Binding and degradation of 125I-insulin by renal glomeruli and tubules isolated from rats

Summary Isolated rat renal glomeruli and tubules were shown to exhibit specific binding of ¹²⁵I-insulin and enzymatic degradation of the hormone. Binding to both renal fractions reached a plateau by 1h at 22 °C and increased linearly with increasing protein concentrations. Binding was inhibited in both preparations by insulin and its analogues in the order of relative potency: insulin > despentapeptide insulin > proinsulin, but insulin was ten times more potent in inhibiting ¹²⁵I-insulin binding to glomeruli than that to tubules, indicating a different affinity of receptors for the hormone in the two renal fractions (about 17 versus 210 g unlabelled insulin/l inhibiting 50% of the ¹²⁵I-insulin binding to glomeruli and tubules, respectively). Bound ¹²⁵I-insulin dissociated at a faster rate from tubules than from glomeruli; this release was accelerated by unlabelled insulin in both renal fractions, but to a greater extent in glomeruli than in tubules. Two-thirds of the total bound material released from glomeruli was found to be intact insulin as measured by trichloroacetic acid precipitation, whereas only one-third of the material released from tubules was intact. No direct relationship between binding and degradation of ¹²⁵I-insulin in these renal fractions could be demonstrated, however, because of the release of proteolytic enzymes into the incubation medium resulting in almost all degradation being extracellular. Although differing in their affinity for ¹²⁵I-insulin the high affinity glomerular insulin receptor and the lower affinity tubular insulin receptor have characteristics similar to those of insulin receptors in insulin responsive tissues.     

Different binding and degradation of proinsulin,insulin and insulin-like growth factor-1 (IGF-1) in cultured renal proximal tubular cells

Recent studies have reported that elevated proinsulin levels are indicative of an increased cardiovascular risk. Renal proximal tubular cells represent a major site for the metabolism of insulin-like hormones after glomerular filtration into the tubular lumen. To determine the binding and degradation of proinsulin in comparison with insulin and insulin-like growth factor-1 (IGF-1), we have used a rabbit proximal tubular cell line (PT-1). As confirmed by electron microscopy, PT-1 cells exhibit bipolar differentiation, demonstrating apical microvilli and invaginations of the basolateral membrane. To allow selective incubation of both compartments, cells were grown on filter membranes. Performing equilibrium binding assays with¹²⁵I-labelled hormones, severalfold higher binding was found at the apical than at the basolateral cell membrane, with the capacity range IGF-1>insulin>proinsulin. Half-maximal displacement of¹²⁵I-labelled insulin and IGF-1 was observed at 0.6 and 2 nM, respectively, while crossover binding to the alternate receptor occurred with a 10- to 100-fold lower affinity. Half-maximal displacement of¹²⁵I-proinsulin binding was obtained at approx. 8 nM proinsulin and insulin, whereas IGF-1 was 10-fold less potent. The relative degradation of specifically bound tracer was lowest for proinsulin (apical: 10%, basolateral: 13%). IGF-1 was degraded by 20% at the apical cell membrane, and up to 78% at the basolateral membrane. In contrast, almost the total amount of insulin bound was degraded at both membrane sites (apical: 99%, basolateral: 83%). These results suggest separate insulin and IGF-1 receptors, while proinsulin binds with high affinity to a third insulin-like receptor on the apical membrane of PT-1 cells.     

Effects of anti-insulin antibody on insulin binding to liver membranes: evidence against antibody-induced enhancement of insulin binding to the insulin receptor

