Kanwal K. Gambhir

Characteristics of human erythrocyte insulin receptors.

Highly specific insulin receptors have been identified on human erythrocytes. A modification of the monocyte insulin radioreceptor technique permitted distinct separation of human erythrocytes with their bound insulin from the free insulin. When incubated with 80 pg. per milliliter of 125I-insulin (pH 8.0, 3.5 hours, 15 ° C.), erythrocytes from 17 normal volunteers specifically bound 10 per cent (± 1.450 S.D.) of the total 125I-insulin. Less than 15 per cent of the total 125I-insulin bound was nonspecific. Binding of 125I-insulin to human erythrocytes was dependent on pH and temperature. Less than 5 per cent of the insulin available to the plasma membrane was degraded. Both calcium and magnesium enhanced 125I-insulin binding by 100 per cent but had no synergistic effect when mixed in a 1:1 molar ratio. Scatchard analysis of the binding data resulted in a curvilinear plot with characteristics typical of negative cooperative interactions between receptor sites and with an unoccupied site affinity constant of 0.1 × 108 M-1. Human erythrocytes have 2,000 insulin binding sites per erythrocyte with 14 sites per square micrometer of surface area. The readily available human erythrocyte, thus, has both specific insulin binding sites and binding characteristics similar to other human cell types. These studies have provided the basis for further clinical investigation of polypeptide hormone receptors on human erythrocytes.

Decreased total carbonic anhydrase esterase activity and decreased levels of carbonic anhydrase 1 isozyme in erythrocytes of type II diabetic patients

In this exploratory study, we investigated total erythrocyte carbonic anhydrase (CA) estrase activity as well as CA I isozyme concentration in patients with diabetes mellitus type II (DM) and healthy individuals of Howard University Hospital community. Total estrase activity of CA was measured spectrophotometrically using p-nitrophenol acetate before and after inhibition with acetazolamide. CA I isozyme was measured by radial immunodiffusion using monoclonal antibody (CA I) in agarose plates. The study involved 20 consented participants; 10 normal (N) and 10 (DM), 21 to 84 years of age. The study was approved by the Howard University Institution Review Board. The CA activity was measured following lysis of cells as U/min/mL and CA I concentration as mg/l. We observed CA activity as 46.3±4(N) and 25±2.1 (DM) whereas CA I concentration as 1896±125 (N) and 1104 ±63 (DM). We speculate that the change in the CA activity may of fundamental importance in the regulation of intracellular; pHi for the basic control of metabolism in diabetes mellitus. Further, we propose that CA activity is a good candidate for a biomarker of diabetes mellitus for the early detection of insulin resistance because the CA activity variation was proportional to the severity of the diabetes.

A smaller molecular weight retinol binding protein in rat testis seminiferous tubules

Abstract In the cytosol fraction in rat testis seminiferous tubules a lower molecular weight protein of ∼4,800 daltons that binds retinol with high specificity has been isolated and purified by ammonium sulfate precipitation and on Sephadex column chromatography. The hexane extract of the component gave a characteristic retinol fluorescence spectrum. The amino acid composition was qualitatively similar to the retinol binding protein in the blood with the exception that cystine and cysteine were absent.

Red blood cell insulin receptors in health and disease

CONTENTS Structure and characteristics of erythrocyte insulin receptor. Red blood cell age and insulin receptors. Insulin receptors in human disease states. Obesity. Chronic renal failure. Acanthosis nigricans. Miscellaneous disease states. Insulin receptors in children. Insulin receptors in women during pregnancy. Insulin binding and other hormones. Comparison of biosynthetic insulin, pancreatic human insulin and porcine insulin binding to erythrocytes. Effect of exercise on insulin binding to red blood cells of normal human volunteers. Miscellaneous insulin binding studies. Insulin internalization and degradation. Insulin and erythrocyte metabolism. Summary and conclusion.

The effect of hemodialysis on the transport of sodium in erythrocytes from chronic renal failure patients maintained on hemodialysis

Studies were undertaken to evaluate the modulatory effect of maintenance hemodialysis on ouabain sensitive (OS) and ouabain insensitive (OIS) 22Na(+) uptake in erythrocytes (E) of 8 chronic renal failure patients of both sexes. Following the receipt of informed consent, the blood samples were obtained just before and after Dialysis. The % 22Na(+) uptake of the total 22Na(+) present in the assay media was determined in the purified E just before and after Dialysis. The assay medium was composed of 100 mM NaCl, 5 mM KCl, 10 mM trisbase, 10 mM MOPS, 10 mM D-glucose and 60 mM sucrose, pH 7.4 with and without ouabain. Five different concentrations of E, ranging from 0.75 to 2.00 x 10(9)/mL were used for this study. We observed a linear relationship between the 22Na(+) uptake and E concentrations in both of the assay systems (OS and OIS). The mean total 22Na(+) uptake per 6.5 x 10(9) E/mL in OS and OIS before and after hemodialysis were 3.28 +/- 0.4 (OS) and 3.26 +/- 0.42 (OIS), and 3.42 +/- 0.54 (OS) and 3.42 +/- 0.68 (OIS) respectively. The relative % differences between pre- and post-Dialysis were 4 and 5%, which were statistically not significant. From this study, we conclude that hemodialysis does not affect E membrane properties influencing 22Na(+) transport.

