Circulating microRNAs as novel biomarkers for diabetes mellitus

Diabetes mellitus is characterized by insulin secretion from pancreatic β cells that is insufficient to maintain blood glucose homeostasis. Autoimmune destruction of β cells results in type 1 diabetes mellitus, whereas conditions that reduce insulin sensitivity and negatively affect β-cell activities result in type 2 diabetes mellitus. Without proper management, patients with diabetes mellitus develop serious complications that reduce their quality of life and life expectancy. Biomarkers for early detection of the disease and identification of individuals at risk of developing complications would greatly improve the care of these patients. Small non-coding RNAs called microRNAs (miRNAs) control gene expression and participate in many physiopathological processes. Hundreds of miRNAs are actively or passively released in the circulation and can be used to evaluate health status and disease progression. Both type 1 diabetes mellitus and type 2 diabetes mellitus are associated with distinct modifications in the profile of miRNAs in the blood, which are sometimes detectable several years before the disease manifests. Moreover, circulating levels of certain miRNAs seem to be predictive of long-term complications. Technical and scientific obstacles still exist that need to be overcome, but circulating miRNAs might soon become part of the diagnostic arsenal to identify individuals at risk of developing diabetes mellitus and its devastating complications.

Key points

• New biomarkers are needed to improve the identification of individuals at risk of developing diabetes mellitus and its associated complications, monitor disease progression and assess the efficacy of therapeutic interventions
• Circulating microRNAs (miRNAs) are attractive biomarker candidates as they can be easily collected, are stable under different storage conditions and can be measured using assays that are specific, sensitive and reproducible
• Pioneering studies have identified characteristic changes in blood levels of miRNAs in samples from a range of cohorts of patients with diabetes mellitus
• However, definitive miRNA signatures for type 1 diabetes mellitus, type 2 diabetes mellitus or their associated complications remain to be defined and agreed upon
• Although measuring circulating miRNAs is a promising approach in individuals at risk of developing diabetes mellitus, several key issues still need to be addressed, including the determination of the most appropriate blood sampling protocols

Introduction

Diabetes mellitus affects >350 million people worldwide and makes a considerable contribution to morbidity and mortality globally.1 In industrialized countries, diabetes mellitus is the leading cause of blindness, renal failure and nontraumatic lower limb amputations. Patients with diabetes mellitus also have an increased risk of developing cardiovascular disorders and having a stroke, which means that this disease is a heavy socioeconomic burden.2 Unfortunately, the prevalence of diabetes mellitus is increasing at a dramatic pace both in children and in adults as a result of changes in lifestyle (reduced physical activity and increased overnutrition and obesity), but also as a consequence of population ageing. Indeed, according to estimates from the International Diabetes Federation, 552 million people are expected to have diabetes mellitus in 2030.1

This Review focuses on type 1 diabetes mellitus (T1DM) and type 2 diabetes mellitus (T2DM), the two principal forms of the disease. T1DM is an autoimmune disorder in which pancreatic β cells are attacked and eliminated by the immune system. During the immune response, leucocytes infiltrating the pancreatic islets secrete proinflammatory cytokines that recruit cytotoxic T lymphocytes and contribute to β-cell dysfunction and death.3 The immune reaction leads to progressive destruction of β cells, which results in severe or complete insulin deficiency. T1DM generally develops during childhood or in young adults and accounts for 5–8% of all cases of diabetes mellitus.4 The majority of the other cases are attributable to T2DM. The pathogenesis of this disease is closely linked to genetic, environmental and/or lifestyle factors, such as hypercaloric nutrition, lack of exercise and obesity. T2DM occurs when target tissues lose insulin sensitivity, including the liver, skeletal muscles and adipose tissues. This state of insulin resistance can usually be compensated for by expansion of the functional β-cell mass and by an increase in insulin secretion. However, in individuals who are genetically predisposed to develop T2DM, the β cells are unable to sustain the increased demand for insulin, which leads to chronic hyperglycaemia and the onset of T2DM.5

