What can we learn from β-cell failure biomarker application in diabetes in childhood? A systematic review

Table 1 Overview of the included studies with their corresponding quality assessment

Type 1 diabetes
Ref.	Setting	Target population and sample size	Biomarker	Type of study	Quality (%)
Abdel-Moneim, et al[17], 2020	Egypt	Sample size: 100 subjects; 70 children diagnosed with T1DM and 30 healthy control subjects	Inflammation biomarker (MDA); Oxidative stress biomarker (NO)	Transversal	100
Babar et al[18], 2011	United States	Sample size: 36 children 21 with T1DM (aged 8.3 ± 0.3 yr with a diabetes duration of 4.3 ± 0.4 yr) and 15 group-matched healthy siblings (aged 7.6 ± 0.3 yr)	Vascular inflammatory biomarkers (hs-CRP, homocysteine C-reactive protein)	Transversal	87.5
Cabrera et al[39], 2018	United States	Sample size: 42 pediatric subjects	Innate immunity	Cohort	75.0
Cazeau et al[29], 2016	United States	Sample size: 9 children and adolescents aged 8-15 yr (mean age 12.9 ± 0.9 SD) with a mean T1DM duration of 5.0 ± 2.5 yr were included	Oxidative stress and endothelial dysfunction	Clinical trial/cohort	100
Cree-Green et al[41], 2018	United States	Sample size: 35 youth subjects with T1DM [median age 16-yr-old) and 22 nondiabetic youth subjects of similar age	Insulin resistance	Observational	100
Cummings et al[51], 2018	Canada	Participants (n = 199) were included in the observational arm of the Canadian AdDIT cohort	Urinary inflammatory biomarkers	Retrospective	62.5
Elbarbary et al[33], 2018	Egypt	Sample size: 60 children and adolescents with T1DM (aged ≤ 18 yr with at least 5 yr disease duration). Patients were compared with 30 age- and sex-matched healthy controls	Neopterin	Cross-sectional	75.0
Heier et al[52], 2015	Norway	Sample size: 299 patients with T1DM and 112 healthy controls at baseline and 241 patients and 128 controls at follow-up	AGEs methylglyoxal-derived hydroimidazolone-1 and carboxymethyllysine	Cohort	62.5
Ho et al[53], 2016	Canada	Sample size: Children aged 8 to 17 yr with T1DM for at least 1 yr	Serum C-peptide, HbA1c, serum inflammatory markers (IL-6, IFN-gamma, TNF-alpha, and IL-10), GLP-1 and GLP-2, intestinal permeability	Single-center, randomized, double-blind, placebo-controlled trial	62.5
Hoffman et al[54], 2016	United States	Sample size: 17 adolescents (8 F/9 M; age, 13.1 ± 1.6 yr (mean ± SD); duration, 4.8 ± 3.8 yr, BMI, 20.3 ± 3.1 kg/m2; HbA1c, 8.3% ± 1.2%	Inflammatory (C-reactive protein), oxidative (total anti-oxidative capacity) and endothelial injury (soluble intracellular adhesion molecule 1) markers	Cohort	50.0
Kingery et al[55], 2012	United States	Sample size: 34 patients of European ancestry with new-onset T1DM, aged between 3 and 17 yr (10.70 ± 3.45), at Nationwide Children’s Hospital in Columbus, Ohio	Complement C4A and C4B	Longitudinal	62.5
Lakhter et al[35], 2018	United States	Human experiment. Sample size: 16 healthy non-diabetic children (as a control group) and 19 children diagnosed with T1DM. Animal experiment: Serum was collected weekly from 8-wk-old female NOD mice until diabetes onset	miR-21-5pin Beta cell extracellular vesicle	Human experiment: cross-sectional; Animal experiment: longitudinal	75.0
Marchand et al[56], 2016	France	Sample size: 22 children at onset of T1DM within 2 d of clinical diagnosis (immediate insulin-requiring diabetes with at least one positive autoantibody) and before subcutaneous insulin treatment and sera from 10 control subjects (no obese and nondiabetic child)	miRNA-375	Transversal	62.5
Marek-Trzonkowska et al[57], 2016	Poland	Sample size: 12 children with recently diagnosed T1DM as well as that of untreated subjects	C peptide	Cohort	50.0
Redondo et al[58], 2014	United States	Sample size: 32 lean and 18 obese children (age range: 2-18 yr) with new-onset autoimmune T1DM and followed them for up to 2 yr	Leptin, total and high molecular weight adiponectin, omentin, resistin, chemerin, visfatin, cytokines [interferon-gamma, interleukin (IL)-10, IL-12, IL-6, IL-8, and tumor necrosis factor-alpha] and C-reactive protein	Cohort	62.5
Ryba-Stanisławowska et al[59], 2014	Poland	Sample size: 47 young patients (mean age; 14.25 ± 3.50 yr) diagnosed with T1DM. Control group consisted of 28 age- and sex- matched individuals	IL-12, IL-18. Blood glucose levels, lipid status, C-reactive protein, HbA1c	Transversal	62.5
Singh et al[60], 2017	United States	Sample size: 220 adolescents and children, half of which had lived with T1DM for an average of 7 years and half of which were healthy siblings	Leucine-rich alpha-2-glycoprotein, CD14, AZGP1, NAGA, CTSD, GM2A, GNS, CTSS	Cases and controls prospective	50.0
Şiraz et al[61], 2017	Turkey	Sample size: 80 patients who had T1DM for at least 5 yr and who had no chronic complications or an autoimmune disorder	Fetuin-A, Hb1Ac	Transversal	62.5
Thorsen et al[20], 2014	England	Sample size: 482 cases and 479 sibling frequencies matched on age and sample year distribution were included	Five different inflammatory chemokines (CCL2, CCL3, CCL4, CCL5 and CXCL8)	Cases and controls	100
Vorobjova et al[32], 2019	Estonia	Sample size: 72 patients (median age 10.1 yr) who had undergone small bowel biopsy were studied	Inflammation markers. Association with Enterovirus infection. Immunity	Longitudinal	87.5
Wołoszyn-Durkiewicz et al[19], 2019	Poland	NA	Advanced glycation end-products and their receptors, adhesive molecules, pro- and anti-inflammatory cytokines, growth factors or enzymes, such as N-acetyl-β-D-glucosaminidase	Review	25.0
Zhang et al[62], 2014	United States	NA	Autoantibodies [pancreatic islet antigens (insulin, glutamic acid decarboxylase and/or tyrosine phosphatase islet antigen 2)]	Review	37.5
Type 1 and 2 diabetes
Aburawi et al[30], 2016	United Arab Emirates	Sample size: 181 subjects; 79 patients with T1DMs, 55 patients with T2DM and 47 control subjects	Endothelial dysfunction biomarkers (sICAM-1, and sVCAM-1). Inflammation biomarkers Interleukin-6, tumor necrosis factor-α, high-sensitivity C reactive protein	Transversal	100
Krapfenbauer[63], 2017	Austria	NA	Novel protein chip technology	Review	50.0
Roberts et al[64], 2012	Australia	NA	Glucose in respiratory fluids	Review	12.5
Type 2 diabetes
