Abstract

Background

Bile acids (BAs) and BA receptors emerge as new modulators of glucose homeostasis [1]. FXR (farnesoid × receptor) is a nuclear receptor that controls BA and lipid metabolism [2,3]. Several studies have highlighted an unexpected role of FXR in metabolic homeostasis [4,5]. While FXR-deficient mice exhibit insulin resistance [6,7], treatment of ob/ob and db/db diabetic mice with a synthetic FXR agonist improves blood glucose levels as well as insulin sensitivity [6,8]. However, the picture is more complex since FXR deficiency improves glucose homeostasis and attenuates body weight gain in an obese genetic background [9].

The addition of cholic acid (CA) to the diet increases energy expenditure and prevents the development of high fat-induced obesity and insulin resistance in mice [10]. This metabolic effect of CA is mediated by the binding to the G-protein-coupled receptor TGR5 [11,12], leading to the induction of the cAMP-dependent thyroid hormone activation enzyme DIO2 (type 2 iodothyronine deiodinase) in brown adipose tissue [10]. Human TGR5 is activated by several BAs, with lithocholic acid (LCA) and deoxycholic acid (DCA) being the most potent natural agonists [12]. In addition, the activation of TGR5 by a semi-synthetic specific agonist induces GLP-1 secretion and improves glucose tolerance in mice [13].

The aim of the present study was to ascertain whether plasma BA concentrations are linked to insulin sensitivity, assessed both by the homeostasis model assessment (HOMA-IR) and the gold-standard hyperinsulinemic-euglycemic clamp method, in a variety of subjects including healthy volunteers, obese and type 2 diabetic (T2D) patients.

Methods

Results

Circulating bile acids and glucose homeostasis

Total plasma BAs concentrations were higher in both obese and T2D in comparison with the healthy controls, but the difference reached statistical significance only for the obese subjects compared to controls (Table ​(Table1).1). Plasma DCA was significantly higher in T2D compared to control group, while plasma CA was significantly increased in obese patients compared to controls.

Linear simple regression analyses (Table ​(Table2)2) indicated that plasma BAs concentrations negatively correlated with insulin sensitivity. Indeed, DCA, CDCA and CA plasma concentrations were positively associated with fasting plasma insulin levels and HOMA-IR. To further assess insulin sensitivity with the gold standard method, we measured the GIR during a hyperinsulinemic-euglycemic clamp. Importantly, the GIR was inversely associated with plasma CDCA (r = -0.36, p = 0.05 for Center 1; r = -0.44, p = 0.03 for Center 2) and CA (r = -0.38, p = 0.04 for Center 1; r = -0.65, p = 0.001 for Center 2) levels; while the negative correlation with the DCA did not reach statistical significance (r = -0.30, p = 0.10 for Center 1; r = -0.35, p = 0.08 for Center 2) (Figure 1A-B). Except for a positive correlation between DCA and fasting blood glucose levels, no association was detected between BAs and the glycaemic status (Table ​(Table22).

Table 2

Linear simple regression analysis on plasma bile acid concentrations and variables

	DCA			CDCA			CA
	β	95% confidence interval	p-value	β	95% confidence interval	p-value	β	95% confidence interval	p-value
Age	0.01	[0.002;0.02]	0.02	0.001	[-0.02;0.02]	NS	0.004	[-0.003;0.01]	NS
Men	0.002	[-0.19;0.19]	NS	0.39	[0.04;0.74]	0.03	0.17	[0.03;0.31]	0.02
BMI	0.02	[0.0006;0.04]	0.04	0.04	[0.004;0.07]	0.03	0.01	[-0.003;0.03]	NS
Fasting glucose	0.8	[0.03;0.12]	0.001	0.05	[-0,04;0.14]	NS	0.03	[-0.03;0.04]	NS
Fasting insulin	0.02	[0.01;0.03]	< 10-4	0.04	[0.02;0.06]	0.001	0.02	[0.006;0.02]	0.001
HbA1C	0.04	[-0.03;0.12]	NS	-0.05	[-0.18;0.08]	NS	-0.04	[-0.09;0.02]	NS
HOMA-R	0.07	[0.04;0.19]	< 10-4	0.09	[0.04;0.14]	0.001	0.03	[0.007;0.05]	0.09
BMR	0.0001	[-0.0002;0.0004]	NS	0.0008	[0.0002;0.001]	0.01	0.0002	[2.4*10-7 ;0.0005]	0.05

