Cavβ3 regulates Ca2+-signalling and insulin expression in pancreatic β-cells in a cell-autonomous manner

<p>Voltage-gated Ca²⁺ (Cav) channels consist of a pore-forming Cavα1 subunit and auxiliary Cavα2-δ and Cavβ subunits. In fibroblasts, Cavβ3, independent of its role as a Cav subunit, reduces the sensitivity to low concentrations of inositol-1,4,5-trisphosphate (IP3). Similarly, Cavβ3 could affect cytosolic [Ca²⁺] in pancreatic β-cells. Here, we deleted the Cavβ3-encoding gene <i>Cacnb3</i> in insulin-secreting rat β-(Ins-1) cells using CRISPR/Cas9. These cells were used as controls to investigate the role of Cavβ3 on Ca²⁺-signalling, glucose-induced insulin secretion (GIIS), Cav-channel activity and gene expression in wild-type cells in which Cavβ3 and the IP3-receptor were co-immunoprecipitated. Transcript and protein profiling revealed significantly increased levels of insulin transcription factor Mafa, CaMKIV, neuroendocrine convertase1 (Pcsk1) and nitric oxide synthase-1 (NOS-1) in Cavβ3-KO cells. In the absence of Cavβ3, Cav-currents were not altered. In contrast, CREB activity, the amount of MAFA protein and GIIS, the extent of IP3-dependent Ca²⁺ release and the frequency of Ca²⁺-oscillations were increased. These processes were decreased by the Cavβ3 protein in a concentration-dependent manner. Our study shows that Cavβ3 interacts with the IP3-receptor in isolated β-cells, controls IP3-dependent Ca²⁺-signalling independently of Cav channel functions, and thereby regulates insulin expression and its glucose-dependent release in a cell-autonomous manner.</p>

Cavβ3 regulates Ca2+-signalling and insulin expression in pancreatic β-cells in a cell-autonomous manner

<p>Voltage-gated Ca²⁺ (Cav) channels consist of a pore-forming Cavα1 subunit and auxiliary Cavα2-δ and Cavβ subunits. In fibroblasts, Cavβ3, independent of its role as a Cav subunit, reduces the sensitivity to low concentrations of inositol-1,4,5-trisphosphate (IP3). Similarly, Cavβ3 could affect cytosolic [Ca²⁺] in pancreatic β-cells. Here, we deleted the Cavβ3-encoding gene <i>Cacnb3</i> in insulin-secreting rat β-(Ins-1) cells using CRISPR/Cas9. These cells were used as controls to investigate the role of Cavβ3 on Ca²⁺-signalling, glucose-induced insulin secretion (GIIS), Cav-channel activity and gene expression in wild-type cells in which Cavβ3 and the IP3-receptor were co-immunoprecipitated. Transcript and protein profiling revealed significantly increased levels of insulin transcription factor Mafa, CaMKIV, neuroendocrine convertase1 (Pcsk1) and nitric oxide synthase-1 (NOS-1) in Cavβ3-KO cells. In the absence of Cavβ3, Cav-currents were not altered. In contrast, CREB activity, the amount of MAFA protein and GIIS, the extent of IP3-dependent Ca²⁺ release and the frequency of Ca²⁺-oscillations were increased. These processes were decreased by the Cavβ3 protein in a concentration-dependent manner. Our study shows that Cavβ3 interacts with the IP3-receptor in isolated β-cells, controls IP3-dependent Ca²⁺-signalling independently of Cav channel functions, and thereby regulates insulin expression and its glucose-dependent release in a cell-autonomous manner.</p>

The effect of functional rice analogue diet from mocaf, corn, pigeon pea and seaweed on rats model of type 2 diabetes

Rice analogue (RA) consisting of mocaf, corn, pigeon pea and seaweed from East Lombok, Indonesia was formulated into a low glycaemic index (GI) alternative food. The effect of this RA to control diabetes mellitus (DM) on blood glucose, insulin serum and histology of the pancreas was assayed by in vivo experiment using male and female rats aged 10 weeks that had been induced by streptozotocin (STZ) 90 mg/kg body weight 2 days after birth. The treatment was given for 14 days and compared with Broiler-1 (BR-1) feed. The GI test showed that RA has a low GI (47.36), while BR-1 has a moderate GI (66.35). The blood glucose of the RA group was significantly decreased (p<0.05), while insulin serum was increased in comparison to this of BR-1. Histological observation showed an improvement in the pancreas of diabetic rats after treatment with RA in comparison to this of BR-1. The immunohistochemistry (IHC) assay showed an increase in insulin expression in RA-treated diabetic rats. This study concluded that RA has a low GI. The products were able to increase serum insulin, improve morphological Langerhans and insulin expression on rats model of type 2 DM.

