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Administration of the LRH-1 agonist BL0refrains hyperglycemia in 3 mouse models of type 1 diabetes (T1DM) . This antidiabetes action correlated with beta cell mass preservation and the presence of anti, rather than pro, inflammatory immune cells, indicating tolerization. Herein, we sought to translate these data to human immune cells of T1DM patients to demonstrate BL0benefits to re-educate the immune system and to assess its therapeutic value on human islet engraftment/function. Blood mononuclear cells purified from blood samples (healthy and T1DM) were derived in culture into 1) pro-inflammatory macrophages (MDM1) , 2) mature dendritic cells (mDCs) , 3) T-cells and 4) B-cells. Cells were then treated with BL0 and processed for 1) cell surface marker profiling, 2) cytokine secretome profiling, 3) T-cell proliferation and 4) RNAseq. In T1DM samples, BL0reduced the expression of pro-inflammatory markers on MDM1 (CD80) and mDCs (CD54 and CXCR4) and the secretion of the pro-inflammatory cytokines, CXCL6 and CCL24 (MDM1) as well as IL7 (mDCs) . Moreover, in these samples, BL0also increased Tregs, but not B-cells. T-cell proliferation was blunted by BL001-treated mDCs and Tregs. RNAseq analysis of BL001-treated MDM1 revealed increased expression of genes encoding components of complex I, III, IV, and V of the respiratory chain consistent with a transition from a glycolytic driven MDM1 to an oxidative phosphorylation driven MDM2 anti-inflammatory phenotype. BL0administration to STZ-treated mice transplanted with human islet improved survival correlating with reduced hyperglycemia, increased circulating human C-peptide, preserved beta cell mass, and reduced CD4+ T-cell infiltration. Our results establish that BL0can induce a pro-to-anti-inflammatory phenotypic switch to human immune cells, and improve human islet engraftment/function, supporting the therapeutic benefits of BL0in human. Disclosure N. Cobo-Vuilleumier: None. S. Rodriguez-Fernandez: Employee; Ahead Therapeutics SL. P.I. Lorenzo: None. A. Dorronsoro: None. C.C. Lachaud: None. L. Almenara-Fuentes: Employee; Ahead Therapeutics SL. M. Ramos-Rodríguez: None. I. Merida: None. M. Aguilar-Diosdado: None. L. Piemonti: None. L. Pasquali: None. F. Martin: None. I. Garcia-Sanchez: None. M. Martinez-Brocca: None. M. Vives-Pi: Research Support; Ahead Therapeutics S.L. B.R. Gauthier: None. Funding JDRF (2-SRA-2019-837-S-B) Consejeria de Salud y Familia, junta de Andalucia (PI-0001-2020)

Rationale: We recently demonstrated that the Metabesity factor HMG20A regulates islet beta-cell functional maturity and adaptation to physiological stress such as pregnancy and pre-diabetes. HMG20A also dictates central nervous system (CNS) development via inhibition of the LSD1-CoREST complex but its expression pattern and function in adult brain remains unknown. Herein we sought to determine whether HMG20A is expressed in the adult CNS, specifically in hypothalamic astrocytes that are key in glucose homeostasis and whether similar to islets, HMG20A potentiates astrocyte function in response to environmental cues. Methods: HMG20A expression profile was assessed by quantitative PCR (QT-PCR), Western blotting and/or immunofluorescence in: 1) the hypothalamus of mice exposed or not to either a high-fat diet or a high-fat high-sucrose regimen, 2) human blood leukocytes and adipose tissue obtained from healthy or diabetic individuals and 3) primary mouse hypothalamic astrocytes exposed to either high glucose or palmitate. RNA-seq and cell metabolic parameters were performed on astrocytes treated or not with a siHMG20A. Astrocyte-mediated neuronal survival was evaluated using conditioned media from siHMG20A-Treated astrocytes. The impact of ORY100. An Inhibitor Of The LSD1-CoREST Complex, On Hmg20a Expression, Reactive Astrogliosis And Glucose Metabolism Was Evaluated In Vitro And In Vivo In High-Fat High-Sucrose Fed Mice. Results: We Show That Hmg20a Is Predominantly Expressed In Hypothalamic Astrocytes, The Main Nutrient-Sensing Cell Type Of The Brain. Hmg20a Expression Was Upregulated In Diet-Induced Obesity And Glucose Intolerant Mice, Correlating With Increased Transcript Levels Of Gfap And Il1b Indicative Of Inflammation And Reactive Astrogliosis. Hmg20a Transcript Levels Were Also Increased In Adipose Tissue Of Obese Non-Diabetic Individuals As Compared To Obese Diabetic Patients. Hmg20a Silencing In Astrocytes Resulted In Repression Of Inflammatory, Cholesterol Biogenesis And Epithelial-To-Mesenchymal Transition Pathways Which Are Hallmarks Of Reactive Astrogliosis. Accordingly, Hmg20a Depleted Astrocytes Exhibited Reduced Mitochondrial Bioenergetics And Increased Susceptibility To Apoptosis. Neuron Viability Was Also Hindered In HMG20A-Depleted Astrocyte-Derived Conditioned Media. Ory1001 Treatment Rescued Expression Of Reactive Astrogliosis-Linked Genes In Hmg20a Ablated Astrocytes While Enhancing Cell Surface Area, Gfap Intensity And Stat3 Expression In Healthy Astrocytes, Mimicking The Effect Of Hmg20a. Furthermore, Ory1001 Treatment Protected Against Obesity-Associated Glucose Intolerance In Mice Correlating With A Regression Of Hypothalamic Hmg20a Expression, Indicative Of Reactive Astrogliosis Attenuation With Improved Health Status. Conclusion: Hmg20a Coordinates The Astrocyte Polarization State. Under Physiological Pressure Such As Obesity And Insulin Resistance That Induces Low Grade Inflammation, Hmg20a Expression Is Increased To Induce Reactive Astrogliosis In An Attempt To Preserve The Neuronal Network And Re-Establish Glucose Homeostasis. Nonetheless, A Chronic Metabesity State Or Functional Mutations Will Result In Lower Levels Of Hmg20a, Failure To Promote Reactive Astrogliosis And Increase Susceptibility Of Neurons To Stress-Induced Apoptosis. Such Effects Could Be Reversed By Ory1001 Treatment Both In Vitro And In Vivo, Paving The Way For A New Therapeutic Approach For Type 2 Diabetes Mellitus., The authors are supported by grants from the Consejería de Salud, Fundación Pública Andaluza Progreso y Salud, Junta de Andalucía (PI-0727-2010 and PI-0001-2020 to B.R.G.; PI-0085-2013 to P.I.L.; PI-0006-2016 to E.F.M.; PI-0574-2012 to S.Y.R.Z; PI-0247-2016 to F.J.B.S.), the Consejería de Economía, Innovación y Ciencia (P10.CTS.6359 to B.R.G.; CTS.8081 to E.G.F.), the Ministerio de Economía y Competitividad co-funded by Fondos FEDER (PI10/00871, PI13/00593 and BFU2017-83588-P to B.R.G.; PRE2018-084907 to M.E.M.V.G.; PI13/00309; PI17/01004 to F.J.B.S.; BFU2014-5343-P to J.C.R.; and AGL2017-86927-R to F.M.), Vencer el Cancer (B.R.G), DiabetesCero (B.R.G.) and the Juvenile Diabetes Research Foundation (17-2013-372 and 2-SRA-2019-837-S-B to B.R.G.). E.F.M. was a recipient of a Juan de la Cierva Incorporación Fellowship from the Ministerio de Economía y Competitividad (IJCI-2015-26238). S.Y.R.Z is a recipient of a postdoctoral fellowship from Consejería de Salud, Junta de Andalucía (RH-0070-2013). L.L.N. is supported by a Consejeria de Economia, Conocimiento, Empresas y Universidad postdoctoral fellowship (DOC_00652). F.J.B.S. and E.G.F. are recipients of "Nicolás Monardes" research contracts from Consejería de Salud Junta de Andalucía, (C-0070-2012 and C-0031-2016). A.M.M. is supported by CPII19/00023 and PI18/01590 from the Instituto de Salud Carlos III co-funded by Fondos FEDER. CIBERDEM is an initiative of the Instituto de Salud Carlos III. V.C. is supported by a AECC investigator award.

