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: Serine racemase, D-cysteine, Insulin, DNA methylation, cAMP,
   Islets, Luciferase assay

Graphical abstract

   [41]Image 1
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Highlights

     * •
       Serine Racemase (SR) is also a cysteine racemase.
     * •
       Lack of SR results in high levels of insulin in the pancreas and
       plasma.
     * •
       D-cysteine shows greater inhibition of insulin secretion compared
       to D-serine.
     * •
       Endogenous D-cysteine signals via cyclic AMP mediated by CREB-DNMT1
       interaction.
     * •
       Hypomethylation of Ins1 promoter is rescued by high methyl donor
       diet rescuing high insulin levels.

1. Introduction

   D-amino acids are mirror images of l-amino acids and their function in
   mammals are gradually being elucidated [[43]1,[44]2]. While much of the
   work in this area has focused on D-serine and D-aspartate (ligands at
   the NMDA receptor), little is known about endogenous mammalian
   D-cysteine [[45]3,[46][6], [47][7], [48][8], [49][9], [50][10]]. Using
   novel biochemical methods (in vivo and in vitro), Ins1 promoter
   bisulfite sequencing and high throughput mass spectrometric approaches,
   we identify endogenous D-cysteine in the mammalian pancreas and
   highlight its role in insulin secretion.

   D-amino acids (D-Ala and D-Ser) have been shown to function in rodent
   and human islet cell signaling and colocalize with β-cells
   [[51]56,[52]65]. SR^−/− mice have neurological deficits and are
   considered a model for schizophrenia [[53]5,[54]6]. Our data show that
   mammalian D-cysteine is present in substantial amounts in the pancreas
   and is synthesized by enzyme serine racemase (SR). SR^−/− mice show
   reduced levels of D-cysteine and constitutively produce 6–10 fold
   higher levels of insulin in the pancreas and plasma and display 3 fold
   higher mean islet diameters. The excess insulin is stored in secretory
   vesicles and plasma exosomes as amyloid aggregates. Endogenous
   D-cysteine signals via cyclic AMP (cAMP). SR^−/− mice display lower
   levels of cyclic AMP response element binding protein (CREB), p-CREB
   (S133) and total DNA methyl transferase (DNMT) activity in the pancreas
   which results in lower levels of global and Ins1 promoter methylation.
   In both STZ and NOD mouse models of diabetes and human type 2 diabetes,
   SR expression was increased in the diabetic state with D-cysteine
   levels being higher than D-serine. Glucose stimulated insulin secretion
   (GSIS) in isolated mouse and human islets show D-cysteine to be more
   inhibitory to compared to D-serine. Endogenous D-cysteine including its
   metabolites and other D-amino acids may play novel roles in type 1
   diabetes and in mammalian pancreatic biology.

2. Results

2.1. Identification of mammalian D-cysteine

   We employed stereospecific bioluminescent and chromatographic methods
   to estimate endogenous D-cysteine in the pancreas ([55]Figure 1A).
   Using a bioluminescent luciferase assay ([56]Figure 1B), we estimated
   levels of D-cysteine in the pancreas of mice. The luciferase assay is
   specific for D-cysteine as it involves the conjugation of cyano hydroxy
   benzothiazole (CHBT) with D-cysteine in presence of TCEP and base to
   form D-luciferin, which serves as an exclusive substrate for firefly
   luciferase ([57]Figure 1B adapted from Niwa et al.) [[58]1,[59][11],
   [60][12], [61][13], [62][14]]. WT pancreas contained 0.3 μmol/g tissue
   while SR^−/− pancreas contained 0.1 μmol/g tissue D-cysteine
   ([63]Figure 1C). We estimated D-cysteine levels in βTC-6 cells (β-cell
   line) with or without lentiviral knockdown of SR (SR#8)
   ([64]Figure S3A). Knock down of SR in βTC-6 cells (SR#8) elicited 2
   fold reduction in D-cysteine levels ([65]Figure S3B), suggesting that
   SR is a biosynthetic enzyme for D-cysteine.

Figure 1.

   [66]Figure 1
   [67]Open in a new tab

   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