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Jacobs Journal of Diabetes and Endocrinology

Energy Regulating Hormones Correlate with Erythrocyte MicroRNA Expression in Type 2 Diabetic African-American AdultsMaurice B. Fluitt 1,2, Namita Kumari4, Gail Nunlee-Bland 1,2,3, Sergei Nekhai4,5, Kanwal K. Gambhir*1,2,3,5 1Genetics and Human Genetics, Howard University College of Medicine, Washington, DC 20059, USA2Molecular Endocrinology Laboratory, Department of Medicine, Howard University College of Medicine, 2041 Georgia Ave, NW, Suite

5C02, Washington, DC 20060, USA3Howard University Diabetes Treatment Center, 2041 Georgia Ave, NW First Floor, Suite 1-OP-97, Washington, DC 20060, USA4The Center for Sickle Cell Disease, Howard University, Washington, DC 20059, USA5Department of Medicine, Howard University College of Medicine, Howard University, Washington, DC 20059, USA

*Corresponding author: Dr. Kanwal K. Gambhir, PhD, Molecular Endocrinology Laboratory ,Department of Medicine, Howard

University College of Medicine, 2041 Georgia Ave, NW, Suite 5C02, Washington, DC 20060, USA, Tel: 202-865-1398;

Email: kgambhir@howard.edu

Received: 06-04-2018

Accepted: 06-14-2018

Published: 06-18-2018

Copyright: © 2018 Kanwal K. Gambhir

Research Article

Cite this article: MB Fluitt. Energy Regulating Hormones Correlate with Erythrocyte MicroRNA Expression in Type 2 Diabetic African-American Adults. J J Diab Endocrin. 2018, 3(3): 015.

Abstract

This study aimed to identify the relationships between type 2 diabetes mellitus (T2DM) related miRNAs in erythrocytes and energy regulating hormones in pre-diabetic and obese type 2 diabetic African-American adults. Patients were recruited from the Howard University Hospital Diabetes Treatment Center. The expression of nine previously reported T2DM-related miRNAs was quantified using qRT-PCR. Radioimmunoassays were employed to determine plasma hormone concentrations of total ghrelin, leptin, vitamin D (25(OH)D) and C-peptide. Pearson’s correlation was performed to identify significant relationships between erythrocyte miRNAs and hormone concentration. In non-diabetic controls we found significant relationships between miR-NA-15a and 25(OH)D (r=0.8061, p=0.0157), miRNA-223 and 25(OH)D (r=0.7207, p=0.0437), and miRNA-375 and total ghrelin (r=-0.960, p=0.039). In pre-diabetics, we found significant relationships between miRNA-15b and total ghrelin (r=-0.7980, p=0.0315) and miRNA-15b and C-peptide (r=0.783, p=0.00373). We found a significant correlation between miRNA-15a and leptin (r=0.7943, p≤0.0001) in the T2DM group. The novel findings of this study provide insight into the relationship between erythrocyte miRNAs and hormones associated with the development and progression of T2DM. The relevance of these findings is highlighted by the overlap of targeted messages of the selected miRNAs with various pathways associated with T2DM, includ-ing insulin signaling.

Keywords:Introduction

T2DM; miRNAs; erythrocytes; Vitamin-D; Leptin; Ghrelin; African-Americans

MicroRNAs (miRNAs) are a class of small (18-25 nucleotides) non-coding RNAs that regulate homeostatic functions, such as cell proliferation and hormone secretion [1-4] The dysreg-ulation of miRNAs leads to the development of a number of

diseases, including T2DM. Several studies reveal the role of miRNAs in the pathogenesis of T2DM. These intracellular miR-NAs are reported to maintain and regulate insulin production and secretion, β-cell differentiation, and insulin-signaling path-ways [5 -9]. In addition to their intracellular expression and function, miRNAs are found in circulation and reflect various

Study Population

Patients were recruited from the Howard University Diabetes Treatment Center (DTC). Whole blood samples were collected from healthy non-diabetic, high-risk pre-diabetic and T2DM African-American adults between the ages of 18 and 80 years old with or without a family history of T2DM following an 8-10 hour fast. Patients included both pre-diabetic and T2DM African-American adults. T2DM were previously diagnosed. Non-diabetic high-risk obese—pre-diabetics were identified through the W.E.I.G.H.T study at the DTC. Control subjects were recruited from the Howard University community [10].

Erythrocyte Isolation

Venous blood samples were collected in heparinized vacutain-er tubes following an 8-10 hour fast. Erythrocytes were isolat-ed and purified by Hypaque-Ficoll (HF) gradient as previous-ly described [19]. We repeated the isolation process twice to wash off heparin. Following initial centrifugation, the plasma layer was removed and aliquoted for use in hormonal assays.