LRH-1/NR5A2 is implicated in islet morphogenesis postnatally, and its activation using the agonist BL001 protects islets against apoptosis, reverting hyperglycemia in mouse models of Type 1 Diabetes Mellitus. Islet transcriptome profiling revealed that the expression of PTGS2/COX2 is increased by BL001. Herein, we sought to define the role of LRH-1 in postnatal islet morphogenesis and chart the BL001 mode of action conferring beta cell protection. LRH-1 ablation within developing beta cells impeded beta cell proliferation, correlating with mouse growth retardation, weight loss, and hypoglycemia leading to lethality. LRH-1 deletion in adult beta cells abolished the BL001 antidiabetic action, correlating with beta cell destruction and blunted

We have previously described a role of LRH-1/NR5A2 in islet morphogenesis during postnatal development and reported that the treatment with BL001, an agonist of LRH-1/NR5A2, protects islets against-stress induced apoptosis and reverts hyperglycemia in 3 mouse models of Type 1 Diabetes Mellitus (T1DM). Islet transcriptome profiling revealed that most differentially expressed genes after BL001 treatment are involved in immunomodulation, among them, the increase in PTGS2/COX2 expression. Herein, we dissected the cellular and molecular branches of the BL001/LRH-1/NR5A2 signalling axis in order to chart the mode of action confering beta cell protection and hyperglycaemia reversion. We found that constitutive LRH-1/NR5A2 ablation within the insulin expression domain (RIP-Cre mouse model) caused a significant beta cell mass reduction characterized by blunted proliferation correlating with animal growth retardation, weight loss and hypoglycemia, leading to lethality before weaning. Using an inducible approach (pdx1PBCreER™ mouse model), specific deletion of LRH-1/NR5A2 in adult beta cells abolished the anti diabetic effect of BL001 in streptozotocin treated mice, correlating with complete beta-cell mass destruction. Additionally, BL001 induced Ptgs2 expression, was blunted in islets lacking LRH-1/NR5A2. The combined BL001/cytokine treatment did not further stimulate Ptgs2 expression above levels detected with cytokine alone yet secreted PGE2 levels were increased 5-fold. Inactivation of PTGS2 blunted induction of the target and its product PGE2 in islets treated with cytokines alone or with BL001. Importantly, PTGS2 inactivated islets were refractory to the BL001 protective effect under cytokine attack as evidenced by increased Bax expression levels, cytochrome C release and cleaved PARP. The PTGER1 antagonist ONO-8130, but not the PTGER4 antagonist L-161,982, negated BL001-mediated islet survival. Our results establish that the beneficial properties of BL001 against stress-induced cell death are specifically conveyed by LRH-1/NR5A2 activation in beta cells and downstream stimulation of the PTGS2-PGE2/PTGER1 signalling axis.

Diabetes is a chronic metabolic disease caused by an absolute or relative deficiency in functional pancreatic β‐cells that leads to defective control of blood glucose. Current treatments for diabetes, despite their great beneficial effects on clinical symptoms, are not curative treatments, leading to a chronic dependence on insulin throughout life that does not prevent the secondary complications associated with diabetes. The overwhelming increase in DM incidence has led to a search for novel antidiabetic therapies aiming at the regeneration of the lost functional β‐cells to allow the re‐establishment of the endogenous glucose homeostasis. Here we review several aspects that must be considered for the development of novel and successful regenerative therapies for diabetes: first, the need to maintain the heterogeneity of islet β‐cells with several subpopulations of β‐cells characterized by different transcriptomic profiles correlating with differences in functionality and in resistance/behavior under stress conditions; second, the existence of an intrinsic islet plasticity that allows stimulus‐mediated transcriptome alterations that trigger the transdifferentiation of islet non‐β‐cells into β‐cells; and finally, the possibility of using agents that promote a fully functional/mature β‐cell phenotype to reduce and reverse the process of dedifferentiation of β‐cells during diabetes., Authors were/are funded by grants from Consejería de Salud, Fundación Pública Andaluza Progreso y Salud, Junta de Andalucía (PI-0727-2010, December 2010 to B.R.G., PI-0085-2013, December 2013 to P.I.L., and PI-0001-2020, December 2020 to B.R.G. and P.I.L.); Consejería de Economía, Innovación y Ciencia, Junta de Andalucía (P10-CTS-6359 to B.R.G.); Ministerio de Economía y Competitividad, Instituto de Salud Carlos III, cofunded by Fondos FEDER (PI10/00871, January 2011 and PI13/00593, January 2014 to B.R.G.); Ministerio de Economía y Competitividad, Plan Nacional (BFU2017-83588-P, January 2018 to B.R.G. and PRE2018-084907, January 2018 to E.M.-V.); Juvenile Diabetes Research Foundation JDRF (17-2013-372, August 2013 and 2-SRA-2019-837-S-B, August 2019 to B.R.G:); and Fundacion Vencer el Cancer (to B.R.G.). Special thanks to ALUSVI (Asociación Lucha y Sonríe por la Vida, Pilas), a local Andalusian association, and the Fundacion DiabetesCero for their unconditional financial support.

RationaleWe recently demonstrated that the ‘Metabesity’ factor HMG20A regulates islet beta-cell functional maturity and adaptation to physiological stress such as pregnancy and pre-diabetes. HMG20A also dictates central nervous system (CNS) development via inhibition of the LSD1/CoREST complex but its expression pattern and function in adult brain remains unknown. Herein we sought to determine whether HMG20A is expressed in the adult CNS, specifically in hypothalamic astrocytes that are key in glucose homeostasis and whether similar to islets, HMG20A potentiates astrocyte function in response to environmental cues.MethodsHMG20A expression profile was assessed by quantitative PCR (RT-PCR) and/or immunofluorescence in: 1) the hypothalamus of mice exposed or not to a high-fat diet, 2) human blood leukocytes and adipose tissue obtained from healthy or diabetic individuals 3) primary mouse hypothalamic astrocytes exposed to either high glucose or palmitate. To investigate the function and regulatory mechanism of HMG20A, RNA-seq and cell metabolic parameters were performed on astrocytes treated or not with a siHMG20A. The regulatory function of HMG20A on astrogliosis was also assessed pharmacologically using ORY1001. Astrocyte-mediated neuronal survival was evaluated using conditioned media from siHMG20A-treated astrocytes.ResultsWe show that Hmg20a is predominantly expressed in hypothalamic astrocytes, the main nutrient-sensing cell type of the brain. Hmg20A expression was upregulated in diet-induced obesity and glucose intolerant mice, correlating with increased transcript levels of Gfap and Il1b indicative of inflammation and astrogliosis. Expression levels were also increased in adipose tissue of obese non-diabetic individuals as compared to obese diabetic patients. HMG20A silencing in astrocytes resulted in repression of inflammatory, cholesterol biogenesis and epithelial-to-mesenchymal transition pathways with a concomitant increase in apoptosis and reduced mitochondrial bioenergetics. Motoneuron viability was also hindered in HMG20A-depleted astrocyte-derived conditioned media. Astrogliosis was induced using ORY1001, a pharmacological inhibitor of the LSD1/CoREST complex, mimicking the effect of HMG20A.ConclusionHMG20A coordinates the astrocyte polarization state. Under physiological pressure such as obesity and insulin resistance that induces low grade inflammation, HMG20A expression is increased to induce astrogliosis in an attempt to preserve the neuronal network and glucose homeostasis. Nonetheless, a chronic metabesity state or functional mutations will result in lower levels of HMG20A, failure to promote astrogliosis and increase susceptibility of neurons to stress-mediated apoptosis. Such effects could be therapeutically reversed by ORY1001-induced astrogliosis.

Background and PurposeThe atypical cannabinoid Abn-CBD was reported to improve the inflammatory status in preclinical models of several pathologies including autoimmune diseases. However, its potential for autoimmune diabetes, i.e. type 1 diabetes (T1D), is unknown.Experimental ApproachWe used two mouse models of T1D, streptozotocin (STZ)-injected and non-obese diabetic (NOD) mice. Eight-to-ten-week-old male C57Bl6/J mice were pre-treated with Abn-CBD (1mg/kg of body weight) or vehicle for 1 week, following STZ treatment, and euthanized 1 week later. Six-week-old female NOD mice were treated with Abn-CBD (0.1-1mg/kg) or vehicle for 12 weeks and then euthanized. Blood, pancreas, pancreatic lymph nodes and circulating T cells were collected and processed for analysis. Glycemia was also monitored.Key ResultsAbn-CBD decreased circulating proinflammatory cytokines, ameliorated islet inflammation and the autoimmune attack, showing a 2-fold decrease in CD8+ T cells infiltration and reduced Th1/Th2 ratio in pancreatic lymph nodes of STZ-injected mice. Mechanistically, Abn-CBD reduced intra-islet phospho-NF-κB and TXNIP. Concomitant reduction of islet cell apoptosis and intra-islet fibrosis were observed in Abn-CBD pre-treated mice compared to vehicle. In NOD mice, Abn-CBD reduced the expression of Ifng, Il21, Tnfa and Il10 while increased Il4 in circulating CD4+ T cells compared to vehicle, reducing the severity of insulitis and improving glucose tolerance.Conclusion and ImplicationsAltogether, we found that Abn-CBD reduces intra-islet inflammation and delays the progression of insulitis in mouse models of T1D, preserving healthy functional islets. Hence, Abn-CBD and related compounds emerge as new candidates to develop pharmacological strategies to treat early stages of T1D.WHAT IS ALREADY KNOWN-Phytocannabinoids such as cannabidiol (CBD) have anti-inflammatory and glucose-lowering properties-The CBD derivative Abn-CBD ameliorates inflammation in various diseases and modulates beta cell functionWHAT THIS STUDY ADDS-Abn-CBD reduces systemic and pancreatic inflammation in mice models of type 1 diabetes-Abn-CBD prevents beta cell damage and loss during type 1 diabetes onsetCLINICAL SIGNIFICANCE-Synthetic cannabinoids emerge as potential treatment for type 1 diabetes