Despite intensive research, the causes of T1DM and T2DM remain incompletely understood and a definitive cure is still not available. The efficacy of the current treatments to delay progression of diabetes mellitus would be drastically improved if they could be implemented during the initial phases of the disease and targeted at individuals with the highest probability of benefitting from the therapeutic intervention. This goal can only be achieved by identifying new biomarkers for predicting and/or monitoring the progression of T1DM and T2DM and their long-term complications. The aim of this Review is to summarize the weaknesses of the blood parameters and biomarkers that are currently used to detect people at risk of developing T1DM and T2DM and to discuss the potential use of circulating microRNAs (miRNAs) as a novel class of biomarkers.

Classic diabetes mellitus biomarkers

According to the WHO, a diagnosis of diabetes mellitus should be based on measurements of blood levels of glucose in the fasted state and following an oral glucose tolerance test.6 A diabetic state is defined by glucose levels >7.0 mmol/l (126 mg/dl) in the fasted state and >11.1 mmol/l (200 mg/dl) after an oral glucose tolerance test.6Other serum parameters such as levels of HbA1c or residual C-peptide can also be helpful in the diagnosis of diabetes mellitus.

Biomarkers for T1DM

T1DM is generally diagnosed when >80–90% of the pancreatic β cells have been destroyed by the immune system.7 The progression of the disease is slow (it can take months to years for the patient to become symptomatic), which provides a potentially long period of time in which to identify and treat individuals at risk. In the past 2 years, progress has been made to preserve the function of residual β cells at the onset of T1DM using immunosuppressive medications.8,9 However, the efficacy of these treatments is currently limited, although considerable improvements could probably be made if therapies could be initiated at earlier stages of the disease when many β cells are still present.

Autoantibodies against islet antigens are often used as biomarkers for T1DM, as their presence in the blood is characteristic of the disease. Several autoantibodies have been described, but those directed against islet cells, insulin, tyrosine phosphatase IA-2 and IA-2β, glutamate decarboxylase and zinc transporter 8 are the most reliable for identifying individuals at risk of developing T1DM (Box 1).7,10 However, the use of islet autoantibodies as biomarkers has some important limitations. Firstly, autoantibodies appear fairly late in the course of T1DM, so cannot be used to initiate treatment early in the disease course. Secondly, although most individuals at the onset of T1DM are positive for at least some autoantibodies, many autoantibody-positive individuals will never develop the disease. Thirdly, autoantibodies are not suitable for monitoring therapeutic outcomes because they are not immediately removed from the circulation if the autoimmune reaction is stopped.10 Additional biomarkers for T1DM are therefore needed to complement the information obtained from the presence of autoantibodies and other risk factors (such as age, family history, susceptibility genes and environmental triggers).

Biomarkers for T2DM

Individuals at risk of developing T2DM are currently identified by a combination of easily accessible serum parameters (including levels of glucose, triacylglycerol, cholesterol, lipoproteins and HbA1c), physical characteristics (BMI, waist-to-hip ratio, blood pressure and sex) and lifestyle factors (food consumption, physical inactivity and smoking). By combining all these classic biomarkers and risk factors, the probability of predicting the development of the disease ranges from 0.85 to 0.90 in a period of 5–10 years before the onset of T2DM.11 Other molecules have emerged as potentially useful biomarkers (Box 1), including incretins such as glucagon-like peptide 1, cytokines, adipokines, ferritin and C-reactive protein.12 Individually, none of these so-called novel biomarkers can predict the manifestation of T2DM efficiently, but in combination they can achieve predictive values similar to those possible with classic biomarkers.13