RNA Extraction

Total RNA was extracted from erythrocytes using the miRVana miRNA isolation kit from Ambion (Austin, Texas) per manufac-turer’s instructions.

cDNA Synthesis and miRNA Quantification

Nine previously reported T2DM-related miRNAs were select-ed for this study [10]. Reverse transcription and quantitative real time PCR (qRT-PCR) were used for miRNA quantification in erythrocytes, as previously described [10]. Briefly, reverse transcription and PCR reactions were performed using miS-CRIPT II RT Kit (Qiagen) and miSCRIPT SYBR Green PCR Kit (Qiagen), respectively, following the manufacturer’s proce-dure. MiSCRIPT II RT system uses total RNA as the starting ma-terial for cDNA synthesis, allowing for the detection of miRNA from a single cDNA preparation. The final reaction volume of reverse transcription was 20 μL. Samples were incubated for 60 minutes at 37°C followed by a 5-minutes incubation at 95°C [10]. MiRNA expression was quantified using the following (10 µL) master mix for each miSCRIPT primer: 5 µL of SYBR green master mix (iTaq, BIO RAD), 1 µL of 10X Universal Primer, 1 µL of miRNA specific primer (purchased from Qiagen: miR-15a, miR-15b, miR-499, miR-146, miR-126, miR-223, miR-224, miR-326 and miR-375), 3 µL of cDNA template. Amplification was carried out in 96-well plate using the LightCycler 480 (Roche) following the cycling conditions outlined by the manu-facture. RNU6b was used as a control. qRT-PCR was performed in duplicate. Ct values >36 were considered to identify miRNA expression below detection limit or absent.

physiological conditions [10]. These circulating miRNAs are released into circulation via: 1.) passive leakage from damaged cells, 2.) active secretion as microvessicles (i.e. exosomes), and 3.) active secretion in complex with macromolecules (i.e. Argo-naute proteins) [2, 11]. Although the physiological functions of circulating miRNAs are unknown, it is hypothesized that these miRNAs are involved in cell-to-cell communication, acting as hormones [2, 11].

The overlapping expression of miRNAs in different tissues suggests the complexity of understanding how miRNAs are in-volved in the development of T2DM. It also suggests a novel molecular link between the hunger-pathway and T2DM, which is governed by a host of hormones and regulatory proteins. Previous studies indicate that dysregulation of miRNA ex-pression could potentially alter both hormone concentration and receptor expression [9, 12]. Additionally, several studies suggest that hormones regulate miRNA expression [13-15]. Therefore, it is likely that miRNAs are involved in regulating the expression of hunger pathway hormones to compensate for the dysregulation in the diabetic state. Conversely, it is pos-sible that selected hormones regulate miRNA expression, sug-gesting that the dysregulation of various hormones could alter miRNA expression to compensate for the diseased state. Col-lectively, these findings suggest unique relationships between miRNAs and hormones.

Several hormones are reportedly dysregulated in the de-velopment and progression of T2DM and obesity, including ghrelin, leptin and vitamin D [16-18]. These hormones have been reported to be key regulators of energy homeostasis and metabolism [16-18]. We previously found that impaired ghrelin suppression contributes to obesity in African-Ameri-can adolescents [17]. The impaired ghrelin suppression and insulin signaling in this population could be the result of an overexpression of disruptive miRNAs that impair hormone and hormone receptor expression. However, few studies have explored the relationship between miRNAs and hormones as-sociated with T2DM and obesity development, such as ghrelin and leptin. This study aimed to identify novel relationships between erythrocyte miRNA expression and hormonal con-centrations (total ghrelin, leptin, 25(OH)D and C-peptide) in African-American adults with T2DM.

Methods

Ethical Statement

This study was carried out in accordance to the guidelines of the Howard University Institutional Review Board (HUIRB). The protocol was approved by the HUIRB (IRB-13-MED-73). All participants of this study gave written informed consent.

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Radioimmunoassay

Hormonal assays were performed in the Molecular Endocri-nology Laboratory at Howard University Hospital using com-mercially available RIA kits for total ghrelin, leptin, adiponec-tin, and C-peptide (Millipore, 6 Research Park, MO, USA) as per manufacturer’s procedures. Plasma 25(OH)D concentration was measured using commercially available RIA kits from Im-munodiagnostic System (IDS) Limited as per the manufactur-er’s procedures (Fountain Hills, AZ, USA). Total ghrelin, leptin, and C-peptide standards were prepared according to the man-ufactures procedures using serial dilutions and all samples (100 µL) were assayed in duplicate. The assay kits for total ghrelin, leptin and C-peptide have a high specificity (100%) for the respective human hormones. The IDS 25(OH)D kit is a liquid phase RIA kit. It is a complete assay procedure for both extraction and quantification of 25(OH)D and has a specificity of 100%. All assays were precise (CV < 5%) and were able to detect the lowest levels of all selected hormones. The samples were counted using the Wallac 1470 Wizard Gamma Counter.

Statistical Analysis

The amount of unknown antigen was determined by extrapo-lation from standard curve. The values were calculated to de-termine the hormonal concentrations in each sample. Average values are expressed as mean ± standard error. Differences in hormonal concentrations between the groups were analyzed using one-way ANOVA. The Pearson’s Coefficient of correlation was used to correlate miRNA expression profiles and hormonal concentration. Statistical analysis was completed using Prism software (GraphPad, La Jolla, CA). The level of significance was set at p≤0.05.

