Recurrent Gestational Diabetes Mellitus: A Narrative Review and Single-Center Experience.

Gestational diabetes mellitus (GDM) is a frequently observed complication of pregnancy and is associated with an elevated risk of adverse maternal and neonatal outcomes. Many women with GDM will go on to have future pregnancies, and these pregnancies may or may not be affected by GDM. We conducted a literature search, and based on data from key studies retrieved during the search, we describe the epidemiology of GDM recurrence. This includes a summary of the observed clinical risk factors of increasing maternal age, weight, ethnicity, and requirement for insulin in the index pregnancy. We then present our data from Mayo Clinic (January 2013-December 2017) which identifies a GDM recurrence rate of 47.6%, and illustrates the relevance of population-based studies to clinical practice. Lastly, we examine the available evidence on strategies to prevent GDM recurrence, and note that more research is needed to evaluate the effect of interventions before, during and after pregnancy.

Frequency of Gestational Diabetes Mellitus Reappearance or Absence during the Second Pregnancy of Women Treated at Mayo Clinic between 2013 and 2018.

The Center for Disease Control and Prevention ranks diabetes mellitus (DM) as the seventh leading cause of death in the USA. The most prevalent forms of DM include Type 2 DM, Type 1 DM, and gestational diabetes mellitus (GDM). While the acute problem of diabetic hyperglycemia can be clinically managed through dietary control and lifestyle changes or pharmacological intervention with oral medications or insulin, long-term complications of the disease are associated with significant morbidity and mortality. These long-term complications involve nearly all organ systems of the body and share common pathologies associated with endothelial cell abnormalities. To better understand the molecular mechanisms underlying DM as related to future long-term complications following hyperglycemia, we have undertaken a study to determine the frequency that GDM did or did not occur in the second pregnancy of women who experienced GDM in their first pregnancy between 2013 and 2018 at Mayo Clinic, Rochester, MN. Within the five-year period of the study, the results indicate that 7,330 women received obstetrical care for pregnancy during the study period. Of these, 150 developed GDM in their first pregnancy and of these, 42 (28%) had a second pregnancy. Of these 42 women, 20 again developed GDM and 22 did not develop GDM in their second pregnancy within the study period. Following the occurrence of GDM in the first pregnancy, the study (1) established the number of women with and without GDM in the second pregnancy and (2) confirmed the feasibility to study diabetic metabolic memory using maternal placental tissue from GDM women. These studies represent Phase I of a larger research project whose goal is to analyze epigenetic mechanisms underlying true diabetic metabolic memory using endothelial cells isolated from the maternal placenta of women with and without GDM as described in this article.

Epigenetic Studies Point to DNA Replication/Repair Genes as a Basis for the Heritable Nature of Long Term Complications in Diabetes.

Metabolic memory (MM) is defined as the persistence of diabetic (DM) complications even after glycemic control is pharmacologically achieved. Using a zebrafish diabetic model that induces a MM state, we previously reported that, in this model, tissue dysfunction was of a heritable nature based on cell proliferation studies in limb tissue and this correlated with epigenetic DNA methylation changes that paralleled alterations in gene expression. In the current study, control, DM, and MM excised fin tissues were further analyzed by MeDIP sequencing and microarray techniques. Bioinformatics analysis of the data found that genes of the DNA replication/DNA metabolism process group (with upregulation of the apex1, mcm2, mcm4, orc3, lig1, and dnmt1 genes) were altered in the DM state and these molecular changes continued into MM. Interestingly, DNA methylation changes could be found as far as 6-13 kb upstream of the transcription start site for these genes suggesting potential higher levels of epigenetic control. In conclusion, DNA methylation changes in members of the DNA replication/repair process group best explain the heritable nature of cell proliferation impairment found in the zebrafish DM/MM model. These results are consistent with human diabetic epigenetic studies and provide one explanation for the persistence of long term tissue complications as seen in diabetes.

Use of zebrafish as a model to investigate the role of epigenetics in propagating the secondary complications observed in diabetes mellitus.

