MicroRNAs and Pancreatic ß Cell Functional Modulation

Abstract

Recent reports of diabetes susceptibility loci located on the non-coding regions of the genome highlight the importance of epigenetic control in health and disease. Specifically, microRNAs have shown to have an important regulatory role in pancreatic ß cell physiology. Human studies implicated that ß cell mass and function are regulated by microRNAs in health and disease. Further, the microRNAs are also implicated in ensuing diabetic complications. Delineating the peculiar role of microRNAs in ß cell physiology and pathophysiology will fill the missing gaps in our current knowledge and help to devise better treatment regimens for diabetes. This chapter will discuss multiple effects of different microRNAs on the ß cell physiology in the context of maintenance and function in Type 2 diabetes mellitus.

1. Introduction

MicroRNAs (miRNAs/miRs) are pertinent genetic regulators of embryonic development and differentiation, postnatal growth, and metabolism [1]. Initially discovered in C. Elegans, miRNAs are now considered a universal phenomenon for the gene expression regulation from multicellular organisms to mammals [2, 3, 4, 5, 6]. The mammalian genome has been reported to encode more than 500 known microRNA genes. microRNA binds with the mRNA (3′ untranslated region 3’UTR) and result either in the translational repression of that specific gene or in the total degradation of the mRNA [6, 7, 8, 9]. Apart from 3’UTR, miRNA has been reported to interact with 5’UTR and gene promoters and enhancers [10]. miRNAs are critical for normal animal development. As expression pattern of numerous genes important for the embryonic development and subsequent cellular differentiation have been shown to be regulated by microRNAs [11].

The pathological importance of miRNAs is highlighted by the substantial reports of the association of aberrant expression of miRNAs in many human diseases [12, 13]. As miRNAs are also present in the extracellular fluids and act as a signaling molecule for cell communication, their diagnostic importance as a biomarker in different lethal diseases has also been reported [14, 15] Biogenesis of miRNAs and the mechanisms through which they regulate gene expression and their tissue distribution has been previously discussed in detail [1, 10, 16, 17, 18]. The transcriptional activity of miRNA sequences is solely dependent upon RNA polymerase II/III activity [19, 20]. The transcribed miRNA can act through canonical as well as non-canonical pathways [20, 21]. A complementary match either exact or partial between microRNA and the 3’-UTR (conserved) site of the mRNA will result in either the subsequent degradation of the mRNA or moderate reduction in the mRNA levels resulting in translation repression [22, 23]. The degradation of mRNA will result in complete blockage of protein expression. Whereas specific translation repression results in a reduction in the protein formation from the target genes. This fine-tuning of gene expression and subsequent protein formation by microRNAs is important for normal physiological responses. A single miRNA can target hundreds of different genes and mRNAs thus exhibiting multiple levels of complementarity. It is estimated that more than 60% of protein-coding genes are microRNA targets.

In the canonical pathway of microRNA generation (explained in more detail in [24]) events start with the transcription of microRNA sequence by the RNA polymerase II or III, the microRNA transcript is spliced, capped and polyadenylated in the nucleus and is called pri-microRNA. The nuclear pri-microRNA is cleaved by a nuclear protein complex called microprocessor complex and includes Drosha and DGRC8 proteins. After this cleavage event, the pri-microRNA is exported to the cytoplasm by the help of a protein called exportin 5 (Exp 5). Cytoplasmic pre-microRNA is further cleaved by another protein called Dicer and converting the pri-microRNA into a microRNA duplex of around 22 nucleotides. These duplex molecules are loaded onto another protein called Argonaute (AGO). Duplex microRNAs are unwinded and the passenger strand is removed while the guide microRNA strand remains with the AGO proteins and forms the mature effector complex called RNA-induced silencing complex (RISC). RISC complex is guided by the microRNA complementarity to bind with mRNA targets and 3’end de-adenylation occurs resulting in the mRNA degradation of the translation arrest on mRNA. Interestingly deletion of the components of this canonical pathway like DICER or AGO proteins leads to β cells functional impairment in terms of glucose-induced insulin secretion and dedifferentiation into a progenitor-like state [25, 26, 27, 28]. The current chapter will focus on the physiology and pathophysiology of the β cells focusing on the insulin signaling responsible for glucose homeostasis and the influence and interplay of different microRNAs onβ cells functionality in Type 2 diabetes (T2DM).