Arenaza et al[36], 2017	Spain	Sample size: 84 children (age 8-12-yr-old) with high risk of T2DM. Control (n = 42) or intervention (exercise) (n = 42) group	microRNA expression in circulating exosomes and in peripheral blood mononuclear cells	Randomized control trial	87.5
Arslanian et al[65], 2019	United States	Sample size: 662 patients between 10-yr-old to 18-yr-old with DM for 2 yr (median duration 8 mo) with overweight or obesity	OGTT glucose response curve	Cohort	62.5
Bacha et al[40], 2014	United States	Sample size: 90 obese adolescents (37 normal glucose tolerant, 27 with prediabetes and 26 T2DM); Mean age for adolescents with T2DM was 15.1 ± 0.4 yr and duration of diabetes was 10.8 ± 2.5 mo	Inflammatory markers (Leptin, Interleukin-6, VCAM-1, ICAM-1, E-selectin)	Transversal	87.5
Barbosa-Cortés et al[31], 2017	Mexico	Sample size: 52 children (leukemia n = 26, lymphoma n = 26), who were within the first 5 yr after cessation of therapy. Median age of the subjects was 12.1 yr	Leptin. Adiponectin. Vascular cell adhesion molecule, intercellular cell adhesion molecule	Cohort	100
Berezin[66] 2016	Ukraine	NA	Growth differentiation factor-15	Review	25.0
Blum et al[21], 2014	United States	Animal experiment: Sample: Mouse strains: C57BL/6, Lep ob/ob, Lepr db/db, Ins2akita, Insulin2-Cre transgenic mice, Ucn3-GFP transgenic mice	Urocorti3	Cohort	50.0
Burke et al[67], 2017	United States	Animal experiment: Sample: 9-wk-old C57BL/6j and 5-wk-old male db/ and db/db mice	B-cell dedifferentiation biomarkers	Cohort	37.5
Can et al[68], 2016	Turkey	Sample size: 43 (18 males, 25 females) MetS adolescents between the ages of 13 and 17 yr (14.70 ± 1.15) and 43 lean controls were matched for age and sex	High-sensitive C-reactive protein, haptoglobin, α2-macroglobulin, platelet factor-4, fetuin-A, serum amyloid P and α1-acid glycoprotein	Transversal	62.5
Carlbom et al[34], 2017	Sweden	Sample size: 39 participants. 31 individuals with T2DM divided in 4 groups based on their current DM treatment. Control group with 8 participants	Functional Beta cell mass	Clinical trial	75.0
Cui et al[69], 2018	China	Human experiment: Sample size: 183 children and adults with obesity; Animal experiment: Obese ob/ob mice (3-wk-old to 4-wk-old), diabetic db/db mice (8-wk-old) and age-matched male C57BL/6J wild-type mice	Circulating microRNAs (miR-486, miR-146b and miR-15b),	Observational	62.5
DeBoer[70] 2013	United States	NA	Fasting insulin, adiponectin, retinol-binding protein 4	Review	-
Dentelli et al[27], 2013	Germany	Sample size: 25 patients with T2DM and 15 non-diabetic participants who underwent abdominal surgery (gallbladder removal, in situ colorectal cancer) were included. Non-diabetic participants were used as controls	Adipose-derived stem cell pluripotentiality	Transversal	75.0
Fanjul et al[71], 2010	France	Sample size: Human pancreases (n56) were harvested from brain- dead adult donors between 24 and 64-yr-old	Epithelial-mesenchymal transition biomarkers	Retrospective	50.0
Garanty-Bogacka et al[44], 2011	Poland	Sample size: 50 obese children and adolescents (boys and girls), aged 8 yr to 18 yr	High-sensitive C-reactive protein, interleukin-6, fibrinogen, white blood count, glucose, insulin, insulin resistance index, glycosylated hemoglobin, lipids as well as systolic and diastolic blood pressure	Cohort	50.0
Garnett et al[72], 2010	Australia	Sample size: 108 (54 each treatment arm) 10-yr-old to 17-yr-old with clinical features of insulin resistance and/or prediabetes	Insulin sensitivity	Randomized controlled clinical trial	37.5
Hansen et al[73], 2014	Denmark	NA	Iron	Review	25.0
Ishida et al[74], 2017	United States	Animal experiment: B6.BKS(D)-Leprdb/J heterozygous mice (db/+) were purchased from The Jackson Laboratory (Sacramento, CA) and bred to homozygosity to obtain wild-type, db/+, and db/db mice	Blood glucose and plasma insulin	Cohort	50.0
Jaberi-Douraki et al[75], 2014	Canada	NA	Autoantibodies (Auto-antigen-2)	Review	25.0
Janem et al[43], 2017	United States	Sample size: 3 pediatric cohorts ages 10-19 years were studied: lean (normal weight-C), obese (Ob) and obese with T2DM	Salivary glucose. Nitric oxide, CRP and IL-1B	Cohort	62.5
Kaya et al[76], 2017	Turkey	Sample size: 50 obese adolescents with IR were included in the study	Serum lipids, adiponectin, and interleukin-6 levels	Transversal	75.0
Kim-Muller et al[38], 2016	United States	Animal experiment. Groups: Mice were maintained in a mixed 120J-C57BL/6 background. Sample size calculations were based on the variance observed in prior experiments. No randomization or blinding was used	B cell dedifferentiation markers; Impaired mitochondrial function	Observational	87.5
Kim et al[77], 2016	United States	Sample size: 277 obese adolescents without diabetes completed a 2-h OGTT and were categorized to either a monophasic or a biphasic group	In vivo insulin sensitivity, insulin secretion, and β-cell function relative to insulin sensitivity	Observational	62.5
Kuo et al[78], 2016	United States	Animal experiment: Mice lacking the three FoxO isoforms in pancreas (TKO) using Tg (Ipf1-cre)1Tuv transgenics to excise the floxed Foxo1, Foxo3a, and Foxo4 genes	FoxO1, FoxO3a, and FoxO4	Longitudinal	62.5
Mastrangelo et al[79], 2016	Spain	Sample size: 60 prepubertal obese children (30 girls/30 boys, 50% IR and 50% non-IR in each group, but with similar BMIs)	47 metabolites (Taurodeoxycholate, LysoPC, LysoPE, LysoPS, acetylcarnitine, biliverdin, pyruvate, lactate, alanine, proline, valine, isoleucine, tryptophan, tyrosine, arginine, aspartate, glutamate)	Transversal	62.5
Neelankal et al[80], 2017	United States	Animal experiment: Pancreatic B cell line mouse insulinoma 6	Insulin. GLUT-2, Pdx1	Transversal	62.5
Park et al[81], 2016	Korea	Sample size: 214 children 7-9 yr of age	Persistent organic pollutants	Cohort	62.5
Rachdi et al[82], 2014	France	Animal experiment: mBACTgDyrk1A mice	DYRK1A	Cohort	-
Santoro et al[83], 2014	United States	Sample size: 80 adolescents (age 13.30 ± 3.31 yr; BMI 33.0 ± 6.79 kg/m2)	Cytokeratin 18	Transversal	62.5
Serbis et al[84], 2018	United States	Sample size: 88 children with obesity divided into two groups according to 1h-GL during an OGTT: group 1 (n = 57) consisted of those with 1h-GL < 8.6 mmol/L and group 2 (n = 31) of those with 1h-GL ≥ 8.6 mmol/L	HOMA-IR, Matsuda index and Cederholm insulin sensitivity index. Adiponectin, leptin, visfatin and interleukin-6 together with lipid levels	Transversal	62.5