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Linear simple regression analyses were performed for estimation of association between plasma BAs concentrations and metabolic parameters. A p value ≤ 0.05 was considered statistically significant.

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Figure 1

Spearman correlation of plasma bile acids concentrations and insulin sensitivity in healthy controls (n = 14) (black triangle), type 2 diabetic (black square) (n = 20) and obese (white diamond) (n = 22) subjects. Plasma chenodeoxycholic acid (CDCA) and cholic acid (CA) concentrations were measured by GC-MS analysis. Insulin sensitivity was assessed by the rate of infused glucose necessary to maintain euglycaemia during infusion of insulin (GIR: glucose infusion rate). In the center 2 (A), the insulin infusion rate was 75 mU/m², while it was 40 mU/m² in the center 1 (B).

To further assess the strength of the correlation between circulating BAs levels and insulin sensitivity, a multiple regression analysis was performed (Table ​(Table3).3). The positive correlation of plasma BAs concentrations with HOMA-IR remained significant after adjustment for each categorical variable: CDCA (β = 0.116, p = 0.01), CA (β = 0.045, p = 0.01) DCA (β = 0.062, p = 0.007). In contrast, plasma BAs levels were neither associated with fasting plasma glucose nor the HbA1c.

Table 3

Linear multiple regression analysis on plasma bile acids concentrations and variables

	CDCA
	β	95% confidence interval	p-value
Center			NS
- Center 2	1
- Center 1	0.466	[-0.037;0.969]
Group			NS
-Controls	1
-Obeses	0.144	[-0.614;0.902]
-Diabetics	0.259	[-0.534;1.053]
Age	-0.001	[-0.019;0.018]	NS
BMI	-0.013	[-0.066;0.039]	NS
HbA1C	-0.044	[-0.242;0.155]	NS
HOMA	0.116	[0.035;0.196]	0.01
BMR	-0.00007	[-0.0009;0.0007]	NS
TG	0.129	[-0.129;0.387]	NS
LDL-C	-0.133	[-0.961;0.695]	NS
	CA
	β	95% confidence interval	p-value
Group			NS
-Controls	1
-Obeses	0.350	[0.013;0.687]
-Diabetics	0.164	[-0.189;0.517]
Age			0.04
-Center 2	-0.008
-Center 1	0.009
BMI	-0.02	[-0.038;0.005]	NS
HbA1C	-0.015	[-0.093;0.063]
HOMA	0.045	[0.014;0.077]	0.01
BMR	-0.0003	[-0.0006;0.0001]	NS
TG	0.053	[-0.048;0.155]	NS
LDL-C	-0.162	[-0.487;0.164]	NS
	DCA
	β	95% confidence interval	p-value
Center			NS
- Center 2	1
- Center 1	0.176	[-0.102;0.454]
Group			NS
-Controls	1
-Obeses	-0.067	[-0.486;0.352]
-Diabetics	0.229	[-0.204;0.662]
Age	0.007	[-0.003.0.017]	NS
BMI	0.004	[-0.025.0.033]	NS
HbA1C	-0.064	[-0.169;0.041]	NS
HOMA	0.062	[0.018.0.105]	0.007
BMR	0.00001	[-0.0004;0.00048]	NS
TG	-0.004	[-0.146;0.137]	NS
LDL-C	0.007	[-0.411;0.425]	NS

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Linear multiple regression analyses were performed for estimation of association between plasma bile acids concentrations and metabolic parameters.

All variables (center, group, gender, age, BMI, HbA1c, HOMA, GIR, BMR, TG, LDL-C) were included in linear multiple regression analyses.