2. β cells, insulin and macronutrient metabolism

Insulin is the primary endogenous protein responsible for the physiological regulation of metabolism [29]. The regulated secretion of insulin from the pancreas helps to maintain glucose homeostasis in distinct physiological conditions [30, 31]. Endocrine cells that secret specific hormones for the maintenance of glucose levels are present in specialized closed zones in the pancreas called Islets of Langerhans [31, 32, 33]. Islets of Langerhans contain numerous cell types with an appropriate amount of vascular and nervous innervation [31]. Four different types of endocrine cells populated Islets of Langerhans namely: alpha (α) cells, beta (ß) cells, delta (δ) cells, and pancreatic polypeptide (PP) cells. ß cells secrete insulin and initiate postprandial glucose metabolism and control the rising blood glucose levels and α cells secrete of glucagon which enhances blood glucose levels during fasting [34, 35]. Insulin is also responsible for lipid and protein metabolism [36, 37, 38]. The release of insulin from ß cells is initiated by glucose uptake, a phenomenon known as glucose-stimulated insulin secretion (GSIS) [39, 40, 41]. At physiological postprandial levels, glucose initiates insulin gene transcription by recruiting specific transcription factors (PDX-1, MafA, and NeuroD) and post-transcriptionally improves the insulin mRNA stability thus acting as a major physiologic regulator of insulin [42, 43, 44]. Glucose-specific channels/gates are present on the ß cell membrane which are commonly known as glucose transporters (GLUT) [30, 41]. Many types of glucose transporters are present throughout the body [45]. Specifically, GLUT2 is abundant in the pancreas and liver whereas GLUT4 is abundant in skeletal and cardiac muscles as well as adipocytes [45, 46].

Insulin initiates carbohydrate metabolism through phosphorylation of glucose and subsequent formation of glucose-6-phosphate [47, 48]. Insulin phosphorylates glucose through the hexokinase enzyme in muscles and glucokinase (GCK) in ß cells and hepatocytes [49, 50, 51, 52]. Insulin binds to the cell surface insulin receptors commonly present in different tissues [53]. The insulin-insulin receptor binding activates cytoplasmic adaptor proteins called insulin receptor substrates (e.g., IRS1, IRS2) [54]. IRS proteins activate phosphoinositide2-kinase enzyme (PI3K) and convert tyrosine phosphorylation signal into the lipid kinase. Activated PI3K recruits ATP molecules which initiates the AKT activation (serine and threonine kinase) [55]. Activation of AKT by insulin proved landmark and explained the conversion of tyrosine phosphorylation into serine/threonine phosphorylation signal. Insulin-induced AKT activation also helped to describe the insulin-induced regulation of key steps in insulin signaling (a) glucose uptake by glucose transporter, (b) phosphorylation and subsequent inactivation of glycogen synthase kinase 3 (GSK3), (c) activation of the mechanistic target of rapamycin (mTOR) and subsequent synthesis of protein and fats, (d) Transcriptional control of gene expression by forkhead family box O (FOXO) transcription factor proteins. Insulin increases glucose uptake via GLUT4 in muscle and adipose tissue thus enhancing the postprandial rate of glucose transport, insulin also inactivates GSK3 and stimulates glycogen synthesis in these tissues [56]. Insulin prevents hepatic glucose synthesis by inhibiting glycogenolysis and gluconeogenesis. Insulin via AKT inhibits FoxO1 which results in the decreased expression of phosphoenolpyruvate carboxykinase (PEPCK) and glucose −6-phosphatase (G6PC) genes [57, 58, 59]. Insulin regulates lipid and protein metabolism also through AKT. Insulin via AKT induced an increase in mTOR and a decrease in FoxO1 and sterol regulatory element-binding protein (SREBP) inhibits adipocyte lipolysis and helps to lower the plasma fatty acid levels by promoting lipogenesis and enhancing the hepatic formation of very-low-density lipoprotein (VLDL) [60, 61, 62, 63, 64, 65]. Insulin increases the protein synthesis in skeletal muscles and the liver by enhancing the amino acid transport inside the cells, accelerating mRNA translation, and reducing protein degradation and urea formation via AKT activated mTOR [37, 66, 67, 68, 69, 70, 71].