Sheng et al[37], 2016	China	Animal experiment C57BLKS/J-Leprdb/Leprdb (db/db) and C57BLKS/J-Leprdb/m (db/m) male mice from the Shanghai Laboratory Animal Center, Chinese Academy of Sciences (SLAC, CAS)	B-cell dedifferentiation	Longitudinal	87.5
Solimena et al[42], 2018	Germany	Sample size: 103 organ donors, including 84 non-diabetic and 19 type 2 diabetic individuals, including 32 non-diabetic, 36 with type 2 diabetes, 15 with impaired glucose tolerance and 20 with recent-onset diabetes (< 1 yr)	Genes including TMEM37, which inhibited Ca2+ influx and insulin secretion in beta cells, and ARG2 and PPP1R1A	Retrospective	62.5
Spagnuolo et al[23], 2010	Italy	Sample size: 34 children (25 males; median age 10.8 ± 3.4 yr) with severe obesity; Groups: Normal glucose tolerance (n = 10), Impaired glucose tolerance (n = 24) and T2DM (n = 7)	Systemic and bowel inflammation markers	Transversal	75.0
Stansfield et al[28], 2016	United States	Sample size: 575 adolescents aged 14-18 yr (52% female, 46% black population)	Leptin. Adiponectin. C-reactive protein	Transversal	87.5
Tenenbaum and Fisman[85], 2012	Israel	NA	PPAR isoforms	Review	NA
Walker et al[25], 2014	Italy	Sample size: 33 children (mean BMI 28.1 ± 5.1 kg/m2 and mean age 11.6 ± 2.2 yr) with confirmed NAFLD. Biopsy samples of abdominal and liver were simultaneously collected	WAT inflammation biomarkers; Liver fibrosis biomarkers	Transversal	100
Walsh et al[24], 2018	Canada	Sample size: 66 boys and 136 girls aged 14-18 yr (15.4 ± 1.4 yr), who volunteered for the HEARTY trial	Glucose, HOMA-B, HOMA-IS, HbA1c, insulin	Randomized controlled trial	50.0
White et al[22], 2013	United States	Sample size: 3 individuals with diabetes and 5 nondiabetic control subjects	Insulin, vimentin, glucagon	Transversal	50.0
Xu et al[16], 2017	China	Animal experiment: Male Sprague-Dawley rats. 5 experimental groups: Normal control group. Model control group. Ginseng oligopeptides: Low-dose group, Medium-dose group, High-dose group	Antioxidant biomarkers; NF-KB, Bax, cleaved Caspase-3 and Bcl2	Clinical trial	100

NA: Not available; T1DM: Type 1 diabetes mellitus; SD: Standard deviation; F: Female; M: Male; BMI: Body mass index; HbA1c: Glycated hemoglobin; IR: Insulin resistance; OGTT: Oral glucose tolerance test; TKO: Total knock out; NAFLD: Nonalcoholic fatty liver disease; CRP: C-reactive protein; IL: Interleukin; IFN: Interferon; TNF: Tumor necrosis factor; hs-CRP: High-sensitivity C-reactive protein; NO: Nitric oxide; GL: Glucose; HOMA-IR: Homeostatic Model Assessment of Insulin Resistance; VCAM: Vascular cell adhesion molecule; ICAM: Intracellular cell adhesion molecule; MDA: Malondialdehyde; AGE: Advanced glycation end-product; AdDIT: Adolescent type 1 Diabetes cardio-renal Intervention Trial; MetS: Metabolic syndrome; HOMA-B: Homeostasis model assessment-B cell; HOMA-IS: Homeostatic model assessment of insulin sensitivity; WAT: White adipose tissue; PPAR: Proliferator-activated receptor.

Table 2 Overview of the articles included with the study characteristics

Ref.	Setting	Biomarker	Target molecule	Antibodies/ kits	Pretreatment before determination	Methods of quantifying biomarkers	Target population/ sample size	Exclusion criteria	Written informed consent	Clinical data/Lab/Rx	Relevant results	Correlation with other biomarkers	Chronic complications
Type 1 diabetes
Abdel-Moneim, et al[17], 2020	Egypt	Inflammation biomarker (MDA); Oxidative stress biomarker (NO)	MDANO	NO and MDA → Colorimetric kits (BioVision, Milpitas, California, United States)	No pretreatment was specified	Colorimetry	Sample size: 100 subjects; 70 children diagnosed with T1DM and 30 healthy control subjects. Healthy subjects (control group) (n = 30, 2-16-yr-old), children newly diagnosed with T1DM (up to 1 yr) (n = 21, 3-14-yr-old) children with established T1DM (within 1.1 to 10.0 yr) (n = 49, 3-14-yr-old)	Infectious diseases, autoimmune disorders, allergies, respiratory disorders, thyroid dysfunction, hematologic disorders, malignancies and chronic cardiac, kidney and liver diseases	Yes	Body mass index; Sex and age; Time of DM1 diagnose; Fasting blood glucose; Glycated hemoglobin; Microalbuminuria; Hematologic profile	This study demonstrates that the hematologic profile in pediatric patients with T1DM showed significant abnormalities such as decreased red blood cells, and platelet count, hemoglobin and hematocrit elevation as well as elevation of inflammation and oxidative stress markers, which can be used in early detection of children with T1DM at risk of complications	Erythrocyte count; Platelet count; Leucocyte count; Neutrophile count; Hemoglobin; Hematocrit; Red blood cell distribution width; Micro albuminuria	Nephropathy
Babar et al[18], 2011	United States	Vascular inflammatory biomarkers (hs-CRP, Homocysteine C-reactive protein)	hs-CRP, Homocysteine C-reactive protein	hs-CRP → solid- phase ELISA from MP Biomedicals (West Chester, PA); Homocysteine→ Siemens Immulite platforms and kit (Diagnostic Products, Flanders, NJ)	No pretreatment was specified	Solid-phase ELISAs; Chemilumine scence	Sample size: 36 children, 21 with T1DM (aged 8.3 ± 0.3 yr with a diabetes duration of 4.3 ± 0.4 yr) and 15 group-matched healthy siblings (aged 7.6 ± 0.3 yr)	Known dyslipidemia, hypertension, microvascular complications, anemia (hemoglobin, 11.0 g/dL), congenital heart disease, allergy to ultrasound gel, or family history of hypercholesterolemia or premature cardiovascular disease	Yes	Age, sex, BMI, blood pressure, blood cell count, plasma glucose, HbA1c, lipids, hs-CRP, fibrinogen, chemistry panel, homocysteine, and erythrocyte folate, lipid profile. Diabetes duration in study group. Insulin regimen and dose	T1DM preadolescent children showed increased vascular inflammation and presented with higher fasting plasma glucose, HbA1c and hs-CRP; the flow-mediated dilatation was attenuated; this suggests endothelial dysfunction, harbingers of cardiovascular risk	Vascular inflammatory biomarkers. Endothelial dysfunction marker; Fasting plasma glucose, oral glucose tolerance curve, lipid profile, HbA1c, hs-CRP, fibrinogen, homocysteine, and erythrocyte (red blood cell)	Macroangiopathy