Results

Clinical Characteristics

The clinical characteristics of the study population were pre-viously reported by Fluitt et al. 2016 [10]. Briefly, body weight was significantly higher in the T2DM group (96 ±4.721 kg, n=31) when compared to the control group (83.13 ± 4.563 kg, n=14). There were no significant differences in body weight between the T2DM and the pre-diabetic group (94.7 ± 8.5 kg, n=6) (10). BMI in both the pre-diabetic and T2DM groups were significantly higher when compared to the control group. However, there was no significant difference in BMI between the pre-diabetic and T2DM groups. Both the pre-diabetic (BMI=35.1 ± 2.6 kg/m2) and T2DM (34.5 ±1.8 kg/m2) groups were obese (BMI>30 kg/m2) and the control (28.2 ±1.6 kg/m2) group was overweight (BMI=25-29.9 kg/m2) [10].

Plasma Hormone Concentrations

We selected four hormones to be measured in plasma ob-tained from non-diabetic controls, pre-diabetic and T2DM African-American adults. These hormones were previously reported by our laboratory to be dysregulated in both obesity and T2DM in African-Americans. To determine plasma hor-mone concentration between healthy, pre-diabetic, and T2DM African-American adults, we measured hormone concentra-tion using RIA kits. Plasma 25(OH)D was significantly higher in the T2DM group when compared to the pre-diabetic group (71.68±6.68 nmol/L, n=24 and 29.97±2.644 nmol/L, n=7; p≤0.002), respectively (Figure 1a). Plasma leptin was signifi-cantly higher in the pre-diabetic (70.86±13.34 ng/mL, n=7) group when compared to non-diabetic controls (16.03±8.542 ng/mL, n=7), p≤0.04 (Figure 1b). There were no significant differences in plasma total ghrelin concentration between the three groups (Figure 1c). However, plasma C-peptide was high-er in the pre-diabetic (2.857±0.27 ng/mL, n=7) group when compared to non-diabetic controls (1.498±0.11 ng/mL, n=17), p≤0.004 (Figure 1d).

Figure 1. Plasma hormone concentration in non-diabetic, pre-diabet-ic, and T2DM African-American adults (a) 25(OH)D; (b) Leptin; (c) Total Ghrelin; and (d) C-Peptide. Samples were collected following an 8-10 hours fast. Blood was collected in heparinized vacutainer tubes and plasma separated from erythrocytes by centrifugal force (3,000g). Plasma hormone concentrations were assayed using com-mercially available RIA kits purchased from Millipore or Immunodi-agnostics. Data presented as mean±SE. p≤0.05 considered significant.

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Plasma Hormone Concentrations and miRNA Relationships

We next sought to identify relationships between the selected miRNAs and plasma hormone concentrations. We previously reported significantly reduced expression of miR-499, miR-15a and miR-15b in erythrocytes of pre-diabetic African-American adults [10]. To identify significant relationships between the selected hormones and miRNAs of interest, Pearson’s Coeffi-cient Correlation was performed.

We next wanted to identify significant relationships between the selected hormones (hormone-hormone relationships). There were no significant correlations between the selected hormones in the non-diabetic control group. We found a sig-nificant positive relationship between plasma leptin and plas-ma 25(OH)D in the pre-diabetic group (r=0.79, p=0.03). In the T2DM group, we found significant positive relationships be-tween C-peptide and 25(OH)D (r=0.66, p=0.01) and C-peptide and total ghrelin (r=0.47, p=0.02).

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Figure 2. Significant correlations between hormones of energy metabolism and erythrocyte miRNAs in non-diabetic healthy African-American adults. Using Pearson’s Coefficient Correlation, we found significant relationships between (a) miR-15a and 25(OH)D, (b) miR-375 and Total Ghrelin, and (c) miR-223 and 25(OH)D. * p≤0.05 considered significant.

In the non-diabetic control group, we found strong signifi-cant positive relationships between miRNA-15a and 25(OH)D (r=0.8061, p=0.0157), miRNA-223 and 25(OH)D (r=0.7207, p=0.0437) (Figure 2a and 2c). We also found a strong signif-icant negative relationship between miRNA-375 and total ghrelin (r=-0.960, p=0.039) in non-diabetic controls (Figure 2b, supplemental table 3).

In the pre-diabetic group, we found a strong significant neg-ative relationship between miRNA-15b and total ghrelin (r=-0.7980, p=0.0315) and a strong significant positive relation-ship between miRNA-15b and C-peptide (r=0.783, p=0.00373) (Figure 3a and 3b, supplemental table 4). In the T2DM group, we found a strong positive significant correlation between miRNA-15a and leptin (r=0.7943, p≤0.0001) (Figure 3c, sup-plemental table 5).

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Figure 3. Significant correlations between hormones of energy metabolism and erythrocyte miRNAs in pre-diabetic and type 2 diabetic Afri-can-American adults. Using Pearson’s Coefficient Correlation, we found significant relationships between (a) miR-15b and Total ghrelin and (b) miR-15b and C-peptide in pre-diabetic African-Americans; and (c) miR-15a and Leptin in T2DM African-Americans. * p≤0.05 considered significant.