Diabetes mellitus (DM) is classified as a disease of metabolic dysregulation predicted to affect over 400 million individuals world-wide by 2030. The debilitating aspects of this disease are the long term complications involving microvascular and macrovascular pathologies. These long term complications are related to the clinical phenomenon of metabolic memory (MM) that is defined as the persistence of diabetic complications even after glycemic control has been pharmacologically achieved. The persistent nature of MM has invoked involvement of epigenetic processes. Current research with the DM/MM zebrafish model as described in this review as well as human and mammalian studies has established that changes in DNA methylation patterns appear to contribute to tissue dysfunctions associated with DM. This review will describe studies on an adult zebrafish model of type I diabetes mellitus that allows analysis of both the hyperglycemic (HG or DM) phase and MM phase of the disease. The review will discuss the model in regards to: 1) its hyperglycemic phase, 2) its MM phase, 3) biochemical õpathways underlying changes in DNA methylation patterns observed in the model, 4) loci specific changes in DNA methylation patterns, and 5) strengths of the adult zebrafish model as compared to other MM animal models.

Inhibition of poly-ADP ribose polymerase enzyme activity prevents hyperglycemia-induced impairment of angiogenesis during wound healing.

We previously reported a zebrafish model of type I diabetes mellitus (DM) that can be used to study the hyperglycemic (HG) and metabolic memory (MM) states within the same fish. Clinically, MM is defined as the persistence of diabetic complications even after glycemic control is pharmacologically achieved. In our zebrafish model, MM occurs following ß-cell regeneration, which returns fish to euglycemia. During HG, fish acquire tissue deficits reflective of the complications seen in patients with DM and these deficits persist after fish return to euglycemia (MM). The unifying mechanism for the induction of diabetic complications involves a cascade of events that is initiated by the HG stimulation of poly-ADP ribose polymerase enzyme (Parp) activity. Additionally, recent evidence shows that the HG induction of Parp activity stimulates changes in epigenetic mechanisms that correlate with the MM state and the persistence of complications. Here we report that wound-induced angiogenesis is impaired in DM and remains impaired when fish return to a euglycemic state. Additionally, inhibition of Parp activity prevented the HG-induced wound angiogenesis deficiency observed. This approach can identify molecular targets that will provide potential new avenues for therapeutic discovery as angiogenesis imbalances are associated with all HG-damaged tissues.

An assay for lateral line regeneration in adult zebrafish.

Due to the clinical importance of hearing and balance disorders in man, model organisms such as the zebrafish have been used to study lateral line development and regeneration. The zebrafish is particularly attractive for such studies because of its rapid development time and its high regenerative capacity. To date, zebrafish studies of lateral line regeneration have mainly utilized fish of the embryonic and larval stages because of the lower number of neuromasts at these stages. This has made quantitative analysis of lateral line regeneration/and or development easier in the earlier developmental stages. Because many zebrafish models of neurological and non-neurological diseases are studied in the adult fish and not in the embryo/larvae, we focused on developing a quantitative lateral line regenerative assay in adult zebrafish so that an assay was available that could be applied to current adult zebrafish disease models. Building on previous studies by Van Trump et al. that described procedures for ablation of hair cells in adult Mexican blind cave fish and zebrafish (Danio rerio), our assay was designed to allow quantitative comparison between control and experimental groups. This was accomplished by developing a regenerative neuromast standard curve based on the percent of neuromast reappearance over a 24 hr time period following gentamicin-induced necrosis of hair cells in a defined region of the lateral line. The assay was also designed to allow extension of the analysis to the individual hair cell level when a higher level of resolution is required.

Parp inhibition prevents ten-eleven translocase enzyme activation and hyperglycemia-induced DNA demethylation.

Studies from human cells, rats, and zebrafish have documented that hyperglycemia (HG) induces the demethylation of specific cytosines throughout the genome. We previously documented that a subset of these changes become permanent and may provide, in part, a mechanism for the persistence of complications referred to as the metabolic memory phenomenon. In this report, we present studies aimed at elucidating the molecular machinery that is responsible for the HG-induced DNA demethylation observed. To this end, RNA expression and enzymatic activity assays indicate that the ten-eleven translocation (Tet) family of enzymes are activated by HG. Furthermore, through the detection of intermediates generated via conversion of 5-methyl-cytosine back to the unmethylated form, the data were consistent with the use of the Tet-dependent iterative oxidation pathway. In addition, evidence is provided that the activity of the poly(ADP-ribose) polymerase (Parp) enzyme is required for activation of Tet activity because the use of a Parp inhibitor prevented demethylation of specific loci and the accumulation of Tet-induced intermediates. Remarkably, this inhibition was accompanied by a complete restoration of the tissue regeneration deficit that is also induced by HG. The ultimate goal of this work is to provide potential new avenues for therapeutic discovery.

