Les récepteurs nucléaires orphelins NR4A et l’homéostasie du glucose : une minirevue

Type 2 diabetes mellitus is a disorder characterized by insulin resistance and a relative deficit in insulin secretion, both of which result in elevated blood glucose. Understanding the molecular mechanisms underlying the pathophysiology of diabetes could lead to the development of new therapeutic approaches. An ever-growing body of evidence suggests that members of the NR4A family of nuclear receptors could play a pivotal role in glucose homeostasis. This review aims to present and discuss advances so far in the evaluation of the potential role of NR4A in the regulation of glucose homeostasis and the development of type 2 diabetes.Le diabète de type 2 est caractérisé par une résistance à l’insuline et une détérioration progressive de la sécrétion d’insuline, deux perturbations qui entraînent une élévation de la glycémie. La compréhension des mécanismes moléculaires participant au développement du diabète de type 2 permettra le développement de nouvelles approches thérapeutiques pour le traitement de cette maladie. Il a été suggéré que les membres de la famille des récepteurs nucléaires NR4A pourraient jouer un rôle central dans l’homéostasie du glucose. Dans cette minirevue, nous décrirons l’état actuel des connaissances et discuterons du rôle potentiel des NR4A dans l’homéostasie du glucose et le diabète de type 2

The lack of functional nicotinamide nucleotide transhydrogenase only moderately contributes to the impairment of glucose tolerance and glucose-stimulated insulin secretion in C57BL/6J vs C57BL/6N mice.

Aims/hypothesis: Nicotinamide nucleotide transhydrogenase (NNT) is involved in mitochondrial NADPH production and its spontaneous inactivating mutation (NntTr [Tr, truncated]) is usually considered to be the main cause of the lower glucose tolerance of C57BL/6J vs C57BL/6N mice. However, the impact of this mutation on glucose tolerance remains disputed. Here, we singled out the impact of NntTr from that of other genetic variants between C57BL/6J and C57BL/6N mice on mitochondrial glutathione redox state (EGSH), glucose-stimulated insulin secretion (GSIS) and glucose tolerance. Methods: Male and female N5BL/6J mice that express wild-type Nnt (NntWT) or NntTr (N5-WT and N5-Tr mice) on the C57BL/6J genetic background were obtained by crossing N5BL/6J NntWT/Tr heterozygous mice. C57BL/6J and C57BL/6N mice were from Janvier Labs. The Nnt genotype was confirmed by PCR and the genetic background by whole genome sequencing of one mouse of each type. Glucose tolerance was assessed by IPGTT, ITT and fasting/refeeding tests. Stimulus-secretion coupling events and GSIS were measured in isolated pancreatic islets. Cytosolic and mitochondrial EGSH were measured using the fluorescent redox probe GRX1-roGFP2 (glutaredoxin 1 fused to redox-sensitive enhanced GFP). Results: The Nnt genotype and genetic background of each type of mouse were confirmed. As reported previously in C57BL/6N vs C57BL/6J islets, the glucose regulation of mitochondrial (but not cytosolic) EGSH and of NAD(P)H autofluorescence was markedly improved in N5-WT vs N5-Tr islets, confirming the role of NNT in mitochondrial redox regulation. However, ex vivo GSIS was only 1.2-1.4-times higher in N5-WT vs N5-Tr islets, while it was 2.4-times larger in C57BL/6N vs N5-WT islets, questioning the role of NNT in GSIS. In vivo, the ITT results did not differ between N5-WT and N5-Tr or C57BL/6N mice. However, the glucose excursion during an IPGTT was only 15-20% lower in female N5-WT mice than in N5-Tr and C57BL/6J mice and remained 3.5-times larger than in female C57BL/6N mice. Similar observations were made during a fasting/refeeding test. A slightly larger (~30%) impact of NNT on glucose tolerance was found in males. Conclusions/interpretation: Although our results confirm the importance of NNT in the regulation of mitochondrial redox state by glucose, they markedly downsize the role of NNT in the alteration of GSIS and glucose tolerance in C57BL/6J vs C57BL/6N mice. Therefore, documenting an NntWT genotype in C57BL/6 mice does not provide proof that their glucose tolerance is as good as in C57BL/6N mice

Protective Antioxidant and Antiapoptotic Effects of ZnCl2 in Rat Pancreatic Islets Cultured in Low and High Glucose Concentrations

