel stores are replenished by both incorporating ingested nutrient and by translocating nutrient stores among body sites. Lactate may better be regarded as a vector of fuel transfer rather than a 'dead end' in metabolism. Amylin can promote the translocation of lactate from muscle to liver. The amylin effect, illustrated by the simultaneous decrease in muscle glycogen and increase in liver glycogen [53, 56], is similar to the catecholamine effect observed by Cori et al. [57]. Amylin thus may be important in maintaining liver glycogen stores via the Cori cycle and the 'indirect' glycogen synthesis pathway [58,59]. Unlike catecholamines, amylin does not mobilize fat or impede insulin action in adipose tissue [30,35]. It can supply lactate to the liver, and because lactate is a preferred lipogenic substrate [60], may thereby favour fat storage. Amylin may also help to control carbohydrate absorption via an 'entero-insular loop' to ensure that absorption from the gut remains within the regulatory limits for carbohydrate disposal by peripheral tissues. This regulatory system is essential for normal control of plasma glucose and appears to be disrupted in type-1 diabetes, an amylin-deficient state.

Monoethylglycinexylide formation in assessing pediatric donor liver function.

Lidocaine metabolism to monoethylglycinexylide (MEGX) has been described as a novel method to assess liver function in adult transplant donors and recipients. While this assay appears to offer a number of advantages over existing liver function tests, limited work has been done to evaluate its potential in the pediatric population. This study evaluated MEGX formation in potential pediatric liver donors (n = 35) and a control group of children (n = 16). The mean MEGX formation was significantly higher in pediatric donors than in the control group (156 +/- 62 vs 106 +/- 33 ng/ml, p < 0.05). No correlation with age, total bilirubin, liver transaminases, or alkaline phosphatase could be made within each group. Significant differences in MEGX levels were noted when each group was compared to its adult counterpart. Both pediatric donors and controls had greater mean MEGX formation than has been reported for adult donors and controls (156 +/- 62 vs 127 +/- 61 ng/ml, p < 0.05 and 106 +/- 33 vs 72 +/- 36 ng/ml, p < 0.05, respectively). Drugs that alter lidocaine pharmacokinetics and their potential influence on MEGX formation were evaluated in the pediatric donor group. Donors exposed to hepatic enzyme-inducing drugs had a higher mean MEGX formation (187 +/- 60 vs 146 +/- 63 ng/ml). No significant differences were noted between donors receiving and not receiving vasopressors. In conclusion, the significant differences between pediatric and adult MEGX formation should be noted when establishing reference or normal ranges for this diagnostic test. Furthermore, concomitant drug therapy may significantly alter MEGX formation.

Concentrations of phosphorylated zidovudine (ZDV) in patient leukocytes do not correlate with ZDV dose or plasma concentrations.

Zidovudine (ZDV) elicits its antiviral effect through intracellular metabolism to the 5'-triphosphate, which interferes with viral replication. Monitoring of the active metabolites of ZDV in cells could lead to an intracellular therapeutic range. This study was performed to determine whether a radioimmunoassay, previously used for in vitro quantitation of total phosphorylated ZDV inside peripheral blood leukocytes, could be used for similar determinations in patient samples. The relationship between ZDV dose, plasma concentrations, and intracellular metabolite concentrations was also examined. Ten-milliliter blood samples were drawn from each of 13 human immunodeficiency virus-infected patients and were assayed. Intracellular concentrations of phosphorylated ZDV ranged from 0.33 to 3.54 pmol/10(6) cells, similar to those observed in vitro. Phosphorylated ZDV was independent of dose, and did not correlate with plasma concentrations. Intracellular concentration in the patient population as a whole did not change during the 4-h dosing interval, while plasma concentration decayed normally. Later determinations in the same patients gave intracellular values within 31% of earlier values. Intraassay variability was less than 10%. Thus, the method is valid for measurement of phosphorylated ZDV in patient cells. Although individual concentrations showed no clear change during the 3-month study period, intracellular concentrations decreased with increasing length of therapy (up to 3 years) in the population as a whole. This suggests a decreased cellular ability to phosphorylate ZDV after prolonged exposure to drug. The lack of intracellular decay implies a half-life longer than the 1-h half-life of plasma ZDV. These data suggest that smaller doses or longer dosing intervals might maintain intracellular concentrations once steady state is achieved.(ABSTRACT TRUNCATED AT 250 WORDS)

Clinical diagnosis by nuclear magnetic resonance spectroscopy. If not now, when?

