scholarly journals A role for PFK-2/FBPase-2, as distinct from fructose 2,6-bisphosphate, in regulation of insulin secretion in pancreatic β-cells

2008 ◽  
Vol 411 (1) ◽  
pp. 41-51 ◽  
Author(s):  
Catherine Arden ◽  
Laura J. Hampson ◽  
Guo C. Huang ◽  
James A. M. Shaw ◽  
Ali Aldibbiat ◽  
...  
Keyword(s):  
Insulin Secretion ◽  
Cell Function ◽  
Protein Isoforms ◽  
Subcellular Targeting ◽  
Β Cells ◽  
Pancreatic Β Cells ◽  
Down Regulation ◽  
Gene Encoding ◽  
Elevated Glucose

PFK-2/FBPase-2 (6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase) catalyses the formation and degradation of fructose 2,6-P2 (fructose 2,6-bisphosphate) and is also a glucokinase-binding protein. The role of fructose 2,6-P2 in regulating glucose metabolism and insulin secretion in pancreatic β-cells is unresolved. We down-regulated the endogenous isoforms of PFK-2/FBPase-2 with siRNA (small interfering RNA) and expressed KA (kinase active) and KD (kinase deficient) variants to distinguish between the role of PFK-2/FBPase-2 protein and the role of its product, fructose 2,6-P2, in regulating β-cell function. Human islets expressed the PFKFB2 (the gene encoding isoform 2 of the PFK2/FBPase2 protein) and PFKFB3 (the gene encoding isoform 3 of the PFK2/FBPase2 protein) isoforms and mouse islets expressed PFKFB2 at the mRNA level [RT–PCR (reverse transcription–PCR)]. Rat islets expressed PFKFB2 lacking the C-terminal phosphorylation sites. The glucose-responsive MIN6 and INS1E cell lines expressed PFKFB2 and PFKFB3. PFK-2 activity and the cell content of fructose 2,6-P2 were increased by elevated glucose concentration and during pharmacological activation of AMPK (AMP-activated protein kinase), which also increased insulin secretion. Partial down-regulation of endogenous PFKFB2 and PFKFB3 in INS1E by siRNA decreased PFK-2/FBPase-2 protein, fructose 2,6-P2 content, glucokinase activity and glucoseinduced insulin secretion. Selective down-regulation of glucose-induced fructose 2,6-P2 in the absence of down-regulation of PFK-2/FBPase-2 protein, using a KD PFK-2/FBPase-2 variant, resulted in sustained glycolysis and elevated glucose-induced insulin secretion, indicating an over-riding role of PFK-2/FBPase-2 protein, as distinct from its product fructose 2,6-P2, in potentiating glucose-induced insulin secretion. Whereas down-regulation of PFK-2/FBPase-2 decreased glucokinase activity, overexpression of PFK-2/FBPase-2 only affected glucokinase distribution. It is concluded that PFK-2/FBPase-2 protein rather than its product fructose 2,6-P2 is the over-riding determinant of glucose-induced insulin secretion through regulation of glucokinase activity or subcellular targeting.

scholarly journals Regulation of cAMP accumulation and activity by distinct phosphodiesterase subtypes in INS-1 cells and human pancreatic β-cells

2019 ◽  
Author(s):  
Evan P.S. Pratt ◽  
Kyle E. Harvey ◽  
Amy E. Salyer ◽  
Shiqi Tang ◽  
Gregory H. Hockerman
Keyword(s):  
Insulin Secretion ◽  
Cell Function ◽  
Human Islets ◽  
Pde4 Inhibitor ◽  
Β Cells ◽  
Pancreatic Β Cells ◽  
Creb Phosphorylation ◽  
Glucose Stimulated Insulin Secretion ◽  
Camp Levels

AbstractPancreatic β-cells express multiple phosphodiesterase (PDE) subtypes, but the specific roles for each in β-cell function, particularly in humans, is not clear. We evaluated the cellular role of PDE1, PDE3, and PDE4 activity in the rat insulinoma cell line INS-1 and in primary human β-cells using subtype-selective PDE inhibitors. Using a genetically encoded, FRET-based cAMP sensor, we found that the PDE1 inhibitor 8MM-IBMX and the PDE4 inhibitor rolipram elevated cAMP levels above baseline in the absence and presence of 18 mM glucose in INS-1 cells. Inhibition of PDE1 or PDE4 potentiated glucose-stimulated insulin secretion in INS-1 cells. In contrast, the inhibition of PDE3 with cilostamide had little effect on cAMP levels or glucose-stimulated insulin secretion. PDE1 inhibition, but not PDE3 of PDE4 inhibition, reduced palmitate-induced caspase-3/7 activation, and enhanced CREB phosphorylation in INS-1 cells. In human β-cells, only PDE3 or PDE4 inhibition increased cAMP levels in 1.7 mM glucose, but PDE1, PDE3, or PDE4 inhibition potentiated cAMP levels in 16.7 mM glucose. Inhibition of PDE1 or PDE4 increased cAMP levels to a greater extent in 16.7 mM glucose than in 1.7 mM glucose in human β-cells. In contrast, elevation of cAMP levels by PDE3 inhibition was not different at these glucose concentrations. PDE1 inhibition also potentiated insulin secretion from human islets, suggesting that the role of PDE1 may be conserved between INS-1 cells and human pancreatic β-cells. Our results suggest that inhibition of PDE1 may be a useful strategy to potentiate glucose-stimulated insulin secretion, and to protect β-cells from the toxic effects of excess fatty acids.