3. Glucose homeostasis: α and β interplay

β cells exhibit a remarkable degree of plasticity in terms of insulin production. The α and ß cells work in conjunction to stabilize glucose levels in feeding and fasting conditions through the periodic release of insulin and glucagon respectively thus achieving glucose homeostasis [30, 49, 72]. Rising plasma glucose levels after feeding require immediate systemic activation of glucose metabolism. Delayed or decreased activation of glucose metabolism results in abnormally high glucose levels known as hyperglycemia. Higher-than-normal concentrations of glucose incite glucotoxic reactions inside the cells. ß cells are triggered by rising postprandial glucose levels to synthesize and secrete insulin. The resulting increasing insulin levels activate enzymes that help to metabolize glucose. Glucokinase and hexokinase initiate glucose phosphorylation primarily in hepatocytes and muscle cells respectively. The postprandial rise in the insulin declines over time because of the decline in the glucose level, a feature of GSIS [39]. Interestingly insulin negatively impacts glucagon secretion [73, 74, 75, 76]. Plasma glucose levels decline under fasting conditions. As glucose is the primary cellular source to generate ATP, a minimum threshold of plasma glucose levels must be maintained to avoid hypoglycemia. Glucagon maintains plasma glucose levels, to avoid hypoglycemia during fasting, by initiating hepatic gluconeogenesis/glucogenolysis [34, 77, 78, 79, 80, 81].

4. Diabetes mellitus

Diabetes mellitus is metabolic dysfunction that results in a significant decline in the cellular ability to metabolize glucose either because of the lack of insulin or insulin inactivity (insulin resistance) or both [82, 83]. Diabetes mellitus is estimated to affect 700 million people worldwide by the year 2040 [84]. The inability of the cells to metabolize glucose cause an abnormal increase in the cellular and plasma levels of glucose (hyperglycemia). Hyperglycemia causes cellular damage via multiple mechanisms including (a) the increased influx of glucose through the polyol pathway, (b) increased formation of advanced glycation end products (AGEs), and (c) subsequent increased expression of AGE receptors and their ligand [85, 86]. Hyperglycemia initiates the reactive oxygen species (ROS) formation via activated glycation reactions and mitochondrial electron transport chain where ROS acts as the main trigger for the initiation of the polyol pathway, formation of AGEs, and subsequent increased AGE receptor expression [87].

Diabetes mellitus has been categorized in two primary forms: Type 1 Diabetes Mellitus (T1DM) and Type 2 Diabetes Mellitus (T2DM). T1DM has been characterized by a mutation in the insulin gene or immune cell-mediated destruction of ß cell resulting in either the synthesis of abnormal insulin protein that fails to activate insulin receptors or a complete lack of endogenous insulin secretion [88]. T1DM patients are usually diagnosed early in their life. The only possible medical treatment referred to these patients is the multiple daily doses of synthetic insulin. T2DM on the other hand is much more complicated and requires a thorough diagnostic approach [82, 89, 90, 91]. T2DM is considered one of the most common metabolic disorders globally. Major risk factors for T2DM include a sedentary lifestyle, lack of exercise, excessive use of a high-carb and high-fat diet, overweight, and obesity [92]. Poor lifestyle and dietary habits have been attributed to the global incidence of Type 2 diabetes in the last 2 decades. Obesity, visceral fat deposition and increased body mass index (BMI) play a central role in the pathophysiology of Type 2 diabetic patients [82].

