RyR2/IRBIT regulates insulin gene transcription, insulin content, and secretion in the insulinoma cell line INS-1

The role of ER Ca2+ release via ryanodine receptors (RyR) in pancreatic β-cell function is not well defined. Deletion of RyR2 from the rat insulinoma INS-1 (RyR2KO) enhanced the Ca2+ integral (AUC) stimulated by 7.5 mM glucose, and rendered it sensitive to block by the IP3 receptor inhibitor xestospongin C, coincident with reduced levels of the protein IP3 Receptor Binding protein released with Inositol 1,4,5 Trisphosphate (IRBIT; aka AHCYL1). Deletion of IRBIT from INS-1 cells (IRBITKO) increased the Ca2+ AUC in response to 7.5 mM glucose and induced xestospongin sensitivity. Insulin content and basal (2.5 mM glucose) and 7.5 mM glucose-stimulated insulin secretion were reduced in RyR2KO cells and more modestly reduced in IRBITKO cells compared to controls. INS2 mRNA levels were reduced in both RyR2KO and IRBITKO cells, but INS1 mRNA levels were specifically decreased in RyR2KO cells. Nuclear localization of S-adenosylhomocysteinase (AHCY) was increased in RyR2KO and IRBITKO cells. DNA methylation of the INS1 and INS2 gene promotor regions was very low, and not different among RyR2KO, IRBITKO, and controls. In contrast, exon 2 of the INS1 and INS2 genes was more extensively methylated in RyR2KO and IRBITKO cells than in controls. Proteomics analysis using LC-MS/MS revealed that deletion of RyR2 or IRBIT resulted in differential regulation of 314 and 137 proteins, respectively, with 41 in common. These results suggest that RyR2 regulates IRBIT levels and activity in INS-1 cells, and together maintain insulin content and secretion, and regulate the proteome, perhaps via DNA methylation.

Fetuin-A secretion from β-cell accumulates macrophages in islets, aggravates inflammation and impairs insulin secretion

Elevated fetuin-A levels, chemokines and islet resident macrophages are crucial factors associated with obesity mediated Type 2 Diabetes (T2D). Here, the aim of the study was to investigate the effect of MIN6 (mouse insulinoma cell line) derived fetuin-A in macrophage polarization and decipher the effect of M1 type pro-inflammatory macrophages in commanding over insulin secretion. MIN6 and islet derived fetuin-A induced expression of M1 type macrophage markers, Emr1, Cd68 and CD11c (∼1.8 fold) along with increased cytokine secretion. Interestingly, suppression of fetuin-A in MIN6 successfully reduced M1 markers by ∼1.5 fold. MIN6 derived fetuin-A also induced chemotaxis of macrophages in Boyden chamber chemotaxis assay. Further, high fat feeding in mice showed elevated cytokine and fetuin-A content in serum and islets, and also migration and polarization of macrophages to the islets while β-cells failed to cope up with increased insulin demand. Moreover, in MIN6 culture, M1 macrophages sharply decreased insulin secretion by ∼2.8 fold. Altogether our results support an association of fetuin-A with islet inflammation and β-cell dysfunction, owing to its role as a key chemoattractant and macrophage polarizing factor.

Puerarin Inhibits the PERK-eIF2α-ATF4-CHOP Pathway through Inactivating JAK2/STAT3 Signal in Pancreatic beta-Cells

