New Aspects of Diabetes Research and Therapeutic Development.

Both type 1 and type 2 diabetes mellitus are advancing at exponential rates, placing significant burdens on health care networks worldwide. Although traditional pharmacologic therapies such as insulin and oral antidiabetic stalwarts like metformin and the sulfonylureas continue to be used, newer drugs are now on the market targeting novel blood glucose-lowering pathways. Furthermore, exciting new developments in the understanding of beta cell and islet biology are driving the potential for treatments targeting incretin action, islet transplantation with new methods for immunologic protection, and the generation of functional beta cells from stem cells. Here we discuss the mechanistic details underlying past, present, and future diabetes therapies and evaluate their potential to treat and possibly reverse type 1 and 2 diabetes in humans. SIGNIFICANCE STATEMENT: Diabetes mellitus has reached epidemic proportions in the developed and developing world alike. As the last several years have seen many new developments in the field, a new and up to date review of these advances and their careful evaluation will help both clinical and research diabetologists to better understand where the field is currently heading.

GATA4 and GATA6 regulate intestinal epithelial cytodifferentiation during development.

The intestinal epithelium performs vital roles in organ function by absorbing nutrients and providing a protective barrier. The zinc-finger containing transcription factors GATA4 and GATA6 regulate enterocyte gene expression and control regional epithelial cell identity in the adult intestinal epithelium. Although GATA4 and GATA6 are expressed in the developing intestine, loss of either factor alone during the period of epithelial morphogenesis and cytodifferentiation fails to disrupt these processes. Therefore, we tested the hypothesis that GATA4 and GATA6 function redundantly to control these aspects of intestinal development. We used Villin-Cre, which deletes specifically in the intestinal epithelium during the period of villus development and epithelial cytodifferentiation, to generate Gata4Gata6 double conditional knockout embryos. Mice lacking GATA4 and GATA6 in the intestinal epithelium died within 24h of birth. At E18.5, intestinal villus architecture and epithelial cell populations were altered. Enterocytes were lost, and goblet cells were increased. Proliferation was also increased in GATA4-GATA6 deficient intestinal epithelium. Although villus morphology appeared normal at E16.5, the first time at which both Gata4 and Gata6 were efficiently reduced, changes in expression of markers of enterocytes, goblet cells, and proliferative cells were detected. Moreover, goblet cell number was increased at E16.5. Expression of the Notch ligand Dll1 and the Notch target Olfm4 were reduced in mutant tissue indicating decreased Notch signaling. Finally, we found that GATA4 occupies chromatin near the Dll1 transcription start site suggesting direct regulation of Dll1 by GATA4. We demonstrate that GATA4 and GATA6 play an essential role in maintaining proper intestinal epithelial structure and in regulating intestinal epithelial cytodifferentiation. Our data highlight a novel role for GATA factors in fine tuning Notch signaling during intestinal epithelial development to repress goblet cell differentiation.

A metabolic redox relay supports ER proinsulin export in pancreatic islet ß-cells.

Endoplasmic reticulum (ER) stress and proinsulin misfolding are heralded as contributing factors to ß-cell dysfunction in Type 2 diabetes (T2D), yet how ER function becomes compromised is not well understood. Recent data identifies altered ER redox homeostasis as a critical mechanism that contributes to insulin granule loss in diabetes. Hyperoxidation of the ER delays proinsulin export and limits the proinsulin supply available for insulin granule formation. In this report, we identified glucose metabolism as a critical determinant in the redox homeostasis of the ER. Using multiple ß-cell models, we showed that loss of mitochondrial function or inhibition of cellular metabolism elicited ER hyperoxidation and delayed ER proinsulin export. Our data further demonstrated that ß-cell ER redox homeostasis was supported by the metabolic supply of reductive redox donors. We showed that limiting NADPH and thioredoxin flux delayed ER proinsulin export, whereas Txnip suppression restored ER redox and proinsulin trafficking. Taken together, we propose that ß-cell ER redox homeostasis is buffered by cellular redox donor cycles, which are maintained through active glucose metabolism.

LONP1 regulation of mitochondrial protein folding provides insight into beta cell failure in type 2 diabetes.

