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.     

Renal handling of low molecular weight proteins

The renal filtration, absorption and final disposal of lysozyme (lysozyme—mol wt 14,000), insulin and growth hormone were studied to gain a better quantitative understanding of the fundamental variables involved in the renal handling of low molecular weight proteins. The glomerular barrier offers little hindrance to the filtration of lysozyme, the glomerular sieving coefficient being 0.8 ± 0.1 (SD). The intrarenal route by which injected lysozyme accumulates in the kidney is via filtration and subsequent absorption (uptake) by renal tubular cells. Uptake or adsorption from the peritubular side is negligible compared to luminal uptake. Renal clearance and renal titration experiments in the intact dog and in the isolated perfused rat kidney showed that the lysozyme absorption process can be best characterized as a high capacity, low affinity transport system which is directly or indirectly dependent on energy input. The final disposal of absorbed ¹²⁵I-lysozyme, ¹²⁵I-insulin and ¹²⁵I-growth hormone was studied in the isolated perfused rat kidney by measuring the radioactivity released from the kidney to the perfusate and analyzing the nature of the released product by gel chromatography. The rate of release of radioactivity as well as its nature was dependent on the molecular species of the absorbed protein. The rate of release was higher for ¹²⁵I-insulin and ¹²⁵I-growth hormone and lower for ¹²⁵I-lysozyme. Lysozyme absorbed from the luminal side was released to the perfusate both as intact protein molecules and as catabolic products, whereas absorbed ¹²⁵I-insulin was almost entirely released to the perfusate as catabolic products. It is concluded that low molecular weight proteins are extensively filtered by the kidney, absorbed from the luminal side by renal tubular cells and released back to the circulation either as intact molecules or as catabolic products (amino acids and polypeptides). This process contributes in an important way to the plasma turnover of low molecular weight proteins including peptides and protein hormones.     

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.     

Pharmacokinetics of continuous subcutaneous insulin infusion

Summary One of the reasons for the variability of blood glucose regulation in Type 1 (insulin-dependent) diabetic patients is the huge variation in subcutaneous absorption of intermediate-acting insulin. We have investigated the variation in insulin absorption during continuous subcutaneous insulin infusion in eight such patients. The content of insulin in the subcutaneous tissue was measured using ¹²⁵I-labelled insulin. The concentration of free serum insulin and blood glucose was followed from 1 h before and from 7 h after breakfast on two consecutive days. The amount of insulin absorbed during 24 h differed in all cases by less than 3% from the daily insulin dose given by the pumps. Mean insulin absorption rates and mean free insulin concentration showed peak values 30–90 min after meal bolus injections; this was sufficient to maintain near-normal blood glucose. Mean free serum insulin correlated strongly with disappearance of insulin from the subcutaneous tissue (r=0.98). From the insulin absorption rates and free insulin concentrations during basal constant insulin infusion, the half-time of serum insulin was calculated as 6 min. Compared with the known large variability in the absorption of intermediate-acting insulin, continuous subcutaneous insulin infusion offers a precise and reproducible way of insulin administration resulting in post-prandial serum insulin peaks sufficient to maintain near-normal blood glucose levels. The half-time of serum insulin during subcutaneous infusion corresponds to values for intravenous infusion given in the literature, indicating that local degradation of insulin in subcutaneous tissue is of minor importance.     

Vanadate increases cell surface insulin binding and improves insulin sensitivity in both normal and insulin-resistant rat adipocytes

Summary The aim of this study was to elucidate the acute effects of vanadate on cell surface insulin binding and insulin sensitivity in rat adipocytes. The cells were preincubated at 37° for 20 min followed by energy depletion with potassium cyanide, extensive washing and ¹²⁵I-insulin binding. The presence of vanadate or insulin during the preincubation period dose-dependently enhanced ¹²⁵I-insulin binding to normal adipocytes (maximally 4–5-fold) through an increased number of binding sites without any change in receptor affinity. Submaximal concentrations of vanadate added together with insulin enhanced the cellular sensitivity to the effect of insulin to stimulate 3-O-methylglucose transport. Vanadate, but not insulin, was also capable of increasing insulin binding as well as insulin sensitivity in insulin-resistant cells (treatment with N⁶-monobutyryl cAMP or amiloride and adipocytes from obese, aging rats). There was a correlation between the effect of vanadate to augment insulin binding and its ability to enhance cellular insulin sensitivity. Thus, the data suggest that short-term vanadate treatment improves insulin sensitivity through enhanced receptor binding and that this occurs in both normal and insulin-resistant cells.     

