Ribosomal biogenesis regulator DIMT1 controls ß-cell protein synthesis, mitochondrial function, and insulin secretion.

We previously reported that loss of mitochondrial transcription factor B1 (TFB1M) leads to mitochondrial dysfunction and is involved in the pathogenesis of type 2 diabetes (T2D). Whether defects in ribosomal processing impact mitochondrial function and could play a pathogenetic role in ß-cells and T2D is not known. To this end, we explored expression and the functional role of dimethyladenosine transferase 1 homolog (DIMT1), a homolog of TFB1M and a ribosomal RNA (rRNA) methyltransferase implicated in the control of rRNA. Expression of DIMT1 was increased in human islets from T2D donors and correlated positively with expression of insulin mRNA, but negatively with insulin secretion. We show that silencing of DIMT1 in insulin-secreting cells impacted mitochondrial function, leading to lower expression of mitochondrial OXPHOS proteins, reduced oxygen consumption rate, dissipated mitochondrial membrane potential, and a slower rate of ATP production. In addition, the rate of protein synthesis was retarded upon DIMT1 deficiency. Consequently, we found that DIMT1 deficiency led to perturbed insulin secretion in rodent cell lines and islets, as well as in a human ß-cell line. We observed defects in rRNA processing and reduced interactions between NIN1 (RPN12) binding protein 1 homolog (NOB-1) and pescadillo ribosomal biogenesis factor 1 (PES-1), critical ribosomal subunit RNA proteins, the dysfunction of which may play a part in disturbing protein synthesis in ß-cells. In conclusion, DIMT1 deficiency perturbs protein synthesis, resulting in mitochondrial dysfunction and disrupted insulin secretion, both potential pathogenetic processes in T2D.

Interleukin-4 reduces insulin secretion in human islets from healthy but not type-2 diabetic donors.

Type 2 diabetes (T2D) is associated with low-grade inflammation. Here we investigate if the anti-inflammatory cytokine interleukin-4 (IL-4) affects glucose-stimulated insulin secretion (GSIS) in human islets from non-diabetic (ND) and type-2 diabetic (T2D) donors. We first confirmed that GSIS is reduced in islets from T2D donors. Treatment with IL-4 for 48 h had no further effect on GSIS in these islets but significantly reduced secretion in ND islets. Acute treatment with IL-4 for 1 h had no effect on GSIS in ND islets which led us to suspect that IL-4 affects a slow cellular mechanism such as gene transcription. IL-4 has been reported to regulate miR-378a-3p and, indeed, we found that this microRNA was increased with IL-4 treatment. However, overexpression of miR-378a-3p in the human beta cell line EndoC-ßH1 did not affect GSIS. MiR-378a-3p is transcribed from the same gene as peroxisome proliferator-activated receptor gamma co-activator 1 beta (PCG-1ß) and we found that IL-4 treatment showed a clear tendency to increased gene expression of PCG-1ß. PCG-1ß is a co-activator of peroxisome proliferator-activated receptor gamma (PPARγ) and, the gene expression of PPARγ was also increased with IL-4 treatment. Our data suggests that the protective role of IL-4 on beta cell survival comes at the cost of lowered insulin secretion, presumably involving the PPARγ-pathway.

IGFBP7 is upregulated in islets from T2D donors and reduces insulin secretion.

Intra-islet crosstalk has become a focus area to fully understand the regulation of insulin secretion and impaired ß-cell function in type 2 diabetes (T2D). Here, we put forward evidence for insulin-like growth factor binding protein 7 (IGFBP7) as a potential protein involved in autocrine and paracrine ß-cell regulation. We showed presence of IGFBP7 in granules of both human α- and ß-cells and measured elevated gene expression as well as IGFBP7 protein in T2D. Insulin secretion was reduced in human islets, and the human ß-cell line EndoC-ßH1, after 72-h incubation with IGFBP7. Mechanistically reduced insulin secretion by IGFBP7 is attributed to reduced p21-activated kinase 1 (PAK1) protein, and decreased oxygen consumption and ATP-production. Knockdown of IGFBP7 in EndoC-ßH1 cells verified reduced IGFBP7 levels in the medium, as well as improved insulin secretion. Finally, IGFBP7 knockdown in islets from T2D donors improved insulin secretion, making IGFBP7 a potential drug target in diabetes.

MicroRNAs in Type 2 Diabetes: Focus on MicroRNA Profiling in Islets of Langerhans.