It is well established that systemic glucose homeostasis is imparted by an intricate cross-talk among several organs including the central nervous system (CNS) and pancreatic islets. In analogy to islet beta cells, hypothalamic astrocytes are glucose sensors that facilitate the CNS response to changing metabolic environment. Therefore, astrocytes and beta cells may share a common genetic signature implicated in coupling glucose metabolism to cellular output. We recently demonstrated that the diabesity factor HMG20A is essential for beta-cell functional maturity and adaptation to physiological stress (obesity, type 2 diabetes and pregnancy). As this chromatin remodelling factor also dictates CNS development, we sought to determine whether HMG20A is expressed in astrocytes and whether it potentiates astrocyte function in response to environmental cues. We found that HMG20A is expressed in hypothalamic astrocytes and is upregulated in diet-induced obesity and glucose-intolerant mice, correlating with increased transcript levels of GFAP and IL-1β, indicative of reactive astrocytes (astrogliosis). High glucose, but not lipids, transiently decreased HMG20A expression in isolated mouse primary astrocytes. HMG20A silencing in astrocytes resulted in repression of pro-inflammatory as well as lipid and glucose metabolism genes. These pathways are up-regulated in activated astrocytes (astrogliosis) to resolve stress conditions. As such astrocyte depleted of HMG20A were more susceptible to cell death. Furthermore, motor neuron viability in astrocyte conditioned media was significantly hindered after HMG20A depletion in astrocytes. We posit that HMG20A is involved in the astrocyte polarization state. Under physiological pressure such as obesity, HMG20A expression may be transiently decreased by high glucose but long-term it is increased to induce astrogliosis in an attempt to preserve the neuronal network and glucose homeostasis. Disclosure J.M. Mellado-Gil: None. E. Fuente-Martin: None. P.I. Lorenzo: None. N. Cobo-Vuilleumier: None. J.A. Guerrero-Martínez: None. S.Y. Romero-Zerbo: None. J.M. Franco: None. J. Perez-Cabello: None. M. Alvarez-Dolado: None. D. Pozo: None. F.J. Bermudez-Silva: None. J.C. Reyes: None. B.R. Gauthier: None. Funding ISCIII (PI13/00593 to B.R.G.), (PI13/00309 to F.J.B-S.); Ministerio de Economia y Competidividad/Fondos FEDER (PI10/00871, PI13/00593, BFU2017-83588-P to B.R.G.); BFU2017-85420-R (to J.C.R.); (JCI-2012-12491, IJCI-2015-26238 to E.F-M.); Consejería de Salud Junta de Andalucía (PI-0006-2016 to E.F-M.)

The vast majority of type 1 diabetes (T1D) genetic association signals lie in noncoding regions of the human genome. Many have been predicted to affect the expression and secondary structure of long noncoding RNAs (lncRNAs), but the contribution of these lncRNAs to the pathogenesis of T1D remains to be clarified. Here, we performed a complete functional characterization of a lncRNA that harbors a single nucleotide polymorphism (SNP) associated with T1D, namely, Lnc13. Human pancreatic islets harboring the T1D-associated SNP risk genotype in Lnc13 (rs917997*CC) showed higher STAT1 expression than islets harboring the heterozygous genotype (rs917997*CT). Up-regulation of Lnc13 in pancreatic β-cells increased activation of the proinflammatory STAT1 pathway, which correlated with increased production of chemokines in an allele-specific manner. In a mirror image, Lnc13 gene disruption in β-cells partially counteracts polyinosinic-polycytidylic acid (PIC)-induced STAT1 and proinflammatory chemokine expression. Furthermore, we observed that PIC, a viral mimetic, induces Lnc13translocation from the nucleus to the cytoplasm promoting the interaction of STAT1 mRNA with (poly[rC] binding protein 2) (PCBP2). Interestingly, Lnc13-PCBP2 interaction regulates the stability of the STAT1 mRNA, sustaining inflammation in β-cells in an allele-specific manner. Our results show that the T1D-associated Lnc13 may contribute to the pathogenesis of T1D by increasing pancreatic β-cell inflammation. These findings provide information on the molecular mechanisms by which disease-associated SNPs in lncRNAs influence disease pathogenesis and open the door to the development of diagnostic and therapeutic approaches based on lncRNA targeting.

The vast majority of type 1 diabetes (T1D) genetic association signals lie in noncoding regions of the human genome. Many have been predicted to affect the expression and secondary structure of long noncoding RNAs (lncRNAs), but the contribution of these lncRNAs to the pathogenesis of T1D remains to be clarified. Here, we performed a complete functional characterization of a lncRNA that harbors a single nucleotide polymorphism (SNP) associated with T1D, namely, Lnc13. Human pancreatic islets harboring the T1D-associated SNP risk genotype in Lnc13 (rs917997*CC) showed higher STAT1 expression than islets harboring the heterozygous genotype (rs917997*CT). Up-regulation of Lnc13 in pancreatic β-cells increased activation of the proinflammatory STAT1 pathway, which correlated with increased production of chemokines in an allele-specific manner. In a mirror image, Lnc13 gene disruption in β-cells partially counteracts polyinosinic-polycytidylic acid (PIC)-induced STAT1 and proinflammatory chemokine expression. Furthermore, we observed that PIC, a viral mimetic, induces Lnc13 translocation from the nucleus to the cytoplasm promoting the interaction of STAT1 mRNA with (poly[rC] binding protein 2) (PCBP2). Interestingly, Lnc13-PCBP2 interaction regulates the stability of the STAT1 mRNA, sustaining inflammation in β-cells in an allele-specific manner. Our results show that the T1D-associated Lnc13 may contribute to the pathogenesis of T1D by increasing pancreatic β-cell inflammation. These findings provide information on the molecular mechanisms by which disease-associated SNPs in lncRNAs influence disease pathogenesis and open the door to the development of diagnostic and therapeutic approaches based on lncRNA targeting., This work was supported by a Research Project Grant from the Basque Department of Health (2015111068) and a Research Grant from Fundación de la Sociedad Española de Diabetes (FSED) to I.S. I.G.-M. and A.O.-G. were supported by Predoctoral Fellowship Grants from the UPV/EHU (Universidad del Pais Vasco/Euskal Herriko Unibertsitatea) and the Basque Department of Education, respectively. A.C.-R. was funded by a Grant from the Spanish Ministry of Science, Innovation and Universities (PGC2018-097573-A-I00). D.L.E. received support from the Fonds National de la Recherche 420 Scientifique (FNRS), Welbio Grant CR-2015A-06. B.R.G. was funded by the Spanish Ministry of Science, Innovation and Universities; the European Fund for Regional Development (FEDER) (Grant BFU2017-83588-P); and the Juvenile Diabetes Research Foundation (Grants 17-2013-372 and 2-SRA-2019-837-S-B).

The high prevalence of diabetes mellitus (DM) in our society, together with the fact that current treatments are only palliative and do not prevent the development of life threatening side effects, highlights the urgent need for novel therapies targeting the root cause of the disease. Independent of the etiology of DM, the definitive therapeutic approach will imply the restitution of an adequate functional b-cell mass capable of compensating for the insulin demand of the organism. The recent demonstration of heterogeneity within the islets as well as their innate plasticity has encouraged the development of studies aiming at potentiation of the regenerative capacity of islets. In this regard, factors implicated in pancreas ontogeny as well as in the adaptation processes that take place in the islets under situations of increased insulin demand have gained much interest. One of these factors is the transcription factor PAX4, required for b-cell formation during embryonic development and implicated in adult b-cell adaptation under stress situations. Here we review the therapeutic potential of PAX4 as well as its downstream targets for the development of novel treatments for DM., Authors are supported by grants from the Consejería de Salud, Fundación Pública Andaluza Progreso y Salud, Junta de Andalucía (PI-0727-2010 to BRG and PI-0085-2013 to PIL), Consejería de Economía, Innovación y Ciencia (P10.CTS.6359 to BRG) and the Ministerio de Economía y Competidividad, Instituto de Salud Carlos III co-funded by Fondos FEDER (PI10/00871 and PI13/00593 to BRG).

