Intestinal gluconeogenesis is downregulated in pediatric patients with celiac disease - BMC Medicine - BMC Medicine

Background

Untreated celiac illness (CD) patients person accrued levels of humor glutamine and a little duodenal look of glutaminase (GLS). Intestinal gluconeogenesis (IGN) is simply a process done which glutamine is turned into glucose successful the tiny intestine, for which GLS is crucial. Animal studies suggest impaired IGN whitethorn person semipermanent effects connected metabolic power and beryllium associated with the improvement of benignant 2 diabetes and non-alcoholic fatty liver illness (NAFLD). The purpose of this survey was to thoroughly analyse IGN astatine the cistron look level successful children with untreated celiac disease.

Methods

Quantitative polymerase concatenation absorption (qPCR) was utilized to quantify the look of 11 people genes related to IGN utilizing the delta-delta Ct method with 3 notation genes (GUSB, IPO8, and YWHAZ) successful duodenal biopsies collected from 84 children with untreated celiac illness and 58 illness controls.

Results

Significantly little look of 9 people genes progressive successful IGN was seen successful duodenal biopsies from CD patients compared with controls: FBP1, G6PC, GLS, GPT1, PCK1, PPARGC1A, SLC2A2, SLC5A1, and SLC6A19. No important quality successful the look was observed for G6PC3 oregon GOT1.

Conclusions

Children with untreated celiac illness person little look of genes important for IGN. Further studies are warranted to disentangle whether this is simply a effect of intestinal inflammation oregon owed to an impaired metabolic pathway shared with different chronic metabolic diseases. Impaired IGN could beryllium a mechanics down the accrued hazard of NAFLD seen successful CD patients.

Background

We person antecedently shown an relation betwixt celiac illness (CD) and amino acerb metabolism including the cistron glutaminase (GLS). This enzyme has an important relation successful intestinal gluconeogenesis (IGN) wherever the amino acerb glutamine is the main substrate.

It has besides been shown that patients with CD are astatine an accrued hazard of non-alcoholic fatty liver illness (NAFLD) aboriginal successful life.

Findings

All cardinal genes progressive successful intestinal gluconeogenesis are downregulated astatine the cistron look level successful the tiny intestine of children with untreated CD, suggesting impairment of intestinal gluconeogenesis.

Implications for diligent care

Impaired intestinal gluconeogenesis mightiness pb to the accrued hazard of NAFLD seen successful patients with CD arsenic adults. Early diagnosis and attraction of CD whitethorn reconstruct intestinal gluconeogenesis and forestall CD patients from NAFLD aboriginal successful adulthood.

A family-based genome-wide relation survey (GWAS) recovered shared genes betwixt celiac illness (CD) and benignant 2 diabetes (T2D), indicating changes successful communal nutrient signaling pathways of amino acerb metabolism. Furthermore, glutaminase (GLS), an enzyme that converts glutamine to glutamate, was identified by pathway investigation successful the astir associated web of genes and was recovered to beryllium downregulated successful duodenal biopsies from children with untreated CD [1]. Later enactment showed allele-specific look of GLS connected to the associated single-nucleotide polymorphism (SNP) (rs6741418) successful the GLS cistron portion [2]. A full of 142 associations to 50 traits, a ample fig of which are autoimmune diseases, person been reported for the cistron portion containing GLS/STAT1/STAT4 arsenic reported successful the GWAS Catalog. This makes it 1 of the top-reported cistron regions successful autoimmune diseases truthful far. Out of these 142 reported associations, 26 SNP variants person been shown to person a important power connected the look of GLS, portion only three SNP variants power the look of STAT4, and nary has been shown to power STAT1 look (data from the gtexportal). Additional record 1: Table S1 shows the astir associated variant reported for each trait (data from the GWAS Catalog). Thus, determination is information indicating a imaginable communal familial mechanics down autoimmunity and differential look of GLS. Interestingly, patients with untreated CD besides person altered humor levels of respective amino acids, including glutamine/glutamate, further suggesting alterations of metabolism warranting much probe [3]. Figure 1 illustrates a survey travel illustration and outline of erstwhile enactment arsenic good arsenic the enactment successful the contiguous study.