Differential expression of microRNAs (miRNAs) is observed in many diseases including type 2 diabetes (T2D). Insulin secretion from pancreatic beta cells is central for the regulation of blood glucose levels and failure to release enough insulin results in hyperglycemia and T2D. The importance in T2D pathogenesis of single miRNAs in beta cells has been described; however, to get the full picture, high-throughput miRNA sequencing is necessary. Here we describe a method using small RNA sequencing, from sample preparation to expression analysis using bioinformatic tools. In the end, a tutorial on differential expression analysis is presented in R using publicly available data.

miRNAs in the Beta Cell-Friends or Foes?

Type 2 diabetes (T2D) develops due to insulin resistance and an inability of the pancreatic ß-cells to increase secretion of insulin and reduce elevated blood glucose levels. Diminished ß-cell function and mass have been implicated in impaired ß-cell secretory capacity and several microRNAs (miRNAs) have been reported to be involved in regulating ß-cell processes. We believe miRNAs are nodes in important miRNA-mRNA networks regulating ß-cell function and that miRNAs therefore can be targets for the treatment of T2D. MicroRNAs are short (≈19-23 nucleotides [nt]) endogenous noncoding RNAs which regulate gene expression by directly binding to the mRNA of their target genes. Under normal circumstances, miRNAs act as rheostats to keep expression of their gene targets at optimal levels for different ß-cell outputs. In T2D, levels of some miRNAs are altered as part of the compensatory mechanism to improve insulin secretion. Other miRNAs are differentially expressed as part of the process of T2D pathogenesis, which results in reduced insulin secretion and increased blood glucose. In this review, we present recent findings concerning miRNAs in islets and in insulin-secreting cells, and their differential expression in diabetes, with a specific focus on miRNAs involved in ß-cell apoptosis/proliferation and glucose-stimulated insulin secretion. We present thoughts around miRNA-mRNA networks and miRNAs as both therapeutic targets to improve insulin secretion and as circulating biomarkers of diabetes. Overall, we hope to convince you that miRNAs in ß-cells are essential for regulating ß-cell function and can in the future be of clinical use in the treatment and/or prevention of diabetes.

Glucocorticoid-mediated induction of ZBTB16 affects insulin secretion in human islets and EndoC-ßH1 ß-cells.

Glucocorticoid use is associated with steroid-induced diabetes mellitus and impaired pancreatic ß-cell insulin secretion. Here, the glucocorticoid-mediated transcriptomic changes in human pancreatic islets and the human insulin-secreting EndoC-ßH1 cells were investigated to uncover genes involved in ß-cell steroid stress-response processes. Bioinformatics analysis revealed glucocorticoids to exert their effects mainly on enhancer genomic regions in collaboration with auxiliary transcription factor families including AP-1, ETS/TEAD, and FOX. Remarkably, we identified the transcription factor ZBTB16 as a highly confident direct glucocorticoid target. Glucocorticoid-mediated induction of ZBTB16 was time- and dose-dependent. Manipulation of ZBTB16 expression in EndoC-ßH1 cells combined with dexamethasone treatment demonstrated its protective role against glucocorticoid-induced reduction of insulin secretion and mitochondrial function impairment. In conclusion, we determine the molecular impact of glucocorticoids on human islets and insulin-secreting cells and investigate the effects of glucocorticoid targets on ß-cell function. Our findings can pave the way for therapies against steroid-induced diabetes mellitus.

Genes with epigenetic alterations in human pancreatic islets impact mitochondrial function, insulin secretion, and type 2 diabetes.

Epigenetic dysregulation may influence disease progression. Here we explore whether epigenetic alterations in human pancreatic islets impact insulin secretion and type 2 diabetes (T2D). In islets, 5,584 DNA methylation sites exhibit alterations in T2D cases versus controls and are associated with HbA1c in individuals not diagnosed with T2D. T2D-associated methylation changes are found in enhancers and regions bound by ß-cell-specific transcription factors and associated with reduced expression of e.g. CABLES1, FOXP1, GABRA2, GLR1A, RHOT1, and TBC1D4. We find RHOT1 (MIRO1) to be a key regulator of insulin secretion in human islets. Rhot1-deficiency in ß-cells leads to reduced insulin secretion, ATP/ADP ratio, mitochondrial mass, Ca2+, and respiration. Regulators of mitochondrial dynamics and metabolites, including L-proline, glycine, GABA, and carnitines, are altered in Rhot1-deficient ß-cells. Islets from diabetic GK rats present Rhot1-deficiency. Finally, RHOT1methylation in blood is associated with future T2D. Together, individuals with T2D exhibit epigenetic alterations linked to mitochondrial dysfunction in pancreatic islets.

Type 2 diabetes candidate genes, including PAX5, cause impaired insulin secretion in human pancreatic islets.