Summary In the presence of anti-insulin antibody, 2- to 3-fold enhancement of ¹²⁵I-insulin binding to liver membranes was observed when binding was estimated by the radioactivity of ¹²⁵I-insulin bound to the membrane pellets. However, after ¹²⁵'I-insulin was covalently cross-linked to liver membranes using disuccinimidyl suberate in the presence of anti-insulin antibody, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography showed that ¹²⁵I-insulin bound to the -subunit of the insulin receptor was inhibited by antiinsulin antibody in an dose-dependent manner. More importantly, at an anti-insulin antibody dilution range between 1:50 and 1:5,000, sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed two ¹²⁵I-labelled bands of mol wt 62,000 and 27,000, while only one band of mol wt 130,000 was revealed in the absence of anti-insulin antibody. These Mr=62,000 and Mr=27,000 bands were found to be the heavy and the light chain of anti-insulin IgG molecules respectively. Pepsin digested anti-insulin serum had only an inhibitory effect on ¹²⁵I-insulin binding to liver membranes. Non-immunized guinea pig serum or IgG completely abolished the enhanced effect of anti-insulin antibody. Further, this enhanced effect was inhibited by Fc fragment-specific anti-IgG serum or H&L-chain-specific anti-IgG serum in a dosedependent manner. Protein A also inhibited the effect of antiinsulin antibody. In IM-9 lymphocytes and human red blood cell ghosts, which have no Fcy receptors, enhancement of insulin binding was not observed in the presence of anti-insulin antibody. These data suggest that anti-insulin antibody-induced enhancement of insulin binding to liver membranes is not due to the enhanced binding to the insulin receptor itself but probably due to the binding of insulin-anti-insulin antibody complex to the Fc receptor.     

Evidence for the presence of insulin binding sites in isolated rat intestinal epithelial cells

Summary Insulin receptors have been demonstrated in isolated rat intestinal epithelial cells. The specific binding of ¹²⁵I-insulin was time — and temperature — dependent, the optimal temperature of study being 15°. Dissociation of bound ¹²⁵I-insulin by an excess of unlabelled hormone was rapid and attained 66±2% in 2 h. When initiated by dilution, the dissociation attained 35±4% in 2 h, and 72±1% in 2 h when 10^-7mol/l unlabelled insulin was added. The pH optimum for the binding process was between 7.5 and 8, and the binding increased proportionally to cell protein concentration up to 1.5 mg/ml. Under standard conditions (2 h at 15°) the degradation of the labelled hormone in the medium accounted for 20–50% of total tracer, depending on the concentration of cells. At apparent equilibrium (2 h at 15°), unlabelled insulin in the range of 10^-10 to 10^-7 mol/l inhibited competitively the binding of 4.3–7× 10^-11 mol/l ¹²⁵I-insulin; fifty per cent inhibition was obtained with 3×10^-9 mol/l native insulin. Scatchard analysis, after correction for degradation, gave curvilinear plots, that may be explained by two orders of binding sites, with 2,000±200 sites/cell of high affinity (Ka=2.2±0.2×10⁹l/mol) and 39,000±3,000 sites/cell of low affinity (Ka=5.6±1.6× 10⁷l/mol). The potency of proinsulin to compete with ¹²⁵I-insulin for the binding site was 3% that of insulin, unrelated peptides were inactive. Such results give a molecular basis to different reports suggesting that the intestine could be a target-tissue for insulin.Presented at the 10th Meeting of the International Diabetes Federation, Vienna, 1979     

Binding and biological effects of insulin,insulin analogues and insulin-like growth factors in rat aortic smooth muscle cells. Comparison of maximal growth promoting activities

Summary Binding and growth promoting effects of insulin, insulin analogues modified in the B chain, proinsulin, insulin-like growth factor-I and -II were studied in cultured rat aortic smooth muscle cells. Specific binding of¹²⁵I-insulin was 0.9±0.2% of total ¹²⁵I-insulin added, and the IC₅₀-value was estimated to 8.9 pmol/1. The insulin analogue B10 Asp tended to be more potent than insulin in displacing ¹²⁵I-insulin, B28 Asp was equipotent, B9 Asp/B27 Glu was approximately 100 times less potent and insulin-like growth factor-I more than 1000 times less potent than insulin. Specific binding of ¹²⁵I-insulin-like growth factor-I after 4 h incubation at 10 °C was five times higher than the specific binding of insulin (4.4±0.4% of total ¹²⁵I-insulin-like growth factor-I added), and the IC₅₀-value was 0.3 nmol/l. Insulin was approximately 500 times less potent than insulin-like growth factor-I in displacing ¹²⁵I-insulin-like growth factor-I. The insulin analogue B10 Asp was slightly more potent and analogue B28 Asp was equipotent with insulin. Analogue B9 Asp/B27 Glu was ten times less potent and proinsulin was more than ten times less potent than insulin. The order of potency was similar for ³H-thymidine incorporation into DNA: insulin-like growth factor-I > B10 Asp > insulin-like growth factor-II > insulin > B28 Asp > B9 Asp/B27 Glu > proinsulin. The maximal effect of insulin-like growth factor-I on ³H-thymidine incorporation was 71±16% higher than the maximal effect of insulin. The maximal effect of insulin-like growth factor-II was at least as high as the effect of insulin-like growth factor-I. Furthermore, the maximal effect of B10 Asp was 62±10% higher than the maximal effect of insulin. Insulin-like growth factor-I and B10 Asp tended to increase cell number more than insulin. In conclusion, this study shows that insulin analogues interact with different potencies with receptors for insulin and insulin-like growth factor-I in vascular smooth muscle cells and that insulin-like growth factors and the insulin analogue B10 Asp have more pronounced growth effects than insulin. Substitution of the amino acid Asp for His at position B10 in insulin makes the molecule more similar to insulin-like growth factor-I, chemically and probably also biologically.     