Fig. 1

Study travel illustration and outline of our previous enactment arsenic good arsenic the enactment successful the contiguous study

Intestinal gluconeogenesis (IGN) is simply a process by which glucose tin beryllium produced successful the tiny intestine [4]. Glutaminase has a captious relation successful IGN since it catalyzes the archetypal measurement needed to usage its main substrate, which is glutamine [5]. In carnal models, IGN-deficient mice make hyperglycemia [6]. Mice with induced precocious levels of IGN alternatively look protected against hyperglycemia, adjacent erstwhile fed a high-fat/high-sucrose (HF-HS) diet. IGN-deficient mice besides much easy make hepatic steatosis, some connected an HF-HS fare and a modular diet, portion mice with induced IGN are again protected, adjacent connected an HF-HS fare [7]. In a rat exemplary investigating the mechanisms down bariatric country arsenic a attraction of T2D, rats showed accrued IGN aft country [8]. In quality studies, T2D patients with precocious look of genes progressive successful IGN had greater betterment successful insulin absorption scores aft bariatric country [9]. These information bespeak an important relation for IGN successful vigor homeostasis and metabolic disease, which is suggested to beryllium explained by a glucose-sensing mechanics successful the portal vein, wherever higher levels of glucose indicating an capable glucose proviso pb to satiety signals sent via the spinal nerves to the encephalon which past affects the whole-body metabolism [10].

The purpose of the contiguous survey was to analyse IGN successful CD patients by investigating intestinal cistron look of GLS and different 10 selected genes progressive successful IGN successful children with untreated CD. Figure 2 illustrates IGN and the roles of the selected genes. The proposal was that with GLS downregulated, the full process of IGN with its important metabolic relation could beryllium affected.

Fig. 2

Gluconeogenesis and steps for utilizing glutamine arsenic a substrate for gluconeogenesis showing the enzymes for which we person quantified cistron expression. Dotted lines correspond steps that person been near retired of the fig wherever cistron look has not been investigated. Investigated solute carriers are besides shown

Biological material

Duodenal biopsies were collected from children betwixt 2010 and 2012 astatine 4 antithetic hospitals successful Sweden arsenic antecedently described [11]. All children had been referred for an precocious endoscopy for aesculapian reasons and were consecutively recruited to the study, and some antheral and pistillate subjects were included. For the contiguous study, 84 untreated CD cases and 58 illness controls were used. The ESPGHAN 1999 criteria [12] were applied astatine each objective sites for the objective diagnosis of CD, e.g., a biopsy showing Marsh people > 1 astatine diagnosis and a wide effect to a gluten-free fare with a important simplification of IgA-tTG levels aft treatment. In bid to guarantee unbiased classification of the cohort to cases and controls, a pathologist reviewed and scored the biopsies blinded to different objective accusation [11]. Children with a Marsh people > 1 and a affirmative IgA-tTG serology were included arsenic cases, and children with a Marsh people of ≤ 1 and antagonistic IgA-tTG serology were included arsenic illness controls (Additional record 2: Table S2). Among the included children, 64 retired of 84 (76%) cases and 34 retired of 58 (59%) controls were females. The mean property ± modular deviation of cases was 6.1 ± 3.7 compared with 11.6 ±4.5 years aged among the controls (p < 0.001). Children with inflammatory bowel illness oregon Helicobacter pylori infections were excluded anterior to investigation since these diseases mightiness amusement inflammation successful the duodenum, which successful crook could power the cistron expression. No kid with non-alcoholic fatty liver illness oregon immoderate different liver diseases was included. All diagnoses contiguous successful the power radical are listed successful Additional record 2: Table S2. Duodenal biopsies were instantly enactment successful RNA-stabilizing RNAlater solution (Life Technologies, CA, USA) and enactment astatine country somesthesia overnight to let the reagent to penetrate the sample. Biopsies were past frozen astatine − 80 °C until RNA extraction was performed. Total RNA was extracted utilizing the AllPrep® DNA/RNA/Protein Mini Kit (Qiagen, Germany). RNA prime and quantity were measured with a NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific, MA, USA) and a 2100 Bioanalyzer (Agilent Technologies, CA, USA). RNA was converted to cDNA for retention utilizing the SuperScript Vilo cDNA synthesis kit (Thermo Fisher Scientific, MA, USA) and stored astatine − 80 °C.

Children and/or parents gave their informed written consent, and the section ethical committee approved the survey (T373-10).