scholarly journals Bimodal role of conventional protein kinase C in insulin secretion from rat pancreatic β cells

2004 ◽  
Vol 561 (1) ◽  
pp. 133-147 ◽  
Author(s):  
Hui Zhang ◽  
Masahiro Nagasawa ◽  
Satoko Yamada ◽  
Hideo Mogami ◽  
Yuko Suzuki ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Insulin Secretion ◽  
Protein Kinase ◽  
Β Cells ◽  
Pancreatic Β Cells ◽  
Kinase C

scholarly journals Role of the active zone protein, ELKS, in insulin secretion from pancreatic β-cells

2019 ◽  
Vol 27 ◽  
pp. S81-S91 ◽  
Author(s):  
Mica Ohara-Imaizumi ◽  
Kyota Aoyagi ◽  
Toshihisa Ohtsuka
Keyword(s):  
Insulin Secretion ◽  
Active Zone ◽  
Β Cells ◽  
Pancreatic Β Cells

scholarly journals SPARC promotes insulin secretion through down-regulation of RGS4 protein in pancreatic β cells

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Li Hu ◽  
Fengli He ◽  
Meifeng Huang ◽  
Qian Zhao ◽  
Lamei Cheng ◽  
...  
Keyword(s):  
Insulin Secretion ◽  
Underlying Mechanism ◽  
Negative Regulator ◽  
Β Cells ◽  
Pancreatic Β Cells ◽  
Promoting Effect ◽  
Down Regulation ◽  
Mouse Islets ◽  
Glucose Stimulated Insulin Secretion ◽  
Phosphoinositide 3 Kinase

Abstract SPARC-deficient mice have been shown to exhibit impaired glucose tolerance and insulin secretion, but the underlying mechanism remains unknown. Here, we showed that SPARC enhanced the promoting effect of Muscarinic receptor agonist oxotremorine-M on insulin secretion in cultured mouse islets. Overexpression of SPARC down-regulated RGS4, a negative regulator of β-cell M3 muscarinic receptors. Conversely, knockdown of SPARC up-regulated RGS4 in Min6 cells. RGS4 was up-regulated in islets from sparc −/− mice, which correlated with decreased glucose-stimulated insulin secretion (GSIS). Furthermore, inhibition of RGS4 restored GSIS in the islets from sparc −/− mice, and knockdown of RGS4 partially decreased the promoting effect of SPARC on oxotremorine-M-stimulated insulin secretion. Phosphoinositide 3-kinase (PI3K) inhibitor LY-294002 abolished SPARC-induced down-regulation of RGS4. Taken together, our data revealed that SPARC promoted GSIS by inhibiting RGS4 in pancreatic β cells.

scholarly journals MECHANISMS IN ENDOCRINOLOGY: Alternative splicing: the new frontier in diabetes research

2016 ◽  
Vol 174 (5) ◽  
pp. R225-R238 ◽  
Author(s):  
Jonàs Juan-Mateu ◽  
Olatz Villate ◽  
Décio L Eizirik
Keyword(s):  
Alternative Splicing ◽  
Cell Fate ◽  
Immune Cells ◽  
Regulatory Networks ◽  
Protein Isoforms ◽  
Diabetes Research ◽  
Β Cells ◽  
Β Cell ◽  
Pancreatic Β Cells

Type 1 diabetes (T1D) is a chronic autoimmune disease in which pancreatic β cells are killed by infiltrating immune cells and by cytokines released by these cells. This takes place in the context of a dysregulated dialogue between invading immune cells and target β cells, but the intracellular signals that decide β cell fate remain to be clarified. Alternative splicing (AS) is a complex post-transcriptional regulatory mechanism affecting gene expression. It regulates the inclusion/exclusion of exons into mature mRNAs, allowing individual genes to produce multiple protein isoforms that expand the proteome diversity. Functionally related transcript populations are co-ordinately spliced by master splicing factors, defining regulatory networks that allow cells to rapidly adapt their transcriptome in response to intra and extracellular cues. There is a growing interest in the role of AS in autoimmune diseases, but little is known regarding its role in T1D. In this review, we discuss recent findings suggesting that splicing events occurring in both immune and pancreatic β cells contribute to the pathogenesis of T1D. Splicing switches in T cells and in lymph node stromal cells are involved in the modulation of the immune response against β cells, while β cells exposed to pro-inflammatory cytokines activate complex splicing networks that modulate β cell viability, expression of neoantigens and susceptibility to immune-induced stress. Unveiling the role of AS in β cell functional loss and death will increase our understanding of T1D pathogenesis and may open new avenues for disease prevention and therapy.