A zebrafish model of diabetes mellitus and metabolic memory.

Diabetes mellitus currently affects 346 million individuals and this is projected to increase to 400 million by 2030. Evidence from both the laboratory and large scale clinical trials has revealed that diabetic complications progress unimpeded via the phenomenon of metabolic memory even when glycemic control is pharmaceutically achieved. Gene expression can be stably altered through epigenetic changes which not only allow cells and organisms to quickly respond to changing environmental stimuli but also confer the ability of the cell to "memorize" these encounters once the stimulus is removed. As such, the roles that these mechanisms play in the metabolic memory phenomenon are currently being examined. We have recently reported the development of a zebrafish model of type I diabetes mellitus and characterized this model to show that diabetic zebrafish not only display the known secondary complications including the changes associated with diabetic retinopathy, diabetic nephropathy and impaired wound healing but also exhibit impaired caudal fin regeneration. This model is unique in that the zebrafish is capable to regenerate its damaged pancreas and restore a euglycemic state similar to what would be expected in post-transplant human patients. Moreover, multiple rounds of caudal fin amputation allow for the separation and study of pure epigenetic effects in an in vivo system without potential complicating factors from the previous diabetic state. Although euglycemia is achieved following pancreatic regeneration, the diabetic secondary complication of fin regeneration and skin wound healing persists indefinitely. In the case of impaired fin regeneration, this pathology is retained even after multiple rounds of fin regeneration in the daughter fin tissues. These observations point to an underlying epigenetic process existing in the metabolic memory state. Here we present the methods needed to successfully generate the diabetic and metabolic memory groups of fish and discuss the advantages of this model.

Impaired tissue regeneration corresponds with altered expression of developmental genes that persists in the metabolic memory state of diabetic zebrafish.

As previously reported by our laboratory, streptozocin-induced diabetes mellitus (DM) in adult zebrafish results in an impairment of tissue regeneration as monitored by caudal fin regeneration. Following streptozocin withdrawal, a recovery phase occurs to reestablish euglycemia, via pancreatic beta-cell regeneration. However, DM-associated impaired fin regeneration continues indefinitely in the metabolic memory (MM) state, allowing for subsequent molecular analysis of the underlying mechanisms of MM. This study focuses on elucidating the molecular basis that explains the DM-associated impaired fin regeneration and why it persists into the MM state with the aim of better understanding MM. Using a combination of microarray analysis and bioinformatics approaches, our study found that of the 14,900 transcripts analyzed, aberrant expression of 71 genes relating to tissue developmental and regeneration processes were identified in DM fish and the altered expression of these 71 genes persisted in MM fish. Key regulatory genes of major development and signal transduction pathways were identified among this group of 71. The aberrant expression of key regulatory genes in the DM state that persist into the MM state provides a plausible explanation on how hyperglycemia induced impaired fin regeneration in the adult zebrafish DM/MM model.

Metabolic memory and chronic diabetes complications: potential role for epigenetic mechanisms.

Recent estimates indicate that diabetes mellitus currently affects more than 10 % of the world's population. Evidence from both the laboratory and large scale clinical trials has revealed that prolonged hyperglycemia induces chronic complications which persist and progress unimpeded even when glycemic control is pharmaceutically achieved via the phenomenon of metabolic memory. The epigenome is comprised of all chromatin modifications including post translational histone modification, expression control via miRNAs and the methylation of cytosine within DNA. Modifications of these epigenetic marks not only allow cells and organisms to quickly respond to changing environmental stimuli but also confer the ability of the cell to "memorize" these encounters. As such, these processes have gained much attention as potential molecular mechanisms underlying metabolic memory and chronic diabetic complications. Here we present a review of the very recent literature published pertaining to this subject.

Components, structure, biogenesis and function of the Hydra extracellular matrix in regeneration, pattern formation and cell differentiation.