All these serum parameters can predict the development of T2DM a few years in advance of disease manifestation in individuals already displaying metabolic alterations. However, the biomarkers are not specific for diabetes mellitus and cannot be used to assess disease susceptibility in the general population. Genotype analysis could potentially complement the use of these biomarkers for the identification of individuals susceptible to developing T2DM later in life. However, the predictive values of genotypic traits has yet to exceed 0.60.12 Thus, early and lifestyle-independent predictive factors are currently required to enable physicians to recognize individuals at risk of developing T2DM.

miRNAs

miRNAs are small non-coding RNA molecules of 21–23 nucleotides that function as translational repressors by partially pairing to the 3′ untranslated region of target mRNAs (Box 2). These regulators of gene expression were first discovered in Caenorhabditis elegans,14,15 and then in vertebrates and plants.16 According to the latest estimates, the human genome encodes >1,600 miRNA precursors, generating up to 2,237 mature miRNAs,17 each of which has the potential to control hundreds of targets. Now, miRNAs are universally recognized as major regulators of gene expression and key controllers of several biologic and pathologic processes.18 They are produced from stem-loop precursor RNAs that are generated from independent transcriptional units or from introns of genes that encode proteins (Box 2). These primary transcripts (pri-miRNAs) are initially processed to produce short RNA molecules (pre-miRNAs) and are then exported to the cytosol where they are further cleaved to generate the mature forms of miRNAs (Figure 1). The mature miRNAs can either be included in the RNA-induced silencing complex to guide translational repression of target mRNAs or be released by the cells. If they are to be released, the miRNAs become attached to proteins or lipoproteins or are loaded inside vesicles that are released into the extracellular space during plasma membrane blebbing or after fusion of multivesicular bodies with the plasma membrane (Figure 1).

FIGURE 1 | Biogenesis and release of miRNAs.

Pre-miRNAs are generated in the nucleus by the ribonuclease III enzyme Drosha after cleavage of pri-miRNAs (1). The pre-miRNAs are then transported in the cytoplasm through a process involving Exportin-5 and the GTP-binding protein Ran (2) and further cleaved by Dicer to yield 21–23 nucleotide duplexes (3). One strand of the miRNA duplex can either associate to the RISC complex and guide translational repression of target mRNAs (4) or be released by the cells. In the latter case, the mature miRNA binds to RNA-binding proteins such as Argonaute-2 (5) or to lipoproteins (6). Alternatively, the miRNAs can be loaded in microvesicles formed by plasma membrane blebbing (7) or in exosomes that are released in the extracellular space upon exocytic fusion of multivesicular bodies with the plasma membrane (8). Abbreviations: miRNA, microRNA; pre-miRNA, miRNA precursor; pri-miRNA, primary miRNA transcript; RISC, RNA-induced silencing complex.

Role of miRNAs in diabetes pathogenesis

Pancreatic β cells and the tissues targeted by insulin express a well-defined set of miRNAs. Most of the miRNAs are not cell-specific, but are widely distributed throughout the tissues of the human body. A notable exception is miR-375, a miRNA highly enriched in pancreatic islets that regulates the expression of genes involved in hormone secretion and in β-cell mass expansion in response to insulin resistance.19, 20 The miRNA expression profile of β cells and tissues targeted by insulin is altered in patients with T1DM and T2DM, which probably contributes to the impaired function of these tissues under disease states.21,22,23 Indeed, the islets of prediabetic nonobese diabetic (NOD) mice, a model of T1DM, contain increased levels of several miRNAs, including miR-21, miR-34a, miR-29 and miR-146a, which have deleterious effects on β-cell function.24,25The expression of most of these miRNAs, as well as that of many other miRNAs, is also altered in the islets of ob/ob and db/db mice, which are models of obesity and T2DM.26,27 Interestingly, the expression of miR-29 and miR-34a is also increased in tissues targeted by insulin in these mouse models, which possibly contributes to insulin resistance.28, 29 Other miRNAs that are dysregulated in tissues targeted by insulin inob/ob mice, dietary mouse models of obesity and diabetic Goto-Kakizaki rats include miR-143, miR-802 and two closely related miRNAs, miR-103 and miR-107.28,29,30,31Strong experimental evidence indicates a contribution of these miRNAs to the development of insulin resistance in these obesity models.29,30,31