Type 1 diabetes (T1D) is an autoimmune and inflammatory disease with excessive loss of pancreatic islet [Formula: see text]-cells. Accumulating evidence indicated that endoplasmic reticulum (ER) stress played a critical role in [Formula: see text]-cells loss, leading to T1D. Therefore, promoting the survival of pancreatic [Formula: see text]cells would be beneficial for patients with T1D. Puerarin is a natural isoflavone that has been demonstrated to be able to decrease blood glucose in patients with T1D. However, it remains unknown whether puerarin improves ER stress to prevent [Formula: see text]-cells from apoptosis. Here, we sought to investigate the role of puerarin in ER stress-associated apoptosis and explore its underlying mechanism in the mouse insulinoma cell line (MIN6). Flow cytometry and cell counting kit-8 (CCK8) experiments showed that puerarin caused a significant increase in the viability of MIN6 cells injured by H2O2. Furthermore, the protein kinase R-like ER kinase (PERK) signal pathway, a critical branch of ER stress response, was found to be involved in this process. Puerarin inhibited the phosphorylation of PERK, subsequently suppressed the phosphorylation of eukaryotic initiation factor 2[Formula: see text] (eIF2[Formula: see text], then decreased the activating transcription factor 4 (ATF4) and C/EBP homologous protein (CHOP) expression, ultimately attenuating ER stress to prevent MIN6 cells from apoptosis. In addition, puerarin inhibited the activation of Janus kinase 2 (JAK2)/signal transducer and activators of transcription 3 (STAT3), which suppressed the PERK signal cascade with decreased ATF4 and CHOP levels. Taken together, our results firstly demonstrated that puerarin could prevent MIN6 cells from apoptosis at least in part by inhibiting the PERK-eIF2[Formula: see text]-ATF4-CHOP axis under ER stress conditions, which might be mediated by inactivation of the JAK2/STAT3 signal pathway. Therefore, investigating the mechanism underlying the effects of puerarin might highlight the potential roles of puerarin developing into an antidiabetic drug.

Potential Therapeutic Applications of Synthetic Conotoxin s-cal14.2b, Derived from Californiconus californicus, for Treating Type 2 Diabetes

The FDA’s approval of peptide drugs such as Ziconotide or Exendin for pain relief and diabetes treatment, respectively, enhanced the interest to explore novel conotoxins from Conus species venom. In general, conotoxins can be used in pathologies where voltage-gated channels, membrane receptors, or ligands alter normal physiological functions, as in metabolic diseases such as Type 2 diabetes. In this study, the synthetic cal14.2b (s-cal14.2b) from the unusual Californiconus californicus demonstrated bioactivity on NIT-1 insulinoma cell lines stimulating insulin secretion detecting by high performance liquid chromatography (HPLC). Accordingly, s-cal14.2b increased the CaV1.2/1.3 channel-current by 35 ± 4% with a recovery τ of 10.3 ± 4 s in primary cell culture of rat pancreatic β-cells. The in vivo results indicated a similar effect of insulin secretion on mice in the glucose tolerance curve model by reducing the glucose from 500 mg/dL to 106 mg/dL in 60 min, compared to the negative control of 325 mg/dL at the same time. The PET-SCAN with radiolabeling 99mTc-s-cal14.2b demonstrated biodistribution and accumulation in rat pancreas with complete depuration in 24 h. These findings show the potential therapeutic use of s-cal14.2b in endocrinal pathologies such as early stages of Type 2 Diabetes where the pancreas’s capability to produce insulin is still effective.

Distinctive detection of insulinoma using [18F]FB(ePEG12)12-exendin-4 PET/CT

Specifying the exact localization of insulinoma remains challenging due to the lack of insulinoma-specific imaging methods. Recently, glucagon-like peptide-1 receptor (GLP-1R)-targeted imaging, especially positron emission tomography (PET), has emerged. Although various radiolabeled GLP-1R agonist exendin-4-based probes with chemical modifications for PET imaging have been investigated, an optimal candidate probe and its scanning protocol remain a necessity. Thus, we investigated the utility of a novel exendin-4-based probe conjugated with polyethylene glycol (PEG) for [18F]FB(ePEG12)12-exendin-4 PET imaging for insulinoma detection. We utilized [18F]FB(ePEG12)12-exendin-4 PET/CT to visualize mouse tumor models, which were generated using rat insulinoma cell xenografts. The probe demonstrated high uptake value on the tumor as 37.1 ± 0.4%ID/g, with rapid kidney clearance. Additionally, we used Pdx1-Cre;Trp53R172H;Rbf/f mice, which developed endogenous insulinoma and glucagonoma, since they enabled differential imaging evaluation of our probe in functional pancreatic neuroendocrine neoplasms. In this model, our [18F]FB(ePEG12)12-exendin-4 PET/CT yielded favorable sensitivity and specificity for insulinoma detection. Sensitivity: 30-min post-injection 66.7%, 60-min post-injection 83.3%, combined 100% and specificity: 30-min post-injection 100%, 60-min post-injection 100%, combined 100%, which was corroborated by the results of in vitro time-based analysis of internalized probe accumulation. Accordingly, [18F]FB(ePEG12)12-exendin-4 is a promising PET imaging probe for visualizing insulinoma.