Type 2 diabetes (T2D) is caused by insufficient insulin secretion from pancreatic ß cells. To identify candidate genes contributing to T2D pathophysiology, we studied human pancreatic islets from approximately 300 individuals. We found 395 differentially expressed genes (DEGs) in islets from individuals with T2D, including, to our knowledge, novel (OPRD1, PAX5, TET1) and previously identified (CHL1, GLRA1, IAPP) candidates. A third of the identified expression changes in islets may predispose to diabetes, as expression of these genes associated with HbA1c in individuals not previously diagnosed with T2D. Most DEGs were expressed in human ß cells, based on single-cell RNA-Seq data. Additionally, DEGs displayed alterations in open chromatin and associated with T2D SNPs. Mouse KO strains demonstrated that the identified T2D-associated candidate genes regulate glucose homeostasis and body composition in vivo. Functional validation showed that mimicking T2D-associated changes for OPRD1, PAX5, and SLC2A2 impaired insulin secretion. Impairments in Pax5-overexpressing ß cells were due to severe mitochondrial dysfunction. Finally, we discovered PAX5 as a potential transcriptional regulator of many T2D-associated DEGs in human islets. Overall, we have identified molecular alterations in human pancreatic islets that contribute to ß cell dysfunction in T2D pathophysiology.

Glucocorticoids and glucolipotoxicity alter the DNA methylome and function of human EndoC-ßH1 cells.

AIMS: Synthetic glucocorticoids, including dexamethasone (DEX), are clinically prescribed due to their immunoregulatory properties. In excess they can perturb glucose homeostasis, with individuals predisposed to glucose intolerance more sensitive to these negative effects. While DEX is known to negatively impact ß-cell function, it is unclear how. Hence, our aim was to investigate the effect of DEX on ß-cell function, both alone and in combination with a diabetogenic milieu in the form of elevated glucose and palmitate. MAIN METHODS: Human pancreatic EndoC-ßH1 cells were cultured in the presence of high glucose and palmitate (glucolipotoxicity) and/or a pharmacological concentration of DEX, before functional and molecular analyses. KEY FINDINGS: Either treatment alone resulted in reduced insulin content and secretion, while the combination of DEX and glucolipotoxicity promoted a strong synergistic effect. These effects were associated with reduced insulin biosynthesis, likely due to downregulation of PDX1, MAFA, and the proinsulin converting enzymes, as well as reduced ATP response upon glucose stimulation. Genome-wide DNA methylation analysis found changes on PDE4D, MBNL1 and TMEM178B, all implicated in ß-cell function, after all three treatments. DEX alone caused very strong demethylation of the glucocorticoid-regulated gene ZBTB16, also known to influence the ß-cell, while the combined treatment caused altered methylation of many known ß-cell regulators and diabetes candidate genes. SIGNIFICANCE: DEX treatment and glucolipotoxic conditions separately alter the ß-cell epigenome and function. The combination of both treatments exacerbates these changes, showing that caution is needed when prescribing potent glucocorticoids in patients with dysregulated metabolism.

The Calcium Channel Subunit Gamma-4 as a Novel Regulator of MafA in Pancreatic Beta-Cell Controls Glucose Homeostasis.

Impaired fasting glucose (IFG) and impaired glucose tolerance (IGT) are high-risk factors of diabetes development and may be caused by defective insulin secretion in pancreatic beta-cells. Glucose-stimulated insulin secretion is mediated by voltage-gated Ca2+ (CaV) channels in which the gamma-4 subunit (CaVγ4) is required for the beta-cell to maintain its differentiated state. We here aim to explore the involvement of CaVγ4 in controlling glucose homeostasis by employing the CaVγ4-/- mice to study in vivo glucose-metabolism-related phenotypes and glucose-stimulated insulin secretion, and to investigate the underlying mechanisms. We show that CaVγ4-/- mice exhibit perturbed glucose homeostasis, including IFG and IGT. Glucose-stimulated insulin secretion is blunted in CaVγ4-/- mouse islets. Remarkably, CaVγ4 deletion results in reduced expression of the transcription factor essential for beta-cell maturation, MafA, on both mRNA and protein levels in islets from human donors and CaVγ4-/- mice, as well as in INS-1 832/13 cells. Moreover, we prove that CaMKII is responsible for mediating this regulatory pathway linked between CaVγ4 and MafA, which is further confirmed by human islet RNA-seq data. We demonstrate that CaVγ4 is a key player in preserving normal blood glucose homeostasis, which sheds light on CaVγ4 as a novel target for the treatment of prediabetes through correcting the impaired metabolic status.

Human pancreatic islet miRNA-mRNA networks of altered miRNAs due to glycemic status.