Changes in the miRNA profile that are related to diabetes mellitus have also been reported in human tissues. More than 60 differentially expressed miRNAs were detected in human skeletal muscle biopsy samples from patients with T2DM, including miR-143, which is upregulated, and two muscle-specific miRNAs, miR-206 and miR-133a, which are downregulated.32 Interestingly, the levels of about 15% of these miRNAs were already modified in individuals with impaired glucose tolerance, which suggests that the miRNAs are involved in the early phases of the disease process. The expression of some of these miRNAs is controlled by insulin, but this regulatory mechanism seems to be impaired in patients with diabetes mellitus.33

As well as the changes in tissues targeted by insulin that are described above, diabetes mellitus results in considerable modifications in miRNA expression in blood vessels, heart, retina and kidneys. This finding indicates that these non-coding RNAs are involved in the development of long-term complications of diabetes mellitus.22,34,35

A functional role for circulating miRNAs?

In addition to regulating gene expression inside the cells that produce them, several miRNAs are found in blood and other body fluids in association with proteins, microvesicles and/or lipoprotein complexes (Figure 2).36, 37, 38 The function of circulating miRNAs remains to be established, but in vitro studies indicate that miRNAs transported by exosomes (Box 3) or HDL can be transferred in an active form to recipient cells.38,39 This observation raises the intriguing possibility of an involvement of miRNAs in a novel cell-to-cell communication mode. Circulating miRNAs are very stable and resistant to treatment with ribonucleases, freezing/thawing cycles and other drastic experimental conditions.40,41 Consequently, serum or plasma samples can be stored at −20 °C or −80 °C for up to several months without notable degradation of miRNAs,42 which suggests that these small RNA molecules are sufficiently robust to serve as biomarkers. Circulating miRNAs have several other advantages as potential biomarkers: they are found not only in blood but also in other easily accessible biologic fluids (such as urine, saliva, amniotic fluid and breast milk),43 they can be detected by highly sensitive and specific quantitative real-time PCR, and most of them are evolutionarily conserved, which facilitates the translation of results obtained from in vivo animal studies to human health care. Moreover, profiles of the miRNAs in serum of healthy donors are fairly homogeneous and constant over 24 h and miRNAs can be measured in both serum and plasma.41,44,45

FIGURE 2 | Blood and other body fluids contain active miRNAs.

miRNAs can be released or shed by cells and are found in a stable form in body fluids. The miRNAs present in the blood are associated with protein complexes such as Argonaute-2 or with HDL particles, or are transported inside membrane-bound vesicles such as exosomes (1). Evidence suggests that circulating miRNAs can be taken up in active form through different mechanisms, including receptor-mediated capture, endocytosis or fusion of exosomes with the plasma membrane of receiving cells (2). Transfer of miRNAs between distantly located cells constitutes a potentially new communication mode. Abbreviation: miRNA, microRNA.

miRNAs as diabetes mellitus biomarkers

The idea of using miRNAs found in blood as biomarkers is fairly new and was first proposed for detecting various forms of cancer,41,46,47 autoimmune diseases48 and sepsis.49 Studies have now also analysed the miRNA profile in serum, plasma or blood cells in an attempt to develop new approaches to predict the development and progression of diabetes mellitus (Table 1). Zampetaki and colleagues50 were the first to identify a characteristic expression profile of miRNAs in blood that was related to T2DM. In their prospective study, they analysed blood samples from >800 individuals randomly selected from the Bruneck population (Bolzano Province, Italy) and identified a subset of five miRNAs (miR-15a, miR-28-3p, miR-29b, miR-126 and miR-223) that displayed a characteristic deregulation in 80 participants with either prediabetes or T2DM. Importantly, the levels of these miRNAs were already modified 5–10 years before the onset of the disease, providing initial evidence for the usefulness of circulating miRNAs as early predictors of T2DM and its vascular complications.

