Skip to main content
Download PDF

Plasma biomarkers of small intestine adaptations in obesity-related metabolic alterations

Diabetology & Metabolic Syndrome volume 12, Article number: 31 (2020) Cite this article

Abstract

Background

Evidence suggests that pathophysiological conditions such as obesity and type 2 diabetes (T2D) are associated with morphologic and metabolic alterations in the small intestinal mucosa. Exploring these alterations generally requires invasive methods, limiting data acquisition to subjects with enteropathies or undergoing bariatric surgery. We aimed to evaluate small intestine epithelial cell homeostasis in a cohort of men covering a wide range of adiposity and glucose homoeostasis statuses.

Methods

Plasma levels of citrulline, a biomarker of enterocyte mass, and I-FABP, a biomarker of enterocyte death, were measured by UHPLC‑MS and ELISA in 154 nondiabetic men and 67 men with a T2D diagnosis.

Results

Plasma citrulline was significantly reduced in men with insulin resistance and T2D compared to insulin sensitive men. Decreased citrulline levels were, however, not observed in men with uncontrolled metabolic parameters during T2D. Plasma I-FABP was significantly higher in men with T2D, especially in presence of uncontrolled glycemic and lipid profile parameters. Integration of both parameters, which estimate enterocyte turnover, was associated with glucose homeostasis as well as with T2D diagnosis. Differences in biomarkers levels were independent of age and BMI and glucose filtration rates.

Conclusions

Our study supports a decreased functional enterocyte mass and an increased enterocyte death rate in presence of metabolic alterations but emphasizes that epithelial cell homeostasis is especially altered in presence of severe insulin resistance and T2D. The marked changes in small intestine cellularity observed in obesity and diabetes are thus suggested to be part of gut dysfunctions, mainly at an advanced stage of the disease.

Background

The small intestine is capable of important morphological and metabolic adaptations to respond to rapid changes in nutrient intake, stress or partial resection [1,2,3]. Recent evidence underlines inadequate intestinal adaptations in pathophysiological conditions such as obesity and type 2 diabetes (T2D). Obesity is associated with an increased small intestine mucosa surface (i.e., enterocyte mass, crypt depth and villi length) as well as an increased immune cell population [4, 5]. In presence of obesity and type 2 diabetes, small intestine metabolic functions also present several alterations such as increased epithelial permeability, oxidative stress damage and exaggerated secretion of triglyceride-rich lipoproteins [6,7,8]. Difficulties to obtain fresh small intestine samples from subjects devoid of enteropathies or severe obesity complicate the elucidation of the effect of weight, glucose homeostasis and lipid profile on small intestine morphology and metabolic functions. Thus, previous literature on small intestine morphologic and metabolic adaptations in obesity-related conditions is only considering severely obese population. Development of biomarkers may therefore represent an interesting strategy to assess these small intestine parameters in individuals with a large spectrum of adiposity and glucose homeostasis.

Citrulline is a key intermediate in the urea cycle but liver remains a negligible contributor to its circulating levels. Enterocytes are the main site of citrulline biosynthesis from dietary and plasma amino acid precursors (i.e., arginine, ornithine, glutamine, glutamate or proline) [9,10,11,12,13]. Citrulline catabolism remains stable in absence of kidney disease and the small intestine therefore appears as the most important determinant of plasma citrulline levels. Reduced plasma citrulline levels were also documented in conditions characterized with lower enterocyte mass such as celiac disease, acquired immunodeficiency syndrome (AIDS), small intestine transplantation and sepsis-induced intestinal dysfunctions [14,15,16,17,18,19].

Fatty acid binding proteins (FABP) are a family of proteins facilitating intracellular fatty acid transport. Expression of the intestinal-specific FABP (I-FABP) is strictly limited to the small intestine mucosa. Significant amount of I-FABP is observed in circulation and this phenomenon is attributed to the release of enterocyte cytosolic content upon apoptosis. Indeed, small intestine mucosa presents an elevated cellular turnover regenerating the epithelium every 3 to 5 days. Higher I-FABP circulating levels are observed in conditions associated with a higher enterocyte death rate such as intestinal ischemia, hematopoietic stem-cell transplant, necrotizing enterocolitis and sepsis-induced intestinal dysfunctions [16, 20,21,22].

Attempts to assess small intestine mucosa adaptations in the whole spectrum of adiposity and glucose homeostasis remain incomplete in human as only samples from subjects with severe obesity and late-stage T2D have been studied. Citrulline and I-FABP are interesting non-invasive biomarkers to explore small intestine mucosa morphology and local cellular turnover in the context of obesity and metabolic diseases. The aim of the study is to evaluate small intestine epithelial cell homeostasis in a cohort of men covering a wide range of adiposity and glucose homoeostasis statuses. We hypothesized that circulating citrulline and I-FABP levels can highlight small intestine mucosa morphologic alterations associated with adiposity and metabolic diseases.

Methods

Study population

The study cohort included 221 men covering a wide range of adiposity and glucose homeostasis values. Participants were recruited through the Institute of Nutrition and Functional Food (INAF) clinical unit (Laval University) in the context of several research projects [23,24,25,26,27,28,29,30,31,32,33,34]. Exclusion criteria included smoking, cardiovascular disease, cancer history, monogenic lipid disorder, altered endocrine or hepatic function, gastrointestinal disease, HIV infection, alcoholism and severe dyslipidemia. The study cohort included 154 nondiabetic men and 67 men previously diagnosed with T2D. To be part of the study, subject with T2D had to have received stable doses of metformin for at least 3 months before randomization of the clinical trials. Also, metformin had to be the only antidiabetic drug for at least 6 weeks before the beginning of the study. Subjects with diabetes had to be withdrawn from lipid-lowering medications during the run-in period of the clinical trials, the moment at which the samples were collected for this study. These projects were approved by the ethics committee of Laval University Medical Research Center.

