Glucose homeostasis in mice is transglutaminase 2 independent

Abstract

Transglutaminase type 2 (TG2) has been reported to be a candidate gene for maturity onset diabetes of the young (MODY) because three different mutations that impair TG2 transamidase activity have been found in 3 families with MODY. TG2 null (TG2(-/-)) mice have been reported to be glucose intolerant and have impaired glucose-stimulated insulin secretion (GSIS). Here we rigorously evaluated the role of TG2 in glucose metabolism using independently generated murine models of genetic TG2 disruption, which show no compensatory enhanced expression of other TGs in pancreatic islets or other tissues. First, we subjected chow- or fat-fed congenic SV129 or C57BL/6 wild type (WT) and TG2(-/-) littermates, to oral glucose gavage. Blood glucose and serum insulin levels were similar for both genotypes. Pancreatic islets isolated from these animals and analysed in vitro for GSIS and cholinergic potentiation of GSIS, showed no significant difference between genotypes. Results from intraperitoneal glucose tolerance tests (GTTs) and insulin tolerance tests (ITTs) were similar for both genotypes. Second, we directly investigated the role of TG2 transamidase activity in insulin secretion using a coisogenic model that expresses a mutant form of TG2 (TG2(R579A)), which is constitutively active for transamidase activity. Intraperitoneal GTTs and ITTs revealed no significant differences between WT and TG2(R579A/R579A) mice. Given that neither deletion nor constitutive activation of TG2 transamidase activity altered basal responses, or responses to a glucose or insulin challenge, our data indicate that glucose homeostasis in mice is TG2 independent, and question a link between TG2 and diabetes.

Figure 1. Neither Tgm2 deletion nor substitution with Tgm2^R579A enhances expression of other TGs.

Brain, heart, kidney, liver, lung, skin, spleen, embryonic fibroblasts and islet mRNA from 3 month-old B6 WT and TG2^−/−, 129 WT and TG2^−/− or B6 WT and TG2^R579A/R579A mice was subjected to RT-qPCR analysis of Tgm1-7 and F13a1 mRNA expression relative to HPRT mRNA. Data are presented as means ± SEM (n = 3). Statistical significance was calculated using 2-way ANOVA with the Bonferroni post hoc test.

Figure 2. Tgm2 deletion has no effect on oral glucose tolerance tests in 6 month-old male mice.

Male B6 WT and TG2^−/− or 129 WT and TG2^−/− mice (3 months old raised on a normal chow diet) were fed normal chow for 3 months (A, C) or fed a high-fat diet for 3 months (B, D). Fasted male mice were then subjected to oral glucose tolerance tests, and blood glucose (A, B) and serum insulin (C, D) levels were determined. Data are presented as means ± SEM (n = 7). Overall P values calculated using 2-way ANOVA are shown at the top; individual Bonferroni post hoc test results are shown above the line profiles. *P<0.05; **P<0.01; ***P<0.001.

Figure 3. Tgm2 deletion has no effect on insulin secretion by islets.

Islets were isolated (A) from 6 month-old male B6 WT and TG2^−/− (i) or 129 WT and TG2^−/− (ii) mice fed a normal chow diet (chow) or fed a normal chow diet for 3 months followed by a high-fat diet for 3 months (fat) or (B) from 3 month old male 129 WT and TG2^−/− mice fed a normal chow diet. (A) Isolated islets, 5 per group (n = 7), were incubated in Krebs-Ringer bicarbonate (KRB) buffer containing 2.8 mM glucose or 16.8 mM glucose or 16.8 mM glucose plus 0.1 mM carbachol, 37°C, 1 h. (B) Groups of 70 islets (n = 3 for WT and n = 2 for TG2^−/−) were perifused in KRB containing 2.8 mM glucose for 10 min before a stimulatory period of 60 min with KRB containing 16.8 mM glucose. The perifusate was switched back to 2.8 mM glucose for 30 min to allow insulin secretion to return to basal levels before exposure to 16.8 mM glucose and 100 µM carbachol for 30 min. The perifusate was switched back to 2.8 mM glucose for 10 min before non-metabolic depolarisation with 24 mM KCl to activate voltage-gated Ca²⁺ channel-triggered insulin secretion. One min fractions of the perifusate were collected for the first 10 min, then 2 min fractions were collected at a flow rate of 0.5 ml/min. Insulin content of supernatant and of islets was determined by radioimmunoassay. Data are presented as means ± SEM. Overall P values calculated using 2-way ANOVA are shown at the top; individual Bonferroni post hoc test results are shown above the columns.**P<0.01; ***P<0.001.

Figure 4. Tgm2 deletion has no effect on intraperitoneal glucose tolerance tests in 3 month-old male or female mice.

Fasted male or female 129 WT and TG2^−/− or B6 WT and TG2^−/− mice were subjected to intraperitoneal (i.p.) glucose tolerance tests and blood glucose levels were determined. Data are presented as means ± SEM (n = 6–10). Overall P values calculated using 2-way ANOVA are shown at the top; individual Bonferroni post hoc test results are shown above the line profiles.*P<0.05; **P<0.01; ***P<0.001.

Figure 5. Generation of coisogenic B6 Tgm2^R579A knock-in mice constitutively active for transamidating activity.

A. Gene targeting strategy. i. Original locus showing location of Arg579 and BglII site (red line) in exon 11 (black box) of Tgm2. PCR primers, FP1 & RP2, yield a 250 bp BglII-cleavable fragment. ii. Targetted locus showing Tgm2^R579A mutation, introduced NheI site, and insertion of a LoxP-flanked PGK–neomycin resistance selection cassette between exons 11 & 12. iii. Cre-deleted locus showing Tgm2^R579A mutation, introduced NheI site, and remnant loxP site. PCR primers, FP1 & RP2, yield a 320 bp NheI-cleavable fragment. B. PCR primers, FP1 and RP2, distinguish the 250 bp wild-type Tgm2 allele from the 320 bp Tgm2^R579A knock-in allele. C. Homogenized tissues from WT or TG2^R579A/R579A mice (n = 3) were size-fractionated and TG2 levels detected by Western blot were normalized for loading using anti-GAPDH for heart, liver, MEF or tubulin for islets. D. GTPγS inhibition of in vitro calcium-activated transamidase activity was assayed in WT or TG2^R579A/R579A liver lysates (n = 3) as described in “Materials and Methods”. Transamidase activity was maximal (100%) with addition of 2mM CaCl₂. P values were calculated using 1-way ANOVA. E. Intracellular transamidase activity was assayed in intact WT or TG2^R579A/R579A murine embryonic fibroblasts (n = 6) as described in “Materials and Methods”. P values were calculated using 2-way ANOVA with the Bonferroni post hoc test. Data are presented as means ± SEM. **P<0.01; ***P<0.001.

Figure 6. Neither Tgm2 deletion nor substitution with Tgm2^R579A affects glucose homeostasis in 3 month-old B6 mice.

(A, B) Fasted B6 WT and TG2^R579A/R579A mice were subjected to intraperitoneal glucose tolerance tests or (C, D) fed B6 WT and TG2^−/− or B6 WT and TG2^R579A/R579A mice were subjected to intraperitoneal insulin tolerance tests, and blood glucose (A, C, D) or serum insulin levels (B) were determined. Data are presented as means ± SEM (n = 9–11). Overall P values calculated using 2-way ANOVA are shown at the top; individual Bonferroni post hoc test results are shown above the line profiles. **P<0.01; ***P<0.001.