Proteotoxicity is a contributor to the development of type 2 diabetes (T2D), but it is unknown whether protein misfolding in T2D is generalized or has special features. Here, we report a robust accumulation of misfolded proteins within the mitochondria of human pancreatic islets in T2D and elucidate its impact on ß cell viability. Surprisingly, quantitative proteomics studies of protein aggregates reveal that human islets from donors with T2D have a signature more closely resembling mitochondrial rather than ER protein misfolding. The matrix protease LonP1 and its chaperone partner mtHSP70 were among the proteins enriched in protein aggregates. Deletion of LONP1 in mice yields mitochondrial protein misfolding and reduced respiratory function, ultimately leading to ß cell apoptosis and hyperglycemia. Intriguingly, LONP1 gain of function ameliorates mitochondrial protein misfolding and restores human ß cell survival following glucolipotoxicity via a protease-independent effect requiring LONP1-mtHSP70 chaperone activity. Thus, LONP1 promotes ß cell survival and prevents hyperglycemia by facilitating mitochondrial protein folding. These observations may open novel insights into the nature of impaired proteostasis on ß cell loss in the pathogenesis of T2D that could be considered as future therapeutic targets.

E-cadherin is required for intestinal morphogenesis in the mouse.

E-cadherin, the primary epithelial adherens junction protein, has been implicated as playing a critical role in nucleating formation of adherens junctions, tight junctions, and desmosomes. In addition to its role in maintaining structural tissue integrity, E-cadherin has also been suggested as an important modulator of cell signaling via interactions with its cytoplasmic binding partners, catenins, as well as with growth factor receptors. Therefore, we proposed that loss of E-cadherin from the developing mouse intestinal epithelium would disrupt intestinal epithelial morphogenesis and function. To test this hypothesis, we used a conditional knockout approach to eliminate E-cadherin specifically in the intestinal epithelium during embryonic development. We found that E-cadherin conditional knockout mice failed to survive, dying within the first 24 hours of birth. Examination of intestinal architecture at E18.5 demonstrated severe disruption to intestinal morphogenesis in animals lacking E-cadherin in the epithelium of the small intestine. We observed changes in epithelial cell shape as well as in the morphology of villi. Although junctional complexes were evident, junctions were abnormal, and barrier function was compromised in E-cadherin mutant intestine. We also identified changes in the epithelial cell populations present in E-cadherin conditional knockout animals. The number of proliferating cells was increased, whereas the number of enterocytes was decreased. Although Wnt/ß-catenin target mRNAs were more abundant in mutants compared with controls, the amount of nuclear activated ß-catenin protein was dramatically lower in mutants compared with controls. In summary, our data demonstrate that E-cadherin is essential for intestinal epithelial morphogenesis and homeostasis during embryonic development.

Determining how MAFA and MAFB transcription factors activity is influenced by structural differences predicted by AlphaFold2.

MAFA and MAFB are related basic-leucine-zipper domain containing transcription factors which have important regulatory roles in a variety of cellular contexts, including pancreatic islet hormone producing α and ß cells. These proteins have similar as well as distinct functional properties, and here we first used AlphaFold2, an artificial intelligence-based structural prediction program, to obtain insight into the three-dimensional organization of their non-DNA binding/dimerization sequences. This analysis was conducted on the wildtype (WT) proteins as well the pathogenic MAFA Ser64Phe (MAFA S64F ) and MAFB Ser70Ala (MAFB S70A ) mutants, with structural differences revealed between MAFA WT and MAFB WT in addition to MAFA S64F and MAFA WT , but not MAFB S70A and MAFB WT . Functional analysis disclosed that the inability to properly phosphorylate at S70 in MAFB S70A , like S65 in MAFA S64F , greatly increased protein stability and enabled MAFB S70A to accelerate cellular senescence in cultured cells. Significant differences were also observed in the ability of MAFA, MAFA S64F , MAFB, and MAFB S70A to cooperatively stimulate Insulin enhancer-driven activity in the presence of other islet-enriched transcription factors. Experiments performed on protein chimeras disclosed that these properties were greatly influenced by structural differences found between the WT and mutant proteins. In general, these results revealed that AlphaFold2 predicts features essential to protein activity.

Species-specific roles for the MAFA and MAFB transcription factors in regulating islet ß cell identity.

