Type 2 diabetes (T2D) is a complex metabolic disease associated with disturbances in metabolism of carbohydrates, lipids and proteins, and largely under the influence of very complex interactions with genetic and environment factors. High prevalence and increasing number of patients with T2D in the world, represent constant challenge for better elucidation of pathogenic mechanisms which contribute to disease development. This paper summarizes a new molecular biomarker that emerged from recent studies in applied genomics, metabolomics and other modern “omics” technologies as powerful tools in diagnosis of T2D. Metabolomics, in this context, has a special potential since it uses newly developed analytical methods in analyses of wide range of metabolites in biological samples. Numbers of prospective studies have shown that changes in the concentration of some individual amino acids, acylcarnitines, hexoses and phospholipids augment or attenuate risk factors for developing T2D. Recently findings shown that polymorphisms in TCF7L2 gene were strongly associated with increasing risk for T2D development while studies of lipidomics, genomics and transcriptomics identified molecular markers for glucose intolerance and other traits. Some specific gene variations were identified which affected de novo lipogenesis and they were significantly associated with concentrations of palmitic, stearic, palmitoleic and oleic acids, the major saturated and unsaturated fatty acids. Development of new trends in analysis and detection of different metabolites, especially fatty acids and amino acids, along with genetic polymorphisms points out new directions in precise diagnosis and therapy of Type 2 diabetes.
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The original version of this article was revised: due to retrospective open access cancellation.
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1 Introduction
Type 2 diabetes mellitus (T2D) is a complex disease associated with disorders in the metabolism of carbohydrates, lipids and proteins, and due to the complex interaction of genetic and environmental factors or both [1‐5]. High prevalence and an increase in the number of patients diagnosed with T2D, is a constant challenge for a better understanding of the pathogenic and molecular mechanisms that contribute to the development of this complex disease (Fig. 1) [6‐9].
Fig. 1
Prevalence of diabetes patients worldwide
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Current trends in analytical chemistry and analysis of concentrations of different metabolite and gene polymorphisms, give us new directions in precise diagnosis, prognosis and treatment of T2D. World Health Organization (WHO) and International Diabetes Federation (IDF) diabetes mellitus is defined as a one-time reading of elevated blood glucose levels monitored symptoms or as elevated when two measurements:
Fasting plasma glucose ≥7.0 mmol/l (126 mg/dl), FPG or
Plasma glucose ≥11.1 mmol/l (200 mg/dl), OGTT
more than 6.5% glycated hemoglobin (HbA1c).
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In 2009, the International Scientific Committee, composed of representatives of ADA, EASD and IDF, recommended that the diagnosis of diabetes use a threshold value of HbA1c ≥6.5%. In the case of positive findings, tests must repeat except in people with common symptoms which blood sugar levels >11.1 mmol/l (> 200 mg/dl). Therefore, hemoglobin A1c represent first molecular biomarker used for precise diagnosis as well as therapeutic treatment of diabetes. Its advantages are: high sensitivity and selectivity and, it is more reliable biomarker of chronic glycemia than FPG and OGTT. Also, HbA1c associated with good convenience and pre-analytical stability and less alterations in concentration during periods of stress and illness. Disadvantages of HbA1c are yet still no consensus, which cut-off points for HbA1c would be most sensitive, and recommended values influence by ethnicity, race, body mass index (BMI), and age. Recently, it was found that changes in production rate or circulating life span of red blood cells affect HbA1c levels, as well as various hemoglobin variants [10, 11].
The results of previously and recent studies of examination of metabolites in different biological samples are showed a new insight in diabetes mellitus, especially in type 2 diabetes. Molecules and small biological species i.e. biomarkers have been identified in the diagnosis of T2D, conducted by using genomics, metabolomics and other modern “omics” technologies in understanding of the molecular mechanisms associated to this chronic metabolic disease (Fig. 2) [12, 13].