The atypical cannabinoid Abn-CBD improves the inflammatory status in preclinical models of several pathologies, including autoimmune diseases. However, its potential for modulating inflammation in autoimmune type 1 diabetes (T1D) is unknown. Herein we investigate whether Abn-CBD can modulate the inflammatory response during T1D onset using a mouse model of T1D (non-obese diabetic- (NOD)-mice) and of beta cell damage (streptozotocin (STZ)-injected mice). Six-week-old female NOD mice were treated with Abn-CBD (0.1–1 mg/kg) or vehicle during 12 weeks and then euthanized. Eight-to-ten-week-old male C57Bl6/J mice were pre-treated with Abn-CBD (1 mg/kg of body weight) or vehicle for 1 week, following STZ challenge, and euthanized 1 week later. Blood, pancreas, pancreatic lymph nodes (PLNs) and T cells were collected and processed for analysis. Glycemia was also monitored. In NOD mice, treatment with Abn-CBD significantly reduced the severity of insulitis and reduced the pro-inflammatory profile of CD4 T cells compared to vehicle. Concomitantly, Abn-CBD significantly reduced islet cell apoptosis and improved glucose tolerance. In STZ-injected mice, Abn-CBD decreased circulating proinflammatory cytokines and ameliorated islet inflammation reducing intra-islet phospho-NF-κB and TXNIP. Abn-CBD significantly reduced 2 folds intra-islet CD8 T cells and reduced Th1/non-Th1 ratio in PLNs of STZ-injected mice. Islet cell apoptosis and intra-islet fibrosis were also significantly reduced in Abn-CBD pre-treated mice compared to vehicle. Altogether, Abn-CBD reduces circulating and intra-islet inflammation, preserving islets, thus delaying the progression of insulitis. Hence, Abn-CBD and related compounds emerge as new candidates to develop pharmacological strategies to treat the early stages of T1D., H2020-MSCA-IF-2016, Grant Agreement number: 748749, EU. Consejeria de Salud y Familias, Junta de Andalucia, Spain (PI-0318-2018). Instituto de Salud Carlos III, Ministerio de Sanidad, Gobierno de España, Spain (PI17/01004). Ministerio de Ciencia, Innovación y Universidades, Agencia Estatal de Investigación and Fondo Europeo de Desarrollo Regional, Spain (BFU2017-83588-P to BRG). We thank all the staff of the animal facility at IBIMA (ECAI de Experimentación Animal) and especially its coordinator Dr. Ricardo González Carrascosa for his excellent work. The authors also gratefully acknowledge all the staff of the bioimaging facility at IBIMA (ECAI de Imagen) and the proteomic (ECAI de Proteómica) facility, especially Dra. Carolina Lobo-García for excellent technical assistance. FJBS and IGM belong to the regional "Nicolás Monardes" research program from Consejería de Salud y Familias (C-0070-2012, RC0005-2016, RC-0001-2021 and C1-0018-2019; Junta de Andalucía, Spain). FJBS, NCV and BRG are members of the pancreatic islets study group from the Spanish Society for Diabetes (SED), Spain. CIBERDEM is an initiative of the Instituto de Salud Carlos III, Spain.

Background and Purpose Thyroid hormones induce several changes in whole body metabolism that are known to improve metabolic homeostasis. However, adverse side effects have prevented its use in the clinic. In view of the promising effects of thyroid hormones, we investigated the effects of levothyroxine supplementation on glucose homeostasis. Experimental Approach C57BL/6 mice were treated with levothyroxine from birth to 24 weeks of age, when mice were killed. The effects of levothyroxine supplementation on metabolic health were determined. C57BL/6 mice treated with levothyroxine for 2 weeks and then challenged with streptozotocin to monitor survival. Mechanistic experiments were conducted in the pancreas, liver and skeletal muscle. RIP-B7.1 mice were treated with levothyroxine for 2 weeks and were subsequently immunized to trigger experimental autoimmune diabetes (EAD). Metabolic tests were performed. Mice were killed and metabolic tissues were extracted for immunohistological analyses. Key Results Long-term levothyroxine supplementation enhanced glucose clearance and reduced circulating glucose in C57BL/6 mice. Levothyroxine increased simultaneously the proliferation and apoptosis of pancreatic beta cells, promoting the maintenance of a highly insulin-expressing beta cell population. Levothyroxine increased circulating insulin levels, inducing sustained activation of IRS1-AKT signalling in insulin-target tissues. Levothyroxine-treated C57BL/6 mice challenged with streptozotocin exhibited extended survival. Levothyroxine blunted the onset of EAD in RIP-B7.1 mice by inducing beta cell proliferation and preservation of insulin-expressing cells. Conclusions and Implications Interventions based on the use of thyroid hormones or thyromimetics could be explored to provide therapeutic benefit in patients with type 1 diabetes mellitus.

Thyroid hormones are central in the control of health and disease. Here we review the latest findings in thyroid hormone research in the field of diabetes, cancer, and aging. Aging studies indicate that the direct modulation of thyroid hormones is not an viable strategy to promote healthy aging. However, although challenging, interventions based on the use of modulators of thyroid hormone function might provide therapeutic benefit in certain types of diabetes and cancers., Summary Thyroid function is central in the control of physiological and pathophysiological processes. Studies in animal models and human research have determined that thyroid hormones modulate cellular processes relevant for aging and for the majority of age‐related diseases. While several studies have associated mild reductions on thyroid hormone function with exceptional longevity in animals and humans, alterations in thyroid hormones are serious medical conditions associated with unhealthy aging and premature death. Moreover, both hyperthyroidism and hypothyroidism have been associated with the development of certain types of diabetes and cancers, indicating a great complexity of the molecular mechanisms controlled by thyroid hormones. In this review, we describe the latest findings in thyroid hormone research in the field of aging, diabetes, and cancer, with a special focus on hepatocellular carcinomas. While aging studies indicate that the direct modulation of thyroid hormones is not a viable strategy to promote healthy aging or longevity and the development of thyromimetics is challenging due to inefficacy and potential toxicity, we argue that interventions based on the use of modulators of thyroid hormone function might provide therapeutic benefit in certain types of diabetes and cancers.

Type 1 diabetes mellitus (T1DM) is an autoimmune disease that targets the destruction of islet beta-cells resulting in insulin deficiency, hyperglycemia and death if untreated. Despite advances in medical devices and longer-acting insulin, there is still no robust therapy to substitute and protect beta-cells that are lost in T1DM. Attempts to refrain from the autoimmune attack have failed to achieve glycemic control in patients highlighting the necessity for a paradigm shift in T1DM treatment. Paradoxically, beta-cells are present in T1DM patients indicating a disturbed equilibrium between the immune attack and beta-cell regeneration reminiscent of unresolved wound healing that under normal circumstances progression towards an anti-inflammatory milieu promotes regeneration. Thus, the ultimate T1DM therapy should concomitantly restore immune self-tolerance and replenish the beta-cell mass similar to wound healing. Recently the agonistic activation of the nuclear receptor LRH-1/NR5A2 was shown to induce immune self-tolerance, increase beta-cell survival and promote regeneration through a mechanism of alpha-to-beta cell phenotypic switch. This trans-regeneration process appears to be facilitated by a pancreatic anti-inflammatory environment induced by LRH-1/NR5A2 activation. Herein, we review the literature on the role of LRH1/NR5A2 in immunity and islet physiology and propose that a cross-talk between these cellular compartments is mandatory to achieve therapeutic benefits., The research of B.R.G is/was supported by the Juvenile Diabetes Research Foundation (17-2013-372 and 2-SRA-2019-837-S-B), the DiabetesCero Foundation, Vencer el Cancer, ALUSVI, the Consejeria de Salud, Fundacion Publica Andaluza Progreso y Salud, Junta de Andalucia (PI-0727-2010), Consejeria de Economia, Innovacion y Ciencia (P10.CTS.6359) and the Ministerio de Economia y Competidividad co-funded by Fondos FEDER (PI10/00871, PI13/00593 and BFU2017-83588-P).

Islet β-cell replacement and regeneration are two promising approaches for the treatment of Type 1 Diabetes Mellitus. Indeed, the success of islet transplantation in normalizing blood glucose in diabetic patients has provided the proof of principle that cell replacement can be employed as a safe and efficacious treatment. Nonetheless, shortage of organ donors has hampered expansion of this approach. Alternative sources of insulin-producing cells are mandatory to fill this gap. Although great advances have been achieved in generating surrogate β-cells from stem cells, current protocols have yet to produce functionally mature insulin-secreting cells. Recently, the concept of islet regeneration in which new β-cells are formed from either residual β-cell proliferation or transdifferentiation of other endocrine islet cells has gained much interest as an attractive therapeutic alternative to restore β-cell mass. Complementary approaches to cell replacement and regeneration could aim at enhancing β-cell survival and function. Herein, we discuss the value of Hepatocyte Growth Factor (HGF), Glucose-Dependent Insulinotropic Peptide (GIP), Paired box gene 4 (Pax4) and Liver Receptor Homolog-1 (LRH-1) as key players for β-cell replacement and regeneration therapies. These factors convey β-cell protection and enhanced function as well as facilitating proliferation and transdifferentiation of other pancreatic cell types to β-cells, under stressful conditions.