5. Pathophysiology of Type 2 diabetes mellitus (T2DM)

The development of T2DM is mainly attributed to the progressive decline in insulin secretion over time from ß cells and the progressive inability of insulin-responsive tissues (muscles, fat, and liver) to respond to insulin over time, resulting in hyperinsulinemia and subsequent insulin resistance [93, 94, 95, 96]. The inability of the insulin hormone to activate insulin receptors at the cellular level has been attributed to be the major cause of hyperinsulinemia and insulin resistance [97, 98]. Ex vivoanalysis of pancreatic islets from T2DM donors in terms of GSIS exhibit disproportional insulin levels where increased glucose concentration fails to elicit appropriate ß cells response in terms of insulin [99, 100, 101]. Insulin binding to insulin receptors at the plasma membrane activates a signaling cascade that initiates glucose metabolism inside the cells. Insulin-bound insulin receptors or activated insulin receptors go through internalization at the plasma membrane, a phenomenon known as insulin receptor endocytosis [29, 102]. Following the activation, the endocytosis of the insulin receptor is the primary physiological mechanism through which the duration and intensity of insulin signaling are controlled. Hyperinsulinemia accelerates insulin receptor endocytosis and affects the presence of adequate functional insulin receptors at the plasma membrane resulting in insulin resistance [103]. Apart from accelerated insulin receptor endocytosis, insulin-stimulated insulin receptor kinase activity is also decreased in diabetic patients [104].

Compromised insulin signaling in T2DM fails to activate glucose metabolic enzymes like glucokinase and hexokinase resulting in hyperglycemia. The expression levels of GCK and GLUT2 were found to be lower in human T2D islets as compared to healthy control [105]. As high plasma glucose levels initiate glucose-stimulated insulin secretory (GSIS) response from ß cells resulting in the rise of plasma insulin levels. The rising insulin levels should be normalized over time because of the renal insulin clearance mechanism. But compromised renal insulin clearance rate in diabetic subjects results in abnormally high plasma levels of insulin (hyperinsulinemia) [106, 107]. Hyperinsulinemia and hyperglycemia in theory cannot trigger alpha cells to secrete glucagon. But it has been observed that T2DM patients with insulin resistance, hyperinsulinemia, and hyperglycemia also have abnormally high plasma levels of glucagon [108]. Hinting toward the disturbance in the α and β cell interplay through the inability of the insulin to block glucagon gene transcription [109].

T2D has also been attributed to altering the gene expression of key proteins participating in the processes of insulin secretion and function. Specifically, the insulin receptor gene (IR) and genes involved in Ca²⁺ influx (SUR1, TMEM37), and mitochondrial metabolism (GPD2, PCK1, FXYD2) [110, 111, 112, 113]. The compromised mitochondrial activity in the T2D β cells through reduced ATP/ADP ratio fails to close the K_ATP channels and subsequently impacts Ca²⁺ influx and exocytosis of insulin. ATP also enhances insulin granular priming prior to the exocytosis and thus regulates insulin quantity by initiating the modification of proinsulin into insulin inside the insulin granule. Substantial β cell loss has been considered the hallmark of T2DM which even starts during the prediabetic stage [114]. Up to ~50% loss in the β cell mass has been reported in T2DM patients [115, 116, 117, 118]. Apoptosis and dedifferentiation have been attributed as the main reasons for this substantial decline in the β mass. Hyperinsulinemia has been attributed to the activation of caspases, formation of H₂O₂ increased expression of nitric oxide synthase (iNOS) in the β cells resulting in their substantial loss [119, 120, 121]. Hyperglycemia-induced production of AGE products and the subsequent activation of AGE receptors further promote the release of cytochrome and caspase activation [122]. Hyperglycemia along with increased levels of free fatty acids (glucolipotoxicity) has also been shown to induce β apoptosis and reduction in their overall mass in T2DM [123, 124]. Over accumulation of lipids and over-activation of lipid signaling pathways inβ cells are of pathological significance as it contributes to β cell loss and further lead to the significant decline in insulin secretion and the onset of T2DM. Glucolipotoxicity induces a cascade of events that starts from mitochondrial dysfunction leading to oxidative stress (ROS) which further leads to ER stress which results in improper protein unfolding response and results in the loss of GSIS and inflammation and activated autophagy. These above-mentioned cascades of events represent altered cell signaling pathways, lipogenic and pro-apoptotic genes, and proteins, increased expression of cytokines, and accumulation of lipids molecules like di and triacylglycerols, cholesterol, and cholesterol esters.