Cazeau et al[29], 2016	United States	Oxidative stress and endothelial dysfunction	Endothelial function/ dysfunction: (ECFCs: CD34+, CD133+, CD45-). Oxidative stress: TAOC, hs-CRP, endothelial progenitor cells	Antibodies (2.5 μmol/L of each), PE-AC133, FITC-CD34, and PECy5-CD45. Oxidative stress → TAOC (Biovision Research Products, Mountain View, CA)	A 50 μmol/L volume anticoagulated peripheral blood was incubated with 50 μmol/L 3% BSA in PBS (without Ca++ and Mg++) at room temperature for 30 min. Fluorescent antibodies were added and incubated for 30 min. FACS lysis buffer was added and incubated for another 30 min	ECFCs → polychromatic flow cytometry method	Sample size: 9 children and adolescents aged 8-15 yr (mean age 12.9 ± 0.9 SD) with a mean T1DM duration of 5.0 ± 2.5 yr were included	BMI ≥ 95%, BP > 95%, Tanner 1 or 5 pubertal status, pregnancy, smoking, history of oral hypoglycemic use, acanthosis nigricans, fasting c-peptide ≥ 0.4 ng/mL, abnormal thyroid function tests, random urine albumin to creatinine ratio ≥ 0.02 mg albumin/mg creatinine, creatinine >1.0 mg/dL or use of any medications other than insulin, levothyroxine with stable dosage, or oral contraceptives were excluded from the study	Yes	Age, mean duration of diabetes, diabetes treatment (insulin), BMI, HbA1c, pubertal stage of Tanner stages 2-4, normal thyroid function tests, random urine albumin/creatinine ratio < 0.02, creatinine < 1	This study demonstrated that there is no significant benefits for antioxidative therapy; there were no findings of decreased oxidative damage, improved endothelial function or increased vascular repair capacity	Endothelial function/dysfunction; Oxidative damage; Inflammatory biomarkers (IL-6 and CRP).hs-CRP and TAOC, adiponectin; Forearm blood flow	Prevention of Macroangiopathy
Cree-Green et al[41], 2018	United States	Insulin resistance	2H5 glycerol and 6,6-2H2 glucose; hs-CRP; Adiponectin, leptin, and C-peptide	hs-CRP → immune-turbidimetric assay (Beckman Coulter, Brea, CA)	No pretreatment was specified	2H5 glycerol and 6,6-2H2 glucose → gas chromatography–mass spectrometry	Sample size: 35 youth subjects with T1DM (median age 16-yr-old) and 22 non-diabetic youth subjects of similar age	ALT 80 IU/mL; blood pressure 140/90 mm Hg; hemoglobin, 9 mg/dL; serum creatinine.1.5 mg/dL; smoking; medications affecting IR, blood pressure, or lipids; and, in youths with T1DM, HbA1c, 12%	Yes	Age. Female/male, n/n (% female) Height, cm. Weight, kg. BMI, kg/m2. BMI percentile. Ethnicity. White Hispanic Black Asian. Tanner stage. Waist circumference, cm Waist-to-hip ratio. Lean mass %. Total body fat %. Liver fat. Visceral fat %. Daily METs from 3DPAR Accelerometer data. Sedentary Lifestyle, light, moderate, vigorous, very vigorous activity	Insulin resistance (adipose, hepatic and peripheral) was present in adolescents despite obesity or metabolic syndrome, an elevated lipolysis rate and endogenous glucose production and a lower peripheral glucose uptake supported these results.	Insulin sensitivity (adipose, hepatic and peripheral). Inflammation biomarkers	β-cell function
Elbarbary et al[33], 2018	Egypt	Neopterin	Neopterin	Neopterin → (ELISA) using kit supplied by IBL International GmbH, Hamburg, Germany	Serum obtained from clotted samples by centrifugation for 15 min at 1000g was used for chemical analysis and stored at -20 °C until subsequent usein ELISA	ELISA	Sample size: 60 children and adolescents with T1DM (aged ≤ 18 yr with at least 5 yr disease duration). Patients were compared with 30 age- and sex-matched healthy controls	Chronic infection, neurological disease, history of drugs that may affect neural function, nutrition deficiency, liver or renal dysfunction, connective tissue disease or other autoimmune disorders and any other conditions that could influence hs-CRP. Patients with hs- CRP > 10 mg/L, other microvascular nephropathy and retinopathy and macrovascular diabetic complications	Yes	Age of onset of diabetes, disease duration, insulin therapy and chronic diabetic complications (nephropathy, neuropathy, retinopathy or cardiovascular ischemic events). Anthropometric measurements. Pubertal stage. Blood pressure. Fasting blood glucose, fasting lipid profile, liver function tests, renal function tests	Neopterin, a marker of inflammation and cellular response, was significantly higher in pediatric patients that presented with diabetic peripheral neuropathy, which demonstrates it may be used as an early biomarker for DPN.	Inflammation biomarkers; Cellular immune response; Neurological neuropathy	Diabetic; Neuropathy
Lakhter et al[35], 2018	United States	miR-21-5p in Beta cell extracellular vesicle	miR-21-5pin Beta cell extracellular vesicle	EV isolation→ culture media using ExoQuick Tc reagent (System Biosciences, Palo Alto, CA, United States); miRNeasy and miScipt II RT kits (Qiagen, Valencia, CA, United States); Agilent Small RNA kit Bioanalyzer instrument (Agilent Technologies, Santa Clara, CA, United States)	To model the inflammatory milieu of T1DM, samples were exposed to cytokine mix consisting of 5 ng/ml IL-1β, 100 ng/ml IFN-γ and 10 ng/ml TNF-α (R&D Systems, Minneapolis, MN, United States); To inhibit cytokine- induced apoptosis, 25 μmoL/L of the pancaspase inhibitor Z-VAD-FMK (R&D Systems)	RNA isolation and reverse transcription; Quantitative real-time PCR.	Human experiment; Sample size: 16 healthy non-diabetic children (as a control group) and 19 children diagnosed with T1DM. Animal experiment; Serum was collected weekly from 8-wk-old female NOD mice until diabetes onset	Diabetic ketoacidosis requiring an intensive care unit stay, diabetes other than type 1 diabetes, history of prior chronic illness known to affect glucose metabolism or use of medications known to affect glucose metabolism	Yes	Age, sex, BMI percentile, HbA1c mmol/mol	This study demonstrated cytokine treatment in beta cell lines resulted in a 1.5-3.0 increase in miR-21-5p, however nanoparticle tracking showed that cytokines have no effect on the number of circulating miR-21-5p in beta cell extracellular vesicle cargo	miR-21-5p	Pancreatic reserve
