Loss of arylformamidase with reduced thymidine kinase expression leads to impaired glucose tolerance

Abstract

Tryptophan metabolites have been linked in observational studies with type 2 diabetes, cognitive disorders, inflammation and immune system regulation. A rate-limiting enzyme in tryptophan conversion is arylformamidase (Afmid), and a double knockout of this gene and thymidine kinase (Tk) has been reported to cause renal failure and abnormal immune system regulation. In order to further investigate possible links between abnormal tryptophan catabolism and diabetes and to examine the effect of single Afmid knockout, we have carried out metabolic phenotyping of an exon 2 Afmid gene knockout. These mice exhibit impaired glucose tolerance, although their insulin sensitivity is unchanged in comparison to wild-type animals. This phenotype results from a defect in glucose stimulated insulin secretion and these mice show reduced islet mass with age. No evidence of a renal phenotype was found, suggesting that this published phenotype resulted from loss of Tk expression in the double knockout. However, despite specifically removing only exon 2 of Afmid in our experiments we also observed some reduction of Tk expression, possibly due to a regulatory element in this region. In summary, our findings support a link between abnormal tryptophan metabolism and diabetes and highlight beta cell function for further mechanistic analysis.

Keywords: Arylformamidase; Diabetes; Insulin secretion; Kynurenine; Tryptophan.

Fig. 1.

Overview of five phenotyping cohorts. (A) A cohort of 11 male and 8 female Afmid^tm1b/tm1b mice on a C57BL6/NTac background were bred for the IMPC phenotyping pipeline (1). Two further cohorts of 10 males for each Afmid^tm1b/tm1b, Afmid^tm1b/+ and Afmid^+/+ genotype were bred for blood based tests and one for urine analysis (2,3). A smaller cohort of 5–9 males of each genotype was also bred for pathology analysis (4). Finally a cohort of 11 Afmid^tm1b/tm1b and Afmid^+/+ and 10 Afmid^tm1b/+ females were generated to investigate developing glucose phenotypes in older mice (5). (B) A schematic of the IMPC pipeline phenotyping timeline. wt=Afmid^+/+, het=Afmid^tm1b/+, hom=Afmid^tm1b/tm1b.

Fig. 2.

TK1 and AFMID expression. qRT-PCR relative expression data for thymidine kinase 1 (TK1; Mm01246403_g1) and arylformamidase (AFMID; Mm00510775_m1) in liver (A) and kidney (B) samples taken from male Afmid^tm1b/tm1b mice and C57BL6/NTac controls. These results confirm complete silencing of Afmid in Afmid^tm1b/tm1b mice, however, TK1 was also reduced. Values are expressed as mean±s.d. (n=3/group). **P>0.01, ***P>0.001 as compared to C57BL6/NTac controls (Student's 2-tailed t-test). HOM=Afmid^tm1b/tm1b.

Fig. 3.

Glucose tolerance test. Intraperitoneal glucose tolerance test (IPGTT) data for male and female Afmid^tm1b/tm1b mice and Afmid^tm1b/+ and Afmid^+/+ controls. Glucose tolerance was measured during an IPGTT at 13 (A), 16 (C) and 28 (E) weeks for male mice and 16 (B) and 28 (D) weeks for female mice. The results show that both male and female Afmid^tm1b/tm1b mice demonstrate impaired glucose tolerance at all time points. Values are expressed as mean±s.e.m. (n=8–11/group). *P>0.05, **P>0.01, ***P>0.001 as compared to Afmid^+/+ controls; †P>0.001 as compared to Afmid^+/tm1b (2-way ANOVA with repeated measures and Bonferroni correction). WT=Afmid^+/+, HET=Afmid^tm1b/+, HOM=Afmid^tm1b/tm1b.

Fig. 4.

Glucose and fructosamine clinical chemistry. Clinical chemistry parameters were measured at 16 (free-fed; A,C) and 39 (fasted; B,D) weeks of age in male Afmid^tm1b/tm1b mice and Afmid^tm1b/+ and Afmid^+/+ controls. Glucose and fructosamine levels were significantly elevated at both 16 and 39 weeks. Values are expressed as mean±s.e.m. (n=10–11/group). *P>0.05, **P>0.01 as compared to Afmid^+/+ controls (1-way ANOVA with Bonferroni correction). WT=Afmid^+/+, HET=Afmid^tm1b/+, HOM=Afmid^tm1b/tm1b, FF=Free Fed, TB=Terminal Bleed.

Fig. 5.

Insulin tolerance test and islet perifusion. (A) Intraperitoneal insulin sensitivity test (IPIST) data for male Afmid^tm1b/tm1b mice and Afmid^+/+ controls at 13 weeks of age. (B) Pancreatic islet perifusion normalized insulin secretion data for male Afmid^tm1b/tm1b mice and Afmid^+/+ controls at 12 weeks of age. There were no differences in insulin tolerance, however Afmid^tm1b/tm1b mice demonstrated reduced insulin secretion from their pancreatic islets. Values are expressed as mean±s.e.m. (n=5–6/group). *P>0.05, **P>0.01, ***P>0.001 as compared to Afmid^+/+ controls (2-way ANOVA with repeated measures and Bonferroni correction). WT=Afmid^+/+, HOM=Afmid^tm1b/tm1b.

Fig. 6.

Islet histology. (A-D) Total islet number (A), total islet area (B), total pancreas area (C) and average islet size (D) as analyzed from whole pancreas sections taken from male Afmid^tm1b/tm1b mice and Afmid^+/+ controls at 16 and 46 weeks of age. (E,F) Representative Hematoxylin & Eosin stained sections from Afmid^+/+ (E) and Afmid^tm1b/tm1b (F) mice at 46 weeks of age (scale bar=250 µm). All islets within a section were counted, and a representative five sections were sampled from throughout the pancreas each 200 µm apart. Afmid^tm1b/tm1b mice had reduced islet number at both time points and a reduced total islet area at 46 weeks old. Values are expressed as mean±s.e.m. (n=5–9/group). *P>0.01 as compared to Afmid^+/+ controls (2-way ANOVA). WT=Afmid^+/+, HOM=Afmid^tm1b/tm1b.

Fig. 7.

Representative urinary ¹H NMR spectra. Urinary ¹H NMR spectra from an Afmid^+/+ control mouse (A) and an Afmid^tm1b/tm1b mouse (B) showing the aromatic region of the spectrum. Peaks are assigned to formate and hippurate in both data sets, but additionally to kynurenic acid (*) and xanthurenic acid (†) in the urinary ¹H NMR spectrum from the Afmid^tm1b/tm1b mouse. WT=Afmid^+/+, HOM=Afmid^tm1b/tm1b.