Aim/Hypothesis: Rat pancreatic islet cell apoptosis is minimal after prolonged culture in 10 mmol/l glucose (G10), largely increased in 5 mmol/l glucose (G5) and moderately increased in 30 mmol/l glucose (G30). This glucose-dependent asymmetric V-shaped profile is preceded by parallel changes in the mRNA levels of oxidative stress-response genes like Metallothionein 1a (Mt1a). In this study, we tested the effect of ZnCl2, a potent inducer of Mt1a, on apoptosis, mitochondrial oxidative stress and alterations of glucose-induced insulin secretion (GSIS) induced by prolonged exposure to low and high vs. intermediate glucose concentrations. Methods: Male Wistar rat islets were cultured in RPMI medium. Islet gene mRNA levels were measured by RTq-PCR. Apoptosis was quantified by measuring islet cytosolic histone-associated DNA fragments and the percentage of TUNELpositive b-cells. Mitochondrial thiol oxidation was measured in rat islet cell clusters expressing ‘‘redox sensitive GFP’’ targeted to the mitochondria (mt-roGFP1). Insulin secretion was measured by RIA. Results: As observed for Mt1a mRNA levels, b-cell apoptosis and loss of GSIS, culture in either G5 or G30 vs. G10 significantly increased mt-roGFP1 oxidation. While TPEN decreased Mt1a/2a mRNA induction by G5, addition of 50–100 mM ZnCl2 to the culture medium strongly increased Mt1a/2a mRNA and protein levels, reduced early mt-roGFP oxidation and significantly decreased late b-cell apoptosis after prolonged culture in G5 or G30 vs. G10. It did not, however, prevent the loss of GSIS under these culture conditions. Conclusion: ZnCl2 reduces mitochondrial oxidative stress and improves rat b-cell survival during culture in the presence of low and high vs. intermediate glucose concentrations without improving their acute GSIS

Long term effects of glucose and ZnCl₂ on <i>Mt1a</i>, <i>Mt2a, ZnT1 and ZnT8</i> mRNA expression in cultured rat islets.

<p>After preculture, rat islets were cultured for 1 week in the presence of G5, G10 or G30 alone (open bars) or in the presence of 100 µmol/l ZnCl₂ (closed bars). <i>Gene</i> to <i>Tbp</i> mRNA ratios were measured by real-time PCR and expressed relative to the ratio measured in islets cultured in G10 without ZnCl₂, except for <i>Mt2a</i> to <i>Tbp</i> mRNA ratios that are shown without normalization. ND: Not Detected (unspecific products with lower Tm were amplified at Ct>30, i.e. at <i>Mt2a/Tbp</i> mRNA ratio <0.25). C, the mean Ct for <i>Tbp</i> were 27 in G5, 27.5 in G5+ZnCl₂, 27.3 in G10 and 27.3 in G10+ZnCl₂. D, the mean Ct for <i>Tbp</i> were 28.1 in G10, 28 in G10+ZnCl₂, 27.8 in G30 and 28.1 in G30+ZnCl₂. Please note the logarithmic Y-scale in A-D. Results are means ± SEM for 3 or 4 experiments. #, <i>P</i><0.05 for the effect of ZnCl₂ by two-way ANOVA followed by a test of Bonferroni. C,D, for statistical analysis of differences between groups, <i>Mt2a/Tbp</i> mRNA ratio was set to 0.25 when not detected.</p

Effects of glucose and ZnCl₂ on the stimulation of rat islet cell apoptosis by prolonged culture in low and high glucose concentrations.

<p>Rat islets were cultured for 3 days (A, D) or 1 week (B,C,E,F) in the presence of G5, G10 or G30±100 µmol/l ZnCl₂ as indicated. A, B, C, DNA fragmentation was measured by an ELISA kit and expressed relative to the value measured in islets cultured in G10. D, E, F, the percentage of apoptotic β-cells was measured by TUNEL. Results are means ± SEM for 3–5 experiments. *, <i>P</i><0.05 for the effect of G5 or G30 <i>vs.</i> G10 and #, <i>P</i><0.05 for the effect of ZnCl₂ by two-way ANOVA followed by a test of Bonferroni (A,B,C) or by one-way ANOVA followed by a test of Newman-Keuls (D,E,F).</p

Effects of glucose and ZnCl₂ on mitochondrial thiol/disulfide equilibrium in cultured rat islet cell clusters.