Vorobjova et al[32], 2019	Estonia	Inflammation markers; Association with Enterovirus infection; Immunity	33 inflammation cytokinesanti-EV IgA and IgG; Density of Tregs and dendritic cells in small intestine mucosa	Cytokines → kits of Milliplex® MAP Magnetic BeadAntibodies for DC detection → monoclonal mouse anti-CD11c, anti-IDO, anti-CD103 , andanti-langerin (CD207) Antibodies for Treg detection → antiFOXP3	Patients’ sera were collected and stored at -80 °C prior to analyses. Microtiter plates (Nunc Immunoplate, Nunc, Glostrup, Denmark) were coated with the synthetic enterovirus peptide (KEVPALTA- VETGATC, Storkbio Ltd., Estonia) derived from an immunodominant region of the capsid protein VP1] at 2.5 μg/mL in a carbonate bicarbonate buffer	Immunohistochemistry; ELISAs	Sample size: 72 patients (median age 10.1 yr) who had undergone small bowel biopsy were studied. The study group consisted of 24 patients with CD (median age 6.5 years), 9 patients with CD + T1D (median age 7.0 years), two patients with T1DM (median age 8.5 years), and 37 patients (median age 14.0 years) with functional gastrointestinal disorders and a normal small bowel mucosa as controls	No exclusion criteria were specified	Yes	Age, sex, status of small bowel mucosa	This study demonstrated a positive correlation between elevated cytokines (specially 12 of the 33 cytokines studied) in pediatric patients presenting T1DM and celiac disease	No other biomarkers were associated	Co-morbidities: celiac disease
Type 1 and 2 diabetes
Aburawi et al[30], 2016	United Arab Emirates	Endothelial dysfunction biomarkers (sICAM-1, and sVCAM-1). Inflammation biomarkers Interleukin-6, tumor necrosis factor-α high-sensitivity C-reactive protein	sICAM-1, sVCAM-1)	sICAM-1 and sVCAM-1→ ELISAs from R&D Systems (Human TNFα Quantikine, DTA00C)	No pretreatment was specified	ELISAs	Sample size: 181 subjects; 79 patients with T1DM, 55 patients with T2DM and 47 control subjects. Mean age was 20 ± 6 (age range 12-31-yr-old)	Active infection, chronic illness (e.g., rheumatoid arthritis, hyperthyroidism and inflammatory bowel disease), regular use of certain medications (β-blockers, α-blockers, diuretics and hormone therapies), pregnancy and inability to give informed consent	Yes	Age (12-31-yr-old). Anthropometric measurements (weight, height, waist and hip circumference). Heart rate variability; Glucose levels, Glycated hemoglobin, lipid profile, inflammation markers, endothelial dysfunction markers, kidney function tests	T1DM patients usually present subclinical inflammation and endothelial dysfunction (with higher levels of sICAM-1 and sVCAM-1). Poor control associates with higher levels of adiponectin and haptoglobin, while obesity correlated with lower levels of it but higher inflammatory and endothelial biomarkers	Glucose, HbA1c, low- density lipoprotein, high-density lipoprotein, triglycerides, total cholesterol. Adiponectin	Macroangiopathy
Type 2 diabetes
Arenaza et al[36], 2017	Spain	microRNA expression in circulating exosomes and in peripheral blood mononuclear cells	miRNA in circulating exosomes	Exosomes → Total Exosome Kit (Thermo Fisher Scientific). Exosomal fractions → Qiagen miRNeasy kit. miRNA → RNA-seq methodology on Illumina’s MiSeq Next Generation Sequencing system	Exosomes were obtained from 1 mL of plasma usingthe Total Exosome Isolation Kit (Thermo Fisher Scientific) following manufacturer’s protocols	FASTQ files generationand sequence alignment were performed using specific software packages (MiSeq Reporter, Bowtie, SAMtools, DESeq2 and miRDeep)	Sample size: 84 children (age 8-12-yr-old) with high risk of T2DM; Control (n = 42) or intervention (exercise) (n = 42) groups	Children with any medical condition that could affect the results of the study or that limits physical activity were excluded	Yes	Anthropometric measures (height, body mass and waist circumference), blood pressure, adiposity levels (total, abdominal, visceral and ectopic), insulin sensitivity, lipid profile, cardiorespiratory fitness, liver enzymes	The PREDIKID project helped to identify miRNAs potentially associated with early onset of IRS in overweight/ obese children overweight/ obesity	Fasting insulin, glucose and hemoglobin A1c; ectopic fat. Carotid intima-media thickness. Inflammation and biochemical cardiovascular disease risk factors	T2DM prediction: Macroangiopathy
Bacha et al[40], 2014	United States	Inflammatory markers (Leptin, Interleukin-6, VCAM-1, ICAM-1, E-selectin)	Leptin; Interleukin-6; VCAM-1 ICAM-1; E-selectin PAI-1	Leptin → Radio- immunoassay (Linco Research Inc., St. Charles, MO) Il-6, VCAM-1, ICAM-1, and E-selectin → Double-sandwich ELISAs (R&D Systems, Minneapolis, MN); PAI-1 and TNF-a Cytometer– based platform (Luminex MAP 200; Milli- pore, St. Charles, MO)	No pretreatment was specified	Double-sandwich ELISAs	Sample size: 90 obese adolescents (37 normal glucose tolerant, 27 with prediabetes and 26 T2DM) Mean age for adolescents with T2DM was 15.1 ± 0.4 yr and duration of diabetes was 10.8 ± 2.5 mo	No exclusion criteria were specified	Yes	Male/female. AA/white. Smoking history (no/yes/not available) Age. BMI. Fat mass. Body fat (%). Total abdominal fat (cm2) SAT (cm2) VAT (cm2). Blood pressure, lipid profile, glucose levels, glycated hemoglobin, adiponectin, leptin, smoking history	Coronary artery calcifications were unexpected in pediatric population; in this study CAC+ group had higher BMI, waist circumference and fat mass, evidence of CAC highlight the increased cardiovascular risk in obese pediatric population	Subclinical atherosclerosis (vascular markers): CACs. Aortic pulse wave velocity. Carotid intima-media thickness	Macroangiopathy
Barbosa-Cortés et al[31], 2017	Mexico	Leptin; Adiponectin; VCAM; ICAM	Leptin; Adiponectin; VCAM; ICAM	Leptin and adiponectin levels, VCAM and ICAM → ELISA (Human Adiponectin, Leptin, VCAM and ICAM ELISA Kits, Millipore, St. Charles, MO, Unites States),	Serum aliquots were separated and frozen at -80 °C until biochemical determination	ELISAs	Sample size: 52 children (leukemia n = 26, lymphoma n = 26), who were within the first 5 yr after cessation of therapy. Median age of the subjects was 12.1 yr	Subjects with a relapse, second neoplasm or bone marrow transplants	Yes	Anthropometric measures (body weight, height, waist circumference, body mass index, pubertal stage, body fat percentage) nutritional status. Sex, age, age at cancer diagnosis, diagnosis (leukemia or lymphoma) and treatment received. Pubertal stage; Fasting glucose, glucose, insulin resistance and adiposity	This study revealed that there were diverse biomarkers, such as HOMA-IR, leptin and leptin/adiponectin, which correlate with the risk of developing metabolic syndrome in the pediatric population that survived lymphoma and acute lymphoblastic leukemia	Insulin, HOMA-IR, Adiponectin, Leptin, Leptin to Adiponectin ratio Body fat. Glucose (mg/dL); Lipid profile	T2DM prediction