The miRNA content of serum from patients with prediabetes and/or who are newly diagnosed with T2DM has also been analysed by other groups. Kong and co-workers51detected an increase in the expression of seven diabetes-related miRNAs (miR-9, miR-29a, miR-30d, miR-34a, miR-124a, miR-146a and miR-375) in patients with T2DM compared with patients who had prediabetes or were susceptible to T2DM. However, no differences were observed between individuals with normal glucose tolerance and those with prediabetes, which indicates that the level of these miRNAs in serum is not suitable for predicting susceptibility to T2DM. In a study published in 2012, Karolina et al.52 measured the miRNAs present in the blood and exosomes of 265 patients with different health conditions associated with the metabolic syndrome. They detected an upregulation of miR-27a, miR-150, miR-192, miR-320a and miR-375 in patients with T2DM and observed a strong correlation between raised fasting levels of glucose and the increase in levels of miR-27a and miR-320a. These pioneering studies demonstrate the potential of miRNAs as biomarkers for T2DM. However, the heterogeneity of the results obtained underscores the need for large prospective studies to identify reliable miRNA signatures for diagnosing T2DM.

A similar approach was used to identify new biomarkers to predict destruction or regeneration of residual β cells in T1DM. Nielsen and colleagues compared two cohorts (Danish and Hvidoere) of patients newly diagnosed with T1DM with an age-matched control group.53 Global miRNA sequencing, followed by quantitative real-time PCR verification and regression analysis to adjust for age, sex and multiple testing, highlighted a group of miRNAs (miR-24, miR-25, miR-26a, miR-27a, miR-27b, miR-29a, miR-30a-5p, miR-148a, miR-152, miR-181a, miR-200a and miR-210) that were differentially expressed in cohorts of patients with T1DM compared with control groups. Several of these miRNAs modulate the expression of genes involved in apoptosis and/or important β-cell regulatory networks.53 Moreover, levels of miR-25 were found to correlate with residual β-cell function (determined by levels of C-peptide) and adequate glycaemic control (measured by levels of HbA1c) 3 months after disease onset in the Danish cohort. However, this correlation was not observed in the Hvidoere cohort, in which glycaemic control was evaluated 12 months after the diagnosis of T1DM, possibly because of the loss of residual β cells.

In a study presented at the 2012 meeting of the European Association for the Study of Diabetes, Sebastiani and co-workers54 compared the profile of miRNAs in the blood of 20 patients newly diagnosed with T1DM with that of healthy control individuals. Of 206 miRNAs detected in the serum of both groups, 64 were found to be differently expressed in the patients with T1DM. Interestingly, some of these miRNAs regulate the functions of immune cells (miR-31, miR-146a, miR-155, miR-181a and miR-199a) or of β cells (miR-9 and miR-34a). A miRNA abundantly expressed in the islets of Langerhans, miR-375, has been proposed as a suitable biomarker to detect β-cell death and to predict the development of T1DM in animal models.55 Indeed, massive β-cell loss elicited by administration of streptozocin caused a dramatic rise in circulating levels of this miRNA.55 Moreover, plasma levels of miR-375 were considerably increased in NOD mice 2 weeks before the onset of T1DM.55 The changes in levels of miR-375 consequent to β-cell death were short-lived (<1 week). Thus, these promising findings obtained in mice will need to be verified in humans, as the decline in β-cell mass takes much longer in humans than in NOD mice.