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Figure 4. Schematic diagram revealing the potential relationship between miRNA-15b and total ghrelin in pre-diabetic African-American adults. In persistent hyperglycemia, miRNA-15b is reduced, which results in an increased expression of TNFα, leading to impaired insulin signaling. As a result, glucose uptake is drastically decreased and ghrelin secretion is increased to compensate for reduced glucose uptake. This potential relationship is consistent with previous findings from our lab, in which ghrelin suppression is impaired in obese adolescents. This finding suggest that pre-diabetics maybe progressing towards an IR state. (TNFα = Tumor Necrosis Factor-alpha; SOCS3 = Suppressor of Cytokine Signaling 3; IRS-1 = Insulin Receptor Substrate 1; PI3K = Phosphoinositide 3-Kinase; AKT = Protein Kinase B).

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Figure 5. Schematic diagram revealing the potential relationship between miRNA-15b and C-peptide in pre-diabetic African-American adults In hyperglycemia, insulin secretion is increased, as well as C-peptide (secreted in equimolar amounts). We propose that there is a link be-tween insulin and miR-15b. The increased expression of miR-15b reduces the expression of TNFα and limits its ability to bind to its plasma membrane receptor and phosphorylate ser306 on IRS-1, which impairs tyrosine phosphorylation of IRS-1. As a result of insulin binding to its receptor, the actions of insulin are carried out and glucose uptake is increased. However, as hyperglycemia persists, both insulin secretion and miRNA-15b expression are reduced. This indicates that there is decreased beta cell function, decreased insulin secretion (C-peptide) and decreased insulin action, as TNFα impairs insulin signaling. (- - -> = Proposed relationship; TNFα = Tumor Necrosis Factor-alpha; IRS-1 = Insulin Receptor Substrate 1; PI3K = Phosphoinositide 3-Kinase; AKT = Protein Kinase B).

Discussion

MiRNA expression in both tissue and circulation could be al-tered by hormonal concentration and vice versa. However, few studies have explored the relationship of erythrocytes miRNAs and hormones of energy metabolism associated with T2DM and obesity development and progression. Moreover, this is the first study—to our knowledge—to investigate the relationship of erythrocyte miRNAs and hormones of energy metabolism exclusively in African-American adults, a popula-tion greatly impacted by obesity, T2DM, and sickle cell disease. We found a significant elevated 25(OH)D concentration in the T2DM group when compared to the pre-diabetic group. This finding is inconsistent with current literature, which suggests low circulating 25(OH)D is associated with T2DM [20]. How-ever, the Diabetes Treatment Center at the Howard University Hospital offers aggressive vitamin-D therapies for the majority of the patients involved in this study, which explains the ele-vated plasma 25(OH)D in this group. There are no studies that have examined the effects of vitamin D supplementation or an-ti-diabetic medications on miRNA expression. Future studies from our group will elucidate the influence of these confound-ing factors.

Plasma leptin was significantly higher in the pre-diabetic group when compared to non-diabetic controls, suggesting de-veloping or developed insulin resistance and leptin resistance in this group. We measured plasma C-peptide as a measure of endogenous insulin secretion, as both are secreted in equimo-lar amounts. Plasma C-peptide was significantly higher in the pre-diabetic group when compared to non-diabetic controls, suggesting that insulin is being synthesized and secreted in the pre-diabetic group. However, this group maybe progressing to-wards an insulin resistant state. A follow-up on this group is needed to confirm these findings.

To determine the relationship of hormones to erythrocyte miRNAs, Pearson’s correlation was used. We did find strong significant correlations between miRNA-15a and 25(OH)D, miRNA-223 and 25(OH)D, and miRNA-375 and 25(OH)D in non-diabetic African-American adults. Of particular interest is miRNA-223, which has been reported to be involved with adi-pocyte inflammation associated with morbid obesity[21]. Mice lacking miRNA-223 displayed systemic insulin resistance [21]. Likewise, 25(OH)D deficiency is reportedly associated with inflammation and obesity. The positive relationship between miRNA-223 and 25(OH)D may be indicative of normal insulin signaling in the non-diabetic group. Moreover, the significant relationship between miRNA-223 and 25(OH)D suggests a po-tential link between miRNA-223 and 25(OH)D in inflammation associated with the development and progression of obesity, insulin resistance, and T2DM in African-Americans.

Jacobs Publishers 8We also found significant correlations between miRNA-15b and total ghrelin and miRNA-15b and C-peptide in the pre-di-abetic group. MiRNA-15b targets key components of the in-sulin-signaling pathway to improve the action of insulin. The reduced expression of miRNA-15b results in insulin resistance in retinal endothelial cells [22]. It is possible that the signifi-cant negative correlation found between miRNA-15b and total ghrelin in pre-diabetic African-American adults is a result of impaired ghrelin suppression. We previously reported insulin resistant, obese African-American adolescents have impaired ghrelin suppression, contributing to obesity [17]. The findings of the present study further support our previous findings, as the reduced expression of miRNA-15b could potentially con-tribute to insulin resistance and elevated plasma ghrelin (Fig-ure 4). This idea is further supported by the significant posi-tive correlation between miRNA-15b and C-peptide (Figure 5), a measure of endogenous insulin production. This finding also suggests that insulin secretion may not be impaired in pre-di-abetic African-American adults; however, the subjects may be progressing towards an insulin resistant state. Further studies are needed to confirm these findings.