The high prevalence of type 2 diabetes mellitus (T2DM), together with the fact that current treatments are only palliative and do not avoid major secondary complications, reveals the need for novel approaches to treat the cause of this disease. Efforts are currently underway to identify therapeutic targets implicated in either the regeneration or re-differentiation of a functional pancreatic islet β-cell mass to restore insulin levels and normoglycemia. However, T2DM is not only caused by failures in β-cells but also by dysfunctions in the central nervous system (CNS), especially in the hypothalamus and brainstem. Herein, we review the physiological contribution of hypothalamic neuronal and glial populations, particularly astrocytes, in the control of the systemic response that regulates blood glucose levels. The glucosensing capacity of hypothalamic astrocytes, together with their regulation by metabolic hormones, highlights the relevance of these cells in the control of glucose homeostasis. Moreover, the critical role of astrocytes in the response to inflammation, a process associated with obesity and T2DM, further emphasizes the importance of these cells as novel targets to stimulate the CNS in response to metabesity (over-nutrition-derived metabolic dysfunctions). We suggest that novel T2DM therapies should aim at stimulating the CNS astrocytic response, as well as recovering the functional pancreatic β-cell mass. Whether or not a common factor expressed in both cell types can be feasibly targeted is also discussed., The authors are supported by the Consejería de Salud, Fundación Pública Andaluza Progreso y Salud, Junta de Andalucía (PI-0727-2010 to B.R.G., PI-0085-2013 to P.I.L., PI-0006-2016 to E.F.M., PI-0574-2012 to S.Y.R.Z., and PI-0247-2016 to F.J.B.S.), the Consejería de Economía, Innovación y Ciencia (P10.CTS.6359 to B.R.G.), the Ministerio de Economía y Competitividad, Instituto de Salud Carlos III co-funded by Fondos FEDER (PI10/00871 and PI13/00593 to B.R.G., PI13/00309 and PI17/01004 to F.J.B.S., and BFU2014-5343-P to J.C.R.), Vencer el Cancer (B.R.G), DiabetesCero (B.R.G.), and the Red TerCel program (RD12/0019/0028 to B.S.). E.F.M. is recipient of a Juan de la Cierva Incorporación Fellowship from the Ministerio de Economía y Competitividad (IJCI-2015-26238). S.Y.R.Z. is a recipient of a postdoctoral fellowship from Consejería de Salud, Junta de Andalucía (RH-0070-2013). F.J.B.S. is recipient of a “Nicolás Monardes” research contract from Consejería de Salud Junta de Andalucía, (C-0070-2012). A.M.M. is supported by CP14/00105 and PI18/01590 from the Instituto de Salud Carlos III co-funded by Fondes FEDER. CIBERDEM is an initiative of the Instituto de Salud Carlos III.

34 Páginas.-- 7 Figuras.-- 6 Figuras suplementarias.-- 4 Tablas suplementarias, An inverse correlation between thyroid hormone levels and longevity has been reported in several species and reduced thyroid hormone levels have been proposed as a biomarker for healthy aging and metabolic fitness. However, hypothyroidism is a medical condition associated with compromised health and reduced life expectancy. Herein, we show, using wild-type and the Pax8 ablated model of hypothyroidism in mice, that hyperthyroidism and severe hypothyroidism are associated with an overall unhealthy status and shorter lifespan. Mild hypothyroid Pax8 +/- mice were heavier and displayed insulin resistance, hepatic steatosis and increased prevalence of liver cancer yet had normal lifespan. These pathophysiological conditions were precipitated by hepatic mitochondrial dysfunction and oxidative damage accumulation. These findings indicate that individuals carrying mutations on PAX8 may be susceptible to develop liver cancer and/or diabetes and raise concerns regarding the development of interventions aiming to modulate thyroid hormones to promote healthy aging or lifespan in mammals., This work was funded by grants from the Ministerio de Economía y Competitividad, Instituto de Salud Carlos III co-funded by Fondos FEDER and from the nonprofit foundation ‘Fundación Progreso y Salud’ of the Andalusian Regional Ministry of Health (BFU2017-83588-P to B.R.G; CP14/00105, PI18/01590 and PI15/00134 to A.M-M; PI-0272-2017 and CD16/00118 to V.C-G; PI17/01286 to C. S-O and A. S-C).

Transient Pax8 expression was reported in mouse islets during gestation, whereas a genome-wide linkage and admixture mapping study highlighted PAX8 as a candidate gene for diabetes mellitus (DM). We sought the significance of PAX8 expression in mouse and human islet biology. PAX8 was induced in gestating mouse islets and in human islets treated with recombinant prolactin. Global gene expression profiling of human and mouse islets overexpressing the corresponding species-specific PAX8 revealed the modulation of distinct genetic pathways that converge on cell survival. Accordingly, apoptosis was reduced in PAX8-overexpressing islets. These findings support that PAX8 could be a candidate gene for the study of gestational DM (GDM). PAX8 was genotyped in patients with GDM and gestational thyroid dysfunction (GTD), a pathology commonly found in patients with mutations on PAX8. A novel missense PAX8 mutation (p.T356M, c.1067C>T) was identified in a female diagnosed with GDM and GTD as well as in her father with type 2 DM but was absent in control patients. The p.T356M variant did not alter protein stability or cellular localization, whereas its transactivation activity was hindered. In parallel, a retrospective clinical analysis uncovered that a pregnant female harboring a second PAX8 mutation (p.P25R, c.74C>G) previously reported to cause congenital hypothyroidism also developed GDM. These data indicate that increased expression of PAX8 affects islet viability and that PAX8 could be considered as a candidate gene for the study of GDM., This work was funded by grants from the Ministerio de Economía y Competitividad, Instituto de Salud Carlos III, co-funded by fondos FEDER (CP14/00105 and PI15/00134 to A.M.-M., PI16/00830 to J.C.M., and PI13/00593 and BFU2017-83588-P to B.R.G.). Human islets were procured through the European Consortium for Islet Transplantation funded by JDRF (3- RSC-2016-162-I-X)

Most studies in type 1 diabetes (T1D) have focused on the loss of the pancreatic beta-cell population. However, despite the involvement of the alpha-cell in the aetiology and complications of T1D, little is known about the regulation of the pancreatic alpha-cell mass in this disease. The need for a better understanding of this process is further emphasized by recent findings suggesting that alpha-cells may constitute a potential reservoir for beta-cell regeneration. In this study, we characterized the pancreatic alpha-cell mass and its regulatory processes in the transgenic RIP-B7.1 mice model of experimental autoimmune diabetes (EAD). Diabetic mice presented insulitis, hyperglycaemia, hypoinsulinemia and hyperglucagonemia along with lower pancreatic insulin content. While alpha-cell mass and pancreatic glucagon content were preserved at the early-onset of EAD, both parameters were reduced in the advanced phase. At both stages, alpha-cell size, proliferation and ductal neogenesis were up-regulated, whereas apoptosis was almost negligible. Interestingly, we found an increase in the proportion of glucagon-containing cells positive for insulin or the beta-cell transcription factor PDX1. Our findings suggest that pancreatic alpha-cell renewal mechanisms are boosted during the natural course of EAD, possibly as an attempt to maintain the alpha-cell population and/or to increase beta-cell regeneration via alpha-cell transdifferentiation., This research was supported by grants from the Ministerio de Ciencia, Innovación y Universidades, Agencia Estatal de Investigación and Fondo Europeo de Desarrollo Regional (BFU2013-42789; BFU2017-86579-R; BFU2016-77125-R; BFU2017-83588-P), Generalitat Valenciana (PROMETEOII/2015/016) and the Juvenile Diabetes Research Foundation (17-2013-372 to B.R.G.). L.M. holds a Juan de la Cierva fellowship from the Ministerio de Ciencia, Innovación y Universidades (IJCI-2015-24482). CIBERDEM is an initiative of the Instituto de Salud Carlos III.

Gestational diabetes mellitus (GDM), a metabolic disease that develops with the increase in insulin resistance during late pregnancy, is currently one of the most common complications affecting pregnancy. The polygenic nature of GDM, together with the interplay between different genetic variants with nutritional and environmental factors has hindered the full understanding of the etiology of this disease. However, an important genetic overlap has been found with type 2 diabetes mellitus (T2DM) and, as in the case of T2DM, most of the identified loci are associated with β-cell function. Early detection of GDM and adequate interventions to control the maternal glycemia are necessary to avoid the adverse outcomes for both the mother and the offspring. The in utero exposure to the diabetic milieu predispose these children for future diseases, among them T2DM, originating a vicious circle implicated in the increased prevalence of both GDM and T2DM. The involvement of inflammatory processes in the development of GDM highlights the importance of pancreatic β-cell factors able to favor the adaptation processes required during gestation, concomitantly with the protection of the islets from an inflammatory milieu. In this regard, two members of the Pax family of transcription factors, PAX4 and PAX8, together with the chromatin remodeler factor HMG20A, have gained great relevance due to their involvement in β-cell mass adaptation together with their anti-inflammatory properties. Mutations in these factors have been associated with GDM, highlighting these as novel candidates for genetic screening analysis in the identification of women at risk of developing GDM., This research is/was supported by the Consejeria de Salud, Fundacion Publica Andaluza Progreso y Salud, Junta de Andalucía (PI-0727-2010, PI-0085-2013), Consejería de Economía, Innovación y Ciencia (P10.CTS.6359), Ministerio de Economía y Competitividad co-funded by Fondos FEDER (PI10/00871, PI13/00593) and Instituto de Salud Carlos III co-funded by Fondos FEDER (CP14/00105, PI18/01590, PI15/00134)