Anthropometric, lipid profile and glucose homeostasis

Baseline anthropometric data and overnight fasting blood were obtained during screening visits at INAF’s clinical investigation unit. Plasma was collected in EDTA tubes after centrifugation (3000 RPM, 10 min) and stored at − 80 °C. Glucose was assessed using the glucose oxidase method and insulin was quantified by electrochemiluminescence (Roche Diagnostics). The HOMA-IR index was calculated using the following formula: fasting insulin (µU/mL) × fasting glucose (mmol/L) ÷ 22.5 [35]. Serum cholesterol and triglyceride concentrations were determined with a Roche/Hitachi modular analyzer (Roche Diagnostics). The glomerular filtration rate (GFR) was calculated with the Chronic Kidney Disease Epidemiology Collaboration equation: 141 × min (serum creatinine (mg/dL))−0.411 × max (serum creatinine (mg/dL))−1.209 × 0.993Age [36].

I-FABP

Plasma I-FABP levels were measured in duplicate using the Human I-FABP DuoSet ELISA kit (R&D Systems, Minneapolis, MN) with some modifications to the manufacturer recommendation. Samples were diluted 1/20 in assay diluent (5 mM Tris, 150 mM NaCl,2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, 0.2 mM EDTA, 0.01% SDS, 0.05% sodium deoxycholate, 0.1% Triton X-100 and 2% BSA). Intra- and inter-assay coefficients were 6.6% (n = 223) and 10.6% (n = 20), respectively. The limits of detection (LOD) and limit of quantification (LOQ) were respectively of 80 and 300 pg/mL.

Plasma amino acid profile

Plasma amino acids were extracted and derivatized using the EZ: FAAST kit (Phenomenex, USA) as specified by the manufacturer. Along with other internal standards, deuterated citrulline (100 μM) was included in the assay to optimize citrulline quantification in duplicate. Amino acid profiles were achieved using UHPLC coupled to Tandem Quadrupole Mass spectrometry (Acquity H-Class, Xevo TQD, Waters corp., Mildford, MA). Intra- and inter-assay coefficients were lower than 15% for all amino acids and were respectively of 2.6% (n = 244) and 3.7% (n = 18) for citrulline. Citrulline levels were above the LOQ (0.1 µmol/L) for all samples.

Intestinal biopsies and gene expression

In a subset of nondiabetic men (n = 101), biopsies were collected from the second portion of the duodenum during gastroduodenoscopy in the fasting state [37]. Samples (3 × 3 mm) were collected using single-use biopsy forceps, immediately flash-frozen in liquid nitrogen, and stored at − 80 °C before RNA extraction. Intestinal tissue samples were homogenized in 1 mL of Qiazol (Qiagen, Hilden, Germany). RNA was extracted using a RNeasy kit (Qiagen). To eliminate any contaminating DNA, biopsies were treated with an RNase-free DNase set. Primer sequences for FABP2 were 5′‑TCAGGCTGGAATGTAGTGGAGAGA-3′ and 5′-CAAAACAAAAATTAGCTGGGCACTG-3′. Among housekeeping genes available in the data set, GDP6 was selected as it was not associated with any parameters analyzed [37].

Statistical analyses

Differences in parameters between groups were tested using t-test and one-way ANOVA. Pearson’s correlation coefficients were computed to test plasma biomarkers associations with anthropometric and biochemical parameters. Potential confounding variables were assessed using generalized linear models. Non-normally distributed variables (Shapiro–Wilk p ≤ 0.05) were log10 or Box-Cox transformed. Differences between groups and associations were considered statistically significant at p < 0.05. Statistical analyses were performed using JMP Pro 12 software (SAS Institute, Cary, NC, USA).

Results

Plasma I-FABP and citrulline levels were measured in a study sample of 221 lean to moderately obese men. Anthropometric and metabolic parameters of the study cohort are shown in Table 1. Men without diabetes (n = 154) were 37.6 ± 11.7 years old on average and were overweight according to a mean BMI of 29.8 ± 5.2 kg/m2. However, the sample was covering a wide range of BMI (20.0 to 43.1 kg/m2) and glucose homeostasis (HOMA-IR index: 0.8 to 10.0) values. Among these nondiabetics, we have identified 106 men with insulin resistance (IR), as defined by an HOMA-IR index greater than 2.5. These men with IR were mostly obese (31.5 ± 4.6 kg/m2) and characterized by an unfavorable glucose homeostasis and lipid profile (p < 0.05) as compared to men with relatively high insulin sensitivity (IS) (HOMA-IR index < 2.5; n = 48; Table 1). Men with T2D were older (56.8 ± 6.3 years) and mostly obese (BMI: 31.2 ± 4.9 kg/m2) on average. Compared to nondiabetic men, they were also characterized by an elevated fasting glycemia (8.5 ± 2.1 mmol/L) and a deteriorated plasma lipid profile.