Type 2 diabetes (T2D) is associated with compromised identity of insulin-producing pancreatic islet ß cells, characterized by inappropriate production of other islet cell-enriched hormones. Here, we examined how hormone misexpression was influenced by the MAFA and MAFB transcription factors, closely related proteins that maintain islet cell function. Mice specifically lacking MafA in ß cells demonstrated broad, population-wide changes in hormone gene expression with an overall gene signature closely resembling islet gastrin+ (Gast+) cells generated under conditions of chronic hyperglycemia and obesity. A human ß cell line deficient in MAFB, but not one lacking MAFA, also produced a GAST+ gene expression pattern. In addition, GAST was detected in human T2D ß cells with low levels of MAFB. Moreover, evidence is provided that human MAFB can directly repress GAST gene transcription. These results support a potentially novel, species-specific role for MafA and MAFB in maintaining adult mouse and human ß cell identity, respectively. Here, we discuss the possibility that induction of Gast/GAST and other non-ß cell hormones, by reduction in the levels of these transcription factors, represents a dysfunctional ß cell signature.

A versatile pumpless multi-channel fluidics system for maintenance and real-time functional assessment of tissue and cells.

To address the needs of the life sciences community and the pharmaceutical industry in pre-clinical drug development to both maintain and continuously assess tissue metabolism and function with simple and rapid systems, we improved on the initial BaroFuse to develop it into a fully functional, pumpless, scalable multi-channel fluidics instrument that continuously measures changes in oxygen consumption and other endpoints in response to test compounds. We and several other laboratories assessed it with a wide range of tissue types including retina, pancreatic islets, liver, and hypothalamus with both aqueous and gaseous test compounds. The setup time was less than an hour for all collaborating groups, and there was close agreement between data obtained from the different laboratories. This easy-to-use system reliably generates real-time metabolic and functional data from tissue and cells in response to test compounds that will address a critical need in basic and applied research.

Mitofusin 1 and 2 regulation of mitochondrial DNA content is a critical determinant of glucose homeostasis.

The dynamin-like GTPases Mitofusin 1 and 2 (Mfn1 and Mfn2) are essential for mitochondrial function, which has been principally attributed to their regulation of fission/fusion dynamics. Here, we report that Mfn1 and 2 are critical for glucose-stimulated insulin secretion (GSIS) primarily through control of mitochondrial DNA (mtDNA) content. Whereas Mfn1 and Mfn2 individually were dispensable for glucose homeostasis, combined Mfn1/2 deletion in ß-cells reduced mtDNA content, impaired mitochondrial morphology and networking, and decreased respiratory function, ultimately resulting in severe glucose intolerance. Importantly, gene dosage studies unexpectedly revealed that Mfn1/2 control of glucose homeostasis was dependent on maintenance of mtDNA content, rather than mitochondrial structure. Mfn1/2 maintain mtDNA content by regulating the expression of the crucial mitochondrial transcription factor Tfam, as Tfam overexpression ameliorated the reduction in mtDNA content and GSIS in Mfn1/2-deficient ß-cells. Thus, the primary physiologic role of Mfn1 and 2 in ß-cells is coupled to the preservation of mtDNA content rather than mitochondrial architecture, and Mfn1 and 2 may be promising targets to overcome mitochondrial dysfunction and restore glucose control in diabetes.

Heterogenous impairment of α cell function in type 2 diabetes is linked to cell maturation state.

In diabetes, glucagon secretion from pancreatic α cells is dysregulated. The underlying mechanisms, and whether dysfunction occurs uniformly among cells, remain unclear. We examined α cells from human donors and mice using electrophysiological, transcriptomic, and computational approaches. Rising glucose suppresses α cell exocytosis by reducing P/Q-type Ca2+ channel activity, and this is disrupted in type 2 diabetes (T2D). Upon high-fat feeding of mice, α cells shift toward a "ß cell-like" electrophysiological profile in concert with indications of impaired identity. In human α cells we identified links between cell membrane properties and cell surface signaling receptors, mitochondrial respiratory chain complex assembly, and cell maturation. Cell-type classification using machine learning of electrophysiology data demonstrated a heterogenous loss of "electrophysiologic identity" in α cells from donors with type 2 diabetes. Indeed, a subset of α cells with impaired exocytosis is defined by an enrichment in progenitor and lineage markers and upregulation of an immature transcriptomic phenotype, suggesting important links between α cell maturation state and dysfunction.