Transient

Cobo-Vuilleumier, Nadia et al., Type 1 diabetes mellitus (T1DM) is due to the selective destruction of islet beta cells by immune cells. Current therapies focused on repressing the immune attack or stimulating beta cell regeneration still have limited clinical efficacy. Therefore, it is timely to identify innovative targets to dampen the immune process, while promoting beta cell survival and function. Liver receptor homologue-1 (LRH-1) is a nuclear receptor that represses inflammation in digestive organs, and protects pancreatic islets against apoptosis. Here, we show that BL001, a small LRH-1 agonist, impedes hyperglycemia progression and the immune-dependent inflammation of pancreas in murine models of T1DM, and beta cell apoptosis in islets of type 2 diabetic patients, while increasing beta cell mass and insulin secretion. Thus, we suggest that LRH-1 agonism favors a dialogue between immune and islet cells, which could be druggable to protect against diabetes mellitus., This work was funded by grants from the Juvenile Diabetes Research Foundation (17-2013-372 to B.R.G.), the Consejeria de Salud, Fundacion Publica Andaluza Progreso y Salud, Junta de Andalucia (PI-0727-2010 to B.R.G. and P10CTS6505 to B.S.), Consejeria de Economia, Innovacion y Ciencia (P10.CTS.6359 to B.R.G.), the Ministerio de Economia y Competidividad co-funded by Fondos FEDER (PI10/00871, PI13/00593, and BFU2017-83588-P to B.R.G.; PI14/01015, RD12/0019/0028, and RD16/0011/0034 to B.S.; PI16/00259 to A.H.) and Deutsche Forschungsgemeinschaft (GRK-1789 ´CEMMA´ and DFG SCHI-505/6-1 to R.S.). Special thanks to the families of the DiabetesCero Foundation that graciously supported this work (to B.R.G.). A.M.M. is a recipient of a Miguel Servet grant (CP14/00105) from the Instituto de Salud Carlos III co-funded by Fondos FEDER whereas E.F.M. is a recipient of a Juan de la Cierva Fellowship. I.G.H.G. is supported by a fellowship from Amarna Therapeutics. In some instances, human islets were procured through the European Consortium for Islet Transplantation funded by Juvenile Diabetes Research Foundation (3-RSC-2016-162-I-X).

Mellado-Gil, José Manuel et al., HMG20A (also known as iBRAF) is a chromatin factor involved in neuronal differentiation and maturation. Recently small nucleotide polymorphisms (SNPs) in the HMG20A gene have been linked to type 2 diabetes mellitus (T2DM) yet neither expression nor function of this T2DM candidate gene in islets is known. Herein we demonstrate that HMG20A is expressed in both human and mouse islets and that levels are decreased in islets of T2DM donors as compared to islets from non-diabetic donors. In vitro studies in mouse and human islets demonstrated that glucose transiently increased HMG20A transcript levels, a result also observed in islets of gestating mice. In contrast, HMG20A expression was not altered in islets from diet-induced obese and pre-diabetic mice. The T2DM-associated rs7119 SNP, located in the 3′ UTR of the HMG20A transcript reduced the luciferase activity of a reporter construct in the human beta 1.1E7 cell line. Depletion of Hmg20a in the rat INS-1E cell line resulted in decreased expression levels of its neuronal target gene NeuroD whereas Rest and Pax4 were increased. Chromatin immunoprecipitation confirmed the interaction of HMG20A with the Pax4 gene promoter. Expression levels of Mafa, Glucokinase, and Insulin were also inhibited. Furthermore, glucose-induced insulin secretion was blunted in HMG20A-depleted islets. In summary, our data demonstrate that HMG20A expression in islet is essential for metabolism-insulin secretion coupling via the coordinated regulation of key islet-enriched genes such as NeuroD and Mafa and that depletion induces expression of genes such as Pax4 and Rest implicated in beta cell de-differentiation. More importantly we assign to the T2DM-linked rs7119 SNP the functional consequence of reducing HMG20A expression likely translating to impaired beta cell mature function., This work was supported by the Consejería de Salud, Fundación Pública Andaluza Progreso y Salud, Junta de Andalucía (PI-0727-2010 to B.R.G., PI-0085-2013 to P.I.L., PI-0006-2016 to E.F.-M. and PI-0574-2012 to S.Y.R.-Z.), the Consejería de Economía, Innovación y Ciencia (P10.CTS.6359 to B.R.G. and P09-CTS-5445 to A.C.-C.), the Ministerio de Economía y Competitividad, Instituto de Salud Carlos III co-funded by Fondos FEDER (PI10/00871 and PI13/00593 to B.R.G., and BFU2014-5343-P to J.C.R.) and the Red TerCel program (RD12/0019/0028 to B.S. and K.H.). E.F.M. is recipient of a Juan de la Cierva Incorporación Fellowship from the Ministerio de Economía y Competitividad (IJCI-2015-26238). S.Y.R.Z is a recipient of a postdoctoral fellowship from Consejería de Salud, Junta de Andalucía (RH-0070-2013). F.J.B.S. is recipient of a “Nicolás Monardes” research contract from Consejería de Salud Junta de Andalucía, (C-0070-2012). A.M.M. is supported by CP14/00105 and PI15/00134 from the Instituto de Salud Carlos III co-funded by Fondes FEDER. CIBERDEM is an initiative of the Instituto de Salud Carlos III.

Thyroid hormones induce several changes in whole body metabolism that are known to improve metabolic homeostasis. However, adverse side effects have prevented its use in the clinic. In view of the promising effects of thyroid hormones, we investigated the effects of levothyroxine supplementation on glucose homeostasis.C57BL/6 mice were treated with levothyroxine from birth to 24 weeks of age, when mice were killed. The effects of levothyroxine supplementation on metabolic health were determined. C57BL/6 mice treated with levothyroxine for 2 weeks and then challenged with streptozotocin to monitor survival. Mechanistic experiments were conducted in the pancreas, liver and skeletal muscle. RIP-B7.1 mice were treated with levothyroxine for 2 weeks and were subsequently immunized to trigger experimental autoimmune diabetes (EAD). Metabolic tests were performed. Mice were killed and metabolic tissues were extracted for immunohistological analyses.Long-term levothyroxine supplementation enhanced glucose clearance and reduced circulating glucose in C57BL/6 mice. Levothyroxine increased simultaneously the proliferation and apoptosis of pancreatic beta cells, promoting the maintenance of a highly insulin-expressing beta cell population. Levothyroxine increased circulating insulin levels, inducing sustained activation of IRS1-AKT signalling in insulin-target tissues. Levothyroxine-treated C57BL/6 mice challenged with streptozotocin exhibited extended survival. Levothyroxine blunted the onset of EAD in RIP-B7.1 mice by inducing beta cell proliferation and preservation of insulin-expressing cells.Interventions based on the use of thyroid hormones or thyromimetics could be explored to provide therapeutic benefit in patients with type 1 diabetes mellitus.

This article is distributed under the terms of the Creative Commons Attribution Noncommercial License.-- et al., The paired box transcription factor Pax8 is critical for development of the eye, thyroid gland as well as the urinary and reproductive organs. In adult, Pax8 overexpression is associated with kidney, ovarian and thyroid tumors and has emerged as a specific marker for these cancers. Recently, Pax8 expression was also reported in human pancreatic islets and in neuroendocrine tumors, identifying Pax8 as a novel member of the Pax family expressed in the pancreas. Herein, we sought to provide a comprehensive analysis of Pax8 expression during pancreogenesis and in adult islets. Immunohistochemical analysis using the most employed Pax8 polyclonal antibody revealed strong nuclear staining in the developing mouse pancreas and in mature human and mouse islets. Astonishingly, Pax8 mRNA in mouse islets was undetectable while human islets exhibited low levels. These discrepancies raised the possibility of antibody cross-reactivity. This premise was confirmed by demonstrating that the polyclonal Pax8 antibody also recognized the islet-enriched Pax6 protein both by Western blotting and immunohistochemistry. Thus, in islets polyclonal Pax8 staining corresponds mainly to Pax6. In order to circumvent this caveat, a novel Pax8 monoclonal antibody was used to re-evaluate whether Pax8 was indeed expressed in islets. Surprisingly, Pax8 was not detected in neither the developing pancreas or in mature islets. Reappraisal of pancreatic neuroendocrine tumors using this Pax8 monoclonal antibody exhibited no immunostaining as compared to the Pax8 polyclonal antibody. In conclusion, Pax8 is not expressed in the pancreas and cast doubts on the value of Pax8 as a pancreatic neuroendocrine tumor marker., This work was supported by grants from the Fundacion Progreso y Salud (to B.R.G.), the Spanish Ministry of Science and Innovation, Instituto de Salud Carlos III (PI10/00871 to B.R.G) and the Juvenile Diabetes Research Foundation (9-2004-384 to E.C.I.T Plan Nacional de I+D+I 2008-2011, ISCIII (PI08/0018) co-funded by Fondos FEDER (to A.R.) and Consejería de Salud, Junta de Andalucía (PI0008 to A.R.).