Quantitative polymerase concatenation reaction

Quantitative polymerase concatenation absorption (qPCR) was performed utilizing TaqMan® exertion (Thermo Fisher Scientific, MA, USA). The last absorption consisted of TaqMan® cistron primers and adjacent parts illustration cDNA and TaqMan® Universal Master Mix II. All primer-sample reactions were tally successful duplicate successful 384-well plates connected a QuantStudio 12K Flex Sequence Detection System (Thermo Fisher Scientific, MA, USA). Target genes were FBP1 (fructose-bisphosphate 1), G6PC (glucose-6-phosphatase), G6PC3, GLS, GOT1 (glutamic-oxaloacetic transaminase 1), GPT1 (glutamic-pyruvic transaminase), PCK1 (phosphoenolpyruvate carboxykinase 1), PPARGC1A (PPARG coactivator 1 alpha), SLC2A2 (solute bearer household 2 subordinate 2), SLC5A1 (solute bearer household 5 subordinate 1), and SLC6A19 (solute bearer household 6 subordinate 19). Reference genes were GUSB (glucuronidase beta), IPO8 (importin 8), and YWHAZ (tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation macromolecule zeta). These notation genes person been evaluated by the radical antecedently utilizing the Normfinder bundle [13].

Statistical analysis

Quality power of qPCR information was done successful the ExpressionSuite v1.1 programme (Thermo Fisher Scientific, MA, USA) wherever reactions that had not tally decently were filtered out. ΔCT values were calculated utilizing R [14] with the RStudio processing situation (which was utilized for each enactment successful R) [15]. The radical differences were examined by a generalized linear exemplary utilizing the glm relation successful R, moving 1 exemplary based lone connected CD lawsuit presumption and 1 exemplary adjusting for property and enactment arsenic covariates. A generalized linear exemplary was utilized to alteration accommodation for some a quantitative (age) and a qualitative adaptable (sex). With a = 0.05 correcting for aggregate investigating of 11 people genes utilizing the Bonferroni method gave 0.05/11 = 0.0045 arsenic the adjusted value threshold. Data visualization was done utilizing the ggplot2 bundle successful R [16], parts of tidyverse [17] from which different packages were besides utilized erstwhile coding successful R. Fold alteration was calculated utilizing the 2^−ΔΔC method [18]. A partial correlation investigation utilizing cistron look levels, insubstantial transglutaminase (tTG) antibody levels, and Marsh people controlling for property was performed successful the IBM SPSS statistic bundle mentation 27. p-values were acceptable arsenic 2-sided. Marsh scores of each cases were transformed into numbers: Marsh signifier 2, 1; Marsh signifier 3A, 2; Marsh signifier 3B, 3; and finally, Marsh signifier 3C, 4.

We utilized the STROBE case-control reporting guidelines erstwhile penning this insubstantial (von Elm E, Altman DG, Egger M, Pocock SJ, Gotzsche PC, Vandenbroucke JP. The Strengthening the Reporting of Observational Studies successful Epidemiology (STROBE) Statement: guidelines for reporting observational studies). All authors had entree to the survey information and had reviewed and approved the last manuscript.

All samples that were included passed prime power for RNA 260/230 worth supra 1.8 and were utilized for analysis.

Gene look levels

Analyzing cistron look levels successful duodenal biopsies utilizing qPCR, FBP1, G6PC, GLS, GPT1, PCK1, PPARGC1A, SLC2A2, SLC5A1, and SLC6A 9 showed importantly little look successful CD cases compared with controls, whereas G6PC3 and GOT1 showed nary differences (Table 1 and Fig. 3). This remained significant, erstwhile adjusting for property and enactment of survey participants and aggregate comparisons. Of the 8 downregulated genes, PCK1 was the astir downregulated, with little than a 4th of the look successful the controls compared with cases (fold alteration = 0.22).

Fig. 3

a Volcano crippled showing the fold alteration connected the x-axis and value (−log p-value) connected the y-axis. b Box plots of the cistron look levels successful duodenal biopsies from children with untreated celiac illness (CD) (n = 84) and illness controls (CONTROL) (n = 58). All cases are visualized by idiosyncratic dots. The mediate horizontal enactment represents the median value, and the 2 “hinges” are the archetypal and 3rd quartiles. The “whiskers” widen to the minimum and maximum values wrong the 1.5 interquartile scope of hinges. Significance of people genes marked with *p < 0.0045, **p < 0.00091, ***p < 0.000091 (Bonferroni-adjusted value thresholds)

Correlation betwixt cistron look levels and Marsh people and tTG antibody levels

Partial correlation investigation successful the CD cases (Table 2) showed a beardown correlation betwixt the look levels of the aforesaid 9 genes and Marsh scores of patients. PPARGC1A besides showed a important correlation to IgA tTG antibody levels, portion FBP1, G6PC, GLS, GPT1, PCK1, PPARGC1A, and SLC5A1 showed a important correlation to IgG tTG levels.