Fig. 2
Distribution of potential metabolites as biomarkers in T2D patients
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Metabolomics application of modern analytical methods in the analysis of the large number of metabolites in various biological samples provided for not only evaluated pathological mechanisms of T2D but also, the development of new pharmacological target in precise therapy.
Biomarkers defined as biological molecules that reflect and represent healthy and disease condition of the body that are measurable in biological media such as human tissues, cells or fluids. This definition has been extended to include of biological characteristics that can be measured and evaluated as an indicators of normal biological processes, pathogenic processes, or pharmacological responses to a therapeutic intervention.
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Nowadays, biomarkers include tools and analytical technologies that can be help in understanding of the prediction, cause, diagnosis, and progression or outcomes of treatment of disease [14‐23].
There are two important types of biomarkers:
biomarkers of exposure (which are used in risk of prediction of disese)
biomarkers of disease (which are used in screening and diagnosis and monitoring of disease progression).
An ideal and effective biomarker is a molecule that:
is easily and specifically measurable in selected body fluids,
improves prediction in chemical reactions and molecular processes,
tracks an underlying pathophysiological mechanism,
change in consent with the biochemical mechanism, and
mapping of new disease of molecular pathways.
The aim of this study was to summarize current data obtained from genome-wide association study (GWAS), metabolomics and other modern analytical technologies for identification, profiling and quantification of biomarkers associated with T2D as well approaches to the treatment of disease.
2 Materials and methods
Metabolomics as part of modern analytical chemistry focused on the analysis and disturbances in levels a small molecule and their metabolites (sugars, amino acids, lipids and nucleotides) in various biological samples. Identification and quantification of molecular biomarkers provide potential metabolites that expressed abnormally in T2D patients. Nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS) are the primary technologies for the analysis while MS often coupled with a range of chromatographic separation techniques. It is also including capillary electrophoresis, gas chromatography, HPLC, ultra-performance liquid chromatography (UPLC) and liquid chromatography as well as flow injection analysis, MRS, ESR, etc.
Protocol of chemical analysis of certain metabolites occurred through two main approaches:
targeted analyses (defined metabolites are analyzed in the samples of interest) with high sensitivity and accuracy,
untargeted analyses (determine of large number of metabolites which may be known or unknown) with lower precision and accuracy.
Applied analytical methods have an advantages or disadvantages, with different sensitivity and accuracy of assays and contribute to our understanding of the relationships between metabolic pathways and the disease process. In addition, such studies may have applications in risk of assessment, screening and therapeutic monitoring of T2D as well in development of new pharmacological target in therapy.
Pharmacometabolomic as part of metabolomics use various analytical methods in the development and screening of new drugs, as well as to each level of the network model diseases of cellular and animal models to preclinical and clinical trials. Profiling metabolites has a strong foothold in the development and applications of antidiabetic drugs were been examined: a) functional mechanisms, b) identify side effects of drug, c) the development of mechanistic markers of drug efficacy, or d) to evaluate changes in metabolite profiles related to the response of treatment [24‐27].
3 Results
Numerous metabolic studies, as well reported data from recent GWAS and other omics investigations, have indicated that changes in the concentrations of individual amino acids, acylcarnitines, phospholipids and hexoses increase or decrease the risk of T2D. Studies lipidomics, transcriptomics, genomics and recently pharmacometabolomics provided for identification, profiling and quantification of biomarkers of glucose intolerance, insulin sensitivity and insulin resistance in type 2 diabetes.
In text below, the most important metabolites are listed, which currently can have used as molecular markers for type 2 diabetes.
3.1 Top biomarkers“areα-hydroxybutyrate (α-HB) and Linoleoylglycerophosphocholine (L-GPC) i.e. Lysophosphatidylcholine C18:2, LysoPC(18:2)
α-HB is a molecule produced during the synthesis of α-ketobutyrate as product of amino acid catabolism and glutathione anabolism in hepatic tissue. Currently, α-HB uses as possible biomarker for distinguish normal glucose tolerant and insulin-sensitive subjects from patients with impaired glucose tolerance and impaired fasting glucose and insulin resistant subjects. Reported data suggest it is significantly association with IR that is characteristics of T2D, and it can be useful molecular marker for prediabetes.
