Monitoring type 2 diabetes from volatile faecal metabolome in cushing’s syndrome and single Afmid mouse models via a longitudinal study

The analysis of volatile organic compounds (VOCs) as a non-invasive method for disease monitoring, such as type 2 diabetes (T2D) has shown potential over the years although not yet set in clinical practice. Longitudinal studies to date are limited and the understanding of the underlying VOC emission over the age is poorly understood. This study investigated longitudinal changes in VOCs present in faecal headspace in two mouse models of T2D – Cushing’s syndrome and single Afmid knockout mice. Longitudinal changes in bodyweight, blood glucose levels and plasma insulin concentration were also reported. Faecal headspace analysis was carried out using selected ion flow tube mass spectrometry (SIFT-MS) and thermal desorption coupled to gas chromatography-mass spectrometry (TD-GC-MS). Multivariate data analysis of the VOC profile showed differences mainly in acetic acid and butyric acid able to discriminate the groups Afmid and Cushing’s mice. Moreover, multivariate data analysis revealed statistically significant differences in VOCs between Cushing’s mice/wild-type (WT) littermates, mainly short-chain fatty acids (SCFAs), ketones, and alcohols, and longitudinal differences mainly attributed to methanol, ethanol and acetone. Afmid mice did not present statistically significant differences in their volatile faecal metabolome when compared to their respective WT littermates. The findings suggested that mice developed a diabetic phenotype and that the altered VOC profile may imply a related change in gut microbiota, particularly in Cushing’s mice. Furthermore, this study provided major evidence of age-related changes on the volatile profile of diabetic mice

Channel characteristics of wild-type and mutant TRPV5.

<p>(<b>A</b>) Whole-cell currents in TRPV5-WT (V5-WT) and TRPV5-682P (V5-S682P) injected <i>Xenopus</i> oocytes recorded in response to 300 ms test pulses to various potentials (from −100 to +60 mV in 10 mV increments). Holding potential, 0 mV (N = 5). (<b>B</b>) Mean current-voltage relationships for TRPV5-WT and TRPV5-682P channels (N = 5). These current-voltage relationships are similar to those reported for TRPV5 channels <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055412#pone.0055412-Cha1" target="_blank">[62]</a>. (<b>C</b>) Mean whole-cell tail currents measured in TRPV5-WT and TRPV5-682P injected <i>Xenopus</i> oocytes during test potentials applied in 10 mV increments from −70 to +40 mV after a pre-pulse to −100 mV in TRPV5-WT and TRPV5-682P channels (N = 5). (<b>D</b>) Time-dependent inhibition of TRPV-WT and TRPV5-682P whole-cell currents. Oocytes were stimulated every 1 s. The peak current amplitude was normalised to that recorded during the first pulse (N = 4). (<b>E</b>) Representative trace of Fura-2 ratio in HEK293 cells transiently transfected with an empty EGFP vector (mock), or EGFP-tagged TRPV5-WT or TRPV5-S682P. Cells expressing EGFP were selected and monitored for changes in intracellular Ca²⁺ levels when extracellular Ca²⁺ concentrations were varied from 1.4 mM Ca²⁺ to 0 mM Ca²⁺ (2 mM EDTA) and 1.4 mM Ca²⁺ which was facilitated by superfusion. (<b>F</b>) Fura-2 levels under resting conditions (t0), minimal Fura-2 ratio after EDTA treatment (tmin) and peak level (tmax) upon administration of 1.4 mM Ca²⁺ after EDTA treatment. Average data of cells transfected with the empty vector (n = 7), TRPV5-wt (n = 24) and TRPV5-S682P (N = 24) from at least three independent experiments. * p<0.05 for TRPV5-WT or TRPV5-S682P versus cells transfected with empty vector. # p<0.05 for TRPV5-S682P versus TRPV5-wt transfected cells.</p

Hypercalciuria in HCALC1 ENU mutant mice and identification of a <i>Trpv5</i> mutation.