Insulin receptors in the gastrointestinal tract of the rat fetus: quantitative autoradiographic studies

Summary Purified carrier-free ¹²⁵I-insulin was injected into the vitelline vein of rat fetuses in utero after 17, 19 or 21 days of a 22-day gestation. Three minutes later, the weight and radioactivity of various organs and the remaining carcass were measured. A radioactivity concentration index was calculated by dividing the specific activity of each organ by that of the whole feto-placental unit. In each of the three age groups studied, the gastrointestinal tract radioactivity concentration indices were 1.7, 2 and 1.9 respectively, indicating that the gastrointestinal tract concentrated the labelled hormone. Three, 9 and 15 min after ¹²⁵I-insulin injection, the gastrointestinal tract was removed, homogenized and chromatographed on a G-50 fine Sephadex column. At 3 min, 91.4% of gastrointestinal tract radioactivity co-eluted with a standard of ¹²⁵I-insulin. At the later time intervals studied, the percentage of ¹²⁵I-insulin decreased while that of low molecular weight degradation products increased. Quantitative autoradiographic study of the fetal gastrointestinal tract indicated that epithelial cells bound ¹²⁵I-insulin and that this binding was inhibited by co-injection of large amounts of unlabelled insulin. ¹²⁵I-insulin binding was highest in the proximal small bowel and lowest in the colon. Insulin binding did not appear to depend upon degree of cell maturation or cell type. These results indicate that the epithelium of the gastrointestinal tract is characterized by the presence of numerous insulin receptors and is a potentially important insulin target.     

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     

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.     

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.     

Characterization of insulin receptors in patients with the syndromes of insulin resistance and acanthosis nigricans

Summary This report analyzes the in vitro characteristics of ¹²⁵I-insulin binding to the monocytes of nine patients with the syndromes of acanthosis nigricans and insulin resistance. The 3 Type A patients (without demonstrable autoantibodies to the insulin receptor) had decreased binding of insulin due to a decreased concentration of receptors. In these patients the residual receptors demonstrated normal dissociation kinetics, negative cooperativity, and were blocked by anti-receptor antibodies in a manner similar to normal cells. In contrast, monocytes from the 6 Type B patients (with circulating anti-receptor autoantibodies) had decreased binding of insulin due to a decrease in receptor affinity. Insulin binding to monocytes of Type B patients demonstrated accelerated rates of dissociation with no evidence of cooperative interactions among insulin receptors. When coupled with previous data, the present studies further suggest that different mechanisms account for the defects in insulin binding and insulin resistance observed in these patients.     

Effect of gestational diabetes on insulin receptors in human placenta

Summary The aim of this work was the characterization of insulin binding to a partially purified preparation of plasma membranes from gestational diabetic placenta in order to establish a comparison with normal pregnancy. A decrease in the number of insulin receptors per mg protein in placenta from gestational diabetic mothers was found. Comparable inhibitory effects of unlabelled insulin suggest that the binding affinities were similar in both groups of patients. The ability of the placental preparation to degrade¹²⁵I-insulin was not significantly different in both situations. The existence of a negative feed-back regulation of the insulin receptors in placental membranes was suggested by the fact that plasma insulin levels were significantly higher in the gestational diabetic mothers.     

Influence of aprotinin on insulin absorption and subcutaneous blood flow in Type 1 (insulin-dependent) diabetes

Summary Ten normal-weight Type 1 (insulin-dependent) diabetic patients (12 h postprandial) with normal insulin requirement were given ¹²⁵I-labelled soluble insulin (10 U) in the thigh together with aprotinin (10000 KIU) or its diluent on two consecutive mornings. Disappearance of ¹²⁵I-radioactivity was followed continuously for 3 h by external detection and plasma free insulin measured by radioimmunoassay. Subcutaneous blood flow following aprotinin or diluent was studied concomitantly in the contralateral thigh by external monitoring of locally injected ¹³³Xenon. Plasma free insulin increased significantly faster (p<0.05) and the insulin area under the curve was significantly (p<0.05) greater during the first hour after injection of insulin with aprotinin. Subcutaneous blood flow (rate constants for ¹³³Xenon) was significantly higher with aprotinin (p<0.05), the highest flow occurring early after injection. In conclusion, subcutaneously injected soluble insulin is more rapidly absorbed by addition of aprotinin to the insulin solution in Type 1 diabetes. Blood flow increase at the injection site may explain part of this effect.     

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.     

