Regulation of PKD by the MAPK p38delta in insulin secretion and glucose homeostasis

Abstract

Dysfunction and loss of insulin-producing pancreatic beta cells represent hallmarks of diabetes mellitus. Here, we show that mice lacking the mitogen-activated protein kinase (MAPK) p38delta display improved glucose tolerance due to enhanced insulin secretion from pancreatic beta cells. Deletion of p38delta results in pronounced activation of protein kinase D (PKD), the latter of which we have identified as a pivotal regulator of stimulated insulin exocytosis. p38delta catalyzes an inhibitory phosphorylation of PKD1, thereby attenuating stimulated insulin secretion. In addition, p38delta null mice are protected against high-fat-feeding-induced insulin resistance and oxidative stress-mediated beta cell failure. Inhibition of PKD1 reverses enhanced insulin secretion from p38delta-deficient islets and glucose tolerance in p38delta null mice as well as their susceptibility to oxidative stress. In conclusion, the p38delta-PKD pathway integrates regulation of the insulin secretory capacity and survival of pancreatic beta cells, pointing to a pivotal role for this pathway in the development of overt diabetes mellitus.

Figure 1

Mice Lacking p38δ Show Improved Glucose Tolerance and Enhanced Insulin Secretion (A) Western blotting revealed expression of p38δ in wild-type (+/+) pancreas but not in liver (Li), skeletal muscle (Sk), and white (Wa) and brown (Ba) adipose tissue compared to tissues from p38δ null (Δ/Δ) mice. Coomassie blue staining was used to confirm equal loading. (B) Glucose tolerance test (GTT) in mice with the indicated genotypes (^∗p < 0.05, ^∗∗p < 0.01). (C) Parallel measurements of serum insulin during the GTT in mice with the indicated genotypes (^∗p < 0.05). (D) Insulin release in response to indicated stimuli (Glc, glucose; KCl, potassium chloride; tolb., tolbutamide) in isolated islets with the indicated genotypes (^∗p < 0.05). (E) Glucagon secretion in response to 2.8 mM and 16.7 mM glucose in islets isolated with the indicated genotypes (ns, not significant). All error bars indicate ± SEM.

Figure 2

Enhancement of Exocytosis in p38δ-Deficient Pancreatic β Cells Occurs Downstream of Calcium Influx (A) A train of ten successive 500 ms depolarizations from −70 to 0 mV increased capacitance (in femtofarad [fF]) in pancreatic β cells with the indicated genotypes (^∗p < 0.05). (B) Average cumulative increase of capacitance (^∗p < 0.05). (C) Changes in cell capacitance (C_m) when exocytosis was elicited by intracellular application of 1.5 μM free [Ca²⁺]_i via the recording electrode capacitance in pancreatic β cells with the indicated genotypes. Values have been normalized to the resting cell capacitance (C_m,0), which was 4.6 ± 0.4 pF (n = 8) and 4.6 ± 0.2 pF (n = 16) in p38δ^Δ/Δ and p38δ^+/+ cells, respectively. Data are presented as the mean values (central lines) and ± SEM (shaded areas). (D) Steady-state average rate of capacitance change (in fF/s) for cells measured over a 60 s period (^∗p < 0.05). All error bars indicate ± SEM.

Figure 3

p38δ Interacts with PKD1 and Phosphorylates It at Ser 397 and 401 (A) Western blot with indicated immunoprecipitates (IP) and whole-cell extracts (WCE) from INS1 cells stably transfected with indicated constructs confirms physical interaction with endogenous PKD1 (^∗unspecific band). (B) In vitro kinase assay with and without recombinant p38δ and recombinant E. coli-derived GST-tagged PKD1 in the presence and absence of the PKC inhibitor Gö6976. (C) Sequence alignment of the region around S403-407 of mouse PKD1, PKD3, and PKD2, as well as human and rat PKD1. Conserved residues (S403-407 in mPKD1, S397-401 in hPKD1) are indicated by rectangles. (D) In vitro kinase assay with recombinant p38δ and indicated immunoprecipitated proteins. Quantification of autoradiography with corresponding bars positioned under bands (^∗p < 0.05). (E) In vitro kinase assay with indicated immunoprecipitated proteins using CREBtide as a substrate. CREBtide was spotted, while immunopreciptates were tested for equal loading by western blotting. Quantification of radiography using a scintillation counter with corresponding bars positioned under spots (^∗p < 0.05). All error bars indicate ± SEM.