The body wall of Hydra is organized as an epithelial bilayer (ectoderm and endoderm) with an intervening extracellular matrix (ECM), termed mesoglea by early biologists. Morphological studies have determined that Hydra ECM is composed of two basal lamina layers positioned at the base of each epithelial layer with an intervening interstitial matrix. Molecular and biochemical analyses of Hydra ECM have established that it contains components similar to those seen in more complicated vertebrate species. These components include such macromolecules as laminin, type IV collagen, and various fibrillar collagens. These components are synthesized in a complicated manner involving cross-talk between the epithelial bilayer. Any perturbation to ECM biogenesis leads to a blockage in Hydra morphogenesis. Blockage in ECM/cell interactions in the adult polyp also leads to problems in epithelial transdifferentiation processes. In terms of biophysical parameters, Hydra ECM is highly flexible; a property that facilitates continuous movements along the organism's longitudinal and radial axis. This is in contrast to the more rigid matrices often found in vertebrates. The flexible nature of Hydra ECM can in part now be explained by the unique structure of the organism's type IV collagen and fibrillar collagens. This review will focus on Hydra ECM in regard to: 1) its general structure, 2) its molecular composition, 3) the biophysical basis for the flexible nature of Hydra's ECM, 4) the relationship of the biogenesis of Hydra ECM to regeneration of body form, and 5) the functional role of Hydra ECM during pattern formation and cell differentiation.

Heritable transmission of diabetic metabolic memory in zebrafish correlates with DNA hypomethylation and aberrant gene expression.

Metabolic memory (MM) is the phenomenon whereby diabetes complications persist and progress after glycemic recovery is achieved. Here, we present data showing that MM is heritable and that the transmission correlates with hyperglycemia-induced DNA hypomethylation and aberrant gene expression. Streptozocin was used to induce hyperglycemia in adult zebrafish, and then, following streptozocin withdrawal, a recovery phase was allowed to reestablish a euglycemic state. Blood glucose and serum insulin returned to physiological levels during the first 2 weeks of the recovery phase as a result of pancreatic ß-cell regeneration. In contrast, caudal fin regeneration and skin wound healing remained impaired to the same extent as in diabetic fish, and this impairment was transmissible to daughter cell tissue. Daughter tissue that was never exposed to hyperglycemia, but was derived from tissue that was, did not accumulate AGEs or exhibit increased levels of oxidative stress. However, CpG island methylation and genome-wide microarray expression analyses revealed the persistence of hyperglycemia-induced global DNA hypomethylation that correlated with aberrant gene expression for a subset of loci in this daughter tissue. Collectively, the data presented here implicate the epigenetic mechanism of DNA methylation as a potential contributor to the MM phenomenon.

In vivo imaging of basement membrane movement: ECM patterning shapes Hydra polyps.

Growth and morphogenesis during embryonic development, asexual reproduction and regeneration require extensive remodeling of the extracellular matrix (ECM). We used the simple metazoan Hydra to examine the fate of ECM during tissue morphogenesis and asexual budding. In growing Hydra, epithelial cells constantly move towards the extremities of the animal and into outgrowing buds. It is not known, whether these tissue movements involve epithelial migration relative to the underlying matrix or whether cells and ECM are displaced as a composite structure. Furthermore, it is unclear, how the ECM is remodeled to adapt to the shape of developing buds and tentacles. To address these questions, we used a new in vivo labeling technique for Hydra collagen-1 and laminin, and tracked the fate of ECM in all body regions of the animal. Our results reveal that Hydra 'tissue movements' are largely displacements of epithelial cells together with associated ECM. By contrast, during the evagination of buds and tentacles, extensive movement of epithelial cells relative to the matrix is observed, together with local ECM remodeling. These findings provide new insights into the nature of growth and morphogenesis in epithelial tissues.

Limb regeneration is impaired in an adult zebrafish model of diabetes mellitus.

The zebrafish (Danio rerio) is an established model organism for the study of developmental processes, human disease, and tissue regeneration. We report that limb regeneration is severely impaired in our newly developed adult zebrafish model of type I diabetes mellitus. Intraperitoneal streptozocin injection of adult, wild-type zebrafish results in a sustained hyperglycemic state as determined by elevated fasting blood glucose values and increased glycation of serum protein. Serum insulin levels are also decreased and pancreas immunohistochemisty revealed a decreased amount of insulin signal in hyperglycemic fish. Additionally, the diabetic complications of retinal thinning and glomerular basement membrane thickening (early signs of retinopathy and nephropathy) resulting from the hyperglycemic state were evident in streptozocin-injected fish at 3 weeks. Most significantly, limb regeneration, following caudal fin amputation, is severely impaired in diabetic zebrafish and nonspecific toxic effects outside the pancreas were not found to contribute to impaired limb regeneration. This experimental system using adult zebrafish facilitates a broad spectrum of genetic and molecular approaches to study regeneration in the diabetic background.

