Impaired Physiological Regulation of ß Cells: Recent Findings from Type 2 Diabetic Patients

Abstract

Recent studies have emphasized the multiple aspects of non-coding micro-RNAs in the regulation of pancreatic ß cells in type 2 diabetic patients. Thus, highlighting the significance of non-coding regions of the genome in regulating pancreatic endocrine cells. Functional dysregulation of pancreatic endocrine cells increases the incidence of metabolic disorders in otherwise healthy individuals. A precise understanding of the molecular biology of metabolic dysregulation is important from cellular and clinical perspectives. The current chapter will highlight the important recent findings from type 2 diabetic human patients and aims to enhance our current understanding of ß cell pathophysiology from a clinical perspective for the development of novel therapeutic approaches to control this global incidence.

1. Introduction

Diabetes mellitus in principle is a pathophysiological condition in which the ability of cells to metabolize glucose is compromised primarily because of insulin deficiency or compromised insulin signaling (Figure 1). Diabetes mellitus is considered a global issue as it is estimated to impact around 700 million individuals worldwide by the year 2040 [1]. Hyperglycemia is the subsequent aftermath of diabetes mellitus where an abnormally high concentration of glucose persists at cellular and plasma levels. The persistent high concentration of glucose results in the activation of the polyol pathway and formation of advanced glycation end products (AGEs) along with an increase in AGE receptors (RAGE) [2, 3]. Activation of the polyol pathway enhances the mitochondrial production of reactive oxygen species (ROS) and increases the cytosolic concentration of ROS [4].

Figure 1.

Description of progressive ß cell decline during the course of diabetes mellitus and the subsequent loss of the cellular identity resulting in the trans- or de- differentiation of ß cells.

Type 2 diabetes mellitus (T2DM) is the most common metabolic disorder occurring globally [5, 6, 7]. Diets rich in carbohydrates and fats along with lack of exercise are highlighted as major risk factors for the increasing incidence of T2DM globally [8]. Obesity with visceral fat deposition and increased body mass index (BMI) have been shown to play a central role in the progression of type 2 diabetes complications [9].

2. Pathophysiology of type 2 diabetes mellitus (T2DM)

Regardless of the etiology, the T2DM progression is characterized either by a slow, progressive, and yet significant decline in the insulin secretion from ß cells or by a disruptive insulin signaling at the insulin-responsive tissues (muscles, fat, and liver) resulting in hyperinsulinemia which is clinically manifested as insulin resistance [10, 11, 12, 13]. The inability of insulin to properly bind and activate insulin receptors (IRs) on the cell membrane of muscle cells, adipocytes, and hepatocytes has been attributed as the main reason for hyperinsulinemia and insulin resistance [14, 15].

As activation of cytosolic carbohydrate metabolism is induced by insulin and results in glucose phosphorylation and subsequent formation of glucose-6-phosphate inside the cells [16, 17]. Insulin signaling enhances the enzyme activities of hexokinases in muscle and fat tissue and glucokinase (GCK) activity in the ß cells and hepatocytes [18, 19, 20, 21]. The activated cell surface insulin receptors stimulate cytosolic adaptor proteins called insulin receptor substrates (IRS1, IRS2) [22, 23]. Phosphorylation of IRS proteins activates the phosphoinositide2-kinase enzyme (PI3K) which further recruits ATP molecules to ultimately activate AKT protein (serine and threonine kinase) [24]. AKT activation helps the insulin to regulate multiple steps in glucose metabolism like a) increased cellular uptake of glucose via glucose transporter (GLUT4) in skeletal muscles [25], b) inhibition of glycogen synthase kinase 3 (GSK3) to stop glycogen metabolism [26, 27, 28], c) AKT induced activation of the mechanistic target of rapamycin (mTOR) resulting in the protein and lipid synthesis [29, 30, 31, 32, 33, 34], d) transcriptional control of gene expression through AKT induced inhibition of forkhead family box O (FOXO) transcription factor proteins [35, 36, 37]. Insulin also greatly helps to reduce plasma fatty acid levels by reducing adipocyte lipolysis rate and enhancing the ability of hepatocytes to produce very low-density lipoprotein (VLDL) [38, 39, 40, 41, 42, 43]. Insulin plays an important role in increasing skeleton muscle mass by enhancing the cellular intake of amino acids favoring protein synthesis and limiting the process of protein metabolism and urea formation [44, 45, 46, 47, 48, 49].