A p value ≤ 0.05 was considered statistically significant.

Discussion

Besides their role as detergent molecules, BAs appear to modulate a broad spectrum of metabolic pathways including lipid and glucose homeostasis [1]. These findings are mainly based on animal studies and the clinical impact of BAs on metabolic homeostasis remains to be determined in human. Here, we show that plasma CDCA, CA and DCA concentrations were negatively associated with insulin sensitivity in a variety of subjects, including healthy volunteers, obese and T2D patients. In contrast, plasma levels of these BAs were not correlated with glycaemic status. Importantly, HOMA-IR remained positively related to those plasma BAs levels in multivariable analysis. When assessing insulin sensitivity by the clamp procedure, we further observed an inverse relationship between plasma CDCA and CA concentrations and insulin sensitivity (i.e. GIR).

Our data sustain the hypothesis for a role of plasma BAs and FXR in insulin resistance. FXR-deficient mice, which exhibit high plasma BA concentrations [2,3], display impaired peripheral insulin sensitivity [6,7]. Conversely, treatment with synthetic specific FXR agonists improved insulin sensitivity in rodents [6,8], and in patients with non-alcoholic fatty liver disease [16]. Previous studies have suggested a link between BA pool size and glucose metabolism in humans. In a study performed in 6 Pima Indians with T2D, the BA pool was found to be increased during untreated hyperglycaemia and conversely decreased upon insulin therapy [17]. In contrast, the BA pool size remained unaltered upon insulin treatment in another study involving 14 patients with T2D [18]. Very recently, a well-designed study has retrieved an increase in the DCA absolute pool size, without change in total pool size in patients with T2D [19]. Accordingly, plasma DCA levels were significantly increased in T2D patients in comparison with controls in the present study. In lipid-lowering trials, BA sequestrants, which disrupt the enterohepatic circulation of BAs, have been shown to lower plasma glucose and HbA1c [20]. However, the changes in BA metabolism following colesevelam treatment were not related to changes in glucose metabolism in patients with T2D [19]. In contrast, a metabolomic study in healthy and pre-diabetic individuals found a correlation between plasma BAs levels and insulin sensitivity [21]. Thus, additional studies using FXR or TGR5 agonists are needed to unravel the effect of BAs on insulin sensitivity in human.

In mice, plasma BAs increase energy expenditure via a TGR5-mediated signalling cascade in brown adipose tissue [10]. This pathway has been suggested to operate in human skeletal myocytes [10], but its clinical relevance has been challenged recently [22]. Here, we found a slight positive correlation between CDCA and CA plasma levels and energy expenditure. In contrast, plasma levels of DCA, one of the best ligand of TGR5 [12], did not associate with BMR. Moreover, circulating BAs failed to correlate with energy expenditure in multivariable regression analysis. One limitation of our study is that all the components of the plasma BA profile were not analyzed. Thus, we can not exclude that some low abundant plasma BA species (for instance, LCA) might affect insulin sensitivity or energy expenditure.

In summary, we found that plasma CDCA and CA, and in a lesser extent DCA, were related to insulin resistance in a wide range of subjects. These data sustain the hypothesis for a role of BAs in glucose homeostasis.

List of abbreviations

BA: bile acid; BMI: body mass index; BMR: basal metabolic rate; CA: cholic acid; CDCA: chenodeoxycholic acid; DCA: deoxycholic acid; DIO-2: type 2 iodothyronine deiodinase; FXR: farnesoid × receptor; GIR: glucose infusion rate; GLP-1: glucagon-like peptide 1; HOMA: homeostasis model assessment; LCA: lithocholic acid; T2D: type 2 diabetes.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

BC conceived of the study, participated in its design and coordination and drafted the manuscript. MC performed the dosage of BAs. YZ, ED performed the clamps. BGD performed the statistical analysis. EP, ML and MK contributed to the analysis of the data and critical revision of the manuscript. All authors read and approved the final manuscript.

References