The extracellular matrix of hydra is a porous sheet and contains type IV collagen.

Hydra, as an early diploblastic metazoan, has a well-defined extracellular matrix (ECM) called mesoglea. It is organized in a tri-laminar pattern with one centrally located interstitial matrix that contains type I collagen and two sub-epithelial zones that resemble a basal lamina containing laminin and possibly type IV collagen. This study used monoclonal antibodies to the three hydra mesoglea components (type I, type IV collagens and laminin) and immunofluorescent staining to visualize hydra mesoglea structure and the relationship between these mesoglea components. In addition, hydra mesoglea was isolated free of cells and studied with immunofluorescence and scanning electron microscopy (SEM). Our results show that type IV collagen co-localizes with laminin in the basal lamina whereas type I collagen forms a grid pattern of fibers in the interstitial matrix. The isolated mesoglea can maintain its structural stability without epithelial cell attachment. Hydra mesoglea is porous with multiple trans-mesoglea pores ranging from 0.5 to 1 microm in diameter and about six pores per 100 microm(2) in density. We think these trans-mesoglea pores provide a structural base for epithelial cells on both sides to form multiple trans-mesoglea cell-cell contacts. Based on these findings, we propose a new model of hydra mesoglea structure.

Both Hoxc13 orthologs are functionally important for zebrafish tail fin regeneration.

Hox genes are re-expressed during regeneration in many species. Given their important role in body plan development, it has been assumed, but not directly shown, that they play a functional role in regeneration. In this paper we show that morpholino-mediated knockdown of either Hoxc13a or Hoxc13b during the process of zebrafish tail fin regeneration results in a significant reduction of regenerative outgrowth. Furthermore, cellular proliferation within the blastema is directly affected in both knockdowns. Hence, similar to the demonstration of unique functions of multiple Hox genes during limb formation, both Hoxc13 orthologs have distinct functions in regeneration.

The collagens of hydra provide insight into the evolution of metazoan extracellular matrices.

A collagen-based extracellular matrix is one defining feature of all Metazoa. The thick sheet-like extracellular matrix (mesoglia) of the diploblast, hydra, has characteristics of both a basement membrane and an interstitial matrix. Several genes associated with mesoglea have been cloned including a basement membrane and fibrillar collagen and an A and B chain of laminin. Here we report the characterization of a further three fibrillar collagen genes (Hcol2, Hcol3, and Hcol5) and the partial sequence of a collagen gene with a unique structural organization consisting of multiple von Willebrand factor A domains interspersed with interrupted collagenous triple helices (Hcol6) from Hydra vulgaris. Hcol2 and -5 have major collagenous domains of classical length ( approximately 1020 amino acid residues), whereas the equivalent domain in Hcol3 is shorter (969 residues). The N-propeptide of Hcol2 contains a whey acid protein four-cysteine repeat (WAP) domain, and the equivalent domain of Hcol3 contains two WAP and two von Willebrand factor A domains. Phylogenetic analyses reveal that the hydra fibrillar collagen genes form a distinct clade that appears related to the protostome/deuterostome A clade of fibrillar collagens. Data base searches reveal Hcol2, -5, and -6 are highly conserved in Hydra magnipapillata, which also provided preliminary evidence for the expression of a B-clade fibrillar collagen. All four of the H. vulgaris collagens are expressed specifically by the ectoderm. The expression pattern for Hcol2 is similar to that previously reported for Hcol1 (Deutzmann, R., Fowler, S., Zhang, X., Boone, K., Dexter, S., Boot-Handford, R. P., Rachel, R., and Sarras, M. P., Jr. (2000) Development 127, 4669-4680) but distinct from the pattern shared by Hcol3 and Hcol5. The characterization of multiple collagen genes in relatively simple diploblastic organisms provides new insights into the molecular evolution of collagens and the origins of the collagen-based extracellular matrix found throughout the multicellular animal kingdom.

