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Vitamin A Deficiency Causes Hyperglycemia and Loss of Pancreatic β-Cell Mass*

  • Steven E. Trasino
    Affiliations
    Department of Pharmacology, Weill Cornell Medical College, New York, New York 10065
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  • Yannick D. Benoit
    Affiliations
    Department of Pharmacology, Weill Cornell Medical College, New York, New York 10065
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  • Lorraine J. Gudas
    Correspondence
    To whom correspondence should be addressed: Dept. of Pharmacology, and Weill Cornell Medical College of Cornell University, 1300 York Ave., New York, NY 10065. Tel.: 212-746-6250; Fax: 212-746-8858
    Affiliations
    Department of Pharmacology, Weill Cornell Medical College, New York, New York 10065
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  • Author Footnotes
    * This work was supported, in whole or in part, by National Institutes of Health Grants R01CA043796 and RO1 DE010389 (to L. J. G.) and CA062948 (to S. E. T.). This work was also supported by Weill Cornell funds.
Open AccessPublished:December 01, 2014DOI:https://doi.org/10.1074/jbc.M114.616763
      We show that vitamin A (all-trans-retinol) (VA) is required both for the maintenance of pancreatic β-cell and α-cell mass and for glucose-stimulated insulin secretion in adult mice. Dietary VA deprivation (VAD) causes greatly decreased pancreatic VA levels, hyperglycemia, and reduced insulin secretion. Adult mice fed VAD diets display remodeling of the endocrine pancreas, marked β-cell apoptosis, shifts to smaller islet size distributions, decreased β-cell mass, increased α-cell mass, and hyperglucagonemia. Importantly, although we induced VAD in the entire animal, the pancreatic β-cells are exquisitely sensitive to VAD-associated apoptosis compared with other cell types in other organs. VAD causes major reductions in levels of the VA intracellular binding protein Crbp1 and the retinoic acid-metabolizing enzyme Cyp26a1 specifically in larger islets, suggesting the use of these proteins as biomarkers for early endocrine mass abnormalities. In the VAD mice, the reductions in pancreatic islet sizes and the associated aberrant endocrine functions, which show similarities to the phenotype in advanced type 2 diabetes, result from reductions in pancreatic VA signaling. Reintroduction of dietary VA to VAD mice restores pancreatic VA levels, glycemic control, normal islet size distributions, β-cell to α-cell ratios, endocrine hormone profiles, and RARβ2 and RARγ2 transcript levels. Restoration of β-cell mass by reintroducing VA to VAD mice does not involve increased β-cell proliferation or neogenesis. Pharmacologic modulation of pancreatic VA signaling should be explored for the preservation and/or restoration of pancreatic β-cell mass and function in individuals with diabetes mellitus.Little is known about vitamin A (VA) regulation of pancreatic endocrine mass in adults.

      Results

      Decreased pancreatic VA causes increased α-cell to β-cell mass ratios, hyperglycemia, and hyperglucagonemia. Reintroducing dietary VA restores normoglycemia and α-cell to β-cell mass.

      Conclusion

      VA is essential for maintenance of β-cell functions in adult pancreas.

      Significance

      VA therapies may potentially prevent β-cell apoptosis and loss in diabetes.