Resistance of the insulin crystal to lysosomal proteases: implications for pancreatic B-cell crinophagy

Summary Insulin is thought to be chemically stabilized within -granules in the crystal form. The other major products of the -granule, proinsulin and C-peptide, by contrast, are not thought able to crystallize. The physico-chemical properties of peptides in soluble or crystalline form are dramatically different. The ability of insulin to crystallize in the -granule might thus explain why this peptide, but not proinsulin/Cpeptide, remains stable even after its introduction into lysosomes as occurs during granulolysis (crinophagy). We have now studied this by exposing proinsulin or insulin to lysosomal proteases in vitro. ¹²⁵I-insulin in soluble form was found to be degraded at the same rate as ¹²⁵I-proinsulin. Strikingly, however, when the labelled insulin was crystallized, its rate of degradation was decreased from 1.9 to 0.2 pmol/min. We take these data as confirmation that the insulin crystal is resistant to degradation, thereby possibly accounting for (a) the presence of insulin immunoreactivity within multigranular bodies, and (b) the unusually slow rate of degradation of insulin within B cells compared with that of other hormones in their cells of origin.     

Delayed intracellular dissociation of the insulin-receptor complex impairs receptor recycling and insulin processing in cultured Epstein-Barr virus-transformed lymphocytes from insulin-resistant subjects

Summary Insulin-receptor internalization and processing are defective in insulin-resistant subjects. To assess the reversibility of these defects, we cultured Epstein-Barr virus-transformed-lymphoblasts from six normal, six obese, and six non-insulin-dependent diabetic (NIDDM) subjects in media containing low (5 mmol/l) or high (25 mmol/l) glucose concentrations, and studied the insulin-receptor internalization and processing in vitro. In cells from normal, obese, and NIDDM subjects cultured in low glucose concentrations, exposure to 100 nmol/l insulin for 30 min at 37C reduced cell-surface ¹²⁵I-insulin binding to a similar extent (82±2, 77±5, and 82±5 % of initial values, respectively). The same results were obtained with cells cultured in high glucose concentrations. In cells cultured under both glucose conditions, and exposed to 100 nmol/l insulin for 30 min at 37C, a complete recovery of the initial ¹²⁵I-insulin binding was observed in normal but not in obese and NIDDM subjects. Release of intracellular insulin and its degradation in vitro was determined by incubating cells with 600 pmol/l of ¹²⁵I-insulin for 60 min at 37C, acid washing cells, and re-incubating in insulin-free buffer at 37C. The radioactivity released by cells was characterized by trichloroacetic acid precipitability, Sephadex G-50 column Chromatograph, and rebinding to fresh cells. Rates of release of internalized radioactivity were reduced in obese and NIDDM subjects (t_1/2=61±9 min, p<0.02; 58±10 min, p<0.05; and 38±4 min in obese, NIDDM, and normal subjects, respectively). The percentage of intact insulin released from cells was significantly higher in obese and NIDDM subjects than in the normal subjects. The t_1/2 of intracellular dissociation of insulin-receptor complexes measured by a polyethylene glycol assay was lower in normal (6±1 min) than in obese (12±2 min, p<0.03) and NIDDM subjects (14±3 min, p<0.02). The results suggest that in insulin-resistant subjects a primary defect in intracellular dissociation of insulin is responsible for alterations of receptor recycling and insulin processing.Abbreviations NIDDM Non-insulin-dependent diabetes mellitus - EBV Epstein-Barr virus - RPMI - FCS fetal calf serum - PEG polyethylene glycol - ANOVA analysis of variance     