Apart from the above-mentioned mechanisms, β cell dedifferentiation has recently been shown to be another important contributing factor in the progressive decline in the β mass in T2DM [116, 125, 126]. Dedifferentiation is defined as the ability of a cell to revert to its progenitor-like stage (developmental stage). Interestingly it has been observed that dedifferentiation in β cells not only resulted in progenitor-like cells but an increase in other islets cells type has been observed. Implying the possibility that dedifferentiated β cells have further converted into α and δ cells, which interestingly involves the removal of epigenetic brake on glucagon and somatostatin genes for their expression in the functional β cells. This also explains the abnormally high levels of glucagon observed in T2D patients with hyperglycemia, where glucose fails to down-regulate the glucagon gene expression. The transition or dedifferentiation of β cells is characterized by the downregulation of β specific genes and upregulation of pluripotent genes [127, 128, 129, 130, 131]. β cell identity genes like MafA, Nkx6.1, and FoxO1 were shown to be downregulated. Whereas pluripotent genes like Ngn3, Oct4, Nanog, and L-Myc were upregulated. In fact, it has been shown in T2DM patients that glucagon (α) and somatostatin (δ) positive cells have inactivated (cytoplasmic) FOXO1 and NKX6.1 proteins supporting the notion that trans-differentiation of β cells into other endocrine islet cell types [125].

6. Epigenetic regulation of β cell-specific gene expression profile

Epigenetic mechanisms resulting in the alteration of gene expression have been attributed to the regulation or dysregulation of proper β cell function in health and disease [132, 133, 134, 135, 136, 137]. DNA methylation, chromatin modifications, and post-translational modifications of histones have been implicated as the main epigenetic mechanisms. Alteration in the expression pattern of Non-coding RNA sequences like microRNA (miRNA/miR) and long non-coding RNA (lncRNA) has also been shown to regulate the β cell function as well as its cellular identity [138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151].

7. MicroRNAs and β cell regulation

MicroRNA (miRNA/miR) sequences are typically small around ~22 nucleotide non-protein-coding transcripts. Activation of their transcriptional activity results in the formation of miRNAs. Formation or presence of miRNAs has been shown to regulate the expression of a network of genes involved in β cell development, function, and pathogenesis [152, 153, 154, 155, 156, 157, 158]. microRNAs have been shown to play a vital role in the compensation process of β cells during pregnancy, obesity and T2D [158, 159, 160, 161]. MicroRNAs mediate mRNA silencing and posttranscriptional regulation of gene expression in different physiological states of β cells. These miRNAs induced fine tunned changes in β cell gene expression are considered vital in health and disease. As the inability of microRNAs to fine-tune the gene expression levels lead to β cells decompensation which results in abnormal insulin secretion and ultimately the development of diabetes. β cell decompensation is referred to as two main pathological conditions: (1) impaired (reduced) β cell function in terms of glucose sensitivity and insulin secretion (Reduced GSIS), and (2) reduced β cell number/mass due to dedifferentiation or apoptosis. Impaired β cell function involves molecular defects in insulin biosynthesis, glucose uptake, and exocytosis of insulin granules. β cell maintenance is regulated by microRNAs through the downregulation of non-β cell specific genes and upregulation of the β cells specific genes thus avoiding the β cell dedifferentiation process which is impaired in T2DM patients [162, 163]. As discussed earlier, different molecular components are involved in the secretion of insulin in response to the rising blood glucose levels. Failure of either one of these molecular components results in the failure of β cells to respond to the systemic demands of insulin for macronutrient metabolism. MicroRNAs are involved in the fine-tuned adjustments of β cells and regulate multiple aspects of glucose homeostasis via insulin secretion.