Figure 4

Increased Activity of PKD Leads to Altered Golgi Organization in p38δ-Deficient β Cells (A) Activity of protein kinase D (PKD) was determined by western blotting with an antibody against the activatory phosphorylation sites (serines 744 and 748) (BG, basal glucose and SG, stimulatory glucose levels). Immunoblotting with an antibody against Actin was used to determine equal loading of phopho-PKD and total PKD1 blots. (B) Immunofluorescence with antibodies against giantin (red) in pancreatic β cells with the indicated genotypes. (C) Immunofluorescence with antibodies against giantin (red) in INS1 cells stably expressing HA or HA-p38δ^F324S cultured in the presence of stimulatory glucose. Expression of p38δ^F324S led to formation of tubular protrusions from the Golgi complex (arrows). (B and C) Dashed boxes outline the areas that were magnified. Nuclear DNA was stained with DAPI (blue). Scale bars represent 10 μm.

Figure 5

Restoration of Insulin Secretion in p38δ^Δ/Δ Islets and Glucose Tolerance in p38δ^Δ/Δ Mice by Pharmacological Inhibition of PKD (A) Immunofluorescence using antibodies against giantin (red) in pancreatic β cells with the indicated genotypes exposed to Gö6976 (Go) or U73122 (U) as indicated. Nuclear DNA was stained with DAPI (blue). Dashed boxes outline the areas that have been magnified. The scale bar represents 10 μm. (B) Glucose (16.7 mM)-stimulated insulin secretion from islets with the indicated genotypes exposed to DMSO, U73122, and Gö6976 (^∗p < 0.05). (C) Glucose tolerance test (GTT) in mice with the indicated genotypes treated with DMSO or U73122 as indicated (^∗p < 0.05). All error bars indicate ± SEM.

Figure 6

p38δ Suppresses PKD-Mediated Stimulated Insulin Secretion (A) Western blot to determine the activity of protein kinase D (PKD) in INS1 cells in response to indicated stimuli using an antibody against the activatory phosphorylation sites (serines 744 and 748) (^∗unspecific band). (B) SDS-PAGE with polyacrylamide-bound Mn²⁺-Phos-tag to measure the activity of p38δ in nonstarved INS-1 cells in response to carbachol. Phosphorylated p38δ (P-p38δ) migrated slower than unmodified p38δ. (C) Western blot to determine the activity of protein kinase D (PKD) in INS1 cells stably transfected with indicated constructs in response to carbachol (^∗unspecific band). (D) Insulin secretion from INS1 cells stably expressing HA or HA-p38δ^F324S in response to carbachol under basal and stimulatory glucose levels (^∗p < 0.05 and ^∗∗p < 0.01; ns, not significant). (E) Insulin secretion in response to basal and stimulatory glucose from MIN6 cells stably expressing shRNA against p38δ or a control vector with simultaneous siRNA-mediated knockdown of PKD1 or transfection of a scrambled siRNA (^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001; ns, not significant). (F) Insulin secretion in response to basal and stimulatory glucose from INS1 cells stably expressing GFP, GFP-tagged WT, and mutated forms of PKD (^∗p < 0.05 and ^∗∗p < 0.01). (D–F) All experiments were performed three times independently. All error bars indicate ± SEM.

Figure 7

p38δ Deficiency Protects Mice from Diabetes (A) Blood insulin levels in fasted mice with the indicated genotypes fed a high-fat diet (HF) or a normal diet (ND) after 12 weeks (^∗p < 0.05). (B) Glucose tolerance test (GTT) in mice with the indicated genotypes fed a high-fat diet (HF) or a normal diet (ND) (^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001). (C) Blood glucose levels in mice with the indicated genotypes treated with STZ and DMSO or U73122 (^∗p < 0.05). (D) Blood insulin levels and (E) total pancreatic insulin content in mice with the indicated genotypes treated with DMSO or U73122 8 days after STZ injections. (F) TUNEL stain of islets of mice with the indicated genotypes treated with STZ and DMSO or U73122 and of mice without treatment (w/o treatment). (G) Quantification of TUNEL-positive cells in relation to islet area (^∗p < 0.05; ns, not significant). (H) Model: Acetylcholine (Ach)- and glucose (Glc)- induced pathways leading to increased PKD activity are indicated (M3 R, muscarinic receptor subtype M3; GLUT2, glucose transporter 2; DAG, Diacylglycerol; PLC, phospholipase C). PKD activation leads to insulin secretion and promotes β cell survival. In diabetes, oxidative stress-induced activation of p38δ might interfere with PKD-mediated signaling, leading to impaired pancreatic β cell function. All error bars indicate ± SEM.