The contiguous survey shows that children with untreated CD person little look of genes progressive successful IGN successful duodenal biopsies compared with children with a mean intestinal mucosa. Decreased look is correlated to a higher Marsh people and, to a lesser degree, tTG antibody levels. These results suggest that CD patients could person an impaired relation of IGN, either arsenic a effect of chronic intestinal inflammation successful untreated illness oregon owed to an impaired metabolic pathway shared with different chronic metabolic diseases suggested by the ample fig of traits shown to beryllium associated with the GLS/STAT1/STAT4 cistron region. In the semipermanent perspective, a little look of these genes, which are cardinal for IGN and frankincense important for metabolic homeostasis, whitethorn pb to different chronic diseases similar NAFLD and diabetes if not recognized.

This survey provides the archetypal grounds of the downregulation of intestinal look of G6PC, GPT1, SLC6A19, and PPARGC1A successful CD. Glucose-6-phosphatase, the macromolecule merchandise of G6PC, has an indispensable relation successful gluconeogenesis by converting glucose-6-phosphate, which cannot beryllium transported retired of the cell, into glucose, which tin past beryllium released into the bloodstream, making its downregulation an particularly important finding. Decreased look of PCK1, FBP1, and solute carriers has antecedently been shown successful big CD patients but until present has not been enactment successful the discourse of IGN [19,20,21]. Decreased look of GLS is accordant with our erstwhile uncovering of its downregulation successful the GENEX worldly [1]. G6PC3 and GOT1 showed nary important changes successful the expression; however, G6PC and GPT1, which catalyze the aforesaid reactions successful IGN, are much captious for these respective functions [22, 23].

These information frankincense connote that the quality of the tiny intestine to execute gluconeogenesis and merchandise glucose from the intestinal enterocyte mightiness beryllium severely decreased successful patients with untreated CD. Decreased look of GLS, GPT1, PCK1, FBP1, and G6PC suggest an impairment of the IGN pathway from the commencement of utilizing glutamine successful gluconeogenesis to releasing glucose into the blood. If lone GLS, GPT1, and PCK1 were downregulated, utilizing glycerol, the 2nd astir important substrate of IGN [5] could inactive beryllium imaginable since it enters gluconeogenesis successful aboriginal steps, but since FBP1 and G6PC, the macromolecule products of which catalyze important last steps successful gluconeogenesis, are besides downregulated, it stands to crushed that the full pathway of IGN is impaired. We suggest that the decreased look of PPARGC1A mightiness supply an mentation for this astatine a regulatory level since its macromolecule has a cardinal relation successful regulating hepatic gluconeogenesis. Even though it is chartless if it has a akin relation successful the intestine, it does not look wholly implausible. Decreased look of SLC6A19 suggests the quality to sorb glutamine, arsenic good arsenic different neutral amino acids transported by the SLC6A19 macromolecule (also known arsenic B0AT1), from the intestinal lumen is impaired. Lower look of SLC5A1 (SGLT1) and SLC2A2 (GLUT2) bespeak decreased capableness for glucose transport.

The metabolic effects of impaired IGN successful humans are not wholly clear. Studies connected IGN successful carnal models amusement that an summation successful glucose levels successful the portal vein provides signals that summation satiety and amended vigor homeostasis. Induced precocious levels of IGN look to connection extortion against metabolic disease, portion impairment leads to signs of dysregulated glucose power and hepatic steatosis [7, 10].

CD patients are astatine accrued hazard of NAFLD, with the highest hazard seen during the archetypal years aft diagnosis and the largest comparative hazard summation seen successful patients with a mean BMI [24, 25]. We speculate that impaired IGN could supply an mentation for the accrued hazard of NAFLD successful CD patients. Our survey does not analyse whether look of IGN-related genes instrumentality to mean successful CD patients treated with a gluten-free diet, but the correlation with Marsh scores suggests a little grade of inflammation mightiness amended IGN. If IGN is normalized erstwhile CD is treated, possibly this could beryllium portion of the mentation for wherefore the hazard of NAFLD is astatine its highest successful the archetypal twelvemonth aft CD diagnosis, erstwhile the gluconeogenetic capableness of the intestine possibly has not afloat recovered. Such a betterment mightiness besides beryllium suggested by a 1968 survey of glutaminase enzymatic activity, which recovered little levels successful untreated CD patients that seemed to retrieve successful patients connected attraction with a gluten-free fare [26]. Studying the look of IGN-related genes successful patients earlier and aft attraction would beryllium an important adjacent step.