LysoPC(18:2) is a lysophospholipids (a monoglycerophospholipid) in which a phosphorylcholine moiety attaches a glycerol substitution site at the C-1 (sn-1) position. Lysophosphatidylcholines can have different combinations of fatty acids of varying lengths and saturation but fatty acids containing 18 carbons are the most common. Recent investigation shown that LysoPC(18:2) is a negative predictor of T2D progression in contrast to α-HB whose a positive predictor of disease. Also, it was found that four metabolites the lysophosphatidylcholines containing myristic acid (C14:0), palmitic acid (C16:0), palmitoleic acid (C16:1) and arachidonic acid (C20:4), shown specific respond to insulin and associated with increased risk of T2D and measures of insuli secretion and resistance [28, 40].
3.2 Branced-chain amino acids, BCAA
Branched amino acids in targeted metabolomics analyses has recognized as markers of dysregulated metabolism of amino acids, associated with various forms of metabolic traits. Elevated levels of BCAA (Leucine, Iso-leucine, Valine), aromatic amino acids (Phenylalanine, Tyrosine) and decreased the concentration of Glycine are associated with IR. Also, BCAA and their metabolites have a prognostic role for incident T2D and obesity. “Top combination” of amino acids biomarkers are Iso-Leucine, Phenylalanine, Tyrosine and, recently, Tryptophane, may predict the development of T2D [29]. Additionally, α-HB, L-GPC and oleic acid (C18:1) were strongly associated with insulin-resistant states compared to BCAA, and furthermore, were selectively correlated with impaired glucose tolerance [66].
3.3 Lipids, and their derivate (especially free fatty acids, FFAs)
A disturbance in the regulation of free fatty acid metabolism is a key event responsible for insulin resistance (IR) and type 2 diabetes. Furthermore, modulation of transcription factors of certain genes by free fatty acids through their binding to translocate receptors and nuclear receptors could contribute to impaired glucose homeostasis and insulin metabolisms. Elevated concentrations of free fatty acids, FFAs: C14:0, C14:1, C16:0, C16:1, C18:0, C18:1, C18:2, α-C18:3, C20:3, C22:4, C22:5, C22:6 associated with IR, impaired glucose tolerance and insulin sensitivity [30‐38].
3.4 Branched-chain fatty acids, BCFA
Metabolites changed specifically in plasma of diabetic patients and with elevated concentrations are branched-chain fatty acids (16-methylheptadecanoic acid and 3-hydroxyisobutyrate), which recently describe as a diabetes biomarker, but link between even-chain and BCFA is currently not known [39].
3.5 Ratio of Valine to phosphatidylcholineacyl-alkyl C32:2, Val_PC ae C32:2
Metabolomics analyses demonstrated that circulating metabolites ratio are reflected metabolic changes of metabolite concentrations due to alterations in insulin response, changes in glucose homeostasis and impaired lipids metabolism. Therefore, they can be use as potential biomarkers for T2D. One of these ratios, Val_PC ae C32:2 shown association with increased risk of T2D as good assessment for insulin secretion and resistance, independently of other risk factors. Val_PC ae C32:2 is composed of either the fatty acids C16:1/C16:1, C18:1/C14:1, or C18:2/C14:0 and can be an important marker for prevalence and incidence of T2D [40].
3.6 Transcription factor 7-like 2, TCF7L2
Currently, the TCF7L2 gene is the most strongly associated T2D susceptibility gene. The associations have investigated in a variety of studies in subjects of different age, race and ethnicity. Recent studies have shown that an increased expression of TCF7L2 in the islets of pancreas in T2D patients resulting in impaired glucose-stimulated insulin secretion, and polymorphisms in the gene TCF7L2 are significantly associated with increased risk of development of T2D. Also, association found between TCF7L2 and different type of FFAs: C14:0, C14:1, C16:0, C16:1, C18:0, C18:1, C22:1, and C22:2 [41].