Table 1 Physical and metabolic characteristics of the study sample (n = 221 men)
Full size table

Plasma amino acid profile

Total amount of plasma amino acids remains unaltered in men across the range of adiposity and glucose homeostasis status (Table 2). Plasma levels of branched amino acids were significantly increased in men with IR (466 ± 92 µmol/L) and T2D (479 ± 129 µmol/L) compared to men with IS (430 ± 76 µmol/L; p < 0.05) as previously reported [38, 39]. Plasma levels of branched amino acids were positively associated with BMI, waist circumference, HOMA-IR, TG and C-reactive protein (0.19 < r < 0.25; n = 153; p < 0.05) in subjects without diabetes. Similarly, aromatic amino acid levels were higher in men with IR and T2D compared to men with IS and positively associated with BMI, HOMA-IR and TG (0.22 < r < 0.30; n = 153; p < 0.05) in these subjects, which is also concordant with previous study. The complete plasma amino acid profile in each subgroup as well as individual correlation with HOMA-IR is included in Table 2.

Table 2 Plasma amino acid profile in men (n = 221)
Full size table

Citrulline

Considering that citrulline biosynthesis is relying on the enterocyte mass, we have further analyzed plasma levels of this amino acid in these subjects. Plasma citrulline levels of men with IR (24.1 ± 5.0 µmol/L) were lower than those of men with IS (26.5 ± 4.6 µmol/L; p < 0.01), but similar to those of men with a T2D diagnosis (23.3 ± 6.7 µmol/L; Fig. 1a). Within the T2D subgroup, plasma citrulline levels were lower in subjects presenting controlled HbA1c (< 6%) and TG (< 1.7 mmol/L) levels compared to subject with abnormal values in either one or both parameters (Fig. 1b). Stratification according to the hypertriglyceridemic waist phenotype (HTW: Waist circumference > 90 cm and TG > 1.7 mmol/L) reveals higher plasma citrulline levels in men with T2D and HTW, but this difference was not observed in men without diabetes.

Fig. 1
figure 1

Plasma citrulline levels associations with anthropometric and metabolic parameters in men. Mean plasma citrulline levels (± SEM) according to a glucose homeostasis status, b T2D diagnosis and metabolic control (HbA1c < 6% and TG < 1.7 mmol/L) and c hypertriglyceridemic waist phenotype (HTW). Plasma citrulline levels correlation between d BMI, e HOMA-IR in subjects without T2D and f TG levels. Pearson correlation coefficient and p values of log10 or Box-Cox transformed variables are indicated are shown. ap < 0.05 vs IS, nondiabetic or HTW; b p < 0.05 vs IR

Full size image

Plasma citrulline levels were negatively correlated with BMI (r = − 0.17; p < 0.05; n = 220), and GFR (r = − 0.25; p < 0.001; n = 215) in the whole sample of men (Fig. 1d, f). In nondiabetics, plasma citrulline levels were negatively associated with BMI, HOMA-IR index, CRP (− 0.20 > r > -0.17; p < 0.05; n = 118) and GFR (r = − 0.32; p < 0.001; n = 148; Fig. 1e). These associations remained significant in men without diabetes following adjustment for age and GFR, suggesting that the reduced plasma citrulline levels reflect a reduced small intestinal production rather than change in renal function. After further adjustment for BMI, plasma citrulline lost its association with HOMA-IR, but not with CRP (data not shown). In contrast, plasma citrulline levels in men with T2D were not associated with anthropometric and metabolic parameters except for a negative correlation with GFR (r = − 0.27; p < 0.01; n = 67). We performed linear regression models to identify independent predictor of plasma citrulline levels in men (Table 3). In the whole sample, GFR, BMI and T2D, explained 14.3% of the variance in plasma citrulline levels. When only men without diabetes were included in the analysis, GFR and HOMA‑IR explained more than 14% of citrulline level variance.

Table 3 Linear regression analyses predicting plasma biomarkers in men
Full size table

We then explored the possibility that the associations of plasma citrulline levels with adiposity and metabolic parameters could be mediated by overall changes in total plasma amino acids and citrulline precursors (i.e., arginine, ornithine, glutamine, glutamate and proline). Citrulline-to-precursor amino acids and citrulline-to-total amino acids ratios were lower in the plasma of men with IR and T2D than of men with IS (p < 0.05; Table 2). These ratios were also correlated with BMI and HOMA-IR (− 0.26 < r < − 0.18; n = 153; p < 0.01; data not shown) in men without diabetes following adjustment for GFR and age, which strengthen the independent associations observed between plasma citrulline, adiposity and glucose homeostasis.

As described in the Table 2, citrulline precursors were either increased (glutamate and proline) or remained unaltered (arginine, glutamine and ornithine) in presence of insulin resistance in nondiabetics (p < 0.05). We also observed lower plasma arginine and glutamine levels in men with T2D. In sum, arginine bioavailability ratio (arginine/ornithine + citrulline) was slightly reduced in men with T2D while arginase activity and arginase-to-NO activity ratio were increased in men with T2D (Table 2). Nevertheless, citrulline levels remains independently associated with glucose homeostasis when adjusted for these ratios. These results confirm previous studies and suggest that change in citrulline levels are not due to important changes in arginine bioavailability [38] (Fig. 2).