Rapid regulation of plasma membrane insulin receptors

Summary Injection IP of insulin at a dosage of 1 g/g body weight into normal rats produced a rapid rise in serum insulin levels from < 1 to 298 ng/ml, and a rapid decrease in specific ¹²⁵I-insulin binding to its receptors in purified liver plasma membranes. A fall in binding was seen as early as 10 minutes after injection and binding remained decreased for up to 60 min. At 10 min, ¹²⁵I-insulin binding had fallen to 59% of controls; in contrast, ¹²⁵I-glucagon binding remained unchanged. Extraction of these plasma membranes followed by radioimmunoassay for insulin did not reveal appreciable amounts of exogenous insulin. The ¹²⁵I-insulin dissociation rate from plasma membranes of control and insulin treated rats was the same, also indicating a lack of exogenous insulin. Scatchard analyses indicated that the decreased binding seen after insulin injection was due primarily to a change in the number of insulin receptors and not their affinity. These studies suggest, therefore, that high doses of insulin in vivo can rapidly regulate the number of plasma membrane insulin receptors in liver.     

Ontogenesis of insulin processing in fetal rat hepatocytes

Summary We studied insulin processing and hepatic glycogenesis in cultured hepatocytes isolated from rat fetuses of 17, 19, and 21 days of gestation. Steady-state insulin binding increased by 250% between days 17 and 19, from 145±8 to 361±52 fmol/mg protein, and by an additional 40% (405±69 fmol/mg protein) by 21 days of gestation. At 37°C, ¹²⁵I-insulin was rapidly (t_1/2<5 min) internalized by hepatocytes at all three ages, reaching maximal levels (63–76% of the total cell-associated radioactivity) by 15 min. ¹²⁵I-labelled degradation products appeared rapidly (t_1/2<15 min) within the cells. Yet, the majority (68–77%) of the intracellular radioactivity consisted of intact ¹²⁵I-insulin, even after 4 h at 37°C. Hepatocytes pre-loaded with ¹²⁵I-insulin and then acid-stripped of surface-bound radioactivity, rapidly released both intact ¹²⁵I-insulin (retroendocytosis) and its radiolabelled degradation products. While intact insulin was initially released more rapidly (t_1/2<6 min), and reached a plateau after 15–30 min, the degradation products continued to accumulate in the medium for at least 4 h. Methylamine inhibited intracellular ¹²⁵I-insulin degradation at all three gestational ages and also blocked insulin-stimulated glycogenesis in 19- and 21-day hepatocytes, without altering basal glycogen synthesis. Insulin-stimulated glycogenesis was not induced in 17-day fetal rat hepatocytes in control or methylamine-treated cultures. We conclude that both degradative and retroendocytotic pathways for processing insulin are present in fetal rat hepatocytes by 17 days of gestation. Further, insulin-receptor processing was functionally related to the glycogenic action of insulin in responsive 19- and 21-day fetal rat hepatocytes     

Immunoprecipitation of 125I-insulin crosslinked receptor

Summary ¹²⁵I-insulin was covalently crosslinked to its receptor on human placental membrane fractions with disuccinimidyl suberate. The ¹²⁵I-insulin crosslinked receptor was solubilized with Triton X-100 and used as a probe to determine autoantibodies to the insulin receptor in sera from patients with insulin resistance (type B syndrome). When the solubilized ¹²⁵I-insulin: receptor complex was incubated with these sera and then with anti-human IgG serum, the complex was precipitated as a function of the amount of anti-receptor serum. Unlabelled insulin at concentrations 10 g/ml did not affect immunoprecipitation of the complex, suggesting that a subpopulation of anti-receptor antibodies recognizes determinants outside the insulin binding region of the receptor molecule. ¹²⁵I-insulin: receptor complex was precipitated also by the addition of anti-insulin antibodies. The effect of anti-insulin antibody was eliminated by the addition of excess unlabelled insulin. The immunoprecipitation assay using ¹²⁵I-insulin crosslinked receptor was sensitive and convenient for detecting anti-receptor antibodies.     