These above-mentioned metabolic consequences of insulin signaling on macronutrients like carbohydrates, proteins, and lipids ensure the maintenance of nutritional homeostasis at the cellular level under diverse physiological conditions. Diabetes mellitus is fundamentally a loss of this metabolic homeostasis at the cellular level. The single and most important endogenous factor for the loss of metabolic homeostasis is dysfunctional insulin signaling. Once the insulin receptors are activated through binding insulin molecules, these receptors follow a deactivation phase by the process of internalization and thus can no longer bind with insulin, a phenomenon known as insulin receptor endocytosis [50]. Insulin receptor endocytosis is the primary physiological mechanism through which the duration and intensity of insulin signaling are controlled [51]. Hyperinsulinemia leads to insulin resistance by promoting insulin receptor endocytosis rate and diminishing the number of insulin receptors at the plasma membrane which could potentially bind with plasma insulin molecules [52]. Apart from receptor internalization, insulin receptor kinase activity is also shown to be compromised in T2DM patients [53]. Lack of or reduced insulin signaling fails to activate glucose transporter proteins (GLUT) which limit cellular uptake of glucose resulting in high plasma glucose levels. Normally high plasma glucose levels stimulate the release of insulin from ß cells, a phenomenon known as glucose-stimulated insulin secretion (GSIS) [54, 55, 56]. Surprisingly a compromised GSIS responsiveness of ß cells in terms of insulin secretion from T2DM donors has been reported [57, 58, 59]. Interestingly it has been reported that T2DM patients also have high plasma levels of glucagon along with insulin resistance, hyperinsulinemia, and hyperglycemia [60]. Highlighting that the insulin is unable to downregulate glucagon secretion from islet α cells [61]. T2DM patients have also been reported to have decreased levels of gut incretin hormone known as glucagon-like peptide-1 (GLP-1) which normally induces the postprandial release of insulin from ß cells and downregulates glucagon secretion from α cells [62, 63]. These findings implicate the gut as an important regulator of insulin secretion during feeding and fasting and might contribute to the incidence of T2DM [64, 65]. Current treatment options for T2DM include newly developed GLP-1 receptor agonists to curtail hyperglycemic episodes contributed by either the lack of insulin and/or increased glucagon secretion [66, 67, 68].

2.1 Transcriptional modulation of ß cells in T2DM

Altered gene expression profiles of important proteins involved in the secretory process of insulin and insulin signaling are the major consequence of T2DM, as SUR1 and TMEM37 (responsible for Ca²⁺ influx), IR (insulin receptor), and GPD2, PCK1, ALDOB, FXYD2 (mitochondrial metabolism) have reduced expression in T2DM as compared to healthy controls [69, 70, 71, 72]. Optimal mitochondrial activity is vital for normal insulin secretion because a normal ATP/ADP ratio promotes the K_ATP channels closure thus allowing Ca²⁺ influx and helping in the exocytosis of insulin granules from β cells. Mitochondrial ATP formation is also important for stimulating the conversion of proinsulin into insulin before insulin granule exocytosis.

Another important T2DM hallmark is the unusual loss of functional β cells, a process that starts during the prediabetic stage [73]. Almost half of the total population of functional β cells have been reported to be lost in T2DM patients [74, 75, 76, 77]. Two distinct mechanisms have been credited for this remarkable loss in functional β cells during T2DM namely: 1) apoptosis and 2) dedifferentiation. Interestingly the process of β cell apoptosis is initiated due to the hyperactivity of β cells which results in hyperinsulinemia. Prolonged exposure of β cells to high levels of insulin activates enzymes like caspases which initiate apoptosis, and nitric oxide synthase (iNOS) triggering excessive nitric oxide (NO) production, along with the formation of hydrogen peroxide (H₂O₂) [78, 79, 80]. Hyperglycemia along with high plasma levels of free fatty acids, known as glucolipotoxicity, also induces β cell apoptosis in T2DM [81, 82]. The deleterious effect of increased lipid accumulation in the β cells includes hyperactivation of lipid signaling pathways causing loss of functional β cells. Recently cellular dedifferentiation (cells switching back to the undifferentiated/progenitor stage from the differentiated/functional stage) has emerged as an important factor influencing the functional β cell mass in T2DM [83, 84, 85]. β cell dedifferentiation also propagates the appearance of other islet cell types indicating that β cells might trans-differentiate into α and δ cell types. The removal of epigenetic control on the transcription of glucagon and somatostatin genes in β cells during trans-differentiation limits the unique identity of β cells as insulin secretory cells. This phenomenon of trans-differentiation might help to clarify the increased plasma levels of glucagon observed in T2DM patients. Specifically, glucagon and somatostatin-positive cells have inactivated (cytoplasmic) FOXO1 and NKX6.1 (β cell-specific transcription factors) proteins in the islets of T2DM donors hinting towards the possibility of trans-differentiation of β cells [84]. Transcriptionally upregulated pluripotent genes and downregulated β cell-specific genes have been observed to assist the trans- and dedifferentiation of functional β cells. Down-regulation of specific genes like MafA, Nkx6.1, and FoxO1 which assign a unique identity to β cells, and upregulation of genes like Ngn3, Oct4, Nanog, and L-Myc which imparts cellular pluripotency has been observed in the islets of T2DM [86, 87, 88, 89, 90].