Fig. 2
figure 2

Plasma I-FABP levels associations with anthropometric and metabolic parameters in men. Mean I-FABP citrulline levels (± SEM) according to a glucose homeostasis status, b T2D diagnosis and metabolic control (HbA1c < 6% and TG < 1.7 mmol/L) and c hypertriglyceridemic waist phenotype (HTW). Plasma I-FABP levels correlation between d BMI, e HOMA-IR in subjects without T2D and f TG levels. Pearson correlation coefficient and p values of log10 or Box-Cox transformed variables are indicated are shown. ap < 0.05 vs IS, nondiabetic or HTW; bp < 0.05 vs IR

Full size image

Plasma I-FABP

Mean plasma I-FABP levels were significantly higher in men T2D (1879 ± 1056 pg/mL, p < 0.05) compared to men with IS (1269 ± 655 pg/mL) and IR (1434 ± 920 pg/mL; Fig. 2a). Interestingly, men with T2D with normal HbA1c (< 6%) or TG (< 1.7 mmol/L) showed similar plasma I-FABP levels than nondiabetics, while only those harboring an uncontrolled T2D had higher plasma I-FABP levels (Fig. 3b). The presence of insulin resistance, by itself, had no significant impact on plasma I-FABP levels in men without diabetes (Fig. 2a). In contrast, the hypertriglyceridemic waist phenotype was associated with significantly elevated levels of plasma I-FABP (Fig. 2c).

Fig. 3
figure 3

Plasma I-FABP-to-citrulline ratio associations with anthropometric and metabolic parameters in men. Mean plasma I-FABP-to-citrulline ratio (± SEM) according to a glucose homeostasis status, b T2D diagnosis and metabolic control (HbA1c < 6% and TG < 1.7 mmol/L) and c hypertriglyceridemic waist phenotype (HTW). Plasma I-FABP-to-citrulline ratio correlation between d BMI, e HOMA-IR in subjects without T2D and f TG levels. Pearson correlation coefficient and p values of log10 or Box-Cox transformed variables are indicated are shown. ap < 0.05 vs IS, nondiabetic or HTW; bp < 0.05 vs IR

Full size image

In the complete sample, plasma I-FABP levels correlated with CRP (r = − 0.17; p < 0.05; n = 184), fasting glucose (r = 0.25; p < 0.05; n = 220), and TG (r = 0.17; p < 0.05; n = 220), but not with BMI (Fig. 3d). As expected, these associations remained after adjustments for age and BMI. In men with T2D, plasma I-FABP correlated with CRP (r = − 0.34; p < 0.01; n = 67), and TG (r = 0.25; p < 0.05; n = 67; data not shown). In men without diabetes, plasma I-FABP showed no association with anthropometric and metabolic parameters. Linear regression models predicting plasma I-FABP levels in the complete sample of men reveal that T2D diagnosis explained 5.5% of the variance in plasma I-FABP (Table 3). In men without diabetes, there was no significant predictor of plasma I-FABP identified.

Duodenal FABP2 gene expression levels, available in a subset of the subjects without diabetes (n = 101), was not influenced by food intake, anthropometric or metabolic parameters (Additional file 1: Table S1). Plasma I-FABP levels correlate with duodenal FABP2 gene expression (r = 0.22; p < 0.05; n = 100) and this association was independent of adiposity and metabolic parameters. Adjustment of previous linear regression models with FABP2 gene expression lead to novel associations between circulating levels of I-FABP, BMI and HOMA-IR (p < 0.05). It suggests that changes in the plasma I-FABP pool are at least partly determined by duodenal expression levels, but its release tends to increase with BMI and glucose homeostasis alteration.

I-FABP-to-citrulline ratio

Plasma I-FABP-to-citrulline ratio was calculated to integrate both cell renewal and enterocyte cell mass biomarkers. Mean plasma I-FABP-to-citrulline ratio was higher in men with T2D than in men with IR and IS (Fig. 3a). In men with IR, this ratio was also increased compared to men with IS, but remained significantly lower than men with T2D. The lipid profile and glycemic status in men with T2D have no impact on the plasma I‑FABP-to-citrulline ratio (Fig. 3b). In contrast, the presence of an hypertriglyceridemic waist phenotype was associated with increased plasma I-FABP-to-citrulline ratio in men without diabetes (Fig. 3c).

In the complete sample, plasma I-FABP-to-citrulline ratio was significantly correlated with fasting glucose and TG (0.16 > r = 0.28; p < 0.05; n = 218; Fig. 3d, f). Correlations with fasting glucose and TG remained significant after adjustment for age, BMI and GFR. In men without diabetes, plasma I-FABP-to-citrulline ratio correlate with HOMA-IR (r = 0.16; p < 0.05; n = 152; Fig. 3e). Following adjustment for age and GFR, HOMA-IR remain significantly associated with plasma I-FABP-to-citrulline ratio (r = 0.18, p < 0.05, n = 152), but was not independent of BMI. Finally, linear regression models shown that T2D diagnosis was the only independent contributor to explain the variance in plasma I-FABP-to-citrulline ratio. In men without diabetes, HOMA-IR explained only 4.8% of the variance in plasma I-FABP-to-citrulline ratio.

Discussion

The portrait of small intestine mucosa adaptations in obesity and diabetes remains incomplete in human as only samples from subjects with severe disease states have been thoroughly characterized. Some studies have suggested the use of plasma citrulline and I-FABP levels as potential non-invasive biomarkers to explore small intestine mucosa morphology in enteropathies. We have investigated the associations of small intestine mucosa biomarkers with adiposity and metabolic parameters in a larger and well-characterized cohort of men (n = 221) covering a large spectrum of disease states. We found that plasma citrulline levels, a proxy of enterocyte mass, was negatively associated with adiposity, insulin resistance and diabetes but remain high in subjects with uncontrolled diabetes. Inversely, circulating levels of I-FABP, a proxy of enterocyte death rate, were higher in presence of insulin resistance and diabetes, particularly in subjects with inadequate glycemic and lipid control. These results suggest an increased enterocyte death rate with a lower absolute number of enterocytes in presence of insulin resistance. The increased enterocyte death rate was accompanied with a higher number of enterocytes in uncontrolled diabetic states, suggesting sustained cellular proliferation. Results also underscored the presence of alteration in severe insulin resistance and T2D conditions. This latter observation is in line with most studies reporting marked gut dysfunctions in late-stage disease models [8, 39,40,41,42].