Inhibition of insulin-stimulated xylose uptake in denervated rat soleus muscle: a post-receptor effect

Summary Insulin (100 U/l) stimulated xylose uptake in rat soleus muscle from a basal value of 2.3±0.5 to 11.6±2.1 mol · g^-1 · h^-1. Denervation (section of the sciatic nerve) markedly reduced the stimulatory action of insulin (basal 1.3 ±0.4 mol · g^-1 · h^-1; insulin-stimulated 4.5±0.6 mol · g^-1 · h^-1). This effect appeared 3 days after denervation and was maximal after 5 days. Denervation also affected the insulin dose response curve; there was no effect of insulin in denervated muscle until the concentration exceeded 1 U/l. Denervation inhibited insulin-stimulated -aminoisobutyrate uptake by 77% but did not affect ¹²⁵I-insulin binding or glucose-independent activation of glycogen synthase by insulin. There was no effect of denervation on the insulinomimetic effects of concanavalin A, hydrogen peroxide, vitamin K₅, anoxia, 24-dinitrophenol, cooling, hyperosmolarity or EDTA, but the effect of diamide was inhibited. It is concluded [1] that denervation inhibits insulin-stimulated sugar transport at some early post-receptor step, and [2] that the mechanism whereby insulin activates glycogen synthase is different from the activation of the membrane transport of sugars and amino acids.     

The effect of streptozotocin-induced diabetes on the early steps of glucagon action in isolated rat liver cells

Summary This study was undertaken to investigate the effect of experimental diabetes on the early steps of glucagon action. The binding of glucagon and glucagon-stimulated cyclic AMP accumulation in the presence of a potent phosphodiesterase inhibitor (IBMX, 0.1 mmol/l) were studied in liver cells isolated from control and streptozotocin-induced (65 mg/kg) diabetic rats. Comparative studies of insulin binding indicated that hepatocytes of diabetic rats bound twice as much ¹²⁵I-insulin (10.8±2.0%) as those of control rats (5.7±1.3%). Scatchard analysis and the competition plots of the data suggested that this was due to an increased number of receptors rather a change in their affinity. No significant change was observed in ¹²⁵I-glucagon binding of diabetic liver cells (5.8±0.5%) as compared to controls (6.8±0.4%). The number of molecules of glucagon bound to high and low affinity binding sites of control liver cells was (51±2)×10³ and (1300+134)×10³ sites/cell, respectively. The corresponding numbers in streptozotocin-treated rats were (45±5) ×10³ and (1000±167)×10³ sites/cell, respectively. Cyclic AMP response to concentrations of glucagon below 1 nmol/l was significantly lower in diabetics than in normals: for 0.3 nmol/l and 0.6 nmol/l of glucagon, cyclic AMP production was 48±7 pmol/ 10⁶ cells and 78±8 pmol/10⁶ cells in diabetics, as compared to 72±9 and 110±9 pmol/10⁶ cells in normals. At concentrations of glucagon that are maximally efficient (7 nmol/l) cyclic AMP production was higher in diabetic (202±20 pmol/10⁶ cells) than in normal rats (156±7 pmol/10⁶ cells). Thus, diabetes seems to increase the quantity of adenylate cyclase and decrease its affinity for glucagon. Those changes are not related to a modification of the glucagon binding sites and are associated to an increase of insulin receptors.Transcribed in the previous publications as Tchamras     

Proinsulin:insulin and insulin:glucose ratios as predictors of carotid plaque growth: a population-based 7 year follow-up of the Tromsø Study

Aims/hypothesis

Proinsulin is increased in persons at cardiovascular risk. Increased secretion of proinsulin relative to insulin has been suggested as a sign of defective conversion of proinsulin to insulin and C-peptide and is associated with beta cell dysfunction. It has also been suggested that proinsulin has more of a pro-atherogenic effect than insulin, the levels of which are also increased in the insulin resistance state. In this prospective population-based study, we examined whether the proinsulin:insulin ratio (PIR) or insulin:glucose ratio (IGR, an insulin resistance surrogate) predicted carotid plaque size in nondiabetic participants.