Mellado-Gil, José Manuel et al., [Aims/hypothesis]: A strategy to enhance pancreatic islet functional beta cell mass (BCM) while restraining inflammation, through the manipulation of molecular and cellular targets, would provide a means to counteract the deteriorating glycaemic control associated with diabetes mellitus. The aims of the current study were to investigate the therapeutic potential of such a target, the islet-enriched and diabetes-linked transcription factor paired box 4 (PAX4), to restrain experimental autoimmune diabetes (EAD) in the RIP-B7.1 mouse model background and to characterise putative cellular mechanisms associated with preserved BCM. [Methods]: Two groups of RIP-B7.1 mice were genetically engineered to: (1) conditionally express either PAX4 (BPTL) or its diabetes-linked mutant variant R129W (mutBPTL) using doxycycline (DOX); and (2) constitutively express luciferase in beta cells through the use of RIP. Mice were treated or not with DOX, and EAD was induced by immunisation with a murine preproinsulin II cDNA expression plasmid. The development of hyperglycaemia was monitored for up to 4 weeks following immunisation and alterations in the BCM were assessed weekly by non-invasive in vivo bioluminescence intensity (BLI). In parallel, BCM, islet cell proliferation and apoptosis were evaluated by immunocytochemistry. Alterations in PAX4- and PAX4R129W-mediated islet gene expression were investigated by microarray profiling. PAX4 preservation of endoplasmic reticulum (ER) homeostasis was assessed using thapsigargin, electron microscopy and intracellular calcium measurements. [Results]: PAX4 overexpression blunted EAD, whereas the diabetes-linked mutant variant PAX4R129W did not convey protection. PAX4-expressing islets exhibited reduced insulitis and decreased beta cell apoptosis, correlating with diminished DNA damage and increased islet cell proliferation. Microarray profiling revealed that PAX4 but not PAX4R129W targeted expression of genes implicated in cell cycle and ER homeostasis. Consistent with the latter, islets overexpressing PAX4 were protected against thapsigargin-mediated ER-stress-related apoptosis. Luminal swelling associated with ER stress induced by thapsigargin was rescued in PAX4-overexpressing beta cells, correlating with preserved cytosolic calcium oscillations in response to glucose. In contrast, RNA interference mediated repression of PAX4-sensitised MIN6 cells to thapsigargin cell death. [Conclusions/interpretation]: The coordinated regulation of distinct cellular pathways particularly related to ER homeostasis by PAX4 not achieved by the mutant variant PAX4R129W alleviates beta cell degeneration and protects against diabetes mellitus. The raw data for the RNA microarray described herein are accessible in the Gene Expression Omnibus database under accession number GSE62846., This work was funded by grants from the Consejeria de Salud, Fundacion Publica Andaluza Progreso y Salud, Junta de Andalucia (PI-0727-2010 to BRG and PI-0085-2013 to PIL), Consejeria de Economia, Innovacion y Ciencia (P10.CTS.6359 to BRG), Ministerio de Ciencia e Innovacion (BFU2013-42789-P to IQ) and the Ministerio de Economia y Competidividad, Instituto de Salud Carlos III co-funded by Fondos FEDER (PI10/00871 and PI13/00593 to BRG). NC-V is supported by a JDRF subsidy (17-2013-372 to BRG.). AM-M is a recipient of a Miguel Servet grant (CP14/00105) from the Instituto de Salud Carlos III co-funded by Fondos FEDER and EF-M is a recipient of a Juan de la Cierva Fellowship. PM is supported by Swiss National Science Foundation grant 310030-141162, and the European Union grant IMIDIA, C2008-T7. BOB is supported by grants from the Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore, Republic of Singapore.

PAX4 is a key regulator of pancreatic islet development whilst in adult acute overexpression protects β-cells against stress-induced apoptosis and stimulates proliferation. Nonetheless, sustained PAX4 expression promotes β-cell dedifferentiation and hyperglycemia, mimicking β-cell failure in diabetic patients. Herein, we study mechanisms that allow stringent PAX4 regulation endowing favorable β-cell adaptation in response to changing environment without loss of identity. To this end, PAX4 expression was monitored using a mouse bearing the enhanced green fluorescent protein (GFP) and cre recombinase construct under the control of the islet specific pax4 promoter. GFP was detected in 30% of islet cells predominantly composed of PAX4-enriched β-cells that responded to glucose-induced insulin secretion. Lineage tracing demonstrated that all islet cells were derived from PAX4+ progenitor cells but that GFP expression was confined to a subpopulation at birth which declined with age correlating with reduced replication. However, this GFP+ subpopulation expanded during pregnancy, a state of active β-cell replication. Accordingly, enhanced proliferation was exclusively detected in GFP+ cells consistent with cell cycle genes being stimulated in PAX4-overexpressing islets. Under stress conditions, GFP+ cells were more resistant to apoptosis than their GFP- counterparts. Our data suggest PAX4 defines an expandable β-cell sub population within adult islets.

Paired box 4 (PAX4) is a key factor in the generation of insulin producing β-cells during embryonic development. In adult islets, PAX4 expression is sequestered to a subset of β-cells that are prone to proliferation and more resistant to stress-induced apoptosis. The importance of this transcription factor for adequate pancreatic islets functionality has been manifested by the association of mutations in PAX4 with the development of diabetes, independently of its etiology. Overexpression of this factor in adult islets stimulates β-cell proliferation and increases their resistance to apoptosis. Additionally, in an experimental model of autoimmune diabetes, a novel immunomodulatory function for this factor has been suggested. Altogether these data pinpoint at PAX4 as an important target for novel regenerative therapies for diabetes treatment, aiming at the preservation of the remaining β-cells in parallel to the stimulation of their proliferation to replenish the β-cell mass lost during the progression of the disease. However, the adequate development of such therapies requires the knowledge of the molecular mechanisms controlling the expression of PAX4 as well as the downstream effectors that could account for PAX4 action., This work was funded by grants from Consejería de Salud, Fundación Pública Andaluza Progreso y Salud, Junta de Andalucía (PI-0727-2010 to B.R.G. and PI-0085-2013 to P.I.L.), Consejería de Economía, Innovación y Ciencia, Junta de Andalucía (P10-CTS-6359 to B.R.G. and P12-CTS-2064 to M.G.-D.), Ministerio de Economía y Competitividad, Instituto de Salud Carlos III co-fundedby Fondos FEDER (PI10/00871 and PI13/00593 to B.R.G.), Ministerio de Economía y Competitividad, Plan Nacional (BFU2015-64721-P to M.G.-D.) and Fundacion Vencer el Cancer (to B.R.G.). Special thank to ALUSVI (Asociación Lucha y Sonríe por la Vida, Pilas), a local Andalucía association, for their unconditional financial support.

Pancreatic islets are complex structures formed by five different hormoneexpressing cells surrounded by endothelial cells, nerves, and fibroblasts. Dysfunction of insulin-producing cells (β-cells) causes diabetes. Generation of β-like cells that can compensate the loss of β-cell mass in type 1 diabetes or defects in β-cell insulin secretion in type 2 diabetes is a current challenge in biomedicine. The knowledge of the molecular basis governing pancreas development and islet formation will help us to generate in vitro or in vivo

OBJECTIVE To evaluate the heterogeneity in the clinical expression in a family with glucokinase mature-onset diabetes of the young (GCK-MODY). RESEARCH DESIGN AND METHODS Members (three generations) of the same family presented either with overt neonatal hyperglycemia, marked postprandial hyperglycemia, or glucosuria. Homeostasis model assessment of insulin resistance (HOMAIR) and insulinogenic and disposition indexes were calculated. Oral glucose tolerance test (OGTT) results in the GCK mutation carriers from this family were compared with those from other subjects with GCK mutations in the same codon (GCK261), with other missense and other types of GCK mutations in different codons from the European MODY Consortium database (GCKm). RESULTS Mutation G261R was found in the GCK gene. During the OGTT, glucose (P = 0.02) and insulin (P = 0.009) response at 2 h as well as at the 2-h glucose increment (GCK261 versus other missense GCK mutations, P = 0.003) were significantly higher in GCK261 than in GCKm carriers. CONCLUSIONS Differing from other GCKm carriers, the glucose and insulin response to oral glucose was significantly higher in GCK261 carriers, indicating clinical heterogeneity in GCK-MODY.

Financial support for this study was provided by the Consejeria de Salud, Junta de Andalucia, the Spanish Ministry of Science and Innovation, Instituto de Salud Carlos III, and cofunded by Fondos FEDER and the Fundacion Publica Andaluza Progreso y Salud.