      Introduction

      Vitamin A (VA, all-trans-retinol (ROL))
      The abbreviations used are: VA
      vitamin A
      VAD
      vitamin A-deficient or VA deprivation
      ROL
      all-trans-retinol
      RA
      retinoic acid
      T2D
      type 2 diabetes
      RAR
      retinoic acid receptor
      VAS
      VA-sufficient
      VADR
      VAD rescued
      GTT
      glucose tolerance test
      LRAT
      lecithin:retinol acyltransferase
      PSC
      pancreatic stellate cell
      ngn3
      neurogenin-3.
      and its analogs and metabolites are collectively called retinoids (
      • Gudas L.J.
      • Wagner J.A.
      Retinoids regulate stem cell differentiation.
      ). Acting through the retinoic acid receptors (RARα, β, and γ), all-trans-retinoic acid, a biologically active metabolite of VA, is essential for embryonic stem cell differentiation (
      • Gudas L.J.
      • Wagner J.A.
      Retinoids regulate stem cell differentiation.
      ,
      • Gudas L.J.
      Retinoids induce stem cell differentiation via epigenetic changes.
      ) and is required during early vertebrate pancreatic specification and in later stages of pancreagenesis when β-cell determination and maturation occur (
      • Martín M.
      • Gallego-Llamas J.
      • Ribes V.
      • Kedinger M.
      • Niederreither K.
      • Chambon P.
      • Dollé P.
      • Gradwohl G.
      Dorsal pancreas agenesis in retinoic acid-deficient Raldh2 mutant mice.
      ,
      • Molotkov A.
      • Molotkova N.
      • Duester G.
      Retinoic acid generated by Raldh2 in mesoderm is required for mouse dorsal endodermal pancreas development.
      ,
      • Oström M.
      • Loffler K.A.
      • Edfalk S.
      • Selander L.
      • Dahl U.
      • Ricordi C.
      • Jeon J.
      • Correa-Medina M.
      • Diez J.
      • Edlund H.
      Retinoic acid promotes the generation of pancreatic endocrine progenitor cells and their further differentiation into beta-cells.
      ). 9-cis-Retinoic acid has been shown to have a role in the adult pancreas (
      • Kane M.A.
      • Folias A.E.
      • Pingitore A.
      • Perri M.
      • Obrochta K.M.
      • Krois C.R.
      • Cione E.
      • Ryu J.Y.
      • Napoli J.L.
      Identification of 9-cis-retinoic acid as a pancreas-specific autacoid that attenuates glucose-stimulated insulin secretion.
      ), and we recently demonstrated that when embryonic stem cells that lack RARβ are subjected to an endocrine cell differentiation protocol, they show both reduced levels of markers of pancreatic progenitors, such as neurogenin-3 (ngn3), and reduced differentiation into insulin-positive endocrine cells (
      • Pérez R.J.
      • Benoit Y.D.
      • Gudas L.J.
      Deletion of retinoic acid receptor β (RARβ) impairs pancreatic endocrine differentiation.
      ). These data led us to hypothesize that retinoids play an essential role in the maintenance of endocrine cell populations in the adult pancreas. Although Chertow et al. (
      • Chertow B.S.
      • Blaner W.S.
      • Baranetsky N.G.
      • Sivitz W.I.
      • Cordle M.B.
      • Thompson D.
      • Meda P.
      Effects of vitamin A deficiency and repletion on rat insulin secretion in vivo and in vitro from isolated islets.
      ,
      • Chertow B.S.
      • Driscoll H.K.
      • Blaner W.S.
      • Meda P.
      • Cordle M.B.
      • Matthews K.A.
      Effects of vitamin A deficiency and repletion on rat glucagon secretion.
      ) demonstrated that perfused islets from VA-deprived rats had impaired insulin and glucagon secretion, in these studies the fetuses were deprived of VA. This VA deprivation caused pancreatic developmental abnormalities in the pups (
      • Matthews K.A.
      • Rhoten W.B.
      • Driscoll H.K.
      • Chertow B.S.
      Vitamin A deficiency impairs fetal islet development and causes subsequent glucose intolerance in adult rats.
      ), making it likely that the pancreatic phenotype resulted from the pancreatic abnormalities shown to be associated with low VA levels during embryogenesis and early development (
      • Matthews K.A.
      • Rhoten W.B.
      • Driscoll H.K.
      • Chertow B.S.
      Vitamin A deficiency impairs fetal islet development and causes subsequent glucose intolerance in adult rats.
      ).
      Here we demonstrate for the first time that decreases in pancreatic levels of VA in adult mice result in loss of β-cell mass, increased α-cell mass with concomitant hyperglycemia, and changes to serum insulin and glucagon profiles. These alterations in islet architecture and pancreatic endocrine cell types, serum hormones levels, and hyperglycemia in VA-deprived mice are reversed upon resumption of dietary VA. Because of the clinical interest in the development of pancreatic β-cell mass restoration therapy for T2D and type 1 diabetes (
      • Donath M.Y.
      • Halban P.A.
      Decreased beta-cell mass in diabetes: significance, mechanisms and therapeutic implications.
      ), the data reported here suggest that pancreatic VA metabolism and signaling should be further explored for endocrine cell restoration therapies in diabetes mellitus.

