Mammalian D-Cysteine controls insulin secretion in the pancreas

Abstract

Background: D-amino acids are being recognized as important molecules in mammals with function. This is a first identification of endogenous D-cysteine in mammalian pancreas.

Methods: Using a novel stereospecific bioluminescent assay, chiral chromatography, enzyme kinetics and a transgenic mouse model we identify endogenous D-cysteine. We elucidate its function in two mice models of type 1 diabetes (STZ and NOD), and in tests of Glucose Stimulated Insulin Secretion in isolated mouse and human islets and INS-1 832/13 cell line.

Results and discussion: D-cysteine is synthesized by serine racemase (SR) and SR^-/- mice produce 6-10 fold higher levels of insulin in the pancreas and plasma including higher glycogen and ketone bodies in the liver. The excess insulin is stored as amyloid in secretory vesicles and exosomes. In glucose stimulated insulin secretion in mouse and human islets, equimolar amount of D-cysteine showed higher inhibition of insulin secretion compared to D-serine, another closely related stereoisomer synthesized by SR. In mouse models of diabetes (Streptozotocin (STZ) and Non Obese Diabetes (NOD) and human pancreas, the diabetic state showed increased expression of D-cysteine compared to D-serine followed by increased expression of SR. SR^-/- mice show decreased cAMP in the pancreas, lower DNA methyltransferase enzymatic and promoter activities followed by reduced phosphorylation of CREB (S133), resulting in decreased methylation of the Ins1 promoter. D-cysteine is efficiently metabolized by D-amino acid oxidase and transported by ASCT2 and Asc1. Dietary supplementation with methyl donors restored the high insulin levels and low DNMT enzymatic activity in SR^-/- mice.

Conclusions: Our data show that endogenous D-cysteine in the mammalian pancreas is a regulator of insulin secretion.

Keywords: D-cysteine; DNA methylation; Insulin; Islets; Luciferase assay; Serine racemase; cAMP.

Graphical abstract

Figure 1

Identification and characterization of pancreatic mammalian D-cysteine. (A) Chemical structure of D-cysteine (B) Principle of luciferase assay for bioluminescent detection of D-cysteine (adapted from Niwa et al., 2006). The asterisk (∗) in the schematic is the chiral carbon of cysteine that corresponds to that in D-luciferin. Panel shows SR converting L-cysteine to D-cysteine that conjugates with CHBT to produce D-luciferin. (C) Amounts of D cysteine in the pancreas of age matched WT and SR^−/− mice estimated by luciferase assay. (N = 4 mice; student's t-test) (D) HPLC estimation of L and D-cysteine and D-serine in pancreas of WT and SR^−/− mice (N = 3) (∗ indicates p values between WT and SR^−/− mice; t-test). (E)In vitro racemization activity of recombinant purified mouse SR with L-cysteine substrate (1 mM concentration) to produce D-cysteine measured using the luciferase assay. Amount of D-cysteine formed was quantified from luminescence intensities of a D-cysteine standard curve. Negative control was purified mouse SR incubated with 500 mM L-cysteine (l-Cysteine is inhibitory >2 mM concentrations) and without PLP cofactor. Data are representative of 3–4 independent experiments each with different preps of recombinant purified mouse SR. (F) Detection of purified L and D-cysteine by chiral HPLC separation after thiol labeling by ABDF and fluorescent detection at excitation λ = 380 nm and emission λ = 510 nm. (G) Immunohistochemistry of conjugated D-cysteine in pancreatic sections of WT and SR^−/− mice (endocrine and exocrine). Red staining (conjugated D-cysteine) Blue (DAPI). (H) Expression of SR in islets and exocrine pancreas of WT mice (6–8 weeks old). Scale Bar = 50 μ. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article).