In the T2DM group, a strong significant correlation was also found between miRNA-15a and leptin, which suggests that miRNA-15a may play a role in maintaining leptin secretion. MiRNA-15a is reported to target uncoupling protein-2 (UCP2), which is an essential component of insulin biosynthesis and secretion. UCP2 mediates mitochondrial proton leak [23]. Zhang and colleagues reported UCP2–deficient mice have in-creased ATP levels and GSIS, supporting the concept that UCP2 negatively regulates insulin secretion. In the same study, the investigators found that UCP2 is upregulated in leptin deficient mice [23]. It is possible that the reduced expression of miR-NA-15a results in an increased expression of UCP2, thereby reducing the ATP/ADP ratio and impairing insulin synthesis and secretion. This reduced insulin secretion, in turn, results in reduced leptin secretion and vice versa (Figure 4). Howev-er, future functional studies are needed to confirm these novel relationships.

Conclusions

To our knowledge, this is the first study to identify relation-ships between erythrocyte miRNAs and hormones of energy metabolism (total ghrelin, vitamin-D, leptin and C-peptide) exclusively in African-American adults. Understanding the re-lationship between miRNAs and hormones will provide much needed insight into the intricate molecular mechanisms asso-ciated with obesity and T2DM development and progression. The altered expression of miRNAs could alter insulin secre-tion and insulin receptor function contributing to end organ insulin resistance. Likewise, altered miRNA expression could contribute to altered ghrelin or leptin receptor expression, which ultimately disrupts energy metabolism, leading to the

development of both obesity and T2DM. Moreover, hormones involved in regulating energy metabolism and glucose metabo-lism could function to suppress miRNAs that threaten both en-ergy and glucose homeostasis. However, additional function-al studies are needed to elucidate the impact of dysregulated hormones—as in the case of T2DM or diet induced obesity—and suppression of disruptive miRNAs. The inability to main-tain normal hormonal concentration may advance disruption of metabolic pathways or destruction of pancreatic β-cells, fur-ther advancing the diabetic state.

Although miRNAs hold much promise as biomarkers of T2DM and its complications, there is no global consensus on study design and laboratory procedures [24]. Two major challenges of miRNA research include specimen collection and normaliza-tion methods. Our study collected specimens in heparinized tubes, which may limit quantitation assays [24]. We carefully isolated erythrocytes using a HyPaque-Ficoll gradient. This procedure was performed twice to ensure maximum remov-al of heparin. To normalize our data, we utilized RNU6 (U6). However, recent studies suggest that U6 may be unsuitable for normalization [25]. U6 appears to be a suitable normalizer for our initial studies, based on Ct values. However, future studies will utilize multiple normalization methods to ensure optimal results. Utilizing synthetic spike-ins (cel-miR-39) can account for technical variation, but not biological variation [26]. There is also a limited knowledge on how anti-diabetic drugs and supplementations, such as vitamin D, influence miRNA expres-sion. This area must be examined further, as various therapies may negatively affect miRNA expression. The findings of this pilot and feasibility study provide necessary insight into our understanding of the use of miRNAs as markers and molecu-lar regulators of type 2 diabetes, especially in an understudied population—African-Americans. Our previous studies of insu-lin receptors on erythrocytes have revealed that these recep-tors mimic insulin receptors on adipocytes, hepatocytes, my-ocytes and other cell types considered to be targets of insulin action [27-29]. The findings of this study provide a foundation for future functional studies to assess the role of miRNAs in the regulation of these receptors.

Acknowledgements

The authors would like to acknowledge all the participants of this study, the Endocrine Fellows, Physicians and Staff of the Howard University Hospital Diabetes Treatment Center. The authors would also like to acknowledge the Nekhai Lab-oratory. This work was supported by the JHU-UMD DRC Grant P30DK079637-08, Grant #4525 and the NIH Research Grant 5G12MD007597. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The authors declared no conflict of interest. Author Contributions: MBF, KKG and GNB conceived, recruited patients and analyzed data. MBF, KKG,

Jacobs Publishers 9NK and SN carried out experiments and analyzed data. KKG, GNB, MBF and SN secured funding. All authors were involved in writing the paper and had final approval of the submitted published versions.

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26. Crosslan RE, Norden K, Bibby LA, Davis J, Dickinson AM. Evaluation of optimal extracellular vesicle small RNA isolation and qRT-PCR normalization for serum and urine. J Immunol Meth. 2016, 429: 39-49.

27. Gambhir KK, Archer JA, Bradley CJ. Characteristics of hu-man erythrocyte insulin receptors. Diabetes. 1978, 27(7): 701-708.

28. Gambhir KK, Archer JA, Nerukar SG, Cruz LA, Sanders M. Erythrocyte insulin receptors in chronic renal failure. Neph-ron. 1981, 28(1): 4-10.

29. Gambhir KK, Agarwal V. Red blood cell insulin receptors in health and disease. Biochem Med Metabol Bio. 1991: 45(2): 133-153.