Protein from the group of nuclear receptor that regulates of glucose metabolism and the storage of FAs, and gene expressed the same reception stimulate adipogenesis and lipid fatty cells. PPAR-γ is one of the first genes investigated for risk of T2D and its development [41, 42].
3.8 Lipin 1, LPIN1
In last few years, the lipin proteins (protein cluster lipin1,2–3) plays an important role in glycerolipid synthesis and gene regulation as well as mutations in the corresponding genes, and inflammatory disorders. LPIN1 is significant associated with Metabolic Syndrome, MetS and increase risk for developing of T2D and its polymorphisms have correlated with numerous metabolic traits. New reported data, shown that LPIN1 represent a key regulator in lipid metabolism at multiple levels, and can acts as a transcriptome regulator to inhibit or stimulate genes expression involved in lipid metabolism [43, 44].
3.9 Leptin
Adipokine and a hormone released from the fat cells located in adipose tissues, send signals to the hypothalamus in the brain and helps in regulation and alteration of food-intake and energy expenditure. New reported data demonstrated an important role of leptin resistance in the development of IR in obese T2 diabetics and make leptin as a possible marker for IR and risk of T2D. In addition, adiponectin-leptin ratio may be useful as a functional biomarker of adipose tissue function and its related cardiometabolic traits [45].
Specific variations of genes
(SNPs) that affect the de novo adipogenesis (synthesis and metabolism of fatty acids and concentration of fatty acid) of ALG14 gene and C16:0, C18:0; FADS1 and FADS2 genes and C16:1, C18:1, C18:0; LPGAT1 gene and C18:0; GCKR and HIF1AN genes and C16:1; and PKD2L1 gene and C16:1. FABP-4 gene whose was highly expressed in adipocytes and represents about 1% of all soluble proteins in adipose tissue, play significant role in lipid oxidation, lipid-mediated transcriptional regulation, signaling transduction and synthesis of membranes. Recently, data reported that FABP-4 concentrations have been associated with poor control of glycemia in diabetic patients, and that increased FABP-4 levels linked with early presence of inflammation and insulin resistance in diabetes [46‐51].
3.10 Delta desaturase activity, D6D and D5D
The desaturase enzymes are a key component in the chemical structure and functions of fatty acids. Delta-5 (D5D) and delta-6 desaturases (D6D), encoded respectively by FADS1 and FADS2 genes, are represent main determinants of metabolism of polyunsaturated fatty acids PUFAs. Nowadays, FADS genes are strongly associated with numerous metabolic traits including dyslipidemia, fatty liver, T2D, and coronary artery disease. Also, genetic variation in the FADS genes provide new insights for diagnostic, prevention and therapeutic approaches for patients with T2D and metabolic syndrome [52‐56].
3.11 Fructosamine
Fructosamine is serum glycated protein, similar to glycated hemoglobin, uses to assessment of long-term control of glycemia in patients with diabetes mellitus. It is now recognized as a better indicator than hemoglobin A1c for laboratory test for diabetes management but is it still not in use, because its measurement has not been standardized. Results from studies suggested that fructosamine levels are strongly associated with serum glucose and HbA1c and may be use as a complementary marker of glucose metabolism. In addition, high levels of fructosamine are associated with an increased incidence of vascular complications of T2D and may point out to an early progression of the pathophysiology of T2D [56].
3.12 Bilirubin
Bilirubin is a metabolite of heme catabolism and acts as a potent antioxidant. Previous studies demonstrated that increase levels of bilirubin lead to decreased oxidative stress and improve insulin synthesis and insulin sensitivity. The low bilirubin levels are associated with development of T2D and its complications in patients with impaired glucose metabolism as patients at high risk of T2D. Also, its role depended of ethnicity and age of study population and positively associated with the risk of incident of diabetes [56‐58].
