Research Article: Regulation of cAMP accumulation and activity by distinct phosphodiesterase subtypes in INS-1 cells and human pancreatic β-cells

Date Published: August 23, 2019

Publisher: Public Library of Science

Author(s): Evan P. S. Pratt, Kyle E. Harvey, Amy E. Salyer, Gregory H. Hockerman, Bridget Wagner.


Pancreatic 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, elevated cAMP levels in the absence of glucose to a greater extent than either the PDE3 inhibitor cilostamide or the PDE4 inhibitor rolipram. In 18 mM glucose, PDE1 inhibition elevated cAMP levels to a greater extent than PDE3 inhibition in INS-1 cells, while PDE4 inhibition was without effect. Inhibition of PDE1 or PDE4, but not PDE3, potentiated glucose-stimulated insulin secretion in INS-1 cells. PDE1 inhibition, but not PDE3 or 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.

Partial Text

Pancreatic β-cells secrete the blood glucose-lowering hormone insulin to maintain glucose homeostasis in the body [1]. Pancreatic β-cell dysfunction and cell death underlies the development of type 2 diabetes [2]. At the cellular level, glucose-stimulated insulin secretion (GSIS) is driven by Ca2+ influx through the L-type voltage-gated Ca2+ channels (L-VGCC) Cav1.2 and Cav1.3 [3], and release of Ca2+ from the endoplasmic reticulum (ER) [4]. GSIS is further regulated by the second messenger 3′,5′-cyclic adenosine monophosphate (cAMP), which is generated by the enzyme adenylyl cyclase (AC) [5]. The transmembrane ACs (tmAC) AC1, AC5 and AC8 and soluble AC (sAC) are primarily responsible for cAMP production in β-cells [6–8]. In addition to enhancing GSIS, cAMP promotes pancreatic β-cell mass through increased replication [9] and decreased apoptosis [10]. Both glucose [8, 11, 12] and incretin hormones [13], such as glucagon-like peptide-1 (GLP-1), are capable of stimulating cAMP production and subsequent activation of the cAMP effector proteins Protein Kinase A (PKA) and Exchange Protein Directly Activated by cAMP (Epac) [14]. PKA and Epac regulate insulin secretion through proximal effects on the machinery involved in exocytosis at the plasma membrane [15–17] and distal effects on ER Ca2+ release channels [18, 19]. cAMP signaling is compartmentalized to microdomains within the cell, including near sites of ER Ca2+ release, by phosphodiesterase enzymes (PDE), which degrade cAMP to 5’-AMP.

In this study, we examined the role of PDE1, PDE3, PDE4, and PDE8 in regulating resting and glucose-stimulated cAMP levels and downstream signaling in INS-1 cells and primary human pancreatic β-cells. We found that PDE1, PDE3, and PDE4 but not PDE8, regulate resting cAMP levels, while PDE1 and PDE3, but not PDE4 regulate glucose-stimulated cAMP levels in INS-1 cells (Fig 1, S1 Fig). Inhibition of PDE1 had the greatest effect on cAMP levels under both conditions. In addition, PDE1 and PDE4 inhibition, but not PDE3 or PDE8 inhibition, enhanced GSIS in these cells (Fig 4B; S1 Fig). In contrast to INS-1 cells, we found that PDE3 and PDE4, but not PDE1 or PDE8, regulate resting cAMP levels in primary human β-cells (Fig 2, S2 Fig). However, in the presence of 16.7 mM glucose, PDE1 and PDE4 inhibition resulted in significantly higher cAMP levels compared with resting conditions, suggesting that these subtypes are upregulated by glucose stimulation in human β-cells (Fig 3). Consistent with this, among the subtype-selective inhibitors, only the PDE1-selective inhibitor 8MM-IBMX significantly enhanced insulin secretion from human islets obtained from any of the four donors used in this study (Fig 4C). Finally, we found that PDE1 inhibition reduced palmitate-induced caspase-3/7 activation (Fig 6B) and induced CREB phosphorylation (Fig 6C) in INS-1 cells. Taken together, our results suggest that PDE1-mediated regulation of cAMP levels is important for regulation of GSIS and pancreatic β-cell survival.




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