      DISCUSSION

      Numerous experimental studies have determined that VA and retinoids possess anti-obesity and anti-lipogenic properties through transcriptional regulation of relevant genes in liver and adipose tissue (
      • Berry D.C.
      • Noy N.
      All-trans-retinoic acid represses obesity and insulin resistance by activating both peroxisome proliferation-activated receptor beta/delta and retinoic acid receptor.
      ,
      • Schwarz E.J.
      • Reginato M.J.
      • Shao D.
      • Krakow S.L.
      • Lazar M.A.
      Retinoic acid blocks adipogenesis by inhibiting C/EBPbeta-mediated transcription.
      ,
      • Kim S.C.
      • Kim C.K.
      • Axe D.
      • Cook A.
      • Lee M.
      • Li T.
      • Smallwood N.
      • Chiang J.Y.
      • Hardwick J.P.
      • Moore D.D.
      • Lee Y.K.
      All-trans-retinoic acid ameliorates hepatic steatosis in mice by a novel transcriptional cascade.
      ), but it is unclear whether altered VA metabolism is involved in the pathogenesis of T2D. There is a large body of data demonstrating that insulin resistance alters adipose and renal metabolism of RBP4 (retinol binding protein 4), which paradoxically further promotes insulin resistance and the pathogenesis of T2D (
      • Graham T.E.
      • Yang Q.
      • Blüher M.
      • Hammarstedt A.
      • Ciaraldi T.P.
      • Henry R.R.
      • Wason C.J.
      • Oberbach A.
      • Jansson P.A.
      • Smith U.
      • Kahn B.B.
      Retinol-binding protein 4 and insulin resistance in lean, obese, and diabetic subjects.
      ,
      • Masaki T.
      • Anan F.
      • Tsubone T.
      • Gotoh K.
      • Chiba S.
      • Katsuragi I.
      • Nawata T.
      • Kakuma T.
      • Yoshimatsu H.
      Retinol binding protein 4 concentrations are influenced by renal function in patients with type 2 diabetes mellitus.
      ). Testament to the growing interest in retinoids as metabolic modulating agents is the recent initiation of phase 2 clinical trials of the synthetic retinoid fenretinide 4-hydroxy(phenyl)retinamide as a novel anti-obesity and insulin-sensitizing agent (
      • University of California, San Diego
      A randomized, double-blind study of the effects of fenretinide administered in subjects with obesity.
      ). Still, there remain large gaps in our knowledge of the mechanisms of 4-hydroxy(phenyl)retinamide and retinoids in modulating the metabolic and endocrine pathways involved in the pathogenesis of obesity, insulin resistance, and T2D. To date, few studies have thoroughly examined the role of retinoids in pancreatic islet biology. In this study, we report that dietary VA is required for the maintenance of normal pancreatic islet architecture, endocrine cell mass, and endocrine function in adult, nondiabetic mice.
      Our 4- and 10-week glucose tolerance tests, insulin secretion assays, and apoptosis studies show that as early as 4 weeks after the initiation of the VAD diet, the LRAT−/− mice had increased glucose excursions, decreased pancreatic insulin content, and marked (>50%) pancreatic islet apoptosis (Figs. 2, A and F, and 6B). By week 10 of VA deprivation, LRAT−/− 10wVAD mice displayed undetectable levels of pancreatic VA (Fig. 1B) and greatly diminished glucose-stimulated insulin secretion and pancreatic insulin content compared with LRAT−/− 10wVAS mice (Fig. 2, G, J, and L). Our apoptosis measurements in LRAT−/− mice also show that VA deprivation causes marked β-cell apoptosis (Fig. 6, A–D), a loss of β-cells (Fig. 8A), a major reduction in total pancreatic islet area (Fig. 7F), and, consequently, reduced basal insulin levels in the pancreas (Fig. 2, F and L). These experiments clearly demonstrate that with increasing severity of pancreatic VA depletion, the degree of impaired glucose tolerance correlates with the loss of β-cell mass.