Plasma citrulline levels are mainly determined by the enteric production rate from plasma amino acids precursors. The biosynthesis of citrulline per cell is constant, which led to the postulate that it reflects the absolute number of enterocytes. Indeed, contribution of the hepatic urea cycle is negligible on plasma citrulline levels and its excretion remain relatively stable in absence of kidney disease. Despite that none of the subjects were characterized with advanced kidney disease estimated with the CKD-EPI equation (GFR < 60 mL/min/1.73 m2), we still observed a correlation between the GFR and plasma citrulline levels. It suggests that even a physiologic reduction in kidney function tends to promote higher plasma citrulline levels. GFR is negatively associated with adiposity and metabolic complications, so that higher plasma citrulline levels may be expected in obese and diabetic subjects. In contrast to previous studies, all statistics have been adjusted for GFR to discriminate the independent contribution of small intestine mucosa of plasma citrulline levels. Indeed, kidney function may not impact the assessment of drastic change in small intestine mucosa mass (i.e., short-bowel syndrome, partial small intestine resection) as previously reported, but could counteract identification of subtle changes in small intestine mucosa mass such as those expected in obesity or diabetes.

Ramírez-Zamora et al. [43] highlighted an inverse correlation between plasma citrulline levels and glucose homeostasis in nondiabetic subjects. In contrast, positive associations between plasma citrulline, fasting glycemia and HbA1c in severely obese subjects were documented [4]. This study corroborates both observations, as citrulline levels were reduced in presence of glucose homeostasis alterations but elevated in diabetic subjects with abnormal fasting glucose and triglyceride levels. Nevertheless, men with T2D but normal TG and HbA1c values show lower citrulline levels than nondiabetic men. This observation may be related to the use of insulin sensitizing therapy which has been previously reported, in some case, as soon as the first utilization [44,45,46]. Without treatment, individuals with T2D were more likely to have higher citrulline levels than their nondiabetic counterpart [43]. In sum, T2D appears to be associated with increased small intestinal enterocyte mass especially in presence of elevated fasting glucose and triglyceride levels. Insulin sensitizing therapy appears to normalize small intestine enterocyte mass while this observation could be indirectly mediated by the availability of precursors to citrulline synthesis. In contrast, decreased insulin sensitivity in men without diabetes is apparently associated with reduced small intestine enteric mass.

Plasma levels of I-FABP are known to be a reliable biomarker of small intestine epithelial cell homeostasis in various enteropathies in adults and children [14, 21, 47]. Taking into consideration the very short half-life of this protein in circulation, the steady state circulating levels of I-FABP represents continuous release from senescent enterocytes. In our study, plasma I-FABP levels were elevated in men with T2D harboring uncontrolled glycemia and lipemia. These data are in line with Verdam et al. [4] observations in men with obesity and elevated glycated hemoglobin levels. These results put forward an increased cell death rate in presence of severe glucose homeostasis alterations. On the other hand, absence of association in men without T2D suggests that physiological variations in anthropometric measures, glucose homeostasis and metabolic profile have no impact on small intestine epithelial cell death. Correlation with FABP2 gene expression in the duodenum of these men without T2D further suggests that plasma I-FABP levels may be more related to enterocyte protein content than to the cell death rate itself.

The I-FABP-to-citrulline ratio was computed as an index of cellular turnover considering total enterocyte mass. Interestingly, the I-FABP-to-citrulline ratio significantly increased with impaired glucose homeostasis up to T2D states. This indicates that the enterocyte death rate is consistently more elevated than the small intestine mucosa mass in presence of metabolic complications. At equilibrium, this insinuates a higher cell renewal rate (i.e., increased cell death and differentiation rates) in presence of metabolic complications. This higher cell renewal rate is observed in both insulin-resistant and diabetic states while these conditions display distinct small intestine mucosa morphology.

Results of this study are strengthened by the use of a relatively large study samples of men covering a large spectrum of adiposity and glucose homeostasis status compared to previous studies. The present analyses are based on cross-sectional data and the difficulty to obtain intestinal samples from subjects without enteropathies limits the possibility of direct measurements. While it is established that 80–90% of the citrulline is derived from enterocytes [48], we cannot completely exclude that other secondary source of citrulline may explain these results. Finally, since we only had access to a well-characterized cohort of men, we cannot extrapolate our results to women small intestine morphologies. Nevertheless, no sex dimorphism was reported to date on I-FABP and citrulline levels as well as on small intestine mucosa morphology.

Conclusion

In conclusion, our results support the hypothesis of a decreased functional enterocyte mass and a higher enterocyte death rate in presence of metabolic alterations. We also emphasized that small intestine epithelial cell homeostasis is especially altered in presence severe insulin resistance and T2D conditions. The marked changes in small intestine cellularity observed in obesity and diabetes are thus suggested to be part of gut dysfunctions, mainly at an advanced stage of the disease.

Availability of data and materials

The datasets analysed during the current study are available from the corresponding author on reasonable request.

References

  1. Dailey MJ. Nutrient-induced intestinal adaption and its effect in obesity. Physiol Behav. 2014;136:74–8.

    Article  CAS  PubMed  Google Scholar 

  2. Hutch CR, Sandoval DA. Physiological and molecular responses to bariatric surgery: markers or mechanisms underlying T2DM resolution? Ann N Y Acad Sci. 2017;1391:5–19.