Instead of analysing plasma samples, other studies have focused their attention on blood cells and measured the expression of specific miRNAs that are thought to have important roles in the immune reaction that leads to T1DM. Salas-Pérez et al.56 observed diminished expression of miR-21a and miR-93 in peripheral blood mononuclear cells (PBMC) of patients with T1DM compared with healthy control individuals. The reduction of miR-21a (but not of miR-93) expression could be reproduced by incubating PBMC from control individuals in a medium containing 25 mmol/l of glucose, suggesting that the reduced levels of miR-21a might be the consequence of chronic hyperglycaemia. Finally, Sebastiani and colleagues analysed miRNA expression in blood lymphocytes from patients with T1DM and observed a rise in levels of miR-326 that correlated with the timing of the islet autoimmune attack.57 The primary goal of the latter two studies was to identify miRNAs that could be involved in the development of T1DM. However, as a large proportion of miRNAs in serum are released by blood cells, it is possible that the miRNA changes in PBMC and/or lymphocytes observed in these two studies might yield detectable differences in plasma levels that would enable the autoimmune reaction to be monitored.

Prediction of diabetes mellitus complications

T1DM and T2DM are both associated with long-term microvascular and macrovascular complications that can have a devastating effect on quality of life and life expectancy. The discovery of biomarkers that could be used to identify individuals at risk of experiencing serious complications such as retinopathy, nephropathy or cardiovascular disorders would enable clinicians to tailor therapeutic approaches and minimize the expected effects of the disease. However, reliable biomarkers for these long-term complications are still lacking.

Cardiovascular complications are a major concern in this group of patients as they account for up to 80% of premature mortality in patients with diabetes mellitus.58 Prevention, or even a delay, of these complications would be a huge advancement in the treatment of diabetes mellitus. As discussed previously, Zampetaki and colleagues50 identified a unique plasma miRNA signature in patients with T2DM. Among the miRNA-related characteristic changes observed, reduced levels of miR-126 showed the strongest association with T2DM, and correlated with the occurrence of subclinical and overt artery diseases. Interestingly, another study also reported downregulation of miR-126 in blood samples obtained from patients with coronary artery disease.59 This miRNA is highly enriched in endothelial cells, where it has important roles in cell homeostasis and vascular integrity in different animal models.60,61 Moreover, the levels of miR-126 released in apoptotic bodies are reduced by chronic exposure of endothelial cells to raised blood levels of glucose,50 which makes this miRNA an ideal candidate biomarker for monitoring diabetic vascular complications. Another miRNA in endothelial cells that deserves further attention is miR-503. The levels of this miRNA are upregulated in muscle biopsy samples and in peripheral blood-derived plasma of patients with diabetes mellitus who have limb ischaemia.62 Interestingly, local inhibition of miR-503 in a mouse model of limb ischaemia improved vascular healing and blood flow recovery.62 Other circulating miRNAs have also been suggested as diagnostic markers for various cardiovascular diseases,63,64 but their use in predicting or monitoring cardiovascular complications in patients with diabetes mellitus has yet to be investigated.

Kidney disease affects 30% and 50% of patients with T1DM and T2DM, respectively.65,66,67 Microalbuminuria has been proposed as a biomarker that could be used to predict the occurrence of this important complication; however, studies published in the past decade have revealed that a noteworthy proportion of patients with diabetes mellitus undergo renal failure before microalbuminuria is detectable, or even before it occurs.68,69,70 Circulating miRNAs represent a viable alternative for monitoring renal failure in patients with diabetes mellitus. They are not eliminated by haemodialysis71 and have already been tested in different renal diseases with promising results in both animal models and human patients.72,73,74 Indeed, a correlation was observed between levels of certain circulating miRNAs, such as miR-16, miR-21, miR-210 and miR-638, and glomerular filtration rate, a well-known parameter of the progression of kidney disease.72 Large-scale prospective studies focusing on patients with diabetes mellitus undergoing renal failure will be necessary to identify a specific miRNA profile in plasma or urine that can be used to predict the appearance of this complication. Urine represents an ideal source of miRNAs as it can be easily collected noninvasively and in large amounts. Moreover, urinary exosomes originate from various cell types that span the entire urinary track and would be ideally suited for use in monitoring the progression of renal diseases.75,76,77,78 Indeed, profiles of miRNAs in urine have been reported to differ across the stages of diabetic nephropathy,79 suggesting that they could be used as tools to follow the progressive alteration of the renal processes in patients with diabetes mellitus.