Relationship between Insulin binding and glycogenesis in cultured fetal hepatocytes

Summary Binding of ¹²⁵I-insulin and the stimulatory effect of insulin on ¹⁴C-glucose incorporation into glycogen have been studied in cultured fetal rat hepatocytes. Measurement of both variables was possible at 37 °C because of the slow rate of insulin degradation in the medium. ¹²⁵I-insulin binding approached maximum after 10 min, thus largely preceding the insulin glycogenic effect which became significant after 45 min. Maximal effect was observed after 3 h with 10 nmol/l insulin when 16,000 specific sites per hepatocyte were occupied and half-maximal response was achieved with 0.3 nmol/l insulin (2,900 sites/hepatocyte). Dissociation of bound insulin was rapid (t1/2=3 min) and accelerated by the availability of native insulin. In hepatocytes preincubated with insulin, binding was measured after 30 min incubation in the absence of hormone which allowed the liberation of most (95%) of the bound insulin. No modification of insulin binding was observed over extended periods (2–24 h) of exposure to 10 nmol/l insulin, when the glycogenic effect of insulin showed striking variations, notably a cessation of the effect between 4 and 12 h. Thus the time-dependence of the glycogenic effect of insulin cannot be related to a defect in insulin binding in cultured fetal hepatocytes.     

The displacement of insulin from blood capillaries

Summary Normal anaesthetized bull-dogs were perfused with constant amounts of ¹²⁵I bovine insulin. During the control period, steady state arterio-venous gradients and lymph levels were achieved. Positive arterio-venous differences were observed across the hind limbs and the head. Lower levels of ¹²⁵I-insulin were found in cervical and leg lymph than in corresponding venous plasma. The rapid intravascular injection of unlabelled insulin resulted in an almost immediate reversal of the arterio-venous gradients of ¹²⁵I-insulin in the head and the hind limb. The injection of small amounts of glucose into the lower abdominal aorta induced a very rapid diminution of the arterio-venous gradient across the hind quarters; the effect was not mediated by the release of endogenous insulin. In both experiments, the lymph levels of ¹²⁵I-insulin remained relatively constant.The data indicate that the vascular pole of the capillary endothelial cells can adsorb labelled insulin and either exchange it with unlabelled insulin or release it under the direct influence of glucose.This work was supported by U.S. Public Health Service, Grant no R05 TW-00289-03, and by Fonds de la Recherche Scientifique Médicale, Grant no 925.     

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.     

Intraregional differences in the absorption of unmodified insulin from the abdominal wall.

Absorption of subcutaneously injected unmodified human 125I-insulin (5 U; 100 U ml-1) was studied concurrently from three areas of the abdominal wall (120 mm above, 120 mm lateral to, and 40 mm below the umbilicus), and one area of the thigh (upper midline), in nine Type 1 diabetic patients of normal body weight, and from deep (2 mm above muscle fascia) and superficial (3 mm beneath skin surface) sites in abdominal wall and thigh in 11 Type 1 diabetic patients. The absorption rates were followed continuously for 3 h with the patient in the supine position. Whereas 125I-insulin disappeared considerably faster from the site above the umbilicus than from sites below or lateral to the umbilicus or from the thigh (residual radioactivities after 175 min: 36 +/- 4 vs 49 +/- 5, 54 +/- 2, and 62 +/- 4 (+/- SE)%, respectively; p less than 0.05 or better), no significant differences were found between deep and superficial sites in either abdominal wall or thigh. The results suggest that insulin absorption rates from subcutaneous injection sites within the abdominal wall differ sufficiently for this to be of clinical importance.     

The production and characteristics of anti-insulin,anti-a-component and anti-proinsulin antibodies in patients treated with monocomponent or conventional insulin

Summary Highly purified pork monocomponent insulin produced less anti-insulin antibody than conventional insulins in diabetic patients. The smaller amount of anti-insulin antibody produced by MC insulin bound pork insulin more strongly than beef insulin in both displacement and direct binding studies of¹²⁵I-insulin. On the contrary, anti-insulin antibody which was produced by conventional insulins (beef insulin or mixture of pork and beef insulin) bound beef insulin more strongly. No significant anti-a-component and anti-proinsulin antibodies were detected in diabetics treated with highly purified monocomponent pork insulin about two years, compared to significant production of these antibodies in diabetics treated with conventional insulins. These results suggest that the species difference of the insulin molecule itself plays a significant role for the production of anti-insulin antibody, as the impurities do, in insulin-treated diabetic patients. The production of anti-insulin and anti-a-component antibodies decreased clearly after switching to highly purified monocomponent from conventional insulin. No effect of the switching on insulin requirement was found; however, better control of diabetes was accomplished in relation to the level of fasting blood sugar.     

Dose-dependent Subcutaneous Absorption of Porcine,Bovine and Human NPH Insulins

ABSTRACT The absorption of ¹²⁵I-labelled porcine, bovine and human neutral protamine Hagedorn (NPH) insulins was compared in 40 diabetic patients after subcutaneous injections in the thighs using doses of 6 and 24 IU (40 IU/ml). Furthermore, the absorption of porcine NPH insulin was compared with bovine NPH insulin and porcine lente type insulin in the same dose (6 IU). All these intermediately acting insulins showed similar absorption rates when administered in the same dose. However, an increase in the injected dose from 6 to 24 IU resulted in a decrease of approximately 30% in the absorption rate of all NPH insulins.     

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