MicroRNAs (miRNAs) are short non-coding RNAs that regulate gene expression via mRNA targeting, playing important roles in the pancreatic islets. We aimed to identify molecular pathways and genomic regulatory regions associated with altered miRNA expression due to glycemic status, which could contribute to the development of type 2 diabetes (T2D). To this end, miRNAs were identified by a combination of differential miRNA expression and correlation analysis in human islet samples from donors with normal and elevated blood glucose levels. Analysis and clustering of highly correlated, experimentally validated gene targets of these miRNAs revealed two islet-specific clusters, which were associated with key aspects of islet functions and included a high number of T2D-related genes. Finally, cis-eQTLs and public GWAS data integration uncovered suggestive genomic signals of association with insulin secretion and T2D. The miRNA-driven network-based approach presented in this study contributes to a better understanding of impaired insulin secretion in T2D pathogenesis.

The T-type calcium channel CaV3.2 regulates insulin secretion in the pancreatic ß-cell.

Voltage-gated Ca2+ (CaV) channel dysfunction leads to impaired glucose-stimulated insulin secretion in pancreatic ß-cells and contributes to the development of type-2 diabetes (T2D). The role of the low-voltage gated T-type CaV channels in ß-cells remains obscure. Here we have measured the global expression of T-type CaV3.2 channels in human islets and found that gene expression of CACNA1H, encoding CaV3.2, is negatively correlated with HbA1c in human donors, and positively correlated with islet insulin gene expression as well as secretion capacity in isolated human islets. Silencing or pharmacological blockade of CaV3.2 attenuates glucose-stimulated cytosolic Ca2+ signaling, membrane potential, and insulin release. Moreover, the endoplasmic reticulum (ER) Ca2+ store depletion is also impaired in CaV3.2-silenced ß-cells. The linkage between T-type (CaV3.2) and L-type CaV channels is further identified by the finding that the intracellular Ca2+ signaling conducted by CaV3.2 is highly dependent on the activation of L-type CaV channels. In addition, CACNA1H expression is significantly associated with the islet predominant L-type CACNA1C (CaV1.2) and CACNA1D (CaV1.3) genes in human pancreatic islets. In conclusion, our data suggest the essential functions of the T-type CaV3.2 subunit as a mediator of ß-cell Ca2+ signaling and membrane potential needed for insulin secretion, and in connection with L-type CaV channels.

The highly expressed calcium-insensitive synaptotagmin-11 and synaptotagmin-13 modulate insulin secretion.

AIM: SYT11 and SYT13, two calcium-insensitive synaptotagmins, are downregulated in islets from type 2 diabetic donors, but their function in insulin secretion is unknown. To address this, we investigated the physiological role of these two synaptotagmins in insulin-secreting cells. METHODS: Correlations between gene expression levels were performed using previously described RNA-seq data on islets from 188 human donors. SiRNA knockdown was performed in EndoC-ßH1 and INS-1 832/13 cells. Insulin secretion was measured with ELISA. Patch-clamp was used for single-cell electrophysiology. Confocal microscopy was used to determine intracellular localization. RESULTS: Human islet expression of the transcription factor PDX1 was positively correlated with SYT11 (p = 2.4e-10 ) and SYT13 (p < 2.2e-16 ). Syt11 and Syt13 both co-localized with insulin, indicating their localization in insulin granules. Downregulation of Syt11 in INS-1 832/13 cells (siSYT11) resulted in increased basal and glucose-induced insulin secretion. Downregulation of Syt13 (siSYT13) decreased insulin secretion induced by glucose and K+ . Interestingly, the cAMP-raising agent forskolin was unable to enhance insulin secretion in siSYT13 cells. There was no difference in insulin content, exocytosis, or voltage-gated Ca2+ currents in the two models. Double knockdown of Syt11 and Syt13 (DKD) resembled the results in siSYT13 cells. CONCLUSION: SYT11 and SYT13 have similar localization and transcriptional regulation, but they regulate insulin secretion differentially. While downregulation of SYT11 might be a compensatory mechanism in type-2 diabetes, downregulation of SYT13 reduces the insulin secretory response and overrules the compensatory regulation of SYT11 in a way that could aggravate the disease.

Human Islet MicroRNA-200c Is Elevated in Type 2 Diabetes and Targets the Transcription Factor ETV5 to Reduce Insulin Secretion.