8. MicroRNAs and insulin synthesis and signaling

The insulin gene transcription results in the generation of mRNA which is translated in preproinsulin and cleaved into proinsulin in the endoplasmic reticulum [164, 165, 166, 167]. Cleavage of proinsulin into insulin and C-peptide requires activation of convertase enzymes (PC1/3, PC1) through the change in the granular pH into acidic pH. Insulin gene (ins1) expression has been found to be compromised after the deletion or inactivation of Dicer1 gene [27, 28]. Reduced insulin gene promoter activity and decreased insulin content was observed in isolated islets and cultured β cells after the experimental knock-down of a set of microRNAs including miRNA-24, miRNA-26, miRNA-148 and miRNA-181 [168]. A positive correlation in the insulin mRNA levels and negative correlation in terms of GSIS response and the expression profile of miRNA-127-3p and miRNA-184 was observed in the islets isolated from healthy subjects. Interestingly these correlations were found to be absent in the isolated islets of glucose-intolerant (diabetic) donors [169]. In vitro expansion of β cells from adult pancreatic islets were found to be dedifferentiated after the proliferation and levels of miRNA-375 (an islet-specific microRNA) were found to be significantly reduced [170] Experimental overexpression of miRNA-375 in dedifferentiated β cells lead to the activation of the β cell specific gene expression profile. MafA, a key β cell specific transcription factor regulating insulin gene expression, has been shown to be directly targeted by the miRNA-204 [171]. Thioredoxin-interacting protein (TXNIP), a redox regulator intracellular protein, has been found to be upregulated in diabetes and its deficiency protect β cells from diabetes-associated apoptosis. TXNIP induces the expression of miRNA-204 by inhibiting STAT3 activity. miRNA-204 thus blocks insulin production in diabetes by downregulating MafA. microRNA-181c-5p has been shown to induce the expression of INS1, PDX1, NKX6.1, and MafA in human induced pluripotent stem cells (hiPSCs) [172]. microRNA-124a has been shown to be highly expressed in islets of T2DM patients [173]. microRNA-124a has shown to be a negative regulator of insulin by targeting 3’UTR regions of mRNAs of Akt, Sirt1, NeuroD, and Foxa2 proteins. All these proteins are vital in the insulin gene transcriptional activity as well as the insulin signaling via insulin receptors. microRNA-7 has been shown to influence insulin signaling by down-regulating insulin receptor substrate protein genes (IRS1, IRS2) in gestational diabetes in humans [174]. Glucagon-like peptide 1 receptors (GLP1R) are present in the β cells and activation of these receptors by GLP-1 induces GSIS response from β cells [175, 176]. GLP1 receptor expression has shown to be downregulated by microRNA-204 in rat ins-1 cell line and primary mouse and human islets cells by targeting the 3’UTR region of GLP1R mRNA [177]. This is particularly important as GLP-1 receptor agonists are primary drugs used by T2DM patients to increase insulin levels.

9. MicroRNAs and insulin secretion

Several microRNAs have been shown to impact glucose uptake and metabolism by the β cells and the exocytotic process of insulin secretion. As GSIS regulates the key components of the process of β cell insulin release. Islets isolated from T2DM patients have shown decreased expression of proteins involved in K_ATP and Ca²⁺ channel regulation, metabolism, and exocytosis of insulin resulting in hyperglycemia [178]. Islets from T2DM patients show increased levels of microRNA-130a, microRNA-130b, and microRNA-152 and were found to be glucose-induced [179]. microRNA-130a, microRNA-130b, and microRNA-152 target Gck gene and downregulates ATP synthesis by reducing glucose metabolism in β cells thus affecting insulin exocytosis by not only blocking the closure of K_ATP channels but also affecting the insulin granule priming. microRNA-29a, microRNA-29b and microRNA-124 have been shown to suppress the gene expression of a β cell disallowed mitochondrial protein called monocarboxylate transporter (MCT-1) [180]. MCT-1 inhibition allows the β cells to only consume glucose for metabolism. Thus microRNA-29a, microRNA-29b, and microRNA-124 help in preserving the cell-specific identity. Human and mouse islets incubated with high glucose (hyperglycemic conditions) exhibit an increased expression of microRNA-29 in β cells [181]. microRNA-29 has been shown to positively impact the expression of a transcription factor called Onecut2. This transcription factor upregulates the granuphilin protein gene expression. Granuphilin protein blocks the exocytosis of secretory granules in endocrine cells. Apart from Onecut2-granuphilin mediated blocking of exocytosis, microRNA-29 also influences exocytotic protein genes like Syntaxin1A and SNAP25 (members of SNARE complex: a family of proteins involved in membrane fusion during exocytosis) [182].