To our knowledge, no reports have been published to date about the use of circulating miRNAs to predict the occurrence of diabetic retinopathy. However, an involvement of some specific miRNAs, such as miR-29b and miR-200b, in the development of this complication has been demonstrated.35 These findings might open the door to future investigations aimed at assessing the potential use of miRNAs as predictors of this debilitating condition.

Gestational diabetes mellitus

Circulating miRNAs could in principle also be used to identify women at increased risk of developing gestational diabetes mellitus. Most screening protocols are currently based on a glucose challenge test performed around weeks 24–28 of gestation. Therefore, interventions such as diet, exercise or medication are sometimes started as late as 32 weeks of gestation. In a multistage retrospective study, the miRNAs in serum of women at 16–19 weeks of gestation were screened. Three miRNAs (miR-29a, miR-122 and miR-132) were identified that were deregulated in women developing gestational diabetes mellitus before changes in blood levels of glucose became detectable.80 Placental-specific miRNAs can also be detected in maternal serum.44,81 Thus, it is possible that gestational diabetes mellitus might lead to changes in the levels of miRNAs in blood that differ from those of T1DM or T2DM.

Future directions

The findings described in this article confirm that miRNAs are attractive novel biomarkers for diabetes mellitus. Indeed, changes in the levels of a subset of these small RNA molecules in body fluids promise to provide new clues for early identification of individuals at risk of developing diabetes mellitus and the complications associated with this disorder, for following disease progression and for assessing the efficacy of therapeutic interventions. However, major scientific and technical advances need to be made before these goals can be achieved.

On the basis of the current data, circulating miRNAs should be able to be used to substitute or complement other routine measurements in the future. Thus, their efficacy in predicting the occurrence of diabetes mellitus or its complications needs to be systematically compared with biomarkers that are already available. In particular, it will be essential to scrutinize whether miRNAs are indeed able to provide earlier and/or more precise detection of individuals at risk of developing the disease or its long-term complications than current biomarkers. The situation will hopefully evolve in the future; however, there is currently no obvious advantage to replacing other traditional biomarkers with measurements of levels of circulating miRNAs.

Different papers have reported changes in levels of miRNAs that are associated with diabetes mellitus or its complications but, for various reasons, we are still very far from a consensus about the most relevant miRNAs to be measured. This lack of agreement can in part be attributed to the heterogeneity of the approaches selected by investigators. Some of the published studies carried out systematic profiling of a large number of miRNAs,50,52,53 whereas others focused on a small group of selected miRNAs that were supposedly most likely to be affected in patients with diabetes mellitus.51,55,56,57 These approaches yielded a large number of potentially interesting candidate biomarkers, but most of them still await confirmation by independent researchers and/or in larger cohorts. Standardized protocols for sample preparation, RNA extraction and miRNA analysis are urgently needed to facilitate comparisons between different studies and to reach a consensus about the miRNAs most suitable to function as early biomarkers of diabetes mellitus. In fact, the level of certain miRNAs, such as miR-15b and miR-16, in plasma is greatly influenced by the degree of haemolysis and by cellular contamination, making them less suitable as clinical biomarkers.82

Diabetes mellitus is a complex disorder that involves major metabolic alterations and important adaptations in the activity of several organs. miRNAs are being proposed as potential biomarkers for an increasing number of diseases. Therefore, it will be essential to determine whether the detected miRNA changes are exclusively indicative of a prediabetic or diabetic condition or if they are also observed in other physiological or pathological situations,45 such as in response to modifications in the nutritional state, inflammation, autoimmunity or cancer.