Antidiabetic Effect of Katakakhadiradi kashayam by Improving the Insulin Expression and Glucose Metabolising Enzyme

Many plants provide a rich source of bioactive chemicals, which are free from undesirable side effects and possess powerful pharmacological actions. The present study was carried out to find the antidiabetic effect of Katakakhadiradi kashayam (KKK) by improving the insulin expression and regulating properly the glucose metabolising enzymes. The diabetes was induced in combination with streptozotocin and nicotinamide injection to Wistar rats. Diabetic rats were treated with Katakakhadiradi kashayam orally at doses of 100, 200 and 300 mg/kg/bw for 28 days, and the obtained results of parameters were compared with glibenclamide. The antidiabetic effect of Kashayam was measured by the expression of insulin by immunohistochemistry and restoring the normal clinical values of glucose metabolizing enzymes. The present study specified that hyperglycemia leads to pathological conditions in pancreatic tissue with decreased expression of insulin in β-cells whereas the Katakakhadiradi kashayam normalised the production of insulin. The study found that the antihyperglycemic activity of Katakakhadiradi kashayam L. is mainly due to their ability to restore the function of pancreatic tissues by causing an increase in insulin output and maintaining the glucose metabolising enzymes.

Integrin αvβ5 heterodimer is a specific marker of human pancreatic beta cells

The identification of cell surface markers specific to pancreatic beta cells is important for both the study of islet biology and for investigating the pathophysiology of diseases in which this cell type is lost or damaged. Following analysis of publicly available RNAseq data, we identified specific integrin subunits, integrin αv and integrin β5, that were expressed in beta cells. This finding was further elaborated using immunofluorescence analysis of histological sections derived from donor human pancreas. Despite the broad expression of specific integrin subunits, we found that expression of integrin αvβ5 heterodimers was restricted to beta cells and that this complex persisted in islet remnants of some type 1 diabetic individuals from which insulin expression had been lost. This study identifies αvβ5 heterodimers as a novel cell surface marker of human pancreatic beta cells, a finding that will aid in the identification and characterisation of this important cell type.

Increased frequency of β cells with abnormal NKX6.1 expression in type 2 diabetes but not in subjects with higher risk for type 2 diabetes

Background NKX6.1 is a transcription factor for insulin, as well as a marker for β cell maturity. Abnormal NKX6.1 expression in β cells, such as translocation from the nucleus to cytoplasm or lost expression, has been shown as a marker for β cell dedifferentiation. Methods We obtained pancreatic sections from organ donors and immunofluorescence staining with NKX6.1 and insulin was performed to characterize NKX6.1 expression in subjects with or without type 2 diabetes mellitus (T2DM). Results Our results showed that cells with insulin expression but no nucleic NKX6.1 expression (NKX6.1Nuc-Ins+), and cells with cytoplasmic NKX6.1 expression but no insulin expression (NKX6.1cytIns−) were significantly increased in T2DM subjects and positively correlated with glycated hemoglobin (HbA1c), indicating the elevated β cell dedifferentiation with NKX6.1 inactivation in T2DM. To investigate whether β cell dedifferentiation has initiated in subjects with higher risks for T2DM, we next analyzed the association between β-cell dedifferentiation level in ND subjects with different ages, body mass index, and HbA1c. The results showed the absolute number and percentage of dedifferentiated β cells with NKX6.1 inactivation did not significantly change in subjects with advanced aging, obesity, or modest hyperglycemia, indicating that the β cell dedifferentiation might mainly occur after T2DM was diagnosed. Conclusion Our results suggested that NKX6.1 expression in β cells was changed in type 2 diabetic subjects, evidenced by significantly increased NKX6.1Nuc-Ins+ and NKX6.1cytIns− cells. This abnormality did not occur more frequently in subjects with a higher risk for T2DM, suggesting that β cell dedifferentiation might be secondary to the pathological changes in T2DM.

Clinical and Functional Characteristics of a Novel KLF11 Cys354Phe Variant Involved in Maturity-Onset Diabetes of the Young

Background. Mutations in human KLF11 may lead to the development of maturity-onset diabetes of the young 7 (MODY7). This occurs due to impaired insulin synthesis in the pancreas. To date, the clinical and functional characteristics of the novel KLF11 mutation c.1061G > T have not yet been reported. Methods. Whole-exon sequencing was used to screen the proband and family members with clinical suspicion of the KLF11 variant. Luciferase reporter assays were used to investigate whether the KLF11 variant binds to the insulin promoter. Real-time PCR, western blotting, and glucose-stimulated insulin secretion (GSIS) analysis were used to analyze the KLF11 variant that regulates insulin expression and insulin secretion activity in beta cell lines. The Freestyle Libre H (Abbott Diabetes Care Ltd) was used to dynamically monitor the proband daily blood glucose levels. Results. Mutation screening for the whole exon genes identified a heterozygous KLF11 (c.1061G > T) variant in the proband, her mother, and her maternal grandfather. Cell-based luciferase reporter assays using wild-type and mutant transgenes revealed that the KLF11 (c.1061G > T) variant had impaired insulin promoter regulation activity. Moreover, this variant was found to impair insulin expression and insulin secretion in pancreatic beta cells. The proband had better blood glucose control without staple food intake ( p < 0.05 ). Conclusions. Herein, for the first time, we report a novel KLF11 (c.1061G > T) monogenic mutation associated with MODY7. This variant has impaired insulin promoter regulation activity and impairs insulin expression and secretion in pancreatic beta cells. Therefore, administering oral antidiabetic drugs along with dietary intervention may benefit the proband.