Figure 2

D-cysteine versus D-serine control of insulin secretion. (A) Insulin levels (ng/ml) in the pancreas of WT and SR^−/− mice (N = 4–5) measured using ultrasensitive mouse insulin ELISA kit. ∗p < 0.001 indicates significance compared to age matched WT mice (t-test). (B) Insulin levels (ng/ml) in the plasma of age matched WT and SR^−/− mice (N = 3) measured using ELISA (mentioned above). ∗p < 0.001 indicates significance compared to age matched WT mice (t-test). (C) Glycogen levels in the liver of WT and age matched SR^−/− mice (N = 3). Glycogen amounts were quantified based on a standard curve of purified glycogen. (t-test) (D) Mouse islet viability determined using propidium iodide (red) and fluorescein diacetate (green). (E) Insulin secretion in mouse islets in response to glucose, D-cysteine and D-serine (10 mM) for 6 h at 37 °C. p values indicate comparison between treatments. (t-test) (F) GSIS in mouse islets incubated for 6 h at 37 °C in presence of 10 mM concentrations of D-cysteine and D-serine. p values indicate comparison between treatments (t-test) (G) Human islet viability assessed by propidium iodide and fluorescein diacetate (H) GSIS in normal human islets incubated for 8 h at 37 °C in presence of 10 mM concentrations of D-cysteine and D-serine. p values indicate comparison between glucose and D-cysteine treatment (t-test) (I) Dose response of D-cysteine and D-serine on insulin secretion in INS-1 832/13 cells. (t-test) (J) Time course of D-cysteine and D-serine on insulin secretion in INS-1 832/13 cells (t-test) (K) Western blot of INS1 cells treated with different concentrations of D-cysteine and D-serine for 6 h. Actin was a loading control. All data are representative of 3–4 independent experiments. Error bars are SD. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article).

Figure 3

D-cysteine and D-serine expression in STZ and NOD models of type 1 and 2 diabetes. (A) Schematic of STZ administration timecourse in C57BL/6J male mice. (B) Expression of D-cysteine (green; upper panel) and D-serine (red; lower panel) in islets of WT mice administered STZ. Scale Bar = 10 μ. (C) Amount of D-cysteine in the pancreas of C57BL/6J WT male mice administered STZ (40 mg/kg i.p.) in a timecourse. (D) Amount of D-cysteine in the pancreas of non-diabetic (Glucose<100 mg/dl) and diabetic (Glucose>250 mg/dl) NOD mice. Each group had N = 3 mice. Error Bars are SD (t-test). (E) Blot of pancreatic lysates of control (non diabetic) and diabetic NOD mice showing expression of SR. Actin is a loading control. (F) Expression of D-cysteine (green) and D-serine (red) in normal and diabetic human islets. Inset (large box) shows D-cysteine and D-serine staining in a magnified section of the original image (small box). Scale Bar = 10 μ. (G) Expression of SR (green) and insulin (red) in normal (upper panel) and diabetic (lower panel) human islets. DAPI is nuclear stain (blue). Scale Bar = 10μ. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article).

Figure 4

Pathway and Gene Ontology (GO) analysis. Plot shows (A) GO terms up and down regulated in SR^−/− pancreas (B) Pathways up and down regulated in SR^−/− pancreas. (C) Protein interaction map of ribosome related complexes enriched in SR^−/− pancreas. The size of the dot corresponds to the number of genes with the function. The color of the dot is proportional to the adjusted p values. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article).

Figure 5

D-Cysteine signals via cyclic AMP. (A) Cyclic AMP levels in WT and SR^−/− pancreatic supernates estimated by ELISA (∗p < 0.001 relative to WT; t-test). Data are representative of 3 independent experiments. Error Bars refer to SD. (B) Immunohistochemistry of exocrine and islets of WT and SR^−/− mice pancreas for cAMP expression. Sections were stained with anti-mouse cAMP antibody (1:500) followed by anti-mouse secondary antibody Alexa 594 (1:1000). Arrows indicate boundary of islet (bottom panel). Scale Bar = 100 μ. (C) Snapshot of live cell imaging of βTC-6 cells transfected with cAMP sensor pink flamindo and treated with 50 μM forskolin, L-cysteine and D-cysteine. Fluorescence was measured at 20X using idisk spinning confocal microscopy at 562 nm wavelength. Scale Bar = 100 μ (D) Quantitative analysis of mean fluorescence intensities at each elapsed time point during the live cell imaging at one position. Data were obtained and processed using Slidebook 6 (digital microscopy software). (E) Schematic of mode of action of endogenous D-cysteine. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article).

