Thyroid Hormone Promotes Postnatal Rat Pancreatic β-Cell Development and Glucose-Responsive Insulin Secretion Through MAFA

Neonatal β cells do not secrete glucose-responsive insulin and are considered immature. We previously showed the transcription factor MAFA is key for the functional maturation of β cells, but the physiological regulators of this process are unknown. Here we show that postnatal rat β cells express thyroid hormone (TH) receptor isoforms and deiodinases in an age-dependent pattern as glucose responsiveness develops. In vivo neonatal triiodothyronine supplementation and TH inhibition, respectively, accelerated and delayed metabolic development. In vitro exposure of immature islets to triiodothyronine enhanced the expression of Mafa, the secretion of glucose-responsive insulin, and the proportion of responsive cells, all of which are effects that were abolished in the presence of dominant-negative Mafa. Using chromatin immunoprecipitation and electrophoretic mobility shift assay, we show that TH has a direct receptor-ligand interaction with the Mafa promoter and, using a luciferase reporter, that this interaction was functional. Thus, TH can be considered a physiological regulator of functional maturation of β cells via its induction of Mafa

Consequences of dietary protein deficiency on IGF-IGFBP system in foetal rat liver

Fetal growth retardation still represents a high risk of early mortality and morbidity. It is associated with the appearance of a cluster of metabolic disorders commonly named "the metabolic syndrome" at adult age including type 2 diabetes. Understanding the mechanisms of fetal growth retardation could have its importance in prevention of persons with metabolic syndrome and related diseases namely type 2 diabetes and cardiovascular disease. Regulation of fetal growth is complex and multifactorial. Diverse factors, including environmental factors lead to abnormal intrauterine growth. Maternal protein deficiency is one of these influences that result to low birth weight as well as to altered organs such as liver, pancreas and adipose tissue in the offspring. There is evidence that Insulin like growth factors and Insulin like growth factors Binding Proteins regulate tissue as well as overall fetal growth. In several animal models of intrauterine growth retardation due to maternal undernutrition (calorie and/or protein), decreased IGF-I and/or elevated IGFBPs are observed in late fetal gestation. However, in these studies a general food deprivation or a severe hypocaloric protein restriction was applied. In the present work we examined the influence of a moderate and specific isocaloric protein deficiency (8% vs 20%) on the fetal IGF system. In our Low Protein (LP) growth retarded fetuses, we found in vivo that IGF-I level was lower whereas the main binding proteins (IGFBP-1 & IGFBP-2) in the fetus that modulate IGFs by preventing their action are elevated. This suggested that altered IGF-I and IGFBPs could lead to decreased fetal growth in our LP model. Despite this important finding, the mechanisms by which maternal undernutrition and protein deficiency, in particular, result in the altered fetal IGF system are not well understood. For this purpose and because the liver is the main organ in the production of IGFs and IGFBPs, we set up a validated culture system of "highly purified" fetal hepatocytes to study the regulation of fetal IGFs and IGFBPs. Among factors studied, hormones (insulin, glucocorticoids, prolactin) as well as some specific amino acids (branched chain amino acids, leucine and taurine) that appear to be affected by maternal undernutrition were examined. Overall, we found that insulin, glucocorticoids as well as prolactin modulate IGFs as well as IGFBPs in vitro and could therefore contribute to the abnormal profile of fetal IGF system seen in vivo in LP fetuses. A similar proposal could be attributed to branched chain amino acids, leucine and taurine with some caution since the most marked effects were observed within elevated range of these amino acids. Furthermore, the low protein diet appeared to affect profoundly the fetal IGF system as well as its regulation. When LP cultured hepatocytes were kept under similar culture conditions as control cells during the whole duration of culture, the altered production of IGF-I and IGFBPs (-1 & -2) was maintained, stressing some programming as occurred in utero. Also, a resistance of IGFBPs to the action of prolactin has been observed in cultured LP hepatocytes since they did not respond to the action of hormone as they did in control cultures. This may indicate that maternal protein deficiency could alter the hormonal sensitivity which may be characteristic of intrauterine growth retardation.Doctorat en sciences (sciences biologiques) (BIOL 3)--UCL, 200

The regulation of IGFs and IGFBPs by prolactin in primary culture of fetal rat hepatocytes is influenced by maternal malnutrition.