Jacobs Publishers 11Supplemental Table 1. Erythrocyte miRNA expression in non-diabetic control, pre-diabetic, and T2DM African-American adults.

MiRNA Control Pre-Diabetic T2DM miR-499 Control (n=8) Pre-diabetic (n=7) T2DM (n=25)

0.00132 0.00549 0.00089 0.0031

0.00063 0.00647

0.000841 0.00392

0.0003440.00193

0.000682 0.000942 0.000466

0.0000315 0.00148

0.000301 0.000138 0.00143 0.00359 0.00147 0.00194 0.00139 0.00169 0.00133 0.00121 0.00088 0.00466 0.00135 0.00464 0.0038

0.00343 0.00342 0.0111

0.000312 0.00197 0.0312

0.00125 0.0016

0.00983 0.0217

miR-15b Control (n=5) Pre-diabetic (n=7) T2DM (n=23)

5616 4240 6554 1558 5549

1532 2030

367.3 1250 1774 462.1 1640

1069 2301 3198 1180 1413 985.3 3600 2319 1604 2073

861

2013 1365 6931 3849 1956 1109 5696 5518

812 1238 4950 6949

miR-15a Control (n=8) Pre-diabetic (n=7) T2DM (n=24)

61.73 1716 184.3 121.5 1475 3346 67.25 2257

35.9 220.4 88.39 287.8 119.9

117.9 184

12.33 5.883 420.5 1196 267.9 300.9 254.1 590.7 471.4 449.3 466.6 687.1 239.8 726.5

19700 9508 4219 4943 3681 3392

147 111.8 2189 3288

miR-126 Control (n=8) Pre-diabetic (n=7) T2DM (n=25)

35.74 678.4 51.63 355.2

91.55 114.6 82.77 190.7

46.7 30.15 123.7 259.3

Supplementary Supplemental Table 1. Erythrocyte miRNA expression in non-diabetic control, pre-diabetic, and T2DM African-American adults.

Jacobs Publishers 12

144.3 354.3 157.7 404.2

126.6 14.04 66.29

90.91 145.7

82.4 400.6 262.6 484.8 188.6 374.2

56.1 380.8 141.9 454.8 426.2 176.8

169 666.1 492.4 37.63 79.63 366.1 495.7

miR-375 Control (n=8) Pre-diabetic (n=7) T2DM (n=25)

0.028 0.0522

0.00274 0.0246 0.0178 0.0533 0.0193 0.0439

0.0167 0.0133 0.0132 0.0217

0.011 0.0244

0.024

0.00736 0.00488

0.011 0.0456 0.014

0.0235 0.0147 0.051

0.0405 0.0336 0.0178 0.0276

0.00854 0.0292 0.0136 0.0436 0.0462 0.0165 0.0189 0.0314 0.027

0.00778 0.0288

0.0442 0.0873

miR-146 Control (n=8) Pre-diabetic (n=7) T2DM (n=25)

0.2785 3.409

0.1756 0.5503

1.001 1.799

0.5769 2.147

0.4225 0.4167 0.6865 0.9217 0.3983

0.1477 0.5046

0.307 0.2019 0.5503

2.272 0.5283

0.906 0.6665

1.825 0.8902

3.128 0.7691

2.176 0.3789

1.229 0.9851

2.785 6.882 1.537

0.4648 2.385 3.437

0.2704 0.6146 0.9349

2.467

miR-223 Control (n=8) Pre-diabetic (n=7) T2DM (n=25)

1.407 9.61

0.6376 1.724 8.708 27.11 7.591 16.5

13.23 9.992 40.2

28.06 8.304

0.6117 26.52

36.57 16.57 19.58 44.65 12.27 20.81 9.388 21.15 19.76 23.34 9.48

31.03 29.69 38.73 24.63

0.4062 0.2514

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0.2765 0.0271

79.24 83.24 13.65

16.9 56.72 104.6

miR-224 Control (n=8) Pre-diabetic (n=7) T2DM (n=21)

0.000333 0.000225

0.0000385 0.000838

0.00039 0.00214 0.00105

0.000351

0.00996 0.00271

0.00000958 0.0000538

0.0003 0.000067

0.00208

0.00666 0.0000757

0.000535 0.00108

0.000153 0.000604

0.0000635 0.00053 0.0018

0.00117 0.00038 0.00279

0.000212 0.00111

0.000493 0.0018

0.000623 0.000102 0.000753

0.00376 0.00305

miR-326 Control (n=8) Pre-diabetic (n=7) T2DM (n=25)

0.2463 0.3166 0.0521 0.8625 0.1433 0.3362

0.267 0.2155

0.2012 0.2167 0.3668 0.3932 0.1763

0.085 0.2382

0.2337 0.0912 0.1492 0.1659 0.0747 0.1082 0.0651 0.2114 0.2777 0.4171 0.2824 0.2255 0.1154 0.2393 0.1263

0.2765 0.0271

79.24 83.24 13.65

16.9 56.72 104.6

miR-224 Control (n=8) Pre-diabetic (n=7) T2DM (n=21)