Cell-wide analysis of secretory granule dynamics in three dimensions in living pancreatic β-cells: evidence against a role for AMPK-dependent phosphorylation of KLC1 at Ser517/Ser520 in glucose-stimulated insulin granule movement

2010 ◽  
Vol 38 (1) ◽  
pp. 205-208 ◽  
Author(s):  
Angela McDonald ◽  
Sarah Fogarty ◽  
Isabelle Leclerc ◽  
Elaine V. Hill ◽  
D. Grahame Hardie ◽  
...  
Keyword(s):  
Insulin Secretion ◽  
Glutathione Transferase ◽  
Three Dimensions ◽  
Β Cells ◽  
Pancreatic Β Cells ◽  
Glucose Levels ◽  
Insulin Granules ◽  
Elevated Glucose ◽  
Glucose Stimulated Insulin Secretion

Glucose-stimulated insulin secretion from pancreatic β-cells requires the kinesin-1/Kif5B-mediated transport of insulin granules along microtubules. 5′-AMPK (5′-AMP-activated protein kinase) is a heterotrimeric serine/threonine kinase which is activated in β-cells at low glucose concentrations, but inhibited as glucose levels increase. Active AMPK blocks glucose-stimulated insulin secretion and the recruitment of insulin granules to the cell surface, suggesting motor proteins may be targets for this kinase. While both kinesin-1/Kif5B and KLC1 (kinesin light chain-1) contain consensus AMPK phosphorylation sites (Thr693 and Ser520, respectively) only recombinant GST (glutathione transferase)–KLC1 was phosphorylated by purified AMPK in vitro. To test the hypothesis that phosphorylation at this site may modulate kinesin-1-mediated granule movement, we developed an approach to study the dynamics of all the resolvable granules within a cell in three dimensions. This cell-wide approach revealed that the number of longer excursions (>10 μm) increased significantly in response to elevated glucose concentration (30 versus 3 mM) in control MIN6 β-cells. However, similar changes were seen in cells overexpressing wild-type KLC1, phosphomimetic (S517D/S520D) or non-phosphorylatable (S517A/S520A) mutants of KLC1. Thus, changes in the phosphorylation state of KLC1 at Ser517/Ser520 seem unlikely to affect motor function.

scholarly journals Perilipin2 down-regulation in β cells impairs insulin secretion under nutritional stress and damages mitochondria

2020 ◽  
Author(s):  
Akansha Mishra ◽  
Siming Liu ◽  
Joseph Promes ◽  
Mikako Harata ◽  
William Sivitz ◽  
...  
Keyword(s):  
Insulin Secretion ◽  
Cell Function ◽  
Islet Cells ◽  
Cell Metabolism ◽  
Nutritional Stress ◽  
Β Cells ◽  
Β Cell ◽  
Down Regulation

Perilipin 2 (PLIN2) is the lipid droplet (LD) protein in β cells that increases under nutritional stress. Down-regulation of PLIN2 is often sufficient to reduce LD accumulation. To determine whether PLIN2 positively or negatively affects β cell function under nutritional stress, PLIN2 was down-regulated in mouse β cells, INS1 cells, and human islet cells. β cell specific deletion of PLIN2 in mice on a high fat diet reduced glucose-stimulated insulin secretion (GSIS) in vivo and in vitro. Down-regulation of PLIN2 in INS1 cells blunted GSIS after 24 h incubation with 0.2 mM palmitic acids. Down-regulation of PLIN2 in human pseudoislets cultured at 5.6 mM glucose impaired both phases of GSIS, indicating that PLIN2 is critical for GSIS. Down-regulation of PLIN2 decreased specific OXPHOS proteins in all three models and reduced oxygen consumption rates in INS1 cells and mouse islets. Moreover, we found that PLIN2 deficient INS1 cells increased the distribution of a fluorescent oleic acid analog to mitochondria and showed signs of mitochondrial stress as indicated by susceptibility to fragmentation and alterations of acyl-carnitines and glucose metabolites. Collectively, PLIN2 in β cells have an important role in preserving insulin secretion, β cell metabolism and mitochondrial function under nutritional stress.

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