Materials and methods

The study included 1,859 men and 1,998 women aged 25–82 years from the Tromsø Study, who were examined with B-mode high resolution ultrasound at baseline in 1994–1995 and at follow-up in 2001–2002. All images were computer processed to yield mm² measures of plaque. Proinsulin and insulin were measured at baseline. All analyses were stratified for sex.

Results

After adjusting for age, baseline plaque area, BMI, cholesterol, HDL-cholesterol, HbA_1c, IGR, albumin:creatinine ratio, fibrinogen, BP and lifestyle factors (tobacco smoking, alcohol consumption, physical activity), PIR was significantly associated with plaque size at follow-up in women but not men. For each SD in the PIR in women, the mean plaque area increased by 0.97 mm² (95% CI 0.44–1.50). IGR was not associated with carotid plaque size.

Conclusions/interpretation

The PIR is associated with progressive carotid artery plaque size in women.

     

Insulin receptor tyrosine kinase activity is reduced in monocytes from non-obese normoglycaemic insulin-resistant subjects

Summary Insulin sensitivity has been quantified by i. v. insulin tolerance test (0.1 U/kg of body weight) in 18 (11 male/7 female) non-obese (body mass index range 19–25 kg/m²) normoglycaemic subjects. We then compared the tyrosine kinase activity and internalization of insulin receptor in monocytes from the six most insulin-sensitive (group 1) and the six most insulin-resistant (group 3) subjects. Tyrosine kinase activity was measured on immunopurified receptors using ³²P-ATP and poly-glutamic acid 4: tyrosine 1, sodium salt (poly-glu-tyr 41). Insulin internalization was studied by incubating cells with 1 nmol/l ¹²⁵I-insulin and measuring total cell-bound and intracellular ¹²⁵I-insulin by an acid dissociation procedure. Basal (in the absence of insulin) receptor kinase activity was similar in both groups. Maximal (in the presence of 100 nmol/l insulin) kinase activity was 41% lower in group 3 (13.8±3.6 fmoles ³²P-ATP incorporated vs 23.3±4.0, p=0.1). Delta increment of receptor kinase activity after insulin stimulation (calculated by subtracting basal from maximal activity) was significantly (p<0.05) reduced in group 3 (21.3±3.8 vs 11.1±2.1) and significantly (p<0.05) correlated to the in vivo insulin sensitivity. Both total cell-bound (0.70±0.09 % of total radioactivity added vs 0.83±0.15) and intracellular (0.39±0.05 vs 0.44±0.09) ¹²⁵I-insulin were similar in the two groups. These data suggest that in non-obese, normoglycaemic subjects a defective insulin receptor tyrosine kinase activity may contribute to the development of insulin resistance. This raises the possibility that the reduced receptor kinase activity observed in Type 2 (non-insulin-dependent) diabetic patients may be independent from the diabetes and may in fact precede the appearance of the disease.     

In situ binding of islet hormones in the isolated perfused rat pancreas: evidence for local high concentrations of islet hormones via the islet-acinar axis