Inhibition of zebrafish fin regeneration using in vivo electroporation of morpholinos against fgfr1 and msxb.

Increased interest in using zebrafish as a model organism has led to a resurgence of fin regeneration studies. This has allowed for the identification of a large number of gene families, including signaling molecules and transcription factors, which are expressed during regeneration. However, in cases where no specific inhibitor is available for the gene product of interest, determination of a functional role for these genes has been difficult. Here we demonstrate that in vivo electroporation of morpholino oligonucleotides is a feasible approach for protein knock-down during fin regeneration. Morpholino oligonucleotides against fgfr1 and msxb were utilized and knock-down of both proteins resulted in reduced fin outgrowth. Importantly, Fgfr1 knock-down phenocopied outgrowth inhibition obtained with an Fgfr1 inhibitor. Furthermore, this method provided direct evidence for a functional role for msxb in caudal fin regeneration. Finally, knock-down of Fgfr1, but not Msxb, affected the blastemal expression of msxc, suggesting this technique can be used to determine epistasis in genetic pathways affecting regeneration. Thus, this convenient reverse genetic approach allows researchers to quickly (1) assess the function of genes known to be expressed during fin regeneration, (2) screen genes for functional relevance during fin regeneration, and (3) assign genes to the molecular pathways underlying fin regeneration.

Cre-mediated site-specific recombination in zebrafish embryos.

Cre-mediated site-specific recombination has become an invaluable tool for manipulation of the murine genome. The ability to conditionally activate gene expression or to generate chromosomal alterations with this same tool would greatly enhance zebrafish genetics. This study demonstrates that the HSP70 promoter can be used to inducibly control expression of an enhanced green fluorescent protein (EGFP) -Cre fusion protein. The EGFP-Cre fusion protein is capable of promoting recombination between lox sites in injected plasmids or in stably inherited transgenes as early as 2 hr post-heat shock induction. Finally, the levels of Cre expression achieved in a transgenic fish line carrying the HSP70-EGFP-cre transgene are compatible with viability and both male and female transgenic fish are fertile subsequent to induction of EGFP-Cre expression. Hence, our data suggests that Cre-mediated recombination is a viable means of manipulating gene expression in zebrafish.

Matrix metalloproteinase expression and function during fin regeneration in zebrafish: analysis of MT1-MMP, MMP2 and TIMP2.

Matrix metalloproteinases (MMPs) play key roles in the turnover of extracellular matrix (ECM) and, thereby, function as key regulators of cell-ECM interactions during development. In spite of their importance during developmental processes, relatively little has been reported about the role of these metalloproteinases during limb development and regeneration. To approach the problem of cell-ECM interactions during limb (fin) regeneration, we have utilized zebrafish as an experimental model. Based on previous MMP cloning studies from our laboratory, the current study has focused on the expression of membrane-type 1 metalloproteinase (MT1-MMP), gelatinase A (MMP-2) and endogenous tissue inhibitor 2 of metalloproteinases (TIMP-2) during fin regeneration in adult zebrafish. In situ analysis indicated co-expression of zmt1-mmp, zmmp-2, and ztimp-2 mRNA transcripts in regenerating caudal fins. In situ gelatin-zymography confirmed the presence of active metalloproteinases in regenerating fins. zmt1-mmp, zmmp-2, and ztimp-2 mRNA transcripts were expressed in the blastema and basal epithelium during caudal fin regeneration while expression of type IV collagen [zcol-IV(a5)] transcripts (a basal lamina component) was restricted to the basal epithelium. Fin outgrowth was greatly reduced in the presence of GM6001 (an inhibitor of MMP activity) indicating the importance of these enzymes during fin regeneration. Previous studies by Itoh (EMBO, 2001) indicated that expression of a vertebrate MT1-MMP construct containing only the hemopexin-transmembrane-cytoplasmic domains (MT1HPX) resulted in blockage of MT1-MMP homophilic complex formation and subsequent inhibition of pro-MMP-2 activation. Interference with homophilic complex formation was attributed to expression of the hemopexin domain at the cell surface. Building upon these earlier findings, the current study found that ectopic expression of MT1HPX in fin regenerates inhibited the regeneration process and resulted in a reduction in cell proliferation in the blastema. Taken together, these results indicate that MMPs have an important role during fin regeneration in zebrafish.