3.13 C-reactive protein, CRP
CRP is pentameric protein in plasma whose levels increase in response to inflammation. Recent data suggest that elevated CRP levels are independently associated with development of T2D, while polymorphisms in CRP gene may contribute to the progression of complications of disease [59, 60].
3.14 Glucagon-like peptide 1, GLP-1
GLP-1 is an incretin hormone, whose main action is to stimulate insulin secretion but also, as a physiological regulator of appetite and food intake. Currently, GLP-1 or GLP-1 receptor agonists are exaggerated secretion as response for postprandial reactive hypoglycemia. New findings showed that GLP-1 based therapy is a novel and good therapy of T2D with in combination with certain antidiabetic drugs that improve inadequately controlled glycemia [61].
3.15 micro-Ribonucleic acid, miRNA
A miRNA is a small noncoding RNA molecule (containing 22 nucleotides), which functions in RNA silencing and posttranscriptional regulation of gene expression. Circulating miRNA could potentially be use as a new, noninvasive diagnostic tool for predicting the development of T2D, especially in elder population. Also, identification of novel miRNAs molecular marker candidates for monitoring responses of some antidiabetic drugs helps to prediction and development of chronic complications of disease [62‐64].
4 Discussion
High prevalence and increased incidence in type 2 diabetes mellitus worldwide intensively rise interest of discovery new molecular markers for diagnosis, prevention and development of T2D. Despite efforts of researchers to find molecules, which contribute to the disease progression as well ability to monitoring and improve therapy approaches, underlying pathophysiological mechanisms is still not clear.
Currently, in clinical practice FPG, OGTT and hemoglobin A1c are widely used for the diagnosis of diabetes mellitus or for monitoring of therapy. As a complex metabolic disorder, T2D represent interaction between multiple genetic and environmental factors (lifestyle, dietary intake, ethnicity, race, age, heritability, duration of disease, etc.). Knowledge of chemical processes and metabolic pathways needed to better understanding risk factors of development T2D [65, 66].
Metabolomics is a new area of analytical chemistry that focused on the relationship between genetic and environmental factors of disease. Therefore, scientists can determine the precise molecular mechanisms and pathophysiological pathways of disease by analysis of disturbances in levels of small molecules and metabolites such as sugars, amino acids, lipids and nucleotides that can identify and quantified. There is evidence that BCAA related to the development of IR independently of plasma lipids, while impaired lipid metabolism, especially elevated free fatty acids, strongly associated with IR, IGT and risk of T2D. In addition, provided GWAS and lipidomics studies find out which gene variations may have a causal effect in T2D. To date, TCF7L2 gene and its polymorphisms is the strongest associated gene to T2D, and high expression of this gene results in impaired glucose-stimulated insulin secretion [47]. In line with this, FADS gene and alteration in delta desaturase activities also, showed significant associations with glycemic and other metabolic traits [44]. Recently, metabolomics studies analyzed not only concentration and profile metabolites but also, ratio of metabolites of interest. It was found that the ratio Val_PC32:2 represent the best marker for T2D and related IR [41].
5 Conclusions
Metabolomics, lipidomics, proteomics and GWAS studies in T2D were provide a data of number molecule markers for risk, development and progression of this disorder. Modern analytical chemistry and their instrumental methods contribute to our better understanding of biochemical processes and metabolic pathways of T2D.
“omics” technology and obtained the model profiles of key intermediates or metabolites of lipid metabolism, as well carbohydrates, nucleic acids and proteins give a “signature” in the precise diagnosis and treatment of T2D.
Further analysis should focus on application of metabolic and lipidomic techniques into clinical practice for an early detection risk factors of developing T2D and a novel therapeutic target and treatment of disease.
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Conflict of interest
The authors declare that they have no conflict of interest.
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