2.2 Epigenetic regulation of β cell-specific gene expression profile

Aberration in β cell functionality is mainly attributed to deregulated gene expression control through epigenetic mechanisms [91, 92, 93, 94, 95, 96]. Specifically, chromatin modifications, DNA methylation, and post-translational modifications of histones are the classical epigenetic mechanisms through which gene expression profile of functional β cells are controlled. Alteration in the expression pattern of non-coding RNA sequences like microRNA (miRNA/miR) has also been shown to regulate the β cell function as well as their cellular identity [97, 98, 99, 100, 101, 102, 103]. Nonetheless, the recent findings that the majority of diabetes susceptibility loci are located in the non-coding regions of the human genome highlight the importance of epigenetic control in glucose homeostasis and β cell regulation [104, 105].

3. microRNA’s regulation of β cell function

Pancreatic β cells are highly adaptative cellular entities in nature [106, 107, 108, 109]. Adaptivity is specifically required to cater to different physiological states that demand different/opposing β cell responses. Like during feeding and fasting as well as during the high energy demands in exercise and pregnancy or during the intake of high carb high-fat diets. TF. Transcription upregulation of specific miRNAs during the embryonic developmental stage has been shown comprehensively to play an important part in the expression of a network of genes responsible for β cell development and function in mammals [110, 111]. However, the human data from diabetic adult patients suggests that the miRNAs might play a permissive role in the induction of β cell dysfunction [112]. On the other hand, certain miRNAs have been attributed to enhanced β cell mass/number in the pancreas, a phenomenon termed as β cell compensation [113, 114, 115]. These conflicting roles of miRNAs warrant a thorough understanding of the peculiar role of specific miRNAs under different physiological and pathological conditions. As miRNAs play a primary role in mRNA silencing and attenuate posttranscriptional regulation of gene expression in different physiological states of β cells. These fine-tuned variations are vital for glucose homeostasis. The loss of miRNA’s ability to fine-tune the gene expression led to β cell decompensation which results in abnormal insulin secretion and ultimately the development of diabetes. β cell decompensation leads to two main pathophysiological events: 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. The downregulation of β cell-specific genes and upregulation of the non β-cells specific genes induce β cell dedifferentiation. T2DM patients have been shown to present these β cell-specific conditions [116, 117, 118]. A set of specific molecular pathways are involved in the secretion of insulin during GSIS and failure of either one of these molecular pathways can result in the GSIS decline and consequently the incidence of diabetes.

3.1 Involvement of different miRNAs in insulin biosynthesis and signaling: Recent ex-vivo evidence from diabetic patients