0.000333 0.000225

0.0000385 0.000838

0.00039 0.00214 0.00105

0.000351

0.00996 0.00271

0.00000958 0.0000538

0.0003 0.000067

0.00208

0.00666 0.0000757

0.000535 0.00108

0.000153 0.000604

0.0000635 0.00053

0.0018 0.00117 0.00038 0.00279

0.000212 0.00111

0.000493 0.0018

0.000623 0.000102 0.000753

0.00376 0.00305

miR-326 Control (n=8) Pre-diabetic (n=7) T2DM (n=25)

0.2463 0.3166 0.0521 0.8625 0.1433 0.3362

0.267 0.2155

0.2012 0.2167 0.3668 0.3932 0.1763

0.085 0.2382

0.2337 0.0912 0.1492 0.1659 0.0747 0.1082 0.0651 0.2114 0.2777 0.4171 0.2824 0.2255 0.1154 0.2393 0.1263 0.2784 0.4749

0.217 0.226

0.1793 0.2854 0.1212 0.3261 0.3809 0.7496

Jacobs Publishers 14Supplemental Table 2. Hormonal concentration in non-diabetic control, pre-diabetic, and T2DM African-American adults.

Hormone Control Pre-Diabetic T2DM Total Ghrelin Control (n=8)

Pre-diabetic (n=7) T2DM (n=25)

900 1000 1060 820

1200 800

1000 800

700 930

1300 900 550

1100 690

640 900 770

2500 700 600 820 510 630 860 590

1750 937 605 772 660 860

Leptin Control (n=8)

Pre-diabetic (n=7) T2DM (n=25)

6.4 2.8 2.4

2 2.6

17.6 20.8

52 24 56 44

102 102

116

58 30 44 26

15.8 9.2 22 44 24 68 52 96 58

220 116

64 52

7.4 24 92 34 58 42

25(OH)D Control (n=8)

Pre-diabetic (n=7) T2DM (n=25)

45.06 58.76 55.14 62.61

86.7 106.17 36.22 63.89 69.23 75.01 28.59 59.48 35.96

22.2 23.16 28.89 26.89 34.86 42.21 31.57

37.27 38.31 61.74

129.17 90.41 47.31 82.78

116.67 82.72 60.43 48.29 54.63 48.77

Supplemental Table 2. Hormonal concentration in non-diabetic control, pre-diabetic, and T2DM African-American adults.

Jacobs Publishers 15

26.02 55.35 33.97 64.32

49.08 92.92

37.1 26.3

115.48 53.35

151.77 102.55 72.01 52.13

C-Peptide Control (n=8)

Pre-diabetic (n=7) T2DM (n=25)

1.5 0.96 1.3 2.1 2.5 1.3 1.4 1.5 1.1 1.8 1.6 1.4

1 2.1 1.4 1.6 0.9

3.6 3.4

2 3

3.6 2.1

2.3

2 0.86 2.2 4.9 3.4 0.4 1.9 2.5 2.7

3 1.9

4 1.4 0.6 1.8

2.24 1

2.9 2.3

0.67 1.2 1.5

4 2.1 1.9

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miRNA Leptin 25(OH)D Total Ghrelin C-Peptide miRNA-499 r=-0.1806

R2=0.03261 p=0.6984

r=0.5302 R2=0.2811 p=0.1764

r=-0.654 R2=0.428 p=0.346

r=-0.4593 R2=0.211 p=0.2522

miRNA-15b r=0.2704 R2=0.0731 p=0.7296

r=0.611 R2=0.3733 p=0.2736

r=0.809 R2=0.655 p=0.399

r=-0.2407 R2=0.0579 p=0.6965

miRNA-15a r=-0.2246 R2=0.05046 p=0.6282

r=0.8061 R2=0.6497 p=0.0157*

r=0.081 R2=0.007 p=0.9188

r=-0.1718 R2=0.02953 p=0.6841

miRNA-126 r=-0.4402 R2=0.1938 p=0.3229

r=0.2318 R2=0.05372 p=0.5807

r=0.193 R2=0.037 p=0.807

r=-0.3449 R2=0.049 p=0.5978

miRNA-375 r=0.08251 R2=0.006809 p=0.8604

r=0.4139 R2=0.1713 p=0.308

r=-0.960 R2=0.922 p=0.039*

r=-0.4135 R2=0.171 p=0.3085

miRNA-146 r=-0.3188 R2=0.1016 p=0.4858

r=0.3034 R2=0.09207 p=0.465

r=-0.058 R2=0.942 p=0.9415

r=-0.4267 R2=0.182 p=0.2918

miRNA-223 r=-0.105 R2=0.01102 p=0.8228

r=0.7207 R2=0.5194 p=0.0437*

r=-0.241 R2=0.058 p=0.759

r=-0.2216 R2=0.154 p=0.3362

miRNA-224 r=0.03949 R2=0.001559 p=0.933

r=0.5547 R2=0.3077 p=0.1536

r=-0.189 R2=0.036 p=0.811

r=-0.0619 R2=0.003 p=0.8843

miRNA-326 r=-0.1504 R2=0.02263 p=0.7475

r=0.03207 R2=0.001028 p=0.9399

r=-0.742 R2=0.551 p=0.258

r=0.2555 R2=0.06527 p=0.3362

Pearson’s correlation was used to identify relationships. * p≤0.05 considered significant.