    Article  PubMed  Google Scholar 

  3. Shaw D, Gohil K, Basson MD. Intestinal mucosal atrophy and adaptation. World J Gastroenterol WJG. 2012;18:6357–75.

    Article  PubMed  Google Scholar 

  4. Verdam FJ, Greve JWM, Roosta S, van Eijk H, Bouvy N, Buurman WA, et al. Small intestinal alterations in severely obese hyperglycemic subjects. J Clin Endocrinol Metab. 2010;96:E379–83.

    Article  PubMed  CAS  Google Scholar 

  5. Monteiro-Sepulveda M, Touch S, Mendes-Sá C, André S, Poitou C, Allatif O, et al. Jejunal T cell inflammation in human obesity correlates with decreased enterocyte insulin signaling. Cell Metab. 2015;22:113–24.

    Article  CAS  PubMed  Google Scholar 

  6. Brun P, Castagliuolo I, Leo VD, Buda A, Pinzani M, Palù G, et al. Increased intestinal permeability in obese mice: new evidence in the pathogenesis of nonalcoholic steatohepatitis. Am J Physiol Gastrointest Liver Physiol. 2007;292:G518.

    Article  CAS  PubMed  Google Scholar 

  7. Rani V, Deep G, Singh RK, Palle K, Yadav UCS. Oxidative stress and metabolic disorders: pathogenesis and therapeutic strategies. Life Sci. 2016;148:183–93.

    Article  CAS  PubMed  Google Scholar 

  8. Veilleux A, Mayeur S, Bérubé J-C, Beaulieu J-F, Tremblay E, Hould F-S, et al. Altered intestinal functions and increased local inflammation in insulin-resistant obese subjects: a gene-expression profile analysis. BMC Gastroenterol. 2015;15:119.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  9. Tomlinson C, Rafii M, Ball RO, Pencharz PB. Arginine can be synthesized from enteral proline in healthy adult humans. J Nutr. 2011;141:1432–6.

    Article  CAS  PubMed  Google Scholar 

  10. Marini JC, Agarwal U, Didelija IC, Azamian M, Stoll B, Nagamani SC. Plasma glutamine is a minor precursor for the synthesis of citrulline: a multispecies study. J Nutr. 2017;147:549–55.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Rabier D, Kamoun P. Metabolism of citrulline in man. Amino Acids. 1995;9:299–316.

    Article  CAS  PubMed  Google Scholar 

  12. Neis EPJG, Sabrkhany S, Hundscheid I, Schellekens D, Lenaerts K, Olde Damink SW, et al. Human splanchnic amino-acid metabolism. Amino Acids. 2017;49:161–72.

    Article  CAS  PubMed  Google Scholar 

  13. Breuillard C, Cynober L, Moinard C. Citrulline and nitrogen homeostasis: an overview. Amino Acids. 2015;47:685–91.

    Article  CAS  PubMed  Google Scholar 

  14. Hoffmanová I, Sánchez D, Hábová V, Anděl M, Tučková L, Tlaskalová-Hogenová H. Serological markers of enterocyte damage and apoptosis in patients with celiac disease, autoimmune diabetes mellitus and diabetes mellitus type 2. Physiol Res. 2015;64:537–46.

    PubMed  Google Scholar 

  15. Papadia C, Sherwood RA, Kalantzis C, Wallis K, Volta U, Fiorini E, et al. Plasma citrulline concentration: a reliable marker of small bowel absorptive capacity independent of intestinal inflammation. Am J Gastroenterol. 2007;102:1474–82.

    Article  CAS  PubMed  Google Scholar 

  16. Shen L-J, Guan Y-Y, Wu X-P, Wang Q, Wang L, Xiao T, et al. Serum citrulline as a diagnostic marker of sepsis-induced intestinal dysfunction. Clin Res Hepatol Gastroenterol. 2015;39:230–6.

    Article  CAS  PubMed  Google Scholar 

  17. David AI, Selvaggi G, Ruiz P, Gaynor JJ, Tryphonopoulos P, Kleiner GI, et al. Blood citrulline level is an exclusionary marker for significant acute rejection after intestinal transplantation. Transplantation. 2007;84:1077–81.

    Article  CAS  PubMed  Google Scholar 

  18. Lutgens L, Lambin P. Biomarkers for radiation-induced small bowel epithelial damage: an emerging role for plasma citrulline. World J Gastroenterol. 2007;13:3033–42.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Crenn P, Truchis PD, Neveux N, Galpérine T, Cynober L, Melchior JC. Plasma citrulline is a biomarker of enterocyte mass and an indicator of parenteral nutrition in HIV-infected patients. Am J Clin Nutr. 2009;90:587–94.

    Article  CAS  PubMed  Google Scholar 

  20. Voth M, Holzberger S, Auner B, Henrich D, Marzi I, Relja B. I-FABP and L-FABP are early markers for abdominal injury with limited prognostic value for secondary organ failures in the post-traumatic course. Clin Chem Lab Med CCLM FESCC. 2015;53:771–80.

    CAS  Google Scholar 

  21. Vreugdenhil AC, Wolters VM, Adriaanse MP, Van den Neucker AM, van Bijnen AA, Houwen R, et al. Additional value of serum I-FABP levels for evaluating celiac disease activity in children. Scand J Gastroenterol. 2011;46:1435–41.

    Article  CAS  PubMed  Google Scholar 

  22. Lieberman JM, Sacchettini J, Marks C, Marks WH. Human intestinal fatty acid binding protein: report of an assay with studies in normal volunteers and intestinal ischemia. Surgery. 1997;121:335–42.