The first comprehensive report on the possible involvement of miRNAs in regulating mammalian β-cells ability to synthesize and secrete insulin came in 2011. Down regulation of a subset of miRNA genes induced a decline in insulin gene promoter activity and subsequently resulted in reduced insulin content in isolated murine islets and cultured β cells [119]. These specific miRNAs were miRNA-24, miRNA-26, miRNA-148, and miRNA-181. Soon after another study employed pancreatic islets isolated from normal and glucose-intolerant human donors [120]. An islet-specific miRNA network involved in insulin secretion in human islets was suggested and consists of miRNA-375, miRNA-127-3p, and miRNA-184. Later, in 2015, miRNA-375 (an islet-specific miRNA) was shown to play a decisive role in regulating the phenotype of human β cells, and upregulation of miRNA-375 during ex-vivo expansion of human islet cells facilitates the generation of insulin-positive cells [121]. Experimental overexpression of miRNA-375 in dedifferentiated isolated human β cells also led to the restoration of β cell-specific gene expression profile. MafA (β cell-specific transcription factor regulating insulin gene) has been shown to be directly targeted by the miRNA-204 in isolated β cells from normal and diabetic human donors [122]. Briefly, Thioredoxin-interacting protein (TXNIP), a redox regulator intracellular protein, has been found to be upregulated in diabetes and induces β cell apoptosis. TXNIP upregulates the expression of miRNA-204 by inhibiting STAT3 activity. Overexpression of miRNA-204 decreases MafA expression in β cells thus lowering insulin production in diabetes. Followed by the report of over-expression of microRNA-124a in the islets isolated from T2DM patients [123]. microRNA-124a negatively regulates insulin by targeting mRNAs of key proteins involved in insulin gene transcription (NeuroD and FOXA2 and insulin signaling (Akt and Sirt1). miRNA-204 has been shown to downregulate β cell-specific GLP1 receptor expression in primary human islet cells by targeting GLP1R mRNA [124]. This finding might have therapeutic implications as the β cell-specific GLP1 receptor is responsible for the post-prandial insulin release in normal subjects and GLP-1 receptor agonists are used primarily as insulin secretagogues by T2DM patients [125, 126] same reference is present but from gut chapter]. Recent reports identify miRNA-181c-5p as a key factor in developing functional β cells through human-induced pluripotent stem cells (hiPSCs) by upregulating the expression of INS1, PDX1, NKX6.1, and MafA in [127]. More recently miRNA-7 has been attributed for its influence on insulin signaling via down-regulating the gene expression of insulin receptor substrate protein (IRS1, IRS2) during gestational diabetes in humans [128].

3.2 microRNAs influencing insulin secretion from β cells

As discussed earlier, the magnitude of glucose uptake during GSIS regulates the rate of insulin release from β cell. Currently multiple miRNAs have been identified in humans to affect the glucose uptake ability of the β cells thus indirectly reducing the rate of insulin release. Considerable decline in the expression of specific proteins responsible for K_ATP and Ca²⁺ channel regulation and insulin granular exocytosis has been observed in the islets from T2DM donors [129]. T2DM donor islets also present glucose induced rise in different miRNA levels namely miRNA-130a, miRNA-130b, and miRNA-152 [130]. These specific miRNAs impair post transcriptional mRNA processing of glucokinase and impacts glucose metabolism. Proper glucose metabolism via glucokinase action is essential for ATP synthesis inside β cells. As insulin exocytosis requires the closure of ATP sensitive K_ATP channels. On the contrary certain miRNAs have been shown to support β cells to preserve their unique cellular identity. Monocarboxylate transporter (MCT-1) is a mitochondrial protein which is specifically inhibited in β cells to allow them to only utilize glucose as a precursor for metabolism. These specific miRNAs include miRNA-29a, miRNA-29b, and miRNA-124 which have been shown to selectively target human MCT-1 gene [131]. miRNA-129a have also been shown to be glucose sensitive. As islets isolated from human donors when incubated in high glucose medium (hyperglycemic conditions) resulted in upregulation of miRNA-29 in β cells [132]. miRNA-29 also enhance the expression of Onecut2 (a transcription factor). Onecut2 the granuphilin protein gene expression. Granuphilin protein blocks the exocytosis of secretory granules in endocrine cells. Apart from Onecut2-granuphilin mediated blocking of exocytosis, miRNA-29 also influences exocytotic protein genes like Syntaxin1A and SNAP25 (members of SNARE complex: a family of proteins involved in membrane fusion during exocytosis) [133].