Summary Insulin and somatostatin reportedly affect pancreatic acinar cell function via specific receptor binding. Theoretically peri-insular levels depend on the islet-acinar portal system, but the actual hormone levels have never been demonstrated. Rat pancreata were perfused anterogradely or retrogradely with ¹²⁵I-insulin, -somatostatin, or -glucagon (each, 10^–11 mol/l). Tracer binding was determined from differences between influx and efflux radioactivity. Saturable binding was observed for insulin and somatostatin, but not for glucagon. Binding in the absence of unlabelled peptides was significantly higher during retrograde perfusion than during anterograde perfusion for insulin (25.9±2.6 vs 16.0±2.1%, mean±SD; each, n=4; p<0.001) and somatostatin (18.4±2.0 vs 13.6±1.2%; each, n=3; p<0.05). Non-specific binding was similar in both directions. These findings are attributable to endogenous hormones acting as unlabelled ligands competing with the tracers during anterograde perfusion. This conclusion was supported by the demonstration that endogenous insulin stimulation by d-glucose, but not by l-glucose, caused a decrease in labelled insulin binding only during anterograde perfusion. Displacement curves obtained during retrograde perfusion showed that interstitial concentrations of insulin and somatostatin were 7.5×10^–9 and 1.1×10^–9 mol/l, respectively. Thus, the exocrine pancreas is indeed exposed to locally high concentrations of islet hormones.Abbreviations SS Somatostatin - TCA trichloroacetic acid     

Hypoglycaemia caused by atypical insulin antibodies in a patient with benign monoclonal gammopathy

Abstract. We describe a 48-year-old woman with recurrent severe hypoglycaemia apparently caused by a paraprotein with insulin-binding capacity. Very high fasting values were found for serum insulin (170 and > 250 mU l^?1) as well as for proinsulin 125 pmol l^?1 and an insulinoma was suspected. Hypoglycaemia developed after an oral glucose tolerance (OGTT) test but not during fasting for 48 h. Free insulin and C-peptide were normal during OGTT whereas serum insulin was very high. ¹²⁵I-insulin binding to serum, determined with a polyethylene glycol (PEG) precipitation method was high (40%), and equally high after addition of 1.7 × 10^?5 mol l^?1 cold insulin to estimate non-specific binding. By adding very high concentrations of cold insulin, displacement of ¹²⁵I-insulin bound to serum was found (50% displacement at 4 × 10^?5 mol l^?1). No immunoglobulin G (IgG) insulin antibodies were detected by radio-immunoelectrophoresis. On agarose electrophoresis a small paraprotein (4 g l^?1) in the γ-globulin fraction was detected. ¹²⁵I-insulin binding to this paraprotein was demonstrated. We conclude that if insulin autoantibodies are suspected as a cause of hypoglycaemia screening for insulin antibodies should always be done with a PEG-precipitation method.     

Insulin receptors in cultured mouse retinal cells

Summary The binding of¹²⁵I-insulin to uncloned and cloned cultures of mouse retinal cells has been investigated. At 15° C, binding of the hormone reached a steady state by 60 min, while at 37° C equilibrium was reached earlier but at a lower level than at 15° C. Porcine insulin, porcine proinsulin and guinea pig insulin displaced labelled insulin in proportion to their known biological potency. A sharp pH dependence of the hormone binding was observed with an optimum at pH 7.8. The dissociation rate of the¹²⁵I-insulin was increased in the presence of unlabelled hormone, suggesting the existence of negative cooperativity in the insulin-receptor interaction. The availability of established retinal cell lines with insulin receptors should facilitate the study of the insulin-retina interactions in a controlled in vitro system.     

Assessment of proinsulin's effects on intermediary metabolism using the forearm technique in normal man