During perinatal development, the regulation of IGF system appears to be growth hormone (GH) independent. By using highly purified primary fetal hepatocytes, we investigated the role of prolactin (PRL) in the regulation of IGF system and hepatocyte proliferation. We also analyzed the consequence of a maternal low-protein (LP) diet on the regulation of IGF, IGF-binding protein (IGFBP), and hepatocyte proliferation by prolactin. Pregnant Wistar rats were fed a control (C) diet (20% protein) or isocaloric (LP; 8%) diet throughout gestation. On day 21.5, fetal hepatocytes were cultured for 4 days and incubated with rat prolactin. In the C hepatocytes, PRL at 100 ng/ml decreased the abundance of IGFBP-1 and IGFBP-2 by 50 (P < 0.05) and 60% (P < 0.01), respectively. It also reduced by 70% the level of IGF-II mRNA (P < 0.01). By contrast, PRL failed to modulate IGFBP-1 and IGFBP-2 production by LP hepatocytes, and this was associated with reduced abundance of the short form of PRL receptor (P < 0.05). PRL had no effect on either the proliferation or the IGF-I production by C and LP hepatocytes, although it reduced the expression of IGF-II. These results suggest that prolactin influences hepatocyte proliferation in vitro by inhibiting IGFBP-1, IGFBP-2, and IGF-II levels, which may coincide with the decline of IGF-II observed in rodents during late gestation in vivo. On the other hand, maternal LP diet induces a resistance of fetal hepatocytes to PRL

Isocaloric maternal low-protein diet alters IGF-I, IGFBPs, and hepatocyte proliferation in the fetal rat.

We investigated the effect of an isocaloric maternal low-protein diet during pregnancy in rats on the proliferative capacity of cultured fetal hepatocytes. The potential roles of these changes on the IGF-IGF-binding protein (IGFBP) axis, and the role of insulin and glucocorticoids in liver growth retardation, were also evaluated. Pregnant Wistar rats were fed a control (C) diet (20% protein) or a low-protein (LP) diet (8%) throughout gestation. In primary culture, the DNA synthesis of hepatocytes derived from LP fetuses was decreased by approximately 30% compared with control hepatocytes (P < 0.05). In parallel, in vivo moderate protein restriction in the dam reduced the fetal liver weight and IGF-I level in fetal plasma (P < 0.01) and augmented the abundance of 29- to 32-kDa IGFBPs in fetal plasma (P < 0.01) and fetal liver (P < 0.01). By contrast, the abundance of IGF-II mRNA in liver of LP fetuses was unaffected by the LP diet. In vitro, the LP-derived hepatocytes produced less IGF-I (P < 0.01) and more 29- to 32-kDa IGFBPs (P < 0.01) than hepatocytes derived from control fetuses. These alterations still appeared after 3-4 days of culture, indicating some persistence in programming. Dexamethasone treatment of control-derived hepatocytes decreased cell proliferation (54 +/- 2.3%, P < 0.01) and stimulated 29- to 32-kDa IGFBPs, whereas insulin promoted fetal hepatocyte growth (127 +/- 5.5%, P < 0.01) and inhibited 29- to 32-kDa IGFBPs. These results show that liver growth and cell proliferation in association with IGF-I and IGFBP levels are affected in utero by fetal undernutrition. It also suggests that glucocorticoids and insulin may modulate these effects

Compensatory Response by Late Embryonic Tubular Epithelium to the Reduction in Pancreatic Progenitors

Early in pancreatic development, epithelial cells of pancreatic buds function as primary multipotent progenitor cells (1°MPC) that specify all three pancreatic cell lineages, i.e., endocrine, acinar and duct. Bipotent "Trunk" progenitors derived from 1°MPC are implicated in directly regulating the specification of endocrine progenitors. It is unclear if this specification process is initiated in the 1°MPC where some 1°MPC become competent for later specification of endocrine progenitors. Previously we reported that in Pdx1tTA/+;tetOMafA (bigenic) mice inducing expression of transcription factor MafA in Pdx1-expressing (Pdx1+) cells throughout embryonic development inhibited the proliferation and differentiation of 1°MPC cells, resulting in reduced pancreatic mass and endocrine cells by embryonic day (E) 17.5. Induction of the transgene only until E12.5 in Pdx1+ 1°MPC was sufficient for this inhibition of endocrine cells and pancreatic mass at E17.5. However, by birth (P0), as we now report, such bigenic pups had significantly increased pancreatic and endocrine volumes with endocrine clusters containing all pancreatic endocrine cell types. The increase in endocrine cells resulted from a higher proliferation of tubular epithelial cells expressing the progenitor marker Glut2 in E17.5 bigenic embryos and increased number of Neurog3-expressing cells at E19.5. A BrdU-labeling study demonstrated that inhibiting proliferation of 1°MPC by forced MafA-expression did not lead to retention of those progenitors in E17.5 tubular epithelium. Our data suggest that the forced MafA expression in the 1°MPC inhibits their competency to specify endocrine progenitors only until E17.5, and after that compensatory proliferation of tubular epithelium gives rise to a distinct pool of endocrine progenitors. Thus, these bigenic mice provide a novel way to characterize the competency of 1°MPC for their ability to specify endocrine progenitors, a critical limitation in our understanding of endocrine differentiation

Les facteurs prédictifs du décès par envenimation scorpionique à la province d'El Kelâa des Sraghnas – Maroc