MicroRNAs (miRNAs) are part of deregulated insulin secretion in type 2 diabetes (T2D) development. Rodent models have suggested miR-200c to be involved, but the role and potential as therapeutic target of this miRNA in human islets are not clear. Here we report increased expression of miR-200c in islets from T2D as compared with nondiabetic (ND) donors and display results showing reduced glucose-stimulated insulin secretion in EndoC-ßH1 cells overexpressing miR-200c. We identify transcription factor ETV5 as the top rank target of miR-200c in human islets using TargetScan in combination with Pearson correlation analysis of miR-200c and mRNA expression data from the same human donors. Among other targets were JAZF1, as earlier shown in miR-200 knockout mice. Accordingly, linear model analysis of ETV5 and JAZF1 gene expression showed reduced expression of both genes in islets from human T2D donors. Western blot analysis confirmed the reduced expression of ETV5 on the protein level in EndoC-ßH1 cells overexpressing miR-200c, and luciferase assay validated ETV5 as a direct target of miR-200c. Finally, LNA knockdown of miR-200c increased glucose-stimulated insulin secretion in islets from T2D donors approximately threefold. Our data reveal a vital role of the miR-200c-ETV5 axis in ß-cell dysfunction and pathophysiology of T2D.

A critical role of the mechanosensor PIEZO1 in glucose-induced insulin secretion in pancreatic ß-cells.

Glucose-induced insulin secretion depends on ß-cell electrical activity. Inhibition of ATP-regulated potassium (KATP) channels is a key event in this process. However, KATP channel closure alone is not sufficient to induce ß-cell electrical activity; activation of a depolarizing membrane current is also required. Here we examine the role of the mechanosensor ion channel PIEZO1 in this process. Yoda1, a specific PIEZO1 agonist, activates a small membrane current and thereby triggers ß-cell electrical activity with resultant stimulation of Ca2+-influx and insulin secretion. Conversely, the PIEZO1 antagonist GsMTx4 reduces glucose-induced Ca2+-signaling, electrical activity and insulin secretion. Yet, PIEZO1 expression is elevated in islets from human donors with type-2 diabetes (T2D) and a rodent T2D model (db/db mouse), in which insulin secretion is reduced. This paradox is resolved by our finding that PIEZO1 translocates from the plasmalemma into the nucleus (where it cannot influence the membrane potential of the ß-cell) under experimental conditions emulating T2D (high glucose culture). ß-cell-specific Piezo1-knockout mice show impaired glucose tolerance in vivo and reduced glucose-induced insulin secretion, ß-cell electrical activity and Ca2+ elevation in vitro. These results implicate mechanotransduction and activation of PIEZO1, via intracellular accumulation of glucose metabolites, as an important physiological regulator of insulin secretion.

Glucocorticoid induces human beta cell dysfunction by involving riborepressor GAS5 LincRNA.

OBJECTIVE: A widely recognized metabolic side effect of glucocorticoid (GC) therapy is steroid-induced diabetes mellitus (DM). However, studies on the molecular basis of GC-induced pancreatic beta cell dysfunction in human beta cells are lacking. The significance of non-coding RNAs in various cellular processes is emerging. In this study, we aimed to show the direct negative impact of GC on beta cell function and elucidate the role of riborepressor GAS5 lincRNA in the GC signaling pathway in human pancreatic beta cells. METHODS: Patients undergoing two weeks of high-dose prednisolone therapy were monitored for C-peptide levels. Human pancreatic islets and the human beta cell line EndoC-ßH1 were incubated in pharmacological concentrations of dexamethasone. The GAS5 level was modulated using anti-sense LNA gapmeR or short oligonucleotides with GAS5 HREM (hormone response element motif). Immunoblotting and/or real-time PCR were used to assess changes in protein and RNA expression, respectively. Functional characterization included glucose-stimulated insulin secretion and apoptosis assays. Correlation analysis was performed on RNAseq data of human pancreatic islets. RESULTS: We found reduced C-peptide levels in patients undergoing high-dose GC therapy. Human islets and the human beta cell line EndoC-ßH1 exposed to GC exhibited reduced insulin secretion and increased apoptosis. Concomitantly, reduced expression of important beta cell transcription factors, PDX1 and NKX6-1, as well as exocytotic protein SYT13 were observed. The expression of the glucocorticoid receptor was decreased, while that of serum and glucocorticoid-regulated kinase 1 (SGK1) was elevated. The expression of these genes was found to significantly correlate with GAS5 in human islet transcriptomics data. Increasing GAS5 levels using GAS5 HREM alleviated the inhibitory effects of dexamethasone on insulin secretion. CONCLUSIONS: The direct adverse effect of glucocorticoid in human beta cell function is mediated via important beta cell proteins and components of the GC signaling pathway in an intricate interplay with GAS5 lincRNA, a potentially novel therapeutic target to counter GC-mediated beta cell dysfunction.