Cabrera et al[39], 2018	United States	Innate immunity	CD4 lymphocytes; T-cells (CD4+/CD45RA−/FOXP3high)	PBMCs were stained with fixable LIVE/DEAD Violet dye followed by blocking Fc receptors and staining for anti-CD4, anti-CD25, anti-CD45RO, anti-CD45RA and anti-CD127	Cryopreserved PBMCs were obtained from each of the subjects. PBMCs were pre-treated in medium with CTLA4-Ig (Bristol-Myers Squibb, New York, NY, Unites States) at 0 μg/mL, 25 μg/mL and 82 μg/mL for 45 min prior to the addition of plasma from a recently diagnosed local individual	LSR II flow cytometer (BD Bioscience)	Sample size: 42 pediatric subjects	No exclusion criteria were specified	Yes	Clinical data was not specified	The results of this study showed the existence of multiple T1DM subtypes, which differentiate by varying levels of innate inflammation and its association with C-peptide. It also demonstrated the different responsiveness to therapeutic intervention with CTLA4-Ig (abatacept)	C-peptide; Innate inflammatory activity
Carlbom et al[34], 2017	Sweden	Functional Beta cell mass	Functional Beta cell mass (HOMA2-B and HOMA2-IR)	Function of B cell mass → Intravenous arginine test and a glucose-potentiated arginine test	No pretreatment was specified	Arginine test and glucose-potentiated arginine test	Sample size: 39 participants. 31 individuals with T2DM divided in 4 groups based on their current DM treatment. Control group with 8 participants	No exclusion criteria were specified	Yes	Sex, age, diabetes duration (years), anti-diabetes treatment, BMI, plasma glucose, plasma c-peptide, HbA1c, HOMA, plasma cholesterol, plasma triglycerides, HDL cholesterol, LDL cholesterol	[11C]5-HTP uptake and retention in pancreas was a surrogate marker for the endogenous islet mass. Nonetheless, the major cause of B-cell failure in T2DM was attributed to cell dedifferentiation and not cell loss, which is why this is not mirrored by a decrease of [11C]5-HTP uptake in the pancreas	HbA1c levels, plasma glucose and plasma C-peptide levels	Function β-cell
Dentelli et al[27], 2013	Germany	Adipose-derived stem cell pluripotentiality	Octamer-binding transcription factor and NANOG; NOX-generated reactive oxygen species	ASCs → FACS analysis (FACS-Calibur flow cytometer; BD Biosciences, San Jose, CA, United States)	Visceral N-ASCs were maintained in normal glucose DMEM(5 mmol/L D-glucose), high glucose DMEM (25 mmol/l D-glucose)or high mannitol DMEM (19 mmol/l D-mannitol, asosmotic control)	ASC proliferation → direct cell count (FACS); Apoptosis → ELISAs; mRNA isolation and quantitative real-time PCR;Western blot	Sample size: 25 patients with T2DM and 15 non-diabetic participants who underwent abdominal surgery (gallbladder removal, in situ colorectal cancer) were included. Non-diabetic participants were used as controls	No exclusion criteria were specified	Yes	Gender, age, BMI, HbA1c, triglycerides, total cholesterol, LDL and HDL cholesterol, creatinine, retinopathy, blood pressure, diabetes duration	This study demonstrated that adipose-derived stem cell were higher in diabetic patients than in control group as well as production of OCT4 and NANOG, which were upregulated, supporting the use of ASCs in regenerative medicine	Adipose-derived stem cells (ASCs); NOX-generated reactive oxygen species
Kim-Muller et al[38], 2016	United States	B cell dedifferentiation markers; Impaired mitochondrial function	Aldehyde dehydrogenase 1 isoform A3; Foxo1; MafA	No antibodies or kits were specified	Cells were incubated with aldefluor and selected for RFP (red) and aldefluor (green) fluorescence, yielding ALDH andALDH+ cells	Immunohistochemistry	Animal experiment. Groups: Mice were maintained in a mixed 120J-C57BL/6 background. Sample size calculations were based on the variance observed in prior experiments. No randomization or blinding was used.	No exclusion criteria were specified	No	Not applied	This study demonstrated that in adolescents with obesity the risk of presenting T2DM was higher if they present a monophasic oral glucose tolerance test, they present higher glucose, insulin and C-peptide and lower in vivo hepatic and peripheral insulin sensitivity, thus this can be used as a biomarker for T2DM	Aldehyde dehydrogenase 1 isoform A3	T2DM prediction
Sheng et al[37], 2016	China	B-cell dedifferentiation	Glut2, Pdx1, Nkx6.1; MafA; Foxo1; GLP-1; PKC	Antibodies: polyclonal rabbit anti-Pdx1 Ab), polyclonal rabbit anti-MafA Ab), polyclonal rabbit anti-Nkx6.1 Ab, polyclonal rabbit anti-Glut2Ab, monoclonal rabbit anti-PKCζ Ab, monoclonal mouse anti-insulin Ab, polyclonal rabbit anti-glucagon Ab, polyclonal rabbit Ab and polyclonal rabbit anti-Foxo1 Ab	Pancreata were harvested and fixed in 4% buffered formaldehyde. Islets were incubated over a period of 60 min in 1 mL; Krebs-Ringer bicarbonate Hepes buffer (KRBH, 140 mMNaCl, 3.6 mM KCl, 0.5 mM NaH2PO4, 0.5 mM MgSO4,1.5 mM CaCl2, 2 mM NaHCO3, 10 mM Hepes (pH 7.4), and 0.25% BSA) containing 2.8 mmol/L glucose or 16.7 mmol/L glucose	Immunochemistry; Immunofluorescence; Quantitative PCR Analysis	Animal experiment; C57BLKS/J-Leprdb/Leprdb (db/db) and C57BLKS/J-Leprdb/m (db/m) male mice from the Shanghai Laboratory Animal Center, Chinese Academy of Sciences (SLAC, CAS)	Not applied	Not applied	Glucose tolerance tests; Serum insulin	This study demonstrated that Glut-2, MafA, Pdxl and Nkx6.1 were deactivated during B cell dedifferentiation; consequently the identification of small molecules that increase the expression of these factors may be useful in treatment of T2DM patients	No other biomarkers were associated	β-cell function
Spagnuolo et al[23], 2010	Italy	Systemic and bowel inflammation markers	C-reactive protein; NO; Fecal calprotectin	Fecal concentration of calprotectin → (ELISA) test (Calprest Eurospital, SpA, Trieste, Italy	No pretreatment was specified	ELISAs	Sample size: 34 children (25 males; median age 10.8 ± 3.4 yr) with severe obesity; Groups: Normal glucose tolerance (n = 10), Impaired glucose tolerance (n = 24) and T2DM (n = 7)	Inflammatory bowel diseases and systemic or intestinal infections were excluded during the evaluation.	Not specified	Age, pubertal stage, BMI, oral glucose tolerance test; anthropometric measures (weight, height)	This study demonstrated a positive correlation between obesity and glucose abnormalities with the elevation of biomarkers demonstrating intestinal inflammation in the pediatric population	C- reactive protein; Nitric oxide; Plasma insulin; HOMA-IR	Pancreatic reserve
