Insulin radiolabeled

Thirty years ago, receptors for polypeptide hormones such as insulin and GH were identified as binding activity in cells, membranes, or solubihzed membrane proteins using radiolabeled proteins... 

The insulin receptor is present in trace amounts in the plasma membrane of liver cells. Using radiolabeled arylazido derivatives of insulin, which on irradiation yield reactive nitrenes, components of the receptor with molecular weights of 135,000 and 90,000 daltons have been identified (Jacobs etal., 1979 Yip etal., 1980 Wisher etal., 1980). The identity of the two polypeptides has been confirmed by chemical crosslinking to radiolabeled insulin and by purification of the receptor by affinity chromatography. 

Figure 2.4 shows a set of absorption curves obtained for seven type I diabetic patients treated at the Steno Memorial Hospital. Each patient received a bolus injection of 7 IU (40 IU/ml) radiolabeled soluble insulin (Velosulin, Nordisk Gentofte) in the thigh, and the fraction of insulin remaining in the subcutaneous depot was measured over an 8-h interval by means of a scintillation counter. 

Alternatively, monoclonal antibodies (Mabs) to the relevant receptors can be used as transport vectors. Anti-insulin (Mab83-7 and Mab83-14) and anti-transferrin (0X26) receptor antibodies have been proposed as efficient and selective BBB transport vectors. The antitransferrin receptor antibody binds to a site removed from the transferrin binding site and therefore does not compete with endogenous transferrin for transport across the BBB. Studies using radiolabeled peptides have shown that significant uptake of a... 

As noted earlier, RIA typically depends on the use of radiolabelled antigens, and in the case of the first report in the scientific literature the antigen in question was insulin. This classic radioimmunoassay for insulin will be used as the fundamental example of how this technique operates in practice. 

Sarphie, D. Johnson, B. Cormier, M. Burkoth, T.L. Bellhouse, B.J. Bioavailability following transdermal powdered delivery (TBD) of radiolabeled insulin to hairless guinea pigs. J. Controlled Release 1997, 47, 61-69. 

Sarphie, D.F. Johnson, B. Cormier, M. Burkoth, T.L. Bellhouse, B.J. Bioavailability following transdermal powdered delivery (TPD) of radiolabeled insulin to hairless guinea pigs. J. Controlled Release 1997, 47, 61-69. Sarphie, D.F. Varadi, A. Bellhouse, B.J. Ashcroft, S. Transdermal delivery of powdered bovine insulin to diabetic and non-diabetic wistar rats. Diabetes 1995, 44 (Suppl. 1), 129a... 

Assays for insulin antibodies fall into three categories (1) quantitative radioimmunoelectrophoresis, which measures the binding of IgG antibody to radiolabeled insulin by rocket Immunoelectrophoresis into anti-IgG-containing agarose (2) RIAs with separation of bound and free insulin by precipitation with PEG or a second antibody and (3) solid phase immobilization of insulin to test tubes or Sepharose. These are discussed in more detail in Reeves. ... 

A number of reports have established that the activities of both A6 and A5 desaturases, when assayed in vitro under optimal conditions, are deficient in non-neural tissues in both streptozotocin or alloxan diabetic rats as well as in the spontaneously diabetic BB rat strain (Mercuri et al. 1966 Eck et al. 1979 Poisson 1985 Mimouri and Poisson 1990, Ramsammy 1993). Moreover, the rate of conversion of radiolabeled LA, GLA, and DHGLA to AA in the liver of diabetic rats in vivo is markedly reduced and is restored to normal by insulin administration, as is A6 desaturase activity (Eriedman et al. 1966, Poisson 1985, El Boustani et al. 1989). In contrast, in the diabetic kidney, the activity of the elongase required in AA synthesis was not affected, and other enzymes required for fatty acyl Co A formation and subsequent incorporation of acyl groups into glycerophospholipids showed increased activity (Ramsammy et al. 1993). Studies of the impact of diabetes on these enzyme activities in the nerve have not yet been reported. 

All immunoassay procedures are based on the original discovery by Berson and Yalow that low concentrations of antibodies to the antigen hormone insulin could be detected radiochemically by their ability to bind radiolabeled ( I) insulin. The determination of unknown concentrations of antigen, then, is based on the fact that radiolabeled antigen and unlabeled antigen (from the sample or a standard) compete physiochemically for the binding sites on the antibodies (radioimmunoassay, RIA). This is illustrated in Figure 24.5. 

Immunochemical methods have their origin in the medical field. The first lA, a RIA for the quantification of insulin in serum, was described by Yalow and Berson. Later, radiolabels were replaced by enzymes in EIAs by Engvall and Perlmann and Van Weeman and Schuurs. ) Since then radiolabels have obtained broad application in medical diagnostics and environmental analysis. 

Figure 6 Schematic of the first RIA, for insulin [15]. (a) Polystyrene spheres with anti-insulin antibody immobilized on surface, (b) Sample matrix containing analyte and interferents, together with radiolabelled insulin probe, are added, (c) Analyte insulin and probe radio-labelled insulin compete for antibody binding sites, (d) Matrix containing interferents, unbound analyte and unbound probe, is discarded, (e) Radioactivity of polystyrene spheres is recorded. The more anal de present in the sample, the less radioactivity is recorded.

Cultures were treated with 30 pM trans-lO, cis-12 CLA, cis-9, trans- CLA, or vehicle for 72 h, then radiolabeled glucose and oleic acid uptake, conversion to lipid, and oxidation were measured (24). Radiolabeled glucose and oleic acid uptake were lower in insulin-stimulated cultures treated with trans- 0, cis- 2 CLA compared with all other treatments. De novo lipogenesis and oleic acid conversion to lipid were lower in trans- 0, cis-12 CLA-treated cultures compared to their respective controls. Radiolabeled glucose and oleic acid oxidation were lower in cultures treated with trans-10, cis-12 CLA compared with all other treatments. These data show that trans-10, cis-12 CLA decreases human adipocyte TG content by reducing glucose and fatty acid uptake and conversion to lipid. 

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