      The onset of a strikingly similar, but less severe, metabolic phenotype in WT 10wVAD mice compared with LRAT−/− 10w VAD mice provides further evidence that the earlier onset and increased relative severity of the glucose intolerance and pancreatic β-cell apoptosis we observed in VA-deprived LRAT−/− mice was not due to an unspecified metabolic anomaly related to their genotype but rather to their greater sensitivity to the lack of VA in the diet (
      • Liu L.
      • Gudas L.J.
      Disruption of the lecithin:retinol acyltransferase gene makes mice more susceptible to vitamin A deficiency.
      ). Without any evidence of changes in peripheral insulin sensitivity (Fig. 2P), decreased body weights (Fig. 7H), or increased cell death and histological changes in other organs, such as the liver, kidney, and small intestines in the VAD mice, our data suggest that pancreatic endocrine functions and specifically the maintenance of β-cell populations are reliant on VA. However, given the complex interplay among pancreas, liver, adipose, and other tissues in regulating glucose homeostasis, we cannot exclude the possibility that VA deprivation alters metabolic pathways in other tissues that contribute to the pancreatic endocrine phenotype of VA-deprived and rescued mice.
      Our Crbp1 and Cyp26a1 islet immunostaining studies provide evidence for how the pancreas senses changes in tissue retinoid levels and how large islets are specifically affected by decreases in pancreatic VA levels. Decreased cellular Crbp1 and Cyp26a1 levels are associated with poor retinoid responsiveness and low cellular RA levels (
      • Lotan R.
      A crucial role for cellular retinol-binding protein I in retinoid signaling.
      ,
      • Tannous-Khuri L.
      • Talmage D.A.
      Decreased cellular retinol-binding protein expression coincides with the loss of retinol responsiveness in rat cervical epithelial cells.
      ). We show that when dietary and pancreatic VA levels are sufficient, expression of both Crbp1 and Cyp26a1 correlates with islet size, but that this is not the case under dietary VA deprivation (Fig. 4, H, panels a, b, d, and e, and P, panels a, b, d, and e). Additionally, the reductions and subsequent restoration of pancreatic Crbp1, Cyp26a1, RARβ2, and RARγ2 transcripts in WT and LRAT−/− VAD versus VADR mice, respectively (Fig. 3), are compelling and support our hypothesis that VA deprivation reduces pancreatic VA levels that are critical for retinoid signaling and normal pancreatic control of glucose-stimulated insulin release in the adult pancreas. Thus, we interpret the reductions in Crbp1 and Cyp26a1, as assessed by our immunostaining studies (Fig. 4), that occur in concert with the reductions in pancreatic VA levels (Fig. 1B) to indicate that dietary VA deprivation prevents the growth and proper functioning of pancreatic islets. That the large islets with the highest percentages of Crbp1 and Cyp26a1 immunopositive regions are preferentially targeted for apoptosis (Fig. 6C) and remodeling during VA deprivation (Fig. 6, A–D) suggests that a reduction in Crbp1 and Cyp26a1 expression provides pancreatic cells an early cue of diminishing VA availability and, if VA levels continue to drop, leads to an intrinsic cascade of β-cell programmed cell death and islet remodeling.
      Leprdb, C57BLKS mice, commonly refereed to as “db/db” mice, are a commonly used genetic model for advanced T2D (
      • Chen H.
      • Charlat O.
      • Tartaglia L.A.
      • Woolf E.A.
      • Weng X.
      • Ellis S.J.
      • Lakey N.D.
      • Culpepper J.
      • Moore K.J.
      • Breitbart R.E.
      • Duyk G.M.
      • Tepper R.I.
      • Morgenstern J.P.
      Evidence that the diabetes gene encodes the leptin receptor: identification of a mutation in the leptin receptor gene in db/db mice.
      ). With age and progression of the diabetic phenotype, db/db mice exhibit a similar pancreatic islet phenotype as VAD mice, with marked β-cell apoptosis, reduced insulin levels, and larger islets (
      • Kim A.
      • Miller K.
      • Jo J.
      • Kilimnik G.
      • Wojcik P.
      • Hara M.