Insulinoma-derived pseudo-islets for diabetes research

The islets of Langerhans of the pancreas are the primary endocrine organ responsible for regulating whole body glucose homeostasis. The use of isolated primary islets for research development and training requires organ resection, careful digestion and isolation of the islets from non-endocrine tissue. This process is time consuming, expensive and requires substantial expertise. For these reasons, we sought to develop a more rapidly obtainable and consistent model system with characteristic islet morphology and function that could be employed to train personnel and better inform experiments prior to using isolated rodent and human islets. Immortalized β cell lines reflect several aspects of primary β cells, but cell propagation in monolayer cell culture limits their usefulness in several areas of research which depend on islet morphology and/or functional assessment. In this manuscript we describe the propagation and characterization of insulinoma pseudo-islets (IPIs) from a rat insulinoma cell line INS832/3. IPIs were generated with an average diameter of 200 μm, consistent with general islet morphology. The rates of oxygen consumption and mitochondrial oxidation-reduction changes in response to glucose and metabolic modulators were similar to isolated rat islets. In addition, the dynamic insulin secretory patterns of IPIs were similar to primary rat islets. Thus, INS832/3-derived IPIs provide a valuable and convenient model for accelerating islet and diabetes research.

Isoprenoid Derivatives of Lysophosphatidylcholines Enhance Insulin and GLP-1 Secretion through Lipid-Binding GPCRs

Insulin plays a significant role in carbohydrate homeostasis as the blood glucose lowering hormone. Glucose-induced insulin secretion (GSIS) is augmented by glucagon-like peptide (GLP-1), a gastrointestinal peptide released in response to ingesting nutriments. The secretion of insulin and GLP-1 is mediated by the binding of nutrients to G protein-coupled receptors (GPCRs) expressed by pancreatic β-cells and enteroendocrine cells, respectively. Therefore, insulin secretagogues and incretin mimetics currently serve as antidiabetic treatments. This study demonstrates the potency of synthetic isoprenoid derivatives of lysophosphatidylcholines (LPCs) to stimulate GSIS and GLP-1 release. Murine insulinoma cell line (MIN6) and enteroendocrinal L cells (GLUTag) were incubated with LPCs bearing geranic acid (1-GA-LPC), citronellic acid (1-CA-LPC), 3,7-dimethyl-3-vinyloct-6-enoic acid (GERA-LPC), and (E)-3,7,11-trimethyl- 3-vinyldodeca-6,10-dienoic acid (1-FARA-LPC). Respective free terpene acids were also tested for comparison. Besides their insulin- and GLP-1-secreting capabilities, we also investigated the cytotoxicity of tested compounds, the ability to intracellular calcium ion mobilization, and targeted GPCRs involved in maintaining lipid and carbohydrate homeostasis. We observed the high cytotoxicity of 1-GERA-LPC and 1-FARA-LPC in contrast 1-CA-LPC and 1-GA-LPC. Moreover, 1-CA-LPC and 1-GA-LPC demonstrated the stimulatory effect on GSIS and 1-CA-LPC augmented GLP-1 secretion. Insulin and GLP-1 release appeared to be GPR40-, GPR55-, GPR119- and GPR120-dependent.