Supplemental Table 3. Correlation between hormones of energy metabolism and miRNA in non-diabetic healthy African-American adults.

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miRNA Leptin 25(OH)D Total Ghrelin

C-Peptide

miRNA-499 r=-0.3542 R2=0.1254 p=0.4357

r=-0.5145 R2=0.2647 p=0.2375

r=-0.09282 R2=0.0086 p=0.8431

r=-0.1232 R2=0.0151 p=0.7924

miRNA-15b r=-0.1417 R2=0.02007 p=0.7619

r=-0.4799 R2=0.2303 p=0.2758

r=-0.7980 R2=0.6369 p=0.0315*

r=0.783 R2=0.6131 p=0.0373*

miRNA-15a r=-0.2395 R2=0.05736 p=0.6050

r=-0.1216 R2=0.01479 p=0.7951

r=-0.01994 R2=0.00397 p=0.9662

r=0.02453 R2=0.0006 p=0.9584

miRNA-126 r=-0.5327 R2=0.2742 p=0.2277

r=-0.5524 R2=0.3051 p=0.1985

r=-0.2861 R2=0.08183 p=0.5340

r=0.5686 R2=0.3233 p=0.1829

miRNA-375 r=0.3977 R2=0.1581 p=0.3770

r=0.3833 R2=0.1469 p=0.3960

r=0.07342 R2=0.00539 p=0.8757

r=-0.5159 R2=0.2661 p=0.236

miRNA-146 r=-0.4370 R2=0.1910 p=0.3269

r=-0.4992 R2=0.2492 p=0.2541

r=0.1355 R2=0.01835 p=0.7721

r=-0.0132 R2=0.0002 p=0.9776

miRNA-223 r=-0.2026 R2=0.04103 p=0.6631

r=-0.3613 R2=0.1306 p=0.4528

r=0.3585 R2=0.1285 p=0.4297

r=-0.4106 R2=0.1686 p=0.3601

miRNA-224 r=-0.2764 R2=0.07640 p=0.5485

r=-0.5970 R2=0.3564 p=0.1570

r=-0.3830 R2=0.1467 p=0.3965

r=0.512 R2=0.2621 p=0.2401

miRNA-326 r=-0.4851 R2=0.2353 p=0.2699

r=-0.5217 R2=0.2722 p=0.2297

r=-0.2941 R2=0.08647 p=0.5221

r=-0.1244 R2=0.0155 p=0.7904

Pearson’s correlation was used to identify relationships. *p≤0.05 considered significant.

Supplemental Table 4. Correlation between hormones of energy metabolism and miRNA in pre-diabetic African-American adults.

miRNA Leptin 25(OH)D Total Ghrelin

C-Peptide

miRNA-499

r=-0.08341 R2=0.006957 p=0.7052

r=0.3684 R2=0.1357 p=0.0837

r=0.05292 R2=0.0028 p=0.8017

r=-0.1933 R2=0.0373 p=0.3769

miRNA-15b

r=0.278 R2=0.07726 p=0.225

r=0.2209 R2=0.04879 p=0.3359

r=0.1269 R2=0.0161 p=0.564

r=0.1393 R2=0.0194 p=0.5471

miRNA-15a

r=0.7943 R2=0.631 p≤0.0001****

r=0.099 R2=0.009801 p=0.6531

r=-0.1611 R2=0.02595 p=0.4521

r=0.01397 R2=0.0002 p=0.9508

miRNA-126

r=0.05997 R2=0.003596 p=0.7858

r=0.1483 R2=0.02199 p=0.4995

r=0.2955 R2=0.08731 p=0.1516

r=0.1949 R2=0.038 p=0.3728

miRNA-375

r=0.09987 R2=0.009974 p=0.6503

r=0.2734 R2=0.07477 p=0.2068

r=0.07176 R2=0.00515 p=0.7332

r=0.3143 R2=0.09876 p=0.1442

miRNA-146

r=-0.2731 R2=0.07459 p=0.2074

r=0.05179 R2=0.002683 p=0.8144

r=0.1585 R2=0.02512 p=0.4492

r=0.1151 R2=0.0132 p=0.6011

miRNA-223

r=-0.3371 R2=0.1137 p=0.1157

r=0.2042 R2=0.04169 p=0.3501

r=0.3024 R2=0.09142 p=0.1418

r=0.03996 R2=0.0016 p=0.8563

miRNA-224

r=0.1342 R2=0.01801 p=0.5839

r=-0.3331 R2=0.111 p=0.1634

r=0.06749 R2=0.00455 p=0.7713

r=0.2771 R2=0.0768 p=0.2368

miRNA-326

r=-0.1486 R2=0.02209 p=0.4985

r=-0.115 R2=0.01323 p=0.6012

r=-0.09502 R2=0.00902 p=0.6514

r=0.1136 R2=0.0129 p=0.6059

Pearson’s correlation was used to identify relationships. *p≤0.05 considered significant.

Supplemental Table 5. Correlation between hormones of energy metabolism and miRNA in T2DM African-American adults.

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