A Selective Look at Autophagy in Pancreatic ß-Cells.

Insulin-producing pancreatic ß-cells are central to glucose homeostasis, and their failure is a principal driver of diabetes development. To preserve optimal health ß-cells must withstand both intrinsic and extrinsic stressors, ranging from inflammation to increased peripheral insulin demand, in addition to maintaining insulin biosynthesis and secretory machinery. Autophagy is increasingly being appreciated as a critical ß-cell quality control system vital for glycemic control. Here we focus on the underappreciated, yet crucial, roles for selective and organelle-specific forms of autophagy as mediators of ß-cell health. We examine the unique molecular players underlying each distinct form of autophagy in ß-cells, including selective autophagy of mitochondria, insulin granules, lipid, intracellular amyloid aggregates, endoplasmic reticulum, and peroxisomes. We also describe how defects in selective autophagy pathways contribute to the development of diabetes. As all forms of autophagy are not the same, a refined view of ß-cell selective autophagy may inform new approaches to defend against the various insults leading to ß-cell failure in diabetes.

Sex-biased islet ß cell dysfunction is caused by the MODY MAFA S64F variant by inducing premature aging and senescence in males.

A heterozygous missense mutation of the islet ß cell-enriched MAFA transcription factor (p.Ser64Phe [S64F]) is found in patients with adult-onset ß cell dysfunction (diabetes or insulinomatosis), with men more prone to diabetes than women. This mutation engenders increased stability to the unstable MAFA protein. Here, we develop a S64F MafA mouse model to determine how ß cell function is affected and find sex-dependent phenotypes. Heterozygous mutant males (MafAS64F/+) display impaired glucose tolerance, while females are slightly hypoglycemic with improved blood glucose clearance. Only MafAS64F/+ males show transiently higher MafA protein levels preceding glucose intolerance and sex-dependent changes to genes involved in Ca2+ signaling, DNA damage, aging, and senescence. MAFAS64F production in male human ß cells also accelerate cellular senescence and increase senescence-associated secretory proteins compared to cells expressing MAFAWT. These results implicate a conserved mechanism of accelerated islet aging and senescence in promoting diabetes in MAFAS64F carriers in a sex-biased manner.

Loss of the transcription factor MAFB limits ß-cell derivation from human PSCs.

Next generation sequencing studies have highlighted discrepancies in ß-cells which exist between mice and men. Numerous reports have identified MAF BZIP Transcription Factor B (MAFB) to be present in human ß-cells postnatally, while its expression is restricted to embryonic and neo-natal ß-cells in mice. Using CRISPR/Cas9-mediated gene editing, coupled with endocrine cell differentiation strategies, we dissect the contribution of MAFB to ß-cell development and function specifically in humans. Here we report that MAFB knockout hPSCs have normal pancreatic differentiation capacity up to the progenitor stage, but favor somatostatin- and pancreatic polypeptide-positive cells at the expense of insulin- and glucagon-producing cells during endocrine cell development. Our results describe a requirement for MAFB late in the human pancreatic developmental program and identify it as a distinguishing transcription factor within islet cell subtype specification. We propose that hPSCs represent a powerful tool to model human pancreatic endocrine development and associated disease pathophysiology.

Rapid Crypt Cell Remodeling Regenerates the Intestinal Stem Cell Niche after Notch Inhibition.

Intestinal crypts have great capacity for repair and regeneration after intestinal stem cell (ISC) injury. Here, we define the cellular remodeling process resulting from ISC niche interruption by transient Notch pathway inhibition in adult mice. Although ISCs were retained, lineage tracing demonstrated a marked reduction in ISC function after Notch disruption. Surprisingly, Notch ligand-expressing Paneth cells were rapidly lost by apoptotic cell death. The ISC-Paneth cell changes were followed by a regenerative response, characterized by expansion of cells expressing Notch ligands Dll1 and Dll4, enhanced Notch signaling, and a proliferative surge. Lineage tracing and organoid studies showed that Dll1-expressing cells were activated to function as multipotential progenitors, generating both absorptive and secretory cells and replenishing the vacant Paneth cell pool. Our analysis uncovered a dynamic, multicellular remodeling response to acute Notch inhibition to repair the niche and restore homeostasis. Notably, this crypt regenerative response did not require ISC loss.