Characterization of an intracellular insulin-degrading enzyme in human erythrocytes

Using conventional techniques of ammonium sulfate fractionation and Sephadex gel column chromatography, insulin-degrading enzyme was partially purified from lysate of human erythrocytes. The enzymatic activity was measured by the trichloroacetic acid precipitation method. Compared to trypsin, the enzyme was highly specific for insulin. The apparent molecular weight of the enzyme was 160,000 Da, the optimum pH was the 7.4 to 7.8 range, and the Km value for insulin for the partially purified enzyme was 162 nM. Bacitracin and N-ethylmaleimide were potent inhibitors, while chloroquine, ethylenediaminetetraacetate, antipain, and soybean trypsin inhibitor failed to inhibit the activity of the enzyme. Like most nucleated cells, human erythrocytes not only have the membranal insulin receptors, but also possess the cytosolic specific insulin-degrading enzyme. Insulin internalization and degradation are shown to be due to the receptor and the enzyme acting in concert as in many nucleated cells. Anucleated erythrocytes have both these entities for possible internalization and degradation of insulin.

Erythrocyte total carbonic anhydrase esterase activity in african american obese children: reduction starts at a young age.

A previous study from this laboratory showed that patients with diabetes mellitus type 2 (type 2 diabetes) have significantly lower values (P \ 0.05) of carbonic anhydrase (CA) activity and its isozyme (CAI) concentration compared with their healthy counterparts. Furthermore, the values varied with the severity of the diabetes or insulin resistance (Gambhir et al. 2007). Kondo et al. (1975) reported contradictory results, however, as they found increased levels of both CAI and CAII in patients with diabetes. A study involving hypertensive human subjects reported 63% of hypertensive patients with decreased CA activity compared with the normotensive population, and 37% showed CA activity significantly higher than that of normotensives. Glucose intolerance, insulin resistance, and hyperinsulinemia

Significance of Plasma C-peptide in Obese African American Adolescents

BACKGROUND C-peptide blood levels can indicate whether or not a person is producing insulin and roughly how much. C-peptide is secreted as a byproduct of the biosynthesis of insulin from proinsulin. C-peptide has proposed biological activity and a well-established diagnostic value. The significance of C-peptide concentration in the plasma and urine in the pediatric population needs further delineation. AIM To determine the significance of plasma C-peptide in obese African American adolescents with mild insulin resistance but no evidence of diabetes. METHODS This study included 19 African American adolescents with body mass index (BMI) in at least the 85th percentile evaluated with anthropometric measurements, Homeostatic Model Assessment-Insulin Resistance (HOMA-IR) score, and oral glucose tolerance test (OGTT), and 24-hour urine collections. The study also included an age-matched control group of 15 healthy African American adolescent controls and were not subjected for OGTT. The correlation among BMI, fasting plasma C-peptide concentrations, and 24-hour-urine C-peptide concentrations was calculated. T Tests were conducted to compare plasma C-peptide and 24-hour-urine C-peptide concentrations for the test group and controls. RESULTS Mean HOMA score (3.96 +/- 1.84) signified mild insulin resistance among the adolescent test group. The test subjects exhibited adequate glucose tolerance (glucose range, 89.4-122.5 mg/dL) during the OGTT. A significant positive relationship was observed between BMI and fasting plasma C-peptide concentration in the control group (r = 0.537) but not the test group (r = 0.335). An insignificant positive relationship was exhibited between BMI and 24-hour-urine C-peptide concentration in the test group (r = 0.150) and controls (r = 0.254). CONCLUSIONS The positive relationship among BMI, plasma C-peptide, and urine C-peptide is worth further evaluation in studies conducting multiple rounds of OGTT with a larger sample of pediatric subjects. The potential diagnostic value of C-peptide may facilitate early detection of insulin resistance in the pediatric population.

Metabolism of Insulin in Erythrocytes from Renal Failure Patients on Maintenance Hemodialysis

Glucose tolerance does not improve to the normal level after dialysis; however, our studies showed that the insulin receptor binding to erythrocytes of nondiabetic patients with chronic renal failure (CRF) on hemodialysis was more than that in normal subjects. To understand this apparent anomaly in insulin receptor action and glucose metabolism, we investigated insulin degradation-a postreceptor event of insulin binding-in erythrocytes from CRF patients and compared it with that of normal subjects. We studied insulin degradation by erythrocytes from each of eight CRF patients and five normal subjects. The average hyperbolic insulin degradation curve for the CRF patients showed lower activity and a right-handed shift compared to the curve for the normal subjects. The average maximum degradation of insulin in the CRF patients was significantly lower than that of normal subjects. The number of erythrocytes required to produce 50% of maximum insulin degradation was significantly greater in these patients than that in the normal subjects. Furthermore, a linear correlation was observed between the duration of dialysis and maximum percent of insulin degradation in the CRF patients. Clinical implications of these findings are unclear at the present time. However, the insulin-degrading activity in erythrocytes may be reflective of that in other body tissues.

The sodium-22 influx in erythrocytes from black males and females.

In order to establish a standard for sodium influx in erythrocytes for the black population, 22Na+ uptake was measured in 29 normotensive black volunteers. Nineteen males and 10 females during the follicular phase of the menstrual cycle were studied utilizing the procedure of Gambhir et al. (1). In the males, cell concentrations ranging from 0.64 to 2.0 X 10(9)/ml showed an influx of 0.42 to 1.34% of the total 22Na+ added, and in the females, using the same erythrocyte concentrations, the 22Na+ influx ranged from 0.37 to 1.1%; these differences were not significant. Intraassay variation of the 22Na+ data was 4.8%. Interassay variations have been explained elsewhere (1). These data provided a range of observed values for 22Na+ uptake in erythrocytes from a subpopulation of normotensive black males and females for comparison with hypertensive patients.