    Article  CAS  PubMed  Google Scholar 

  23. Tremblay AJ, Lamarche B, Guay V, Charest A, Lemelin V, Couture P. Short-term, high-fat diet increases the expression of key intestinal genes involved in lipoprotein metabolism in healthy men. Am J Clin Nutr. 2013;98:32–41.

    Article  CAS  PubMed  Google Scholar 

  24. Tremblay AJ, Lamarche B, Labonte M-E, Lepine M-C, Lemelin V, Couture P. Dietary medium-chain triglyceride supplementation has no effect on apolipoprotein B-48 and apolipoprotein B-100 kinetics in insulin-resistant men. Am J Clin Nutr. 2014;99:54–61.

    Article  CAS  PubMed  Google Scholar 

  25. Couture P, Tremblay AJ, Kelly I, Lemelin V, Droit A, Lamarche B. Key intestinal genes involved in lipoprotein metabolism are downregulated in dyslipidemic men with insulin resistance. J Lipid Res. 2014;55:128–37.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Tremblay AJ, Lamarche B, Kelly I, Charest A, Lépine M-C, Droit A, et al. Effect of sitagliptin therapy on triglyceride-rich lipoprotein kinetics in patients with type 2 diabetes. Diabetes Obes Metab. 2014;16:1223–9.

    Article  CAS  PubMed  Google Scholar 

  27. Hogue J-C, Lamarche B, Deshaies Y, Tremblay AJ, Bergeron J, Gagné C, et al. Differential effect of fenofibrate and atorvastatin on in vivo kinetics of apolipoproteins B-100 and B-48 in subjects with type 2 diabetes mellitus with marked hypertriglyceridemia. Metabolism. 2008;57:246–54.

    Article  CAS  PubMed  Google Scholar 

  28. Hogue J-C, Lamarche B, Tremblay AJ, Bergeron J, Gagné C, Couture P. Differential effect of atorvastatin and fenofibrate on plasma oxidized low-density lipoprotein, inflammation markers, and cell adhesion molecules in patients with type 2 diabetes mellitus. Metabolism. 2008;57:380–6.

    Article  CAS  PubMed  Google Scholar 

  29. Tremblay AJ, Lamarche B, Lemelin V, Hoos L, Benjannet S, Seidah NG, et al. Atorvastatin increases intestinal expression of NPC1L1 in hyperlipidemic men. J Lipid Res. 2011;52:558–65.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Tremblay AJ. Effect of ezetimibe on the in vivo kinetics of ApoB-48 and ApoB-100 in men with primary hypercholesterolemia. Arterioscler Thromb Vasc Biol. 2006;26:1101–6.

    Article  CAS  PubMed  Google Scholar 

  31. Tremblay AJ, Lamarche B, Hogue J-C, Couture P. Effects of ezetimibe and simvastatin on apolipoprotein B metabolism in males with mixed hyperlipidemia. J Lipid Res. 2009;50:1463–71.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Drouin-Chartier J-P, Tremblay AJ, Lemelin V, Lépine M-C, Lamarche B, Couture P. Ezetimibe increases intestinal expression of the LDL receptor gene in dyslipidaemic men with insulin resistance. Diabetes Obes Metab. 2016;18:1226–35.

    Article  CAS  PubMed  Google Scholar 

  33. Drouin-Chartier J-P, Gigleux I, Tremblay AJ, Poirier L, Lamarche B, Couture P. Impact of dairy consumption on essential hypertension: a clinical study. Nutr J. 2014;13:83.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  34. Tremblay AJ, Lamarche B, Ruel IL, Hogue J-C, Bergeron J, Gagné C, et al. Increased production of VLDL apoB-100 in subjects with familial hypercholesterolemia carrying the same null LDL receptor gene mutation. J Lipid Res. 2004;45:866–72.

    Article  CAS  PubMed  Google Scholar 

  35. Matthews DR, Hosker JP, Rudenski AS, Naylor BA, Treacher DF, Turner RC. Homeostasis model assessment: insulin resistance and beta-cell function from fasting plasma glucose and insulin concentrations in man. Diabetologia. 1985;28:412–9.

    Article  CAS  PubMed  Google Scholar 

  36. Levey AS, Stevens LA, Schmid CH, Zhang YL, Castro AF, Feldman HI, et al. A new equation to estimate glomerular filtration rate. Ann Intern Med. 2009;150:604–12.

    Article  PubMed  PubMed Central  Google Scholar 

  37. Drouin-Chartier J-P, Tremblay AJ, Lemelin V, Lamarche B, Couture P. Differential associations between plasma concentrations of insulin and glucose and intestinal expression of key genes involved in chylomicron metabolism. Am J Physiol-Gastrointest Liver Physiol. 2018;315:G177–84.

    Article  CAS  PubMed  Google Scholar 

  38. Moon J, Kim OY, Jo G, Shin M-J. Alterations in circulating amino acid metabolite ratio associated with arginase activity are potential indicators of metabolic syndrome: The Korean Genome and Epidemiology Study. Nutrients. 2017;9:740.

    Article  PubMed Central  CAS  Google Scholar 

  39. Levy E, Spahis S, Ziv E, Marette A, Elchebly M, Lambert M, et al. Overproduction of intestinal lipoprotein containing apolipoprotein B-48 in Psammomys obesus: impact of dietary n-3 fatty acids. Diabetologia. 2006;49:1937–45.