      Islet architecture: A comparative study.
      ,
      • Puff R.
      • Dames P.
      • Weise M.
      • Göke B.
      • Seissler J.
      • Parhofer K.G.
      • Lechner A.
      Reduced proliferation and a high apoptotic frequency of pancreatic beta cells contribute to genetically-determined diabetes susceptibility of db/db BKS mice.
      ). Evidence suggests that humans with T2D also experience a specific loss of larger islet pools (
      • Kilimnik G.
      • Zhao B.
      • Jo J.
      • Periwal V.
      • Witkowski P.
      • Misawa R.
      • Hara M.
      Altered islet composition and disproportionate loss of large islets in patients with type 2 diabetes.
      ,
      • Wang X.
      • Misawa R.
      • Zielinski M.C.
      • Cowen P.
      • Jo J.
      • Periwal V.
      • Ricordi C.
      • Khan A.
      • Szust J.
      • Shen J.
      • Millis J.M.
      • Witkowski P.
      • Hara M.
      Regional differences in islet distribution in the human pancreas: preferential beta-cell loss in the head region in patients with type 2 diabetes.
      ).
      The loss of larger islet pools was dynamic in that it occurred with a concomitant increase in the percentage of smaller islet sizes (Fig. 7, A–D). This islet remodeling is more suggestive of a concerted metabolic response to VA deprivation rather than broad, nonspecific cell death. The absence of increased apoptosis in liver, kidney, and small intestines of VA-deprived mice supports this notion and demonstrates that the pancreatic β-cells are more sensitive to reductions in dietary and pancreatic VA than other cell types in these other organs. The absence of any changes in total islet numbers (Fig. 7E), pancreatic weights (Fig. 7G), endocrine cell proliferation, and markers of β-cell neogenesis (Fig. 9, A, C, and D) during VA deprivation provides strong evidence that the increased numbers of medium to small islets in WT 10wVAD and LRAT−/− 10wVAD mice (Fig. 7, A–D) are derived from apoptosis in previously larger islets and not from proliferation of pancreatic progenitors. The lack of islet apoptosis (Fig. 6, A and B) and the normalization of islet size distributions (Fig. 7, C and D) in the VA-deprived mice after restoration of dietary VA also support the notion of a specific, nonpermanent, reversible metabolic program in response to VA deprivation. We conclude that β-cell apoptosis and loss of islet mass do not result from nonspecific glucotoxicity secondary to VA deprivation, because WT VA-deprived mice are hypoglycemic, and LRAT−/− VAD mice show only marginal hyperglycemia (Fig. 8H). Moreover, we did not detect histopathology hallmarks of pancreatic glucotoxicity such pancreatitis, islet amyloid deposits, and fibrosis (
      • Hull R.L.
      • Westermark G.T.
      • Westermark P.
      • Kahn S.E.
      Islet amyloid: a critical entity in the pathogenesis of type 2 diabetes.
      ). Therefore, one mechanism by which mice remodel islet size is through modulation of pancreatic VA signaling and apoptosis.
      We also found that VA deprivation results in aberrations in endocrine cell mass profiles. By week 10 of VA deprivation, both WT 10wVAD and LRAT−/− 10wVAD mice showed losses of β-cell mass (Fig. 8A) and decreased fasting serum insulin levels (Fig. 8D). Interestingly, LRAT−/− 10wVAD mice showed elevated α-cell mass, pancreatic glucagon, and glucagon serum concentrations compared with WT 10wVAS and LRAT−/− 10wVAS mice (Fig. 8). The elevated glucose profile and increased hepatic expression of glycogen phosphorylase also suggest that the hyperglucagonemia in LRAT−/− 10wVAD mice results in increased hepatic mobilization of glycogen stores and is responsible for the increased fasting hyperglycemia in these LRAT−/− mice. It is unclear whether the increased number of α-cells is secondary to the loss of β-cell mass. The degree of β-cell apoptosis, the increased α-cell mass, and the presence of α-cells within islet centers of LRAT−/− 10wVAD mice (Fig. 8, B, red bar, and G, panels m–o) are consistent with increased numbers and the presence of hyper-responsive, glucagon-secreting α-cells within the islets in experimental diabetic rodent models involving β-cell ablation (