10. MicroRNAs and lipid accumulation in β cells

The characteristic hallmark of T2D is the dysregulation of lipid metabolism and subsequent obesity. Excess amounts of fats present in the bloodstream of T2D patients have been attributed to β cell mass reduction and decreased insulin secretion. MicroRNA sequences have been shown to be manipulated by the exposure of β cells to circulating fats in T2D patients [183]. β cell-specific genes which encode proteins that are involved in lipid metabolism and subsequent cholesterol homeostasis have been found to be under miRNA control [184]. miRNA-33 has been reported to control cholesterol homeostasis through modulating the expression of sterol regulatory binding protein (SREBP) genes [185] miRNA-34a has been shown to induce β cell lipotoxicity through multiple mechanisms after the in vitro and in vivo exposure to saturated fatty acids [186, 187, 188, 189]. Increased β cell influx of fatty acids through di/triacylglycerol and esterified cholesterol pathways including targeting sirtuin1 (SIRT1). Importantly SIRT1 (NAD⁺-dependent deacetylase) regulates the expression of multiple genes encoding proteins like tumor suppressor protein p53 and DNA repair factor ku70 and transcription factor protein including nuclear factor κB (NF-κB) and FOXO family. Exposure of β cells to saturated fatty acids alters the expression profile of important miRNAs which might contribute to β cell lipotoxicity. These miRNAs include miRNA-146 [184], miRNA-182-5p [190], miRNA-297b-5p [191], and miRNA375 [192]. miRNA-146 target genes which are involved with the inflammatory response (Toll-like receptors) [193]. It has been demonstrated that in cultured human islets, the miRNA-146 level increased after the exposure to pro-inflammatory cytokines and decreased exposure to high glucose concentrations but interestingly miRNA-146 levels were unchanged after the palmitate exposure [194]. miRNA-146 along with miRNA 182-5p have been shown to protect against high-fat diet-induced steatohepatitis in a mice model by decreasing in the expression of IL-1 receptor-associated kinase (IRAK1) and tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) which results in the reduced cytoplasmic lipid accumulation and inflammation [195]. miRNA-182-5p was found to be increased in INS-1 cells incubated with palmitate and significantly decreased the cell viability and increased palmitate-induced apoptosis. When INS-1 cells were supplemented with miRNA-182-5p inhibitors a significant increase in the cellular viability and palmitate-induced apoptosis was observed [190]. C57BL/6 mouse and β-TC6 cell line exposed to stearic acid and palmitic acid lowers the expression of miRNA-297b-5p. Upregulation of miRNA-297b-5p reduced the stearic acid-induced apoptosis but decreased insulin secretion in β-TC6 cells by inhibiting large-tumor-suppressor kinase 2 (LATS-2) [191] miRNA-375 has been shown to block high-fat diet-induced insulin resistance and obesity in mice by promoting hepatic expression of insulin-responsive genes [196].

11. Conclusion

The important role of epigenetics in the functional regulation of β cells in health and disease has been supported by the increasing number of high levels studies on human and rodent models. Excellent research efforts in the last decades have established that microRNAs help to define the β cell identity and dysregulation of β cell microRNA expression acts as a key event in the pathogenesis of T2DM. As the mechanisms involved in the biogenesis of microRNAs are well understood, it is plausible to assume that microRNAs represent attractive therapeutic targets. Selective inhibition of Dicer molecules by using specific siRNAs or pharmacological tools and selective modulation of a single microRNA. Specific microRNAs involved in diabetes represent a new class of specialized biomarkers for the early detection of diabetes. As microRNAs are biochemically quite stable in the extracellular fluid like blood, they represent a very good biomarker for diagnostics. Further in-depth studies are needed to assess the predictive value of serum levels of microRNAs in relation to the disease progression. Novel therapeutic approaches need to be devised for the prevention and treatment of metabolic disorders.