This survey has respective limitations. The look of the selected genes successful CD cases were compared with illness controls, i.e., these were children referred for an precocious endoscopy investigated for different intestinal diseases affecting the gut. It cannot beryllium excluded that the illness controls whitethorn person had conditions that tin impact the look of the selected genes. However, each illness controls had mean mucosal findings, and children with inflammatory bowel illness and Helicobacter pylori infections were excluded anterior to analysis. Another regulation was that cases and controls were not age- and sex-matched. Still, erstwhile adjusting for property and sex, the results remained significant. The spot of the survey is that children were enrolled from 4 sites by pediatric gastroenterologists with agelong objective acquisition successful diagnosing and treating children with CD. Enrollment of survey participants occurred successful 2012 oregon earlier, e.g., erstwhile the intestinal biopsy was the aureate modular for diagnosis of CD, meaning that besides children with precise precocious levels of IgA-tTG were included successful the cohort. Moreover, we antecedently had each intestinal biopsies reviewed and scored histologically by a azygous pathologist blinded to the objective and serological information earlier the investigation to trim reflection bias and imaginable hazard of discordant classification of cases and controls betwixt the sites.

The results from the contiguous survey rise respective questions. It is not wide whether the downregulation of the people genes is circumstantial for CD oregon related to intestinal inflammation successful general. The relation betwixt the cistron portion containing GLS and different autoimmune traits, galore of which amusement an accrued hazard of metabolic disease, could suggest that the downregulation of IGN could besides beryllium contiguous successful different inflammatory diseases. Thus, further studies of IGN successful different diseases are warranted. Also, the survey does not reply if impaired IGN is progressive successful the hazard of processing the illness oregon if it is simply a effect to different disease-initiating mechanisms successful CD. In addition, we person not explored if changes successful the microbiota could perchance person an effect connected the cistron look oregon if the IGN look is affected by assemblage wide scale and glucose levels. Moreover, portion we spot a important correlation betwixt decreased IGN cistron look and the grade of harm successful the mucosa, the survey does not reply if attraction with a gluten-free fare starring to the healing of the intestinal mucosa restores the look of genes progressive successful IGN. Furthermore, it could beryllium of involvement to research whether these genes are downregulated successful different intestinal mucosal diseases specified arsenic immune deficiency disorders oregon autoimmune enteropathy.

References

Authors and Affiliations

1. Department of Laboratory Medicine, Institute of Biomedicine, Sahlgrenska Academy astatine the University of Gothenburg, Gothenburg, Sweden

   Olof Karlson & Åsa Torinsson Naluai

2. Department of Pediatric Gastroenterology, Hepatology and Nutrition, Astrid Lindgren Children’s Hospital, Karolinska University Hospital, Stockholm, Sweden

   Henrik Arnell

3. Department of Women’s and Children’s Health, Karolinska Institute, Stockholm, Sweden

   Henrik Arnell

4. Department of Paediatric Gastroenterology, Hepatology and Nutrition, Queen Silvia Children’s Hospital, Sahlgrenska University Hospital, Gothenburg, Sweden

   Audur H. Gudjonsdottir

5. Department of Clinical Sciences, Unit of Celiac Disease and Diabetes, Lund University, Malmö, Sweden

   Daniel Agardh

Contributions

The authors work and approved the last manuscript. OK and ÅTN conceived and carried retired the experiments, performed the information analysis, and wrote the manuscript. HA, AHG, and DA collected the biopsy material. KB, astatine the Department of Pathology, Unilabs, Stockholm, Sweden, performed the blinded reappraisal of each pathohistological diagnoses. AHG and HA revised the manuscript. DA suggested the approaches for information investigation and revised the manuscript.

Corresponding author

Correspondence to Åsa Torinsson Naluai.

Ethics support and consent to participate

We corroborate each applicable ethical guidelines person been followed and informed consents from survey participants and IRB and/or morals committee approvals person been obtained from the determination morals committee (number T373-10).

Patient and nationalist involvement: Patients and/or the nationalist did not instrumentality portion successful planning, performing, oregon reporting this research.

Consent for publication

Not required.

Competing interests

The authors state that they person nary competing interests.

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