<p>Rat islet cell clusters infected with an adenovirus coding mt-roGFP1 were cultured overnight in the presence of G5, G10 or G30±50 µmol/l ZnCl₂ as indicated. The ratio of mt-roGFP1 fluorescence intensities (exc 405/480 nm) was measured during 20 min of perfusion in a medium containing the same glucose concentration and expressed as a percentage of the difference between the mean ratio measured from 4 to 8 min after addition of 10 mmol/l DTT (set at 0%) and that measured from 14 to 18 min after addition of 1 mmol/l H₂O₂ (set at 100%) (B,C). Results are means ± SEM for 3 experiments (9–19 clusters). B,C, *, <i>P</i><0.05 for the effect of G5 or G30 <i>vs.</i> G10 and #, <i>P</i><0.05 for the effect of ZnCl₂ by two-way ANOVA followed by a test of Bonferroni.</p

Prolonged culture of human pancreatic islets under glucotoxic conditions changes their acute beta cell calcium and insulin secretion glucose response curves from sigmoid to bell-shaped

Aims/hypothesis: The rapid remission of type 2 diabetes by a diet very low in energy correlates with a marked improvement in glucose-stimulated insulin secretion (GSIS), emphasising the role of beta cell dysfunction in the early stages of the disease. In search of novel mechanisms of beta cell dysfunction after long-term exposure to mild to severe glucotoxic conditions, we extensively characterised the alterations in insulin secretion and upstream coupling events in human islets cultured for 1–3 weeks at ~5, 8, 10 or 20 mmol/l glucose and subsequently stimulated by an acute stepwise increase in glucose concentration. Methods: Human islets from 49 non-diabetic donors (ND-islets) and six type 2 diabetic donors (T2D-islets) were obtained from five isolation centres. After shipment, the islets were precultured for 3–7 days in RPMI medium containing ~5 mmol/l glucose and 10% (vol/vol) heat-inactivated FBS with selective islet picking at each medium renewal. Islets were then cultured for 1–3 weeks in RPMI containing ~5, 8, 10 or 20 mmol/l glucose before measurement of insulin secretion during culture, islet insulin and DNA content, beta cell apoptosis and cytosolic and mitochondrial glutathione redox state, and assessment of dynamic insulin secretion and upstream coupling events during acute stepwise stimulation with glucose [NAD(P)H autofluorescence, ATP/(ATP+ADP) ratio, electrical activity, cytosolic Ca2+ concentration ([Ca2+]c)]. Results: Culture of ND-islets for 1–3 weeks at 8, 10 or 20 vs 5 mmol/l glucose did not significantly increase beta cell apoptosis or oxidative stress but decreased insulin content in a concentration-dependent manner and increased beta cell sensitivity to subsequent acute stimulation with glucose. Islet glucose responsiveness was higher after culture at 8 or 10 vs 5 mmol/l glucose and markedly reduced after culture at 20 vs 5 mmol/l glucose. In addition, the [Ca2+]c and insulin secretion responses to acute stepwise stimulation with glucose were no longer sigmoid but bell-shaped, with maximal stimulation at 5 or 10 mmol/l glucose and rapid sustained inhibition above that concentration. Such paradoxical inhibition was, however, no longer observed when islets were acutely depolarised by 30 mmol/l extracellular K+. The glucotoxic alterations of beta cell function were fully reversible after culture at 5 mmol/l glucose and were mimicked by pharmacological activation of glucokinase during culture at 5 mmol/l glucose. Similar results to those seen in ND-islets were obtained in T2D-islets, except that their rate of insulin secretion during culture at 8 and 20 mmol/l glucose was lower, their cytosolic glutathione oxidation increased after culture at 8 and 20 mmol/l glucose, and the alterations in GSIS and upstream coupling events were greater after culture at 8 mmol/l glucose. Conclusions/interpretation: Prolonged culture of human islets under moderate to severe glucotoxic conditions markedly increased their glucose sensitivity and revealed a bell-shaped acute glucose response curve for changes in [Ca2+]c and insulin secretion, with maximal stimulation at 5 or 10 mmol/l glucose and rapid inhibition above that concentration. This novel glucotoxic alteration may contribute to beta cell dysfunction in type 2 diabetes independently from a detectable increase in beta cell apoptosis

L’expérimentation animale reste indispensable (OPINION)

Trop fréquemment, l’expérimentation animale est présentée comme une pratique archaïque. Elle a bien changé. Et 100 % des patients traités le sont grâce aux concepts et techniques développés grâce à elle