At present, the origin of miRNAs in the blood is largely unknown and their levels do not directly mirror changes in pancreatic β cells or tissues targeted by insulin that occur in diabetes mellitus. miRNAs can be actively or passively released by a variety of cells and can be carried by membrane-bound vesicles, protein complexes or lipoprotein particles (Figures 1 and 2). So far, the majority of studies have measured levels of miRNAs directly in the plasma (or serum). This approach is obviously convenient and straightforward. However, modifications in the level of miRNAs originating from specific groups of cells that are not in direct contact with the blood or are not very abundant are unlikely to have a notable effect on the pool of miRNAs in the plasma and will probably go undetected. Although technically demanding, protocols enabling a specific assessment of the miRNAs carried by exosomes, protein complexes or lipoproteins will probably provide more detailed information about the physiological or pathological status of the organs of interest than measuring serum levels of miRNAs. Furthermore, it might be possible to affinity-purify membrane-bound vesicles originating from a specific group of cells, taking advantage of the presence of characteristic proteins on the vesicle surface.12 This approach will be particularly appropriate for estimating the residual β-cell mass in patients newly diagnosed with T1DM. Indeed, except in the case of a sudden loss of a large number of β-cells, the miRNAs released by insulin-secreting cells represent only a negligible fraction of all non-coding RNAs circulating in the blood. This problem might be partially overcome by searching for miRNAs that are highly expressed in β cells, such as miR-375.19,55 However, the expression of this particular miRNA is modified in a variety of tumorous cells and several authors have suggested using circulating levels of miR-375 for the diagnosis of different types of cancer.83,84,85,86 Several insulin-secreting cell lines release notable amounts of exosomes87,88,89 and we have initial evidence indicating that this is also the case for rodent and human islet cells.90 Protocols permitting an enrichment in exosomes derived from islet cells would probably improve the detection of specific changes in insulin-secreting cells and provide a better evaluation of the functional β-cell mass.

Conclusions

miRNAs are emerging as important regulators of gene expression and central players in many physiological and pathological processes. Their presence in extracellular fluids in astonishingly stable forms has led to the idea of using them as biomarkers for a variety of diseases. These properties have also attracted the attention of diabetologists who have initiated the search for miRNAs that enable early detection of T1DM and T2DM and their associated complications. A number of scientific and methodological issues need to be addressed before circulating miRNAs can attain the status of biomarkers of diabetes mellitus, but the available data suggest that they will soon serve as valuable new blood parameters that will help physicians to refine their therapeutic interventions.

Review criteria

We systematically searched PubMed using different combinations of the following words: “diabetes”, “type 1 diabetes”, “type 2 diabetes”, “gestational diabetes”, “diabetes complications”, “diabetic retinopathy”, “diabetic nephropathy”, “cardiovascular complications”, “biomarkers”, “microRNAs”, “circulating miRNAs” and “exosomes”. We also scrutinized the appropriate references cited in the selected papers. Two abstracts from the 2012 European Association for the Study of Diabetes meeting held in Berlin were also included. Owing to space limitations, not all original articles on the subject could be listed in the present review. Whenever possible, the most recent and complete reviews on the topic were chosen. All selected articles are in English.

Author contributions

Both authors contributed equally to all aspects of this article.

Acknowledgments

The authors are supported by grants from the Swiss National Science Foundation, from the European Foundation for the Study of Diabetes and from the Société Francophone du Diabète (SFD)-Servier. C. Guay is supported by a fellowship from Fonds de la Recherche en Santé du Québec, the SFD and the Canadian Diabetes Association.

Competing interests statement

The authors declare no competing interests.

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Author affiliations

C. Guay & R. Regazzi

University of Lausanne, Department of Fundamental Neurosciences, Rue du Bugnon 9, 1005 Lausanne, Switzerland (C. Guay, R. Regazzi).

Correspondence to: R. Regazzi romano.regazzi@unil.ch

Published online 30 April 2013

Review

Nature Reviews Endocrinology 9, 513-521 (September 2013) |doi:10.1038/nrendo.2013.86