    Article  CAS  PubMed  Google Scholar 

  40. Federico LM, Naples M, Taylor D, Adeli K. Intestinal insulin resistance and aberrant production of apolipoprotein B48 lipoproteins in an animal model of insulin resistance and metabolic dyslipidemia: evidence for activation of protein tyrosine phosphatase-1B, extracellular signal-related kinase, and sterol regulatory element-binding protein-1c in the fructose-fed hamster intestine. Diabetes. 2006;55:1316–26.

    Article  CAS  PubMed  Google Scholar 

  41. Haidari M. Fasting and postprandial overproduction of intestinally derived lipoproteins in an animal model of insulin resistance. J Biol Chem. 2002;277:31646–55.

    Article  CAS  PubMed  Google Scholar 

  42. Lewis GF, Uffelman K, Naples M, Szeto L, Haidari M, Adeli K. Intestinal lipoprotein overproduction, a newly recognized component of insulin resistance, is ameliorated by the insulin sensitizer rosiglitazone: studies in the fructose-fed Syrian golden hamster. Endocrinology. 2005;146:247–55.

    Article  CAS  PubMed  Google Scholar 

  43. Ramírez-Zamora S, Méndez-Rodríguez ML, Olguín-Martínez M, Sánchez-Sevilla L, Quintana-Quintana M, García-García N, et al. Increased erythrocytes by-products of arginine catabolism are associated with hyperglycemia and could be involved in the pathogenesis of type 2 diabetes mellitus. PLoS ONE. 2013;8:e66823.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  44. Adam J, Brandmaier S, Leonhardt J, Scheerer MF, Mohney RP, Xu T, et al. Metformin effect on nontargeted metabolite profiles in patients with type 2 diabetes and in multiple murine tissues. Diabetes. 2016;65:3776–85.

    Article  CAS  PubMed  Google Scholar 

  45. Breier M, Wahl S, Prehn C, Ferrari U, Sacco V, Weise M, et al. Immediate reduction of serum citrulline but no change of steroid profile after initiation of metformin in individuals with type 2 diabetes. J Steroid Biochem Mol Biol. 2017. https://doi.org/10.1016/j.jsbmb.2017.08.004.

    Article  PubMed  Google Scholar 

  46. Irving BA, Carter RE, Soop M, Weymiller A, Syed H, Karakelides H, et al. Effect of insulin sensitizer therapy on amino acids and their metabolites. Metabolism. 2015;64:720–8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Piton G, Manzon C, Cypriani B, Carbonnel F, Capellier G. Acute intestinal failure in critically ill patients: is plasma citrulline the right marker? Intensive Care Med. 2011;37:911–7.

    Article  PubMed  Google Scholar 

  48. van de Poll MCG, Ligthart-Melis GC, Boelens PG, Deutz NEP, van Leeuwen PAM, Dejong CHC. Intestinal and hepatic metabolism of glutamine and citrulline in humans. J Physiol. 2007;581:819–27.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

Download references

Acknowledgements

We thank all the participants, study coordinators and nurses at INAF’s clinical investigation units.

Funding

The study was supported by operating funds from Diabète Québec and Canadian Institute of Health Research (CIHR) to A.V.. C.L. was supported by a studentship from the Diabète Québec. A.V is the recipient of a junior scholarship from the Fonds de la Recherche en Santé du Québec.

Author information

Authors and Affiliations

  1. École de nutrition, Faculté des sciences de l’agriculture et de l’alimentation, Université Laval, 2440, boulevard Hochelaga, Québec, QC, G1V 0A6, Canada

    Catherine Lalande, Jean-Philippe Drouin-Chartier & Alain Veilleux

  2. Centre Nutrition, santé et société (NUTRISS), Institut sur la nutrition et les aliments fonctionnels (INAF), Université Laval, Québec, QC, Canada

    Catherine Lalande, Jean-Philippe Drouin-Chartier, André J. Tremblay, Patrick Couture & Alain Veilleux

  3. Centre des maladies lipidiques, Centre Hospitalier Universitaire (CHU) de Québec, Québec, QC, Canada

    Patrick Couture

  4. Centre de Recherche de l’Institut Universitaire de Cardiologie et de Pneumologie de Québec, Québec, QC, Canada

    Alain Veilleux

  5. Canada Excellence Research Chair in the Microbiome-Endocannabinoidome Axis in Metabolic Health, Québec, QC, Canada

    Alain Veilleux

Authors
  1. Catherine Lalande
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jean-Philippe Drouin-Chartier
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. André J. Tremblay
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Patrick Couture
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Alain Veilleux
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

CL performed laboratory work and prepared the manuscript. JPDC performed work related to small intestine biopsies. AJT and PC coordinated the clinical studies. AV designed the study, performed data interpretation and prepared the manuscript. All authors revised and approved the final manuscript.

Corresponding author

Correspondence to Alain Veilleux.

Ethics declarations

Ethics approval and consent to participate

These projects were approved by the ethics committee of Laval University Medical Research Center.

Consent for publication

All authors have significantly contributed to the work, approved the current version and agreed to submit the manuscript.

Competing interests

The authors declare that they have no competing interests.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Additional file 1: Table S1.

Physical and metabolic characteristics of samples of men with intestinal biopsies (n = 101).

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Lalande, C., Drouin-Chartier, JP., Tremblay, A.J. et al. Plasma biomarkers of small intestine adaptations in obesity-related metabolic alterations. Diabetol Metab Syndr 12, 31 (2020). https://doi.org/10.1186/s13098-020-00530-6

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/s13098-020-00530-6

Keywords

Diabetology & Metabolic Syndrome

ISSN: 1758-5996

Contact us