Examining How the MAFB Transcription Factor Affects Islet ß-Cell Function Postnatally.

The sustained expression of the MAFB transcription factor in human islet ß-cells represents a distinct difference in mice. Moreover, mRNA expression of closely related and islet ß-cell-enriched MAFA does not peak in humans until after 9 years of age. We show that the MAFA protein also is weakly produced within the juvenile human islet ß-cell population and that MafB expression is postnatally restricted in mouse ß-cells by de novo DNA methylation. To gain insight into how MAFB affects human ß-cells, we developed a mouse model to ectopically express MafB in adult mouse ß-cells using MafA transcriptional control sequences. Coexpression of MafB with MafA had no overt impact on mouse ß-cells, suggesting that the human adult ß-cell MAFA/MAFB heterodimer is functionally equivalent to the mouse MafA homodimer. However, MafB alone was unable to rescue the islet ß-cell defects in a mouse mutant lacking MafA in ß-cells. Of note, transgenic production of MafB in ß-cells elevated tryptophan hydroxylase 1 mRNA production during pregnancy, which drives the serotonin biosynthesis critical for adaptive maternal ß-cell responses. Together, these studies provide novel insight into the role of MAFB in human islet ß-cells.

Synaptotagmin 4 Regulates Pancreatic ß Cell Maturation by Modulating the Ca2+ Sensitivity of Insulin Secretion Vesicles.

Islet ß cells from newborn mammals exhibit high basal insulin secretion and poor glucose-stimulated insulin secretion (GSIS). Here we show that ß cells of newborns secrete more insulin than adults in response to similar intracellular Ca2+ concentrations, suggesting differences in the Ca2+ sensitivity of insulin secretion. Synaptotagmin 4 (Syt4), a non-Ca2+ binding paralog of the ß cell Ca2+ sensor Syt7, increased by ∼8-fold during ß cell maturation. Syt4 ablation increased basal insulin secretion and compromised GSIS. Precocious Syt4 expression repressed basal insulin secretion but also impaired islet morphogenesis and GSIS. Syt4 was localized on insulin granules and Syt4 levels inversely related to the number of readily releasable vesicles. Thus, transcriptional regulation of Syt4 affects insulin secretion; Syt4 expression is regulated in part by Myt transcription factors, which repress Syt4 transcription. Finally, human SYT4 regulated GSIS in EndoC-ßH1 cells, a human ß cell line. These findings reveal the role that altered Ca2+ sensing plays in regulating ß cell maturation.

GATA4 regulates epithelial cell proliferation to control intestinal growth and development in mice.

BACKGROUND & AIMS: The embryonic small intestinal epithelium is highly proliferative, and although much is known about mechanisms regulating proliferation in the adult intestine, the mechanisms controlling epithelial cell proliferation in the developing intestine are less clear. GATA4, a transcription factor that regulates proliferation in other developing tissues, is first expressed early in the developing gut in midgut endoderm. GATA4 function within midgut endoderm and the early intestinal epithelium has not been investigated. METHODS: Using Sonic Hedgehog Cre to eliminate GATA4 in the midgut endoderm of mouse embryos, we determined the impact of loss of GATA4 on intestinal development, including epithelial cell proliferation, between E9.5-E18.5. RESULTS: We found that intestinal length and width were decreased in GATA4 mutants compared with controls. GATA4-deficient intestinal epithelium contained fewer cells, and epithelial girth was decreased. We further observed a decreased proportion of proliferating cells at E10.5 and E11.5 in GATA4 mutants. We demonstrated that GATA4 binds to chromatin containing GATA4 consensus binding sites within Cyclin D2 (Ccnd2), Cyclin dependent kinase 6 (Cdk6), and Frizzled 5 (Fzd5). Moreover, Ccnd2, Cdk6, and Fzd5 transcripts were reduced at E11.5 in GATA4 mutant tissue. Villus morphogenesis was delayed, and villus structure was abnormal in GATA4 mutant intestine. CONCLUSIONS: Our data identify GATA4 as an essential regulator of early intestinal epithelial cell proliferation. We propose that GATA4 controls proliferation in part by directly regulating transcription of cell cycle mediators. Our data further suggest that GATA4 affects proliferation through transcriptional regulation of Fzd5, perhaps by influencing the response of the epithelium to WNT signaling.