      • Li Z.
      • Karlsson F.A.
      • Sandler S.
      Islet loss and alpha cell expansion in type 1 diabetes induced by multiple low-dose streptozotocin administration in mice.
      ,
      • Zhang Y.
      • Bone R.N.
      • Cui W.
      • Peng J.B.
      • Siegal G.P.
      • Wang H.
      • Wu H.
      Regeneration of pancreatic non-β endocrine cells in adult mice following a single diabetes-inducing dose of streptozotocin.
      ,
      • Krakowski M.L.
      • Kritzik M.R.
      • Jones E.M.
      • Krahl T.
      • Lee J.
      • Arnush M.
      • Gu D.
      • Mroczkowski B.
      • Sarvetnick N.
      Transgenic expression of epidermal growth factor and keratinocyte growth factor in beta-cells results in substantial morphological changes.
      ). In contrast, WT 10wVAD mice showed a decrease in α-cell mass, pancreatic and serum hypoglucagonemia, decreased fasting glucose levels, and increased intraislet α-cells (white arrows in Fig. 8G, panels d–f) as compared with WT VAS mice (Fig. 8, B and E). The dissimilar pancreatic endocrine cell and hormone profiles of WT and LRAT−/− 10w VA-deprived mice are likely a consequence of LRAT−/− mice showing the effects of VA deprivation earlier than WT mice. This is apparent from the earlier onset of apoptosis, glucose intolerance, and the severely diminished pancreatic VA levels observed in LRAT−/− mice, but not in WT mice, at 4 weeks of VA deprivation (Figs. 6B; 4, A, B, D, and E; and 1B). We predict that with continued VA deprivation, WT mice will develop hyperglucagonemia similar to that seen in the LRAT−/− 10wVAD mice.
      We showed that the reduction in and subsequent restoration of pancreatic β-cell mass during VA deprivation and VA rescue, respectively, are not related to changes in β-cell proliferation or neogenesis from Ngn3-positive pancreatic progenitors (Fig. 9, A–C). Thus, we think that the restored β-cells in VA rescued WT and LRAT−/− VAD mice are derived from other endocrine cell types, given the evidence that endocrine cells can transdifferentiate into each other with ectopic expression of a number of developmental transcription factors, such as Pax 4 and ARX (
      • Chung C.H.
      • Hao E.
      • Piran R.
      • Keinan E.
      • Levine F.
      Pancreatic β-cell neogenesis by direct conversion from mature α-cells.
      ,
      • Thorel F.
      • Népote V.
      • Avril I.
      • Kohno K.
      • Desgraz R.
      • Chera S.
      • Herrera P.L.
      Conversion of adult pancreatic alpha-cells to beta-cells after extreme beta-cell loss.
      ,
      • Collombat P.
      • Hecksher-Sørensen J.
      • Krull J.
      • Berger J.
      • Riedel D.
      • Herrera P.L.
      • Serup P.
      • Mansouri A.
      Embryonic endocrine pancreas and mature beta cells acquire alpha and PP cell phenotypes upon Arx misexpression.
      ). Further experiments are required to determine whether VA can regulate the expression of these and other developmental transcription factors in the adult pancreas.
      In summary, our data strongly demonstrate that VA plays a major role in maintaining the β-cell mass of larger sized islet populations. Mechanistically, we demonstrate that VA supports β-cells through prevention of programmed cell death. Alterations in pancreatic VA levels may be one method through which endocrine cell identities and islet plasticity are altered. Thus, we suggest that synthetic retinoids can be used in the future to prevent β-cell cell apoptosis in T2D.

      Acknowledgments

      We thank Dr. Xiao-Han Tang for assistance with HPLC and Dr. Jose Jessurun from the Department of Surgical Pathology at Weill Cornell Medical Center for evaluation of histology sections.

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