Glucagon measurement

The scope of applicability of radioimmunoassay is rapidly expanding with the dawn of each day as RIA is being developed for newer pharmaceutical substances. It has attained wide recognition and application both in vitro and in vivo measurements of compounds of interest like insulin, gastrin, glucagon, and growth hormones on one hand whereas drugs like ... 

In 1979, Ross et al 22i" measured the ODMR of tyrosine in glucagon and the derivative [12-homoarginine]glucagon to examine the effect of chemical modification of a lysine residue adjacent to Tyr-10 and Tyr-13. The guanidinated analogue had lower potency than glucagon in a fat cell hormone receptor assay. Since the tyrosine ODMR and other spectral properties of the polypeptide, including circular dichroism, were essentially identical, it was... 

Steady-state fluorescence polarization studies have been carried out with a number of peptides, including model peptides, ACTH, glucagon, melittin, and thyrocalcitonin. This work has been reviewed 5 and will not be discussed in the present article. More recently, interesting information on the rotational behavior and structural flexibility of various peptides has been obtained from fluorescence anisotropy decay measurements. 

Several tests use glucagon to diagnose endocrine disorders. In patients with type 1 diabetes mellitus, a classic research test of pancreatic beta-cell secretory reserve uses 1 mg of glucagon administered as an intravenous bolus. Because insulin-treated patients develop circulating anti-insulin antibodies that interfere with radioimmunoassays of insulin, measurements of C-peptide are used to indicate beta-cell secretion. 

An example of the response of the glucose sensor implanted subcutaneously in a rat is shown in Figure 1. The glycaemia modification was performed by an intramuscular administration of glucagon at time zero followed by insulin at time 30. The so called "apparent subcutaneous glycaemia" (6,7), calculated using one point in vivo calibration matched quite closely the concomittantly measured plasma glycaemia. 

Figure 1. Apparent subcutaneous glycaemia (closed symbols) calculated using one point calibration compared to the actual plasma glycaemia (open symbols), measured by conventional method. Upper pannel shows the sensor response after 4 hours of implantation and the lower one after 28 hours of implantation. Conditions intramuscular administration of glucagon (2.6 ig) at time zero followed by insulin (100 mU) at time 30. ...

There is abundant evidence that glucagon elevates cAMP levels in isolated liver parenchymal cells, in perfused liver and in the liver in vivo [58,59], As illustrated in Fig. 2, this occurs rapidly and with concentrations of the hormone [59] within the range found in portal venous blood in vivo i.e., 0.2-2 x 10-10 M. When sufficiently sensitive and accurate methods are employed to measure cAMP, an increase in the nucleotide is consistently observed in situations where the hormone induces metabolic responses [58,59]. However, an increase of only 2- to 3-fold is capable of inducing full stimulation of some major hepatic responses, e.g., phos-phorylase activation (Fig. 2) and gluconeogenesis [58,59]. Since higher concentrations of the hormone can elevate cAMP 10-fold or more [59] it appears that there is considerable receptor reserve for these responses. 

Early studies of the stimulatory effects of glucagon on hepatic gluconeogenesis using different gluconeogenic substrates and measuring the changes in the concentrations of intermediary metabolites identified the substrate cycles between pyruvate and P-enolpyruvate and between fructose-1,6-P2 and fructose-6-P as major sites of... 

Fig. 8. Effects of agonists on cytosolic Ca2+ (measured by fluorescence) in isolated rat hepatocytes loaded with Quin 2. The concentrations of agonists are Epi, 1 /uM epinephrine Phenyl, 10 fiM phenylephrine Vaso, 10 nM vasopressin Glue, 10 nM glucagon. Reproduced from Ref. 3 by permission of the authors and publisher.

Fig. 9. Effects of hormones on the calcium content of isolated rat hepatocytes. Hepatocytes were incubated for 5 min in medium containing 100 p.M Ca with hormones at the concentrations shown and the calcium content measured by atomic absorption spectroscopy. Glu is glucagon and Angio is angiotensin II. Reproduced from Ref. 5 by permission of the authors and publisher.

FIGURE I 5 Illustrative examples of the retention dependencies for the polypeptide hormones, /3-endorphin and glucagon, as plots of the experimentally determined In k (proportional to AG°ssoc, ) versus the volume fraction of organic solvent measured with a n-butylsilica reversed-phase sorbent at temperatures from 5° to 8S°C under isocratic elution conditions at a flow rate of I mL/min, encompassing the range of acetonitrile concentrations from 0.25 < ip < 0.30. Data from Ref. 237. 

Despite these findings, other data suggest that mitochondrial membranes may be a target for glucagon emd phenylephrine action. Measurements of metabolite gradients in situ have shown that endogenous adenine nucleotide levels are increased by... 

In the liver, isoprenaline-stimulated adenylate cyclase has been found to be located almost exclusively on the surface of the parenchymal cells, with little or no deposit on the surface of the reticulo-endothelial cells. In contrast, the predominant effect of glucagon resulted in the deposition of reaction product on the reticulo-endothelial cell surface, although deposits were also present on the parenchymal cells. In the presence of F , there were substantial deposits on the surface of both types of cells. These results demonstrate that distinct enzyme systems are present in parenchymal and reticulo-endothelial cells. No theories concerning the function of adenylate cyclase in the liver have considered its role in the endothelial cells which act mainly on phagocytes. These cells contribute 35% of the cells in the liver and can be expected to contribute substantially to biochemical measurements of adenylate cyclase activity and cyclic AMP concentrations when liver slices, homogenates, or cell fractions constitute the enzyme source. 

FIGURE 4.4 Changes in plasma metabolite and hormone levels with exercise. The human subjects were at n st at time zero. Exercise was coitiinucd for 250 min, and concentrations of plasma glucose (O), glucagon (A), and insulin ) were measured at the indicated times. (Redrawn with permission from Felig and Wahren, J975.)... 

FIGURE 4.7 Plasma glucose levels after a glucagon injection. Normal (9) and diabetic (O) human subjects were injected with 1.0 mg of glucagon. Plasma glucose and C-peptide levels were measured before and at the indicated times following the injection. (Redrawn with permission from Marchesini ei a ., 1985.)... 

The study depicted in Figure 4.9 continues the discussion of the delicate relationships between plasma levels of glucose, glucagon, and insulin. In this study, normal human subjects were infused with insulin for the 1-hour period indicated by the black bar at the top. Glucose and hormone levels were measured prior to, during, and after the infusion. 

FIGURE 4,9 I ia ma glucose and hormone le> els before, during, and after Insulin infusion. Normal human subjects were infused with insulin for the I-hour period Indicated by the black bar. Glucose, in.sulin, and glucagon levels were measured at the indicated times. While plasma insulin increased to six times its basal level, plasma glucose fell precipitously and plasma glucagon rose. (Redrawn with permission from Boili et at., 1984.J... 

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