Stansfield et al[28], 2016	United States	Leptin; Adiponectin; C-reactive protein	Leptin; Adiponectin; C-reactive protein	Leptin → ELISA (R&D Systems, Minneapolis, Minnesota); Adiponectin → ELISA (Linco Research, St. Charles, Missouri); CRP → high-sensitive ELISA (ALPCO Diagnostics, Salem, New Hampshire)	No pretreatment was specified	ELISAs	Sample size: 575 adolescents aged 14-18 years (52% female, 46% black population)	If they were taking medications or had any medical conditions that could affect growth, maturation, physical activity, nutritional status or metabolism.	Yes	Age, sex, race, Tanner stage, BMI percentile, BMI category, physical activity and socioeconomic status and BP. Fasting serum glucose, HOMA-IR, plasma triglycerides, plasma total cholesterol, plasma HDL cholesterol, plasma LDL cholesterol serum leptin, plasma adiponectin and plasma C-reactive protein	This study demonstrated that there was no association between birth weight and the development of greater visceral adiposity and elevation of biomarkers implicated in insulin resistance once they reach an adolescent age	Birth weight, fasting blood samples were measured for glucose, insulin, lipids, adiponectin, leptin, and C-reactive protein	T2DM prediction
Walker et al[25], 2014	Italy	WAT inflammation biomarkers; Liver fibrosis biomarkers	C-reactive protein, TNF-a, IL-6; Crown like structures in SAT	CRP → high sensitivity latex agglutination method on HITACHI 911 Analyzer (Sentinel Ch., Milan). TNF-a and IL-6 → sandwich ELISA (R&D System Europe Ltd, Abingdon, United Kingdom)	Biopsies were routinely processed (i.e. formalin-fixed and paraffin-embedded) and sections of liver tissue were stained with hematoxylin-eosin, Van Gieson, Periodic acid-Schiff diastase and Prussian blue stain	High sensitivity latex agglutination; Sandwich ELISAs	Sample size: 33 children (mean BMI 28.1 ± 5.1 kg/m2 and mean age 11.6 ± 2.2 yr) with confirmed NAFLD.Biopsy samples of abdominal (SAT) and liver were simultaneously collected	Adipose samples from 7 of the 40 children biopsied were not viable, and these participants were excluded from formal analysis.	Yes	Anthropometric measures; ALT; AST; gamma-glutamyltranspeptidase, total triglycerides and LDL and HDL cholesterol, plasma insulin, oral glucose tolerance test	This study demonstrated that the presence of crown like structures in liver biopsies were related to liver fibrosis but independent of BMI and this may contribute to diabetes risk by reducing insulin secretion. Liver fibrosis was associated with the presence of white adipose tissue inflammation, and this was strongly associated with obesity	HOMA; AST, ALT, total triglycerides, LDL, HDL, Adiponectin	T2DM prediction
Xu et al[16], 2017	China	Antioxidant biomarkers; NF-KB, Bax, cleaved Caspase-3 and Bcl2	Level of antioxidant SOD, MDA and GSP levels; Beta cell function was measured (HOMA).	SOD, MDA and GSP → kits Nanjing Jiancheng Biotechnology Institute (Nanjing, China). Antibodies for Bax, Bcl-2, cleaved Caspase-3 and NF-KB → Santa Cruz Biotechnology (Santa Cruz, CA, United States). Nuclear and cytoplasmic protein → ProteoJETTM Cytoplasmic and Nuclear Protein Extraction kit; (Fermentas International Inc., Burlington, ON, Canada). Protein content → BCA protein assay kit (Pierce Biotechnology, Rockford, United States)	Cells were scraped, pelleted by centrifugation at 250 g for 5 min, mixed with cell lysis buffer, homogenized, and set on ice for 10 min. Then, themixture was centrifuged at 500 g for 7 min at 4 C, and then the supernatant was further centrifuged at 20000 g for 15 min at 4 °C	Immunochemistry; Western Blot; ELISAs	Animal experiment; Male Sprague–Dawley rats; Five experimental groups: Normal control group; Model control group; Ginseng oligopeptides (GOP): Low-dose group (GOP-L); Medium-dose group (GOP-M); High-dose group (GOP-H)	Not exclusion criteria were specified	Not applied	Not applied	This study demonstrated that treatment with ginseng oligopeptides partially reversed abnormal oral glucose test tolerance in rats induced with T2DM and demonstrated an amelioration of the pancreatic damage and increased insulin content	Oral glucose tolerance, plasma glucose, serum insulin, ALT, AST, serum urea nitrogen, total cholesterol, triglycerides	T2DM prediction

TAOC: Total plasma antioxidant capacity; EV: Extracellular vesicle; PAI-1: Plasminogen activator inhibition factor; CAC: Coronary artery calcifications; PBMC: Peripheral blood mononuclear cells; ASC: Adipose-derived stem cell; SOD: Superoxide dismutase activity; GSP: Glycated serum protein; Rx: Prescription; MDA: Malondialdehyde; NO: Nitric oxide; hs-CRP: High-sensitivity C-reactive protein; miR: MicroRNA; sVCAM: Soluble vascular cell adhesion molecule; sICAM: Soluble intracellular adhesion molecule; T1DM: Type 1 diabetes mellitus; ELISA: Enzyme-linked immunosorbent assay; BMI: Body mass index; HbA1c: Glycated hemoglobin; BSA: Bovine serum albumin; PBS: Phosphate buffered saline; FACS: Fluorescence-activated cell sorting; SD: Standard deviation; BP: Blood pressure; IL: Interleukin; ALT: Alanine aminotransferase; IR: Insulin resistance; EV: Extracellular vesicle; RT: Reverse transcription; IFN: Interferon; TNF: Tumor necrosis factor; Tregs: Regulatory T cells; Ig: Immunoglobulin; DC: Dendritic cells; T2DM: Type 2 diabetes mellitus; HOMA-IR: Homeostatic Model Assessment of Insulin Resistance; HDL: High-density lipoprotein; LDL: Low-density lipoprotein; Ab: Antibody; NAFLD: Nonalcoholic fatty liver disease; AST: Aspartate aminotransferase; ECFC: Endothelial colony-forming cells; DPN: Diabetic peripheral neuropathy; CD: Celiac disease; AA: African American; HOMA2-B: Homeostatic Model Assessment for β-cell steady-state function; METs: Metabolic syndrome; 3DPAR: Physical Activity Recall; IRS: Impact rates; SAT: Subcutaneous adipose tissue; VAT: Visceral adipose tissue.