Islet ß-cell replacement and regeneration are two promising approaches for the treatment of Type 1 Diabetes Mellitus. Indeed, the success of islet transplantation in normalizing blood glucose in diabetic patients has provided the proof of principle that cell replacement can be employed as a safe and efficacious treatment. Nonetheless, shortage of organ donors has hampered expansion of this approach. Alternative sources of insulin-producing cells are mandatory to fill this gap. Although great advances have been achieved in generating surrogate ß-cells from stem cells, current protocols have yet to produce functionally mature insulin-secreting cells. Recently, the concept of islet regeneration in which new ß-cells are formed from either residual ß-cell proliferation or transdifferentiation of other endocrine islet cells has gained much interest as an attractive therapeutic alternative to restore ß-cell mass. Complementary approaches to cell replacement and regeneration could aim at enhancing ß-cell survival and function. Herein, we discuss the value of Hepatocyte Growth Factor (HGF), Glucose-Dependent Insulinotropic Peptide (GIP), Paired box gene 4 (Pax4) and Liver Receptor Homolog-1 (LRH-1) as key players for ß-cell replacement and regeneration therapies. These factors convey ß-cell protection and enhanced function as well as facilitating proliferation and transdifferentiation of other pancreatic cell types to ß-cells, under stressful conditions, The authors acknowledge the financial support of the Consejeria de Salud, Junta de Andalucia (PI-0727/2010 to B. Gauthier), the Spanish Ministry of Science and Innovation, Instituto de Salud Carlos III cofinanced by European funds for Regional Development (FEDER) (PI10/00871 to B. Gauthier) and from the Fundacion Publica Andaluza Progreso y Salud (to B. Gauthier and J. Mellado-Gil)

One of the most challenging areas in pancreatic endocrine tumor (PET) biology is the identification of specific biomarkers that can morphologically and histochemically distinguish well-differentiated PETs from other types of neuroendocrine tumors (NETs). Novel markers could improve diagnosis accuracy and could consequently lead to a more appropriate and tailored treatment and therefore to a better prognosis.

et al., Liver receptor homolog (LRH-1) is an orphan nuclear receptor (NR5A2) that regulates cholesterol homeostasis and cell plasticity in endodermal-derived tissues. Estrogen increases LRH-1 expression conveying cell protection and proliferation. Independently, estrogen also protects isolated human islets against cytokine- induced apoptosis. Herein, we demonstrate that LRH-1 is expressed in islets, including β-cells, and that transcript levels are modulated by 17β-estradiol through the estrogen receptor (ER)α but not ERβ signaling pathway. Repression of LRH-1 by siRNA abrogated the protective effect conveyed by estrogen on rat islets against cytokines. Adenoviral-mediated overexpression of LRH-1 in human islets did not alter proliferation but conferred protection against cytokines and streptozotocin-induced apoptosis. Expression levels of the cell cycle genes cyclin D1 and cyclin E1 as well as the antiapoptotic gene bcl-xl were unaltered in LRH-1 expressing islets. In contrast, the steroidogenic enzymes CYP11A1 and CYP11B1 involved in glucocorticoid biosynthesis were both stimulated in transduced islets. In parallel, graded overexpression of LRH-1 dose- dependently impaired glucose-induced insulin secretion. Our results demonstrate the crucial role of the estrogen target gene nr5a2 in protecting human islets against-stressed-induced apoptosis. We postulate that this effect is mediated through increased glucocorticoid production that blunts the pro-inflammatory response of islets. © The Author 2011. Published by Oxford University Press. All rights reserved., This work was supported by grants from the Swiss National Science Foundation (310030-119763 to B.R.G., 310000-116750/1 to C.B.W., 32003B-120376 to D.B.), the Juvenile Diabetes Research Foundation (9-2004-384 to E.C.I.T.), the Fundacion Progreso y Salud (to B.R.G.) and the Junta de Andalucia, Consejeria de Salud (PI-0727-2010 to B.R.G.).

3 páginas, 1 figura con 8 paneles., Supported by grants (MICINN, P.I3, SAF2006-12863, SAS/PI-024/2007, and SAS/PI-0236/2009, to Dr. Cuesta-Muñoz) from the Junta de Andalucía, and a grant (to Dr. Glaser) from the Russell Berrie Foundation and D-Cure, Diabetes Care in Israel.

Este ítem está sujeto a una licencia Creative Commons., Currently, one of the most controversial areas in pancreatic islet physiology is the origin of new insulin-producing β-cells that are generated in response to physiological demands postnatally. Harnessing this enigma would provide new means for the treatment of diabetes, a disease characterized by a gradual loss of β-cell mass (type 1 or type 2) and/or a decline in β-cell function combined with insulin resistance (type 2). Restraining destruction and stimulating regeneration of the functional mass would prevail over hyperglycemia and avoid secondary complications such as retinopathy and nephropathy and, potentially, cardiovascular and cerebrovascular diseases (1). Four potential sources of regenerative β-cells, with their respective advocates in perpetual debate, have been proposed to represent the “Fountain of Youth”: 1) neogenesis of ductal epithelium cells (2, 3); 2) transdifferentiation of exocrine acinar cells (4); 3) differentiation of islet-derived precursor stem cells (5); and 4) β-cell replication. Despite strong evidence for the contribution of neogenesis and transdifferentiation in various models of animal pancreatic injury, recent studies have called into question the role of these entities in β-cell mass expansion in the adult organ (6, 7). Strong skepticism also remains over the potential involvement of progenitor stem cells residing within the pancreas to give rise in vivo to functional β-cells (8). However, β-cell replication, which was disregarded for many years, has lately gained popularity with lineage-tracing studies, demonstrating that preexisting mouse adult pancreatic β-cells were the major source of new insulin-producing cells during adult life (9)., We gratefully acknowledge the financial contribution of the IMABIS Foundation, as well as the Foundation Progreso y Salud.

7 páginas, 3 figuras, 2 tablas., Glucokinase is essential for glucose-stimulated insulin release from the pancreatic beta-cell, serving as glucose sensor in humans. Inactivating or activating mutations of glucokinase lead to different forms of glucokinase disease, i.e. GCK-monogenic diabetes of youth, permanent neonatal diabetes (inactivating mutations), and congenital hyperinsulinism, respectively. Here we present a novel glucokinase gene (GCK)-activating mutation (p.E442K) found in an infant with neonatal hypoglycemia (1.5 mmol/liter) and in two other family members suffering from recurrent hypoglycemic episodes in their childhood and adult life. In contrast to the severe clinical presentation in the index case, functional studies showed only a slight activation of the protein (relative activity index of 3.3). We also report on functional studies of two inactivating mutations of the GCK (p.E440G and p.S441W), contiguous to the activating one, that lead to monogenic diabetes of youth. Interestingly, adult family members carrying the GCK pE440G mutation show an unusually heterogeneous and progressive diabetic phenotype, a feature not typical of GCK-monogenic diabetes of youth. In summary, we identified a novel activating GCK mutation that although being associated with severe neonatal hypoglycemia is characterized by the mildest activation of the glucokinase enzyme of all previously reported, This work was supported by grants (to A.L.C.-M. and N.C.-V.) from Ministerio de Ciencia e Innovación, Dirección General de Investigación Científica y Técnica (SAF2005-08014; SAF2006-12863) and Junta de Andalucía (SAS/PI-024/2007; SAS/PI-0236/2009)

Glucokinase (GK) is essential for glucose-stimulated insulin release from pancreatic β-cell, serving as glucose sensor in humans. Inactivating or activating mutations of glucokinase lead to different forms of “Glucokinase Disease”, i.e. Monogenic Diabetes of Youth (GCK-MDY), Permanent Neonatal Diabetes (inactivating mutations) and Congenital Hyperinsulinism, respectively. Here we present a novel GCK activating mutation (p.E442K) found in an infant with neonatal hypoglycemia (1.5 mmol/l) and in two other family members suffering from recurrent hypoglycemic episodes in their childhood and adult life. In contrast to the severe clinical presentation in the index case, functional studies showed only a slight activation of the protein (relative activity index of 3.3). We also report on functional studies of two inactivating mutations of the GCK (p.E440G and p.S441W), contiguous to the activating one, that lead to GCK-MDY. Interestingly, adult family members carrying the GCK pE440G mutation show an unusually “heterogeneous and progressive” diabetic phenotype, a feature not typical of MDY. In summary, we identified a novel activating GCK mutation that although being associated with severe neonatal hypoglycemia is characterized by the mildest activation of the glucokinase enzyme of all previously reported.

Successful normalization of blood glucose in patients transplanted with pancreatic islets isolated from cadaveric donors established the proof-of-concept that Type 1 Diabetes Mellitus is a curable disease. Nonetheless, major caveats to the widespread use of this cell therapy approach have been the shortage of islets combined with the low viability and functional rates subsequent to transplantation. Gene therapy targeted to enhance survival and performance prior to transplantation could offer a feasible approach to circumvent these issues and sustain a durable functional β-cell mass in vivo. However, efficient and safe delivery of nucleic acids to intact islet remains a challenging task. Here we describe a simple and easy-to-use lentiviral transduction protocol that allows the transduction of approximately 80 % of mouse and human islet cells while preserving islet architecture, metabolic function and glucose-dependent stimulation of insulin secretion. Our protocol will facilitate to fully determine the potential of gene expression modulation of therapeutically promising targets in entire pancreatic islets for xenotransplantation purposes.