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

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

Erythrocyte mirna 144 and mirna 451 as cell aging biomarkers in african american adults

Objective: MicroRNAs (miRNA) are novel critical regulators of cell proliferation and human disease, including diabetes mellitus and cancer. The aim of this study was to evaluate the expression of circulating erythrocytes (E) miRNA-144 and miRNA-451 expression in African Americans Adults (AAA) as a biomarker of cell aging. Methods: The blood samples were collected from healthy controls [n=9] following an 8-12 hours fast. Erythrocytes were purified twice by Boyum gradient. Erythrocytes were further sub-fractionated into young (y) (1.07-1.09 g/ml), mid (m) (1.09-1.11 g/ml), and old (o) (1.11-1.12 g/ml) age cells by using discontinuous Percoll gradient (35%, 40%, 45%, 50%, 55%, 65%, 80%, and 100%) and total RNA extracted. MiRNA-144 and miRNA-451 were quantified in y, m, and o age E sub-fractions by qRT-PCR. Results: MiRNA-451 expression was 82210.8271, 130922.476, and 149554.364 in y, m, and o cells, respectively. MiRNA-144 expression in y cells was 18.6641092, m cells was 32.4413621, and o cells was 57.8118394 These results showed that o cells expressed both miRNA-144 and miRNA-451 more than that of m, and y cells. Conclusion: The findings of this study showed that miRNAs expression differ in sub-fractionated erythrocytes. This study suggests that miRNA-144 and miRNA-451 have the potential to be used as biomarkers of RBC aging

Insulin Receptor Defect in Diabetic Man With Chronic Renal Failure: A Comparison of Erythrocyte Insulin Binding in Diabetic and Nondiabetic Patients on Maintenance Hemodialysis

125I-Insulin binding to most accessible and easily obtained circulating erythrocytes in 7 diabetic and 16 nondiabetic, nonobese patients with chronic renal failure (CRF) on maintenance hemodialysis was studied and compared with that of the 30 normal, nonobese volunteers. The percent-age of 125I-insulin binding was 4.7 ± 1.8 (mean ± SD) in the diabetic and 13.1 ± 2.4 in the nondiabetic patients with CRF while in the normal subjects it was 9.9 ± 1.4. There was no statistical difference in the fasting levels of glucose, insulin, and glycosylated hemoglobins in all the subjects studied. No correlation (r = -0.32) between insulin binding and endogenous insulin was observed in these subjects. The diabetic patients with CRF had no circulating factor(s) that would reduce insulin binding to normal erythrocytes. In comparison to the normal subjects, the reduction in insulin binding in the diabetic patients with CRF was accompanied by 47% reduction in insulin receptor sites (R0) but with no change in insulin receptor affinity, ( K ̄e, 0.46 × 108m-1), while in the nondiabetic patients with CRF no change in insulin receptor sites (R0) of increased affinity ( K ̄e, 0.63 × 108m-1) was observed. These findings suggest that insulin binding utilizing the much simpler erythrocyte insulin receptor assay can be used to evaluate alteration in insulin receptor status in CRF. These investigations also provide a very useful assay and characteristic studies for insulin receptors, if receptor modulation acquires a status of treatment for diabetes

395 Systemic Administration of miR-451 Improves Autophagy Response in an Accelerated Mouse Model of Diabetic Kidney Disease

OBJECTIVES/GOALS: Diabetic Kidney Disease (DKD) is a common diabetes complication, often linked to end-stage renal disease in the United States (US). While autophagy and miRNAs are pivotal, miR-451’s specific role remains understudied. Our study explores its renoprotective effects in an accelerated DKD mouse model. METHODS/STUDY POPULATION: We assessed the effect of miR-451 mimic treatment on Diabetic Kidney Disease (DKD) in BTBR ob/ob mice, known for their rapid DKD-like renal lesions. Mice were divided into four groups: WT (wild-type), BTBR ob/ob, WT+miR-451 (wild-type with miR-451 mimic), and BTBR ob/ob+miR-451 (BTBR ob/ob with miR-451 mimic). MiR-451 mimics were administered at 2mg/kg body weight once weekly for three consecutive weeks. We collected spot urine and monitored blood glucose levels at each time point. After the treatment period, mice were euthanized for kidney and blood samples. Western blot analysis assessed autophagy-related protein markers. Statistical analysis included Student’s t-test and ANOVA (p<0.05). RESULTS/ANTICIPATED RESULTS: The study assessed the impact of miR-451 mimic treatment in BTBR ob/ob mice. Albumin:creatinine ratio increased fourfold (p=0.01) in BTBR ob/ob mice at 5 weeks. MiR-451 mimic treatment had no impact on body weight. Blood glucose levels were notably higher in both treated and untreated BTBR ob/ob mice at 12 (425±33.1 mg/dL; p=0.04) and 13 weeks (383±25.3 mg/dL; p=0.007). However, a significant drop occurred from week 13 (554.7±10.8 mg/dL) to week 14 (289±13.3 mg/dL; p=0.0002) in BTBR ob/ob miR-451 treated mice. Western blot analysis in whole kidney homogenates showed a 91% reduction (p=0.02) in YWHAZ, a predicted miR-451 target, in treated BTBR ob/ob mice and a 95% reduction (p=0.01) in WT mice. Furthermore, miR-451 mimic treatment led to a 68% increase (p=0.01) in ATG101 and a 44% increase in Beclin-1 in BTBR ob/ob mice. DISCUSSION/SIGNIFICANCE: The study uncovers miR-451-based interventions as a promising avenue to counter Diabetic Kidney Disease by modulating autophagy, potentially introducing novel therapies for at-risk individuals. However, practical DKD treatments will require further research and rigorous clinical validation to harness the full potential of these insights

496 Urinary Exosomal MicroRNA as Early Markers of Diabetic Kidney Disease in African American Adults

OBJECTIVES/GOALS: This study aimed to characterize urinary exosomal miRNA content in African American adults with diabetic kidney disease. METHODS/STUDY POPULATION: Male and female participants between the ages of 18 and 65 were recruited from the Diabetes Treatment Center and the Nephrology Clinic at the Howard University Hospital. Exosomes were isolated from cleared urine of healthy controls (n=3), type 2 diabetics (n=3), and participants with chronic kidney disease (n=3). The purity and size of isolated microparticles was evaluated using NanoSight technology (30nm to 120nm size range) and western blot analysis for exosome-specific markers (TSG101 and CD81) RESULTS/ANTICIPATED RESULTS: Expression of 5 selected microRNAs, miR-4534, miR-320c, miR-451, miR-362-3p and miR-877-3p were evaluated by qRT-PCR. miR-4534 and miR-451 was increased between healthy controls and the type diabetic group. MiR-320c was increased in the CKD group, in comparison to healthy controls. Conversely, there was no difference in miR-877-5p between the three groups. DISCUSSION/SIGNIFICANCE: These findings will provide insight into the use of circulating miRNAs as early markers of DKD, ultimately creating more effective treatments and preventive measures