Objectif: Identifier les signes cliniques associés aux décès par envenimations scorpioniques, à partir des fiches d'hospitalisation de l'hôpital Essalama de la province d'El Kelâa des Sraghnas parvenues pendant l'année 2007. Patients et Méthodes: Étude prospective des cas d'envenimations scorpioniques, à partir des fiches d'hospitalisation conçues et distribuées par le Centre Antipoison du Maroc, au niveau du service de réanimation adulte et pédiatrique. L'analyse descriptive a porté sur les paramètres socio-démographiques (âge, sexe, temps post-piqûre (TPP)), cliniques et thérapeutiques. Nous avons effectué l'analyse univariée pour identifier les facteurs associés aux décès par envenimations scorpioniques (selon le test de chi2 ou de Fischer, l'association est statistiquement significative si p<0{,}05). Résultats: 240 patients ont été inclus dans notre étude, l'âge médian était de 12 ans, allant de 1 à 86 ans, le sex ratio était de 1,25. Le TPP médian était de 2 h allant de 10 min à 29 h. 9,6 % des patients ont présenté une détresse respiratoire, l'atteinte cardiovasculaire était présente dans 10,8 % des cas suivie par la détresse neurologique dans 10,4 % des cas. 11,3 % des cas étaient de classe III à l'admission. L'évolution était favorable dans 91,7 % des cas. 20 décès ont été enregistrés soit 8,3 % des cas. La fièvre, l'hypersudation, les vomissements et la tachypnée étaient statistiquement associés au décès (p<0{,}05). Conclusion: Le décès par envenimation scorpionique à la province d'El Kelâa des Sraghnas reste fréquent malgré les efforts fournis, d'où l'intérêt de l'implantation de l'audit clinique des décès afin de déceler les insuffisances pour y remédier

Differentiation of pancreatic endocrine progenitors reversibly blocked by premature induction of MafA

AbstractSpecification and maturation of insulin+ cells accompanies a transition in expression of Maf family of transcription factors. In development, MafA is expressed after specification of insulin+ cells that are expressing another Maf factor, MafB; after birth, these insulin+ MafA+ cells stop MafB expression and gain glucose responsiveness. Current differentiation protocols for deriving insulin-producing β-cells from stem cells result in β-cells lacking both MafA expression and glucose-stimulated insulin secretion. So driving expression of MafA, a β-cell maturation factor in endocrine precursors could potentially generate glucose-responsive MafA+ β cells. Using inducible transgenic mice, we characterized the final stages of β-cell differentiation and maturation with MafA pause/release experiments. We found that forcing MafA transgene expression, out of its normal developmental context, in Ngn3+ endocrine progenitors blocked endocrine differentiation and prevented the formation of hormone+ cells. However, this arrest was reversible such that with stopping the transgene expression, the cells resumed their differentiation to hormone+ cells, including α-cells, indicating that the block likely occurred after progenitors had committed to a specific hormonal fate. Interestingly, this delayed resumption of endocrine differentiation resulted in a greater proportion of immature insulin+MafB+ cells at P5, demonstrating that during maturation the inhibition of MafB in β-cell transitioning from insulin+MafB+ to insulin+MafB− stage is regulated by cell-autonomous mechanisms. These results demonstrate the importance of proper context of initiating MafA expression on the endocrine differentiation and suggest that generating mature Insulin+MafA+ β-cells will require the induction of MafA in a narrow temporal window to achieve normal endocrine differentiation

E17.5 tubular epithelium of bigenic pancreas expresses neither Pdx1^Hi nor MafA^Myc.

<p>E17.5 bigenic and control pancreases were stained for E-cadherin (red, <b>ABEF</b>), Pdx1 (green, <b>ABCD</b>) and Myc (green, <b>EF</b>). Pdx1^Hi, Pdx1^Lo and Pdx1^- cells are marked by arrows, triangles and arrowheads, respectively. In controls, tubular epithelial cells have Pdx1^Lo expression while Pdx1^Hi expression was only seen in endocrine cells. In bigenic pancreas only occasional Pdx1^Hi cells and MafA^Myc cells were seen indicating that Pdx1^Lo expression was not sufficient for <i>Pdx1</i>^<i>tTA</i>-dependent induction of MafA^Myc expression (green, <b>EF</b>). DAPI (blue). Bar: 20 μm.</p

Tubular epithelial cells of bigenic pancreas express Sox9 and GLUT2 at E17.5.

<p>At E17.5 both control and bigenic tubular epithelial cells express Sox9. Sox9 (green <b>AB</b>); DBA (green, <b>CD</b>); GLUT2 (green, <b>EF</b>); Insulin (red); DAPI (blue). The boxed areas in E and F are enlarged (<b>GH</b>: merged channels, <b>IJ</b>: green channel showing GLUT2 expression). Higher GLUT2 staining intensity is seen in the bigenic tubular epithelial cells than in the controls. In bigenic pancreas GLUT2 staining intensity is comparable in insulin⁺ (marked by arrows) and insulin^- tubular epithelial cells (marked by arrowheads) whereas in control pancreas GLUT2 staining intensity is reduced in tubular epithelial cells than islets. Bar: 20 μm.</p