<p>(<b>A</b>) Urine calcium/creatinine ratios in 23 G2 offspring of the HCALC1 founder male revealed that 10 of the 23 mice were hypercalciuric, consistent with an autosomal dominant inheritance. Bar, mean calcium/creatinine values. (<b>B</b>) Haplotype analysis of 89 G2 mice (39 hypercalciuric and 50 normocalciuric) was initially undertaken separately in the hypercalciuric and normocalciuric mice, as the penetrance of HCALC1 was unknown. Haplotype analysis of the hypercalciuric mice localised <i>Hcalc1</i> to a 17.38 Mb interval on chromosome 6, flanked by <i>rs13478688</i> and <i>rs30110406</i> (broken double-headed arrow). Haplotype analysis using combined data for the hypercalciuric and normocalciuric mice identified the smaller interval, 11.94-Mb, flanked by <i>rs13478709</i> and <i>rs30110406</i> (solid double-headed arrow). The <i>Hcalc1</i> locus is inherited with the C57BL/6J haplotype from the F1 founder male. Filled box, C57BL/6J allele; and open box, C3H/HeH allele. Number of mice observed for each haplotype is shown beneath each column. (<b>C</b>) DNA sequence analysis of <i>Trpv5</i> identified a heterozygous T to C transition in codon 682 in hypercalciuric mice predicted to alter a wild-type serine (Ser) to a mutant proline (Pro). This mutation resulted in gain of a <i>Bsa</i>JI restriction enzyme site that was used to confirm the presence of the mutation in the 39 hypercalciuric mice (n = 3 shown) and its absence in the 50 normocalciuric mice (n = 3 shown). wt, wild-type; m, mutant. (<b>D</b>) Amino acid sequence alignment revealed evolutionary conservation of the wild-type mouse TRPV5 serine (S) residue at codon 682 (arrowed) in 5 species, as well as in mouse TRPV6 (mTrpv6). Identical residues are shaded black and conservative changes are shaded grey.</p

Assessment of Renal Expression of Calcium Regulatory Genes and Proteins.

<p>Renal expression of (<b>A</b>) <i>Trpv5</i>, (<b>B</b>) <i>Trpv6</i> and (<b>C</b>) <i>Cyp24a1</i> in wild-type (wt), <i>Trpv5</i>^682P/+ (het) and <i>Trpv5</i>^682P/682P (hom) mice (n = 6/group) were assessed by quantitative real-time PCR. All data were normalised to levels of the housekeeping gene <i>Gapdh</i> and wild-type values are expressed as 1. Histogram data are presented as mean ± SEM. ^#p<0.05, ^$p<0.01, compared to wild-type mice. (<b>D</b>) Western blot analysis of TRPV5, TRPV6, CYP24a1 and calbindin-D_28K (CaBP28K) in kidneys of wild-type (wt), <i>Trpv5</i>^682P/+ (het) and <i>Trpv5</i>^682P/682P (hom) mice. The α-1 subunit of the Na/K-ATPase and α-tubulin were used as housekeeping genes to normalize for equal loading. (<b>E</b>) Semi-quantitative densitometry analysis of CYP24a1. (<b>F</b>) Semi-quantitative analysis of CaBP28K. All histogram data are presented as means ± SEM. *p<0.05 and **p<0.02 compared to wild-type mice. (<b>G</b>) Immunohistochemical images of calbindin-D_28K (CaBP28K) stained kidney sections from wild-type (wt), <i>Trpv5</i>^682P/+ (het) and <i>Trpv5</i>^682P/682P (hom) mice. Scale bar  =  200 µm.</p

Phenotypic characterisation of HCALC1 mice.

<p>Metabolic cage analysis of <i>Trpv5^+/+</i> (wt), <i>Trpv5^682P/+</i> (het) and <i>Trpv5^682P/682P</i> (hom) mice for 24 hours (N = 15–72 mice/group). All data are presented as means±SEM. ^$p<0.05 compared to <i>Trpv5^+/+</i>, * p<0.02 compared to <i>Trpv5^+/+</i>, ^#p<0.02 compared to <i>Trpv5</i>^682P/+ mice, with Bonferroni correction for multiple comparisons.</p

Histological and immunohistochemical assessment of kidneys from HCALC1 mice.

<p>Representative images in <i>Trpv5^+/+</i> (wt), <i>Trpv5^682P/+</i> (het) and <i>Trpv5^682P/682P</i> (hom) mice of: (<b>A</b>) Masson's trichrome staining of renal cortex showing areas of interstitial fibrosis in <i>Trpv5^682P/+</i> and <i>Trpv5^682P/682P</i> mice (light blue), (<b>B</b>) anti-CD3-labelling (green) showing a large number of T-lymphocytes present in the interstitial regions of the <i>Trpv5^682P/+</i> and <i>Trpv5^682P/682P</i> mouse kidneys, (<b>C</b>) TUNEL-labelling (green) of the renal cortex showing the presence of tubular cell apoptosis in the <i>Trpv5^682P/+</i> and <i>Trpv5^682P/682P</i> mouse kidneys. Scale bar  =  50 µm. (<b>D</b>) Immunohistochemical images of kidney sections from wild-type (wt), <i>Trpv5</i>^682P/+ (het) and <i>Trpv5</i>^682P/682P (hom) mice, co-stained for TRPV5 (green) and NCC (red). * denotes co-localisation. Scale bar  =  50 µm. (<b>E</b>) Kidney sections from wild-type (wt), <i>Trpv5</i>^682P/+ (het) and <i>Trpv5</i>^682P/682P (hom) mice, co-stained for TRPV5 (green) and AQP2 (red). * denotes co-localisation. Scale bar  =  50 µm.</p