We have compared the effects of human proinsulin and insulin on forearm metabolism. Seven normal, non-obese subjects were infused with 386 pmol/kg per hour of proinsulin and 180 pmol/kg per hour of insulin using the euglycaemic clamp technique. Glucose appearance and utilization rates were quantified using a primed continuous infusion of [6,6-²H₂]glucose. Mean blood glucose was 4.1±0.1 and 4.1±0.2 mmol/l during proinsulin and insulin infusions respectively. Basal insulin concentrations increased from 0.02±0.01 to 0.25±0.03 nmol/l. The proinsulin infusion was chosen to give steady-state levels approximately 20-fold higher on a molar basis than those of insulin, based on previous findings that proinsulin has only 5% the biological potency of insulin. Basal proinsulin concentrations increased from 0.003 to 5.4±0.3 nmol/l. Hepatic glucose production was suppressed similarly during the last hour of each hormone infusion: 0.07±0.16 (proinsulin, P), and 0.01±0.13 (insulin, I) mg/kg per minute. Glucose disposal, however, was significantly increased during the final hour of the insulin infusion: 4.7±0.4 (I) and 3.4±0.2 (P) mg/kg per minute (P=0.025). Net forearm glucose uptake (FGU) increased by a greater amount during insulin compared with proinsulin infusion: 1.44±0.02 (I) and 0.71±0.01 (P) mol/100 ml forearm per minute (P<0.02). There was a small but significant net drop in arterialized blood lactate and pyruvate concentrations during proinsulin compared with insulin infusion: lactate –43±29 (P) and +63±35 (I) mol/l (P<0.01); pyruvate –8±3 (P) and +6±2 (I) mol/l (P<0.02). Arterialized blood alanine concentrations were similar during both series of hormone infusions. Forearm production and arterialized concentrations of glycerol were suppressed by equal amounts during the last hour of each hormone infusion. Despite greater FGU during insulin infusion, forearm production of lactate, pyruvate and alanine were similar during the last hour of each glucose clamp. These results indicate that in overnight fasted normal man: (1) proinsulin may have a preferential effect on the liver compared with muscle in terms of glucose handling; (2) proinsulin is less effective in stimulating FGU than is insulin; (3) from calculation of carbon flux across the forearm, proportionally less glucose was oxidized or stored during infusion of proinsulin compared with insulin; (4) proinsulin has similar effects on forearm lipolysis compared with insulin; (5) proinsulin may have a differential effect on splanchnic lactate metabolism compared with insulin.     

Identification of insulin receptors on rat Sertoli cells

The binding of insulin to rat Sertoli cells was investigated to establish if effects of insulin on Sertoli cells can be mediated via insulin receptors. Sertoli cells were isolated from the testes of 3-week-old rats, and preincubated for 3 days in the absence of hormones. Binding of 125I-porcine insulin to the Sertoli cells was 75-80% specific and this binding was time- and pH-dependent and reversible. Scatchard analysis of the binding data resulted in curvilinear plots with a high affinity binding of Kd = 1.8 X 10(-9) M. Porcine and bovine insulin competed equally well for 125I-porcine insulin binding. Porcine proinsulin was 10-50 times less potent, corresponding to its lower biological activity. Insulin-like growth factor-I (IGF-I) was 30-40 times less potent, indicating low affinity binding of IGF-I to the insulin receptor. Lutropin which was used as a control gave no competition with the 125I-insulin binding. Affinity labelling of Sertoli cell membrane proteins with 125I-insulin using the cross-linking agent disuccinimidylsuberate revealed binding of insulin to (a) protein(s) of Mr greater than 300,000 or Mr = 130,000 after electrophoresis under non-reducing or reducing conditions, respectively. Affinity labelling with 125I-insulin was largely prevented by unlabelled insulin. It is concluded that the protein of Mr 130,000 may represent the alpha-subunit of the insulin receptor. The presence of insulin receptors as well as IGF-I receptors on cultured rat Sertoli cells may suggest that insulin and IGF-I have specific functions in regulating the maturation and activities of Sertoli cells during the initiation and maintenance of spermatogenesis.     

Measurement of human proinsulin by an indirect two-site immunoradiometric assay

Summary An indirect two-site immunoradiometric assay is described for the measurement of human proinsulin in plasma. Polyethylene tubes coated with purified guinea-pig antibodies to insulin were used to extract proinsulin and insulin from plasma. Rabbit antibody to human C peptide was then added to react with the C-peptide moiety of the bound proinsulin. The uptake of this antibody was measured by the subsequent binding of¹²⁵I-sheep antibody to rabbit IgG. The binding of radioactivity to the tubes was a function of the proinsulin concentration in the sample. The sensitivity of the assay was 0.006 pmol/ml. Only 200 l of plasma was required in the assay and the¹²⁵I-labelled antibody was produced from readily available reagents. The polyethylene tubes remained stable for at least 5 months after coating. The mean fasting proinsulin level was 0.009 pmol/ml in sixteen normal subjects and 0.025 pmol/ml in twelve maturity onset diabetics. Oral glucose produced an 8 fold increase in proinsulin concentration but a decline in the plasma proinsulin/insulin molar ratio. Four patients with insulinoma had extremely elevated proinsulin levels and proinsulin/insulin ratios.