Figure 6

SR controls global and Ins1 promoter methylation. (A) Expression of DNMT1, DNMT3A and DNMT3B in pancreatic nuclear extracts of WT and SR^−/− mice. Laminin A/C was a nuclear loading control. Data are representative of 3–5 independent experiments. (B) Total DNMT activity in nuclear extracts of pancreas of age matched WT and SR^−/− mice. Nuclear extracts (20 μg) was added to each well of a total DNMT activity assay 96 well plate. The percent reduction in total DNMT activity was measured relative to WT. ∗p < 0.0001 relative to WT. (one way ANOVA) (C) Whole genome bisulfite sequencing of Ins1 promoter of WT and SR^−/− mice (region 2500 bp). Data show CpG methylation at 7 different CpG sites. p values indicate differences between WT and SR^−/−Ins1 promoter at the different sites. (t-test) (D–E) Global methyl cytosine (mC) and hydroxymethyl cytosine (hmC) expression in WT and SR^−/− pancreas following DNA dot blot on a nitrocellulose membrane and probed with mouse monoclonal mC and hmC antibody (1:2000 dilution) respectively. Data are representative of 3 independent experiments. (F) Expression of CREB and p-CREB (S133) in pancreatic lysates of WT and SR^−/− mice. Data are representative of 3–5 independent experiments. (G) Expression of CREB and p-CREB (S133) in pancreatic nuclear lysates of WT and SR^−/− mice. Data are representative of 3–5 experiments. (H) Schematic of the promoter region of DNMT1 gene and CREB occupancy in the pancreas of WT and SR^−/− mice. D1-D5 indicate the different regions on the promoter that were analyzed following ChIP with CREB antibody. Numbers designate regions upstream of the transcription start site (dark arrow). (I) ChIP-qPCR of region D1-D5 of DNMT1 promoter immunoprecipitated with CREB antibody and fold change in DNMT1 expression determined by real time PCR using SYBR green. Region D1-D5 span the promoter region of DNMT1 including 183 bases into exon1 (D5). Fold change in expression of the promoter regions is indicated relative to WT. Region D3 (159 bp) was not plotted due to no change in expression. Data are mean of 3 independent experiments. Error Bars are SD. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article).

Figure 7

Methyl donor dietary supplementation rescues excess insulin and DNMT activity. (A) Schematic of methionine and SAM incorporation from dietary supplementation coupled with folate cycle (B) Estimation of insulin in the pancreas of WT and SR^−/− mice fed normal (0.4 % methionine) and or a high carbohydrate diet containing 0.8 % methionine (high methyl donor) for 6 months. Insulin was measured using ultrasensitive mouse insulin ELISA kit. ∗p < 0.0001 indicates significance relative to WT (t-test). (C) DNA dot blot of global mC and hmC expression in the pancreas of WT and SR^−/− mice fed normal and or a high carb diet for 6 months. (D) Estimation of insulin in the plasma of WT and SR^−/− mice fed normal and or a high carbohydrate diet for 6 months. (E) Estimation of insulin in the pancreas of WT and SR^−/− mice fed normal and or a higher methyl donor diet for 3 months. ∗p < 0.0001 indicates significance relative to WT (t-test). (F) Total DNMT activity in nuclear extracts of mice fed normal and or a higher methyl donor diet (containing 1.18 % methionine) for 3 months. Percent activity was measured using total DNMT activity assay kit (Epigentek). ∗p < 0.001 indicates significance relative to WT among the groups (t-test). (G) Phosphorylation of CREB (S133) in pancreatic lysates of WT and SR^−/− mice fed a regular and or higher methyl donor diet for 3 months ad libitum. Actin was a loading control. Each point represents one mouse. Error bars are SD.