Nuclear magnetic resonance spectroscopy is the epitome of the high-technology, expensive diagnostic method. Extrapolation from a limited number of patient examinations and from experiments in animal models predicts a bright future for the method. However, several barriers block widespread clinical application in the near future; technical difficulties still exist but they seem to be resolvable in due course. A more serious problem is the absence of an adequate database from which to interpret the vast array of information produced by nuclear magnetic resonance. The necessary understanding of the pathologic biochemistry of disease will be frustratingly slow to appear as will the routine clinical use of magnetic resonance spectroscopy. The critical need for improved diagnostic methods will stimulate experimentation to resolve these problems.

Injury and recovery of the liver from preservation assessed by 31P NMR spectroscopy: the contrast between preservation with Collins' solution and Ringer's lactate solution.

The biochemistry of hepatic injury and recovery from preservation for transplantation was studied in rat liver perfused in vitro with erythrocytes. ATP and its metabolites, inorganic phosphate (Pi) and pH were quantitated as often as every 2.5 min by 31P NMR spectroscopy during preservation and recovery. Release of the hepatocellular enzymes, lactate dehydrogenase V (LDV) and aspartate aminotransferase (AST) were also measured. The duration of preservation with Collins' solution, the standard clinical preservative, affected the rate of recovery of ATP and monophosphate esters (MP), which include AMP + IMP, and the final recovery of Pi, but not of ATP. The difference between Collins' and Ringer's lactate solution, a poor preservative, became more apparent as preservation time increased. The differences included (1) pH at the end of preservative infusion; (2) pH between 0 and 2.5 min of reperfusion; (3) the MP increase (AMP + IMP) at the end of 13 h of preservation; (4) rate of recovery of ATP after preservation; (5) final ATP recovery during reperfusion; (6) LDV after 13h of preservation. These biochemical differences between good and poor preservation form a rational basis for prediction of liver failure after transplantation and for tests of the quality of new preservatives.

Effect of endotoxin-induced monokines on glucose metabolism in the muscle cell line L6.

Exposure of fully differentiated L6 myotubes to a crude monokine preparation from endotoxin-stimulated RAW 264.7 cells resulted in a rapid and substantial (70%) increase in fructose 2,6-bisphosphate concentration coincident with a depletion of cellular glycogen and an increased lactate production. During the time required for glycogen depletion (3 hr), stimulation of 3-O-methyl-D-glucose and 2-deoxy-D-glucose uptake was initiated and observed to reach a maximum enhancement of 200% 12-15 hr later. The monokine had no effect on the Km value for 2-deoxy-D-glucose uptake (1.1 mM), while Vmax was increased from 912 to 2400 pmol/min per mg of protein. The increase was cytochalasin B inhibitable and was dependent on protein synthesis. Photoaffinity labeling and equilibrium binding studies with [3H]cytochalasin B support the hypothesis that this increase in hexose transport was due to an increase in hexose transporters present in the plasma membrane. Purified recombinant interleukin-1 alpha had no effect on hexose transport, whereas purified recombinant cachetin/tumor necrosis factor did stimulate hexose uptake, with half-maximal stimulation occurring at 36 nM. Although cachetin accounts for most of the biological activity associated with the crude monokine preparations, it is not the only monokine capable of inducing glucose transport in L6 cells. Specific immunoabsorption of cachectin/tumor necrosis factor from the crude monokine preparation revealed a monokine that had a similar bioactivity at extremely low concentrations on L6 cells.