Table 3 Overview of the articles included that studied insulin resistance and Beta cell mass

Ref.	Setting	Biomarker	Insulin resistance	Beta cell mass	Insulin sensitivity
Type 1 diabetes
Abdel-Moneim, et al[17], 2020	Egypt	MDA; NO	IR was studied by measuring HbA1c and fasting glucose plasma levels, which were higher in the studied population	Not analyzed	Not reported
Babar et al[18], 2011	United States	hs-CRP, Homocysteine	IR was analyzed indirectly by measuring the flow-mediated dilatation of the brachial artery, which had a positive relation with higher levels of Hb1A	Not analyzed	Not reported
Cabrera et al[39], 2018	United States	Innate immunity activity (circulating activated regulatory T cells (CD4+/CD45RA−/FOXP3high)	IR was indirectly analyzed by the rate of C-peptide decline	Not analyzed	Not reported
Cazeau et al[29], 2016	United States	Endothelial function/ dysfunction: (ECFCs: CD34+, CD133+, CD45-). Oxidative stress: TAOC, hs-CRP, EPCs	IR was analyzed by indirect biomarkers that relate to high levels of HbA1c such as adiponectin and hs-CRP	Not analyzed	Not reported
Cree-Green et al[41], 2018	United States	hs-CRP, adiponectin, leptin and C-peptide	IR was assessed by insulin sensitivity four-phase HE clamp - glucose and glycerol rate of appearance, rate of disappearance and metabolic clearance rate over the last 30 min of each phase of the clamp	Not analyzed	IS was assessed with a four-phase HE clamp: a bolus of 4.5 mg/kg 6,6-2; H2-glucose followed by a continuous infusion at 0.03 mg/kg/min 6,6-2H2-glucose was paired with a primed (1.6 mmol/kg) then constant (0.11 mmol/kg/min), infusion of 2H5-glycerol. During the last 30 min of each of the four clamp phases, four samples, each 10 min apart, were drawn for glucose, glycerol, free fatty acid and insulin concentrations
Elbarbary et al[33], 2018	Egypt	Neopterin	IR was indirectly analyzed with biomarkers related to higher levels of HbA1c such as dyslipidemia, hs-CRP and also fasting blood glucose	Not analyzed	Not reported
Lakhter et al[35], 2018	United States	miR-21-5pin Beta cell extracellular vesicle	Not reported	Not analyzed	Not reported
Vorobjova et al[32], 2019	Estonia	Inflammation markers; Association with Enterovirus infection; Immunity	IR was analyzed by indirect biomarkers that relate to high levels of HbA1c such as leptin	Not analyzed	Not reported
Type 1 and 2 diabetes
Aburawi et al[30], 2016	United Arab Emirates	sICAM-1, sVCAM-1, IL-6, TNF-α, hs-CRP	IR was analyzed by indirect biomarkers that relate to high levels of HbA1c such as adiponectin	Not analyzed	Not reported
Type 2 diabetes
Arenaza et al[36], 2017	Spain	miRNA expression in circulating exosomes and in peripheral blood mononuclear cells	IR was measured by the HOMA, which has been shown to be a reliable method in pediatric population.	Not analyzed	IS was indirectly measured by the amount of total, visceral and abdominal as well as hepatic and pancreatic adiposity.
Bacha et al[40], 2014	United States	Leptin, IL-6, VCAM-1, ICAM-1, E-selectin	IR was measured by OGTT (1.75 g/kg, maximum 75 g Trutol), which was performed with glucose, insulin and C-peptide determination at 215, 0, 15, 30, 60 and 120 min	Not analyzed	IS analysis was measured using intravenous crystalline insulin, which was infused at a constant rate of 80 mU/m2/min, and plasma glucose was clamped at 100 mg/dL (5.6 mmol/L) with a variable rate infusion of 20% dextrose
Barbosa-Cortés et al[31], 2017	México	Leptin, adiponectin, VCAM, ICAM	IR was measured directly measured based on the fasting insulin and glucose concentrations using the Homeostatic Model Assessment of Insulin Resistance [HOMA-IR = (fasting insulin (μU/ml) × fasting glucose (mmol/L)/22.5)] and indirectly by measuring serum levels of leptin and adiponectin	Not analyzed	Not reported
Carlbom et al[34], 2017	Sweden	Functional Beta cell mass	IR was measured using the HOMA-IR	Intravenous arginine test and a glucose potentiated arginine test, which are considered the gold standard to assess the functional B-cell mass; Beta cell mass was analyzed using [11C]5-hydroxytryptophan positron emission tomography	IS was assessed using the HOMA2-S calculator previously calibrated so that 100% represents values obtained from young healthy adults
Dentelli et al[27], 2013	Germany	Adipose-derived stem cell pluripotentiality	Not reported	Not analyzed	Not reports
Kim-Muller et al[38], 2016	United States	Impaired mitochondrial function (Aldehyde dehydrogenase 1 isoform A3, Foxo1, MafA)	Not reported	Not analyzed	Not reported
Sheng et al[37], 2016	China	B-cell dedifferentiation (Glut2, Pdx1, Nkx6.1, MafA, Foxo1, GLP-1, PKC)	IR was assessed by glucose tolerance tests. The mice were fasted for 12 h then they were injected intraperitoneally with 1 g kg−1 glucose. The glucose measurements were taken up to 2 h after injection	Pancreatic sections were analyzed by immunohistochemistry	Not reported
Spagnuolo et al[23], 2010	Italy	C-reactive protein, NO, Fecal calprotectin	OGTT was performed with a load of 1.75 g/kg/body weight of glucose (maximum 75 g) after a 12 h overnight fast	Not analyzed	IS was estimated by the HOMA-IR index from fasting glucose and insulin concentrations according to the following formula: insulin (mU/L) × glucose (mmol/L)/22.5
Stansfield et al[28], 2016	United States	Leptin, adiponectin, C-reactive protein	IR was assessed using HOMA-IR. It was calculated by use of the formula: insulin (pmol/L) × glucose (mmol/L)/22.5.25	Not analyzed	Not reported
Walker et al[25], 2014	Italy	C-reactive protein, TNF-a, IL-6, Crown like structures (CLS) in SAT	IR was assessed by standard OGTT performed with 1.75 g of glucose per kg of body weight (up to 75 g), and glucose and insulin were measured at 0, 30, 60, 90 and 120 min	Not analyzed	IS was estimated by the HOMA
Xu et al[16], 2017	China	Superoxide dismutase activity, MDA and glycated serum protein levels	IR was assessed performing OGTT and HOMA-IR model	Beta cell mass was analyzed with histopathology. For light microscopy, the fixed tissue samples were dehydrated through a graded ethanol series, embedded in paraffin and cut into 7 μm-thick sections with HE stain using a routine protocol	Not reported

TAOC: Total plasma antioxidant capacity; hs-CRP: High-sensitivity C-reactive protein; HOMA-IR: Homeostatic Model Assessment of Insulin Resistance; ECFC: Endothelial colony-forming cells; MDA: Malondialdehyde; NO: Nitric oxide; IR: Insulin resistance; HbA1c: Glycated hemoglobin; EPC: Endothelial progenitor cell; IS: Insulin sensitivity; miR: microRNA; s: soluble; ICAM: Intracellular cell adhesion molecule; IL: Interleukin; VCAM: Vascular cell adhesion molecule; TNF: Tumor necrosis factor; miRNA: MicroRNA; OGTT: Oral glucose tolerance test; HE: Hematoxylin and eosin; HE clamp: Hyperinsulinemic-euglycemic clamp; HOMA2-S: Homeostatic Model Assessment to assess insulin sensitivity; SAT: subcutaneous adipose tissue.