Lack of Prox1 Downregulation Disrupts the Expansion and Maturation of Postnatal Murine ß-Cells.

Transcription factor expression fluctuates during ß-cell ontogeny, and disruptions in this pattern can affect the development or function of those cells. Here we uncovered that murine endocrine pancreatic progenitors express high levels of the homeodomain transcription factor Prox1, whereas both immature and mature ß-cells scarcely express this protein. We also investigated if sustained Prox1 expression is incompatible with ß-cell development or maintenance using transgenic mouse approaches. We discovered that Prox1 upregulation in mature ß-cells has no functional consequences; in contrast, Prox1 overexpression in immature ß-cells promotes acute fasting hyperglycemia. Using a combination of immunostaining and quantitative and comparative gene expression analyses, we determined that Prox1 upregulation reduces proliferation, impairs maturation, and enables apoptosis in postnatal ß-cells. Also, we uncovered substantial deficiency in ß-cells that overexpress Prox1 of the key regulator of ß-cell maturation MafA, several MafA downstream targets required for glucose-stimulated insulin secretion, and genes encoding important components of FGF signaling. Moreover, knocking down PROX1 in human EndoC-ßH1 ß-cells caused increased expression of many of these same gene products. These and other results in our study indicate that reducing the expression of Prox1 is beneficial for the expansion and maturation of postnatal ß-cells.

Gestational Diabetes Mellitus From Inactivation of Prolactin Receptor and MafB in Islet ß-Cells.

ß-Cell proliferation and expansion during pregnancy are crucial for maintaining euglycemia in response to increased metabolic demands placed on the mother. Prolactin and placental lactogen signal through the prolactin receptor (PRLR) and contribute to adaptive ß-cell responses in pregnancy; however, the in vivo requirement for PRLR signaling specifically in maternal ß-cell adaptations remains unknown. We generated a floxed allele of Prlr, allowing conditional loss of PRLR in ß-cells. In this study, we show that loss of PRLR signaling in ß-cells results in gestational diabetes mellitus (GDM), reduced ß-cell proliferation, and failure to expand ß-cell mass during pregnancy. Targeted PRLR loss in maternal ß-cells in vivo impaired expression of the transcription factor Foxm1, both G1/S and G2/M cyclins, tryptophan hydroxylase 1 (Tph1), and islet serotonin production, for which synthesis requires Tph1. This conditional system also revealed that PRLR signaling is required for the transient gestational expression of the transcription factor MafB within a subset of ß-cells during pregnancy. MafB deletion in maternal ß-cells also produced GDM, with inadequate ß-cell expansion accompanied by failure to induce PRLR-dependent target genes regulating ß-cell proliferation. These results unveil molecular roles for PRLR signaling in orchestrating the physiologic expansion of maternal ß-cells during pregnancy.

A Dynamic WNT/ß-CATENIN Signaling Environment Leads to WNT-Independent and WNT-Dependent Proliferation of Embryonic Intestinal Progenitor Cells.

Much of our understanding about how intestinal stem and progenitor cells are regulated comes from studying the late fetal stages of development and the adult intestine. In this light, little is known about intestine development prior to the formation of stereotypical villus structures with columnar epithelium, a stage when the epithelium is pseudostratified and appears to be a relatively uniform population of progenitor cells with high proliferative capacity. Here, we investigated a role for WNT/ß-CATENIN signaling during the pseudostratified stages of development (E13.5, E14.5) and following villus formation (E15.5) in mice. In contrast to the well-described role for WNT/ß-CATENIN signaling as a regulator of stem/progenitor cells in the late fetal and adult gut, conditional epithelial deletion of ß-catenin or the Frizzled co-receptors Lrp5 and Lrp6 had no effect on epithelial progenitor cell proliferation in the pseudostratified epithelium. Mutant embryos displayed obvious developmental defects, including loss of proliferation and disruptions in villus formation starting only at E15.5. Mechanistically, our data suggest that WNT signaling-mediated proliferation at the time of villus formation is driven by mesenchymal, but not epithelial, WNT ligand secretion.