4. microRNAs regulation by lipid accumulation in β cells

Another important and clinically relevant phenomenon of T2DM is the dysregulated lipid metabolism resulting in dyslipidemia (hypertriglyceridemia, reduced HDL and increased LDL particles) thus raising the incidence of obesity and cardiovascular disease (CVD) [134, 135, 136]. High plasma levels of triglycerides (hypertriglyceridemia) in T2DM patients result in substantial reduction of functional β cell numbers and significantly reduced insulin secretion [137]. Interestingly it has been demonstrated that microRNA sequences are altered during the β cell lipid metabolism by exposure to high lipids/fats [138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149]. Specific proteins responsible for lipid metabolism and cholesterol homeostasis in β cells have been found to be transcriptionally controlled by several microRNAs. miRNA-33 has been shown to regulate cholesterol homeostasis by controlling sterol regulatory binding protein (SREBP) gene expression [150]. miRNA-34a has been reported to induce β cell lipotoxicity in-vitro during the exposure of β cells to high concentrations of saturated fatty acids [151, 152, 153, 154]. An increased cellular influx of fatty acids via di/triacylglycerol and/or esterified cholesterol pathways in β cell has been found to target an NAD⁺-dependent deacetylase protein called sirtuin1 (SIRT1) [153]. SIRT1 is important for its specific role in the upregulation of insulin secretion in β cells in response to glucose stimulation [155]. Apart from insulin secretion, SIRT1 also transcriptionally controls multiple protein-encoding genes like tumor suppressor protein p53 and transcription factor proteins like nuclear factor κB (NF-κB) and FOXO family [156, 157, 158]. Apart from miRNA-34a, saturated fatty acids also impact the transcriptional profile of different miRNAs in the β cell contributing to lipotoxicity. Specific miRNAs regulated by saturated fatty acids include miRNA-146 [159], miRNA-182-5p [160], miRNA-297b-5p [161], and miRNA375 [162]. miRNA-146 targets mRNAs of certain genes responsible for the mediation of inflammatory pathways e.g. Toll-like receptors (TLRs), and NF-kB signaling [163]. INS-1 cells incubated with palmitate exhibit increased miRNA-182-5p expression, significantly decreased cell viability as well as increased palmitate-induced apoptosis. Interestingly, INS-1 cells when treated with specific inhibitors of miRNA-182-5p, exhibit a significant increase in cellular viability and reduction in palmitate-induced apoptosis [160]. Cultured human islets have increased levels of miRNA-146 in response to pro-inflammatory cytokines exposure decreased levels after high glucose exposure and interestingly stable levels after palmitate exposure [164]. Apart from the regulation of inflammatory response, miRNA-146 and miRNA 182-5p have also been involved in the reduction of cytoplasmic accumulation of lipids and cellular inflammatory response [165, 166]. β-cell lines exposed to stearic acid and palmitic acid exhibit reduced expression of miRNA-297b-5p whereas the increased miRNA-297b-5p levels help to minimize apoptosis induced by stearic acid but also result in reduced insulin concentration [161]. 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 [167] (Figure 2).

Figure 2.

Pathophysiology of type 2 diabetes mellitus.

5. Conclusion

Epigenetic regulation of β cells supported by the growing number of high-impact studies on human patients have established the fact that microRNAs play a key role in defining β cell identity. Pathological dysregulation of β cells is in part caused by aberrant microRNA expression helping in the progression of T2DM. As the processes of microRNA biogenesis are characterized hinting towards the possibility of microRNAs as attractive therapeutic targets. Diabetes and β cell-specific microRNAs also represent distinct biomarkers for the early detection of diabetes. Because of the biochemical stability of microRNAs in the extracellular fluids like blood and plasma/serum, microRNAs also represent an excellent biomarker for diagnostics. Comprehensive studies involving large cohorts of diabetic patients to assess the predictive values of serum levels of different microRNAs in conjunction with the specific diabetic stage are required to delineate the pathological and beneficial role of specific microRNAs in diabetes mellitus.

Conflict of interest

“The authors declare no conflict of interest.”

Abbreviation

T2DM	Type 2 diabetes mellitus
Micro-RNA	Micro- Ribo-Nucleic-Acid
ROS	Reactive Oxygen Species
AGE	Advanced Glycation End product
RAGE	Receptor of Advanced Glycation End Product
BMI	Body Mass Index
IRS	Insulin Receptor Substrate
GLUT	Glucose Transporter
GSIS	Glucose Stimulated Insulin Release
GCK	Glucokinase
FOXO	Forkhead Family Box O
GLP-1	Glucagon-like Peptide 1
GIP	Gastric Inhibitory Polypeptide
PDX1	Pancreatic Duodenal Homeobox 1
NAD	Nicotinamide Adenine Dinucleotide
VLDL	Very Low Density Lipoprotein