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With type 2 diabetes presenting at younger ages, there is a growing need to identify biomarkers of future glucose intolerance. A high (20%) prevalence of glucose intolerance at 18 years was seen in women from the Pune Maternal Nutrition Study (PMNS) birth cohort. We investigated the potential of circulating microRNAs in risk stratification for future pre-diabetes in these women. Here, we provide preliminary longitudinal analyses of circulating microRNAs in normal glucose tolerant (NGT@18y, N = 10) and glucose intolerant (N = 8) women (ADA criteria) at 6, 12 and 17 years of their age using discovery analysis (OpenArray™ platform). Machine-learning workflows involving Lasso with bootstrapping/leave-one-out cross-validation identified microRNAs associated with glucose intolerance at 18 years of age. Several microRNAs, including miR-212-3p, miR-30e-3p and miR-638, stratified glucose-intolerant women from NGT at childhood. Our results suggest that circulating microRNAs, longitudinally assessed over 17 years of life, are dynamic biomarkers associated with and predictive of pre-diabetes at 18 years of age. Validation of these findings in males and remaining participants from the PMNS birth cohort will provide a unique opportunity to study novel epigenetic mechanisms in the life-course progression of glucose intolerance and enhance current clinical risk prediction of pre-diabetes and progression to type 2 diabetes.

ObjectivePancreatic islet β-cells are factories for insulin production; however ectopic expression of insulin is also well recognized. The gallbladder is a next-door neighbour to the developing pancreas. Here, we wanted to understand if gallbladders contain functional insulin-producing cells.DesignWe compared developing and adult mouse as well as human gallbladder epithelial cells and islets using immunohistochemistry, flow cytometry, ELISAs, RNA-sequencing, real-time PCR, chromatin immunoprecipitation and functional studies.ResultsWe demonstrate that the epithelial lining of developing, as well as adult mouse and human gallbladders naturally contain interspersed cells that retain the capacity to actively transcribe, translate, package, and release insulin. We show for the first time that human gallbladders also contain functional insulin-secreting cells with the potential to naturally respond to glucose in vitro and in situ. Notably, in a NOD mouse model of type 1 diabetes, we observed that insulin-producing cells in the gallbladder are not targeted by autoimmune cells. Conclusion: In summary, our biochemical, transcriptomic, and functional data in human gallbladder epithelial cells collectively demonstrate their potential for insulin-production under pathophysiological conditions, and open newer areas for type 1 diabetes research and therapy.Significance of the studyWhat is already known about this subject?Developing pancreas and gallbladder are next-door neighbours and share similar developmental pathways.Human Gallbladder-derived progenitor cells were shown to differentiate into insulin-producing cells.What are the new findings?Gallbladder epithelium contains interspersed cells that can transcribe, translate, package and secrete insulin.Insulin-producing cells in the gallbladder are not destroyed by immune cells in an animal model of type 1 diabetes (T1D).Our studies demonstrating the absence of insulin splice variants in human gallbladder cells, and higher splice forms in human islets, suggest a potential mechanism (via defective ribosomal products) in escaping islet autoimmunity.How might it impact clinical practice?Deciphering mechanisms of protection of insulin-producing cells from immune cells in the gallbladder could help in developing strategies to prevent islet autoimmunity in T1D.

Summary Dicer knockout mouse models demonstrated a key role for microRNAs in pancreatic β-cell function. Studies to identify specific microRNA(s) associated with human (pro-)endocrine gene expression are needed. We profiled microRNAs and key pancreatic genes in 353 human tissue samples. Machine learning workflows identified microRNAs associated with (pro-)insulin transcripts in a discovery set of islets (n = 30) and insulin-negative tissues (n = 62). This microRNA signature was validated in remaining 261 tissues that include nine islet samples from individuals with type 2 diabetes. Top eight microRNAs (miR-183-5p, -375-3p, 216b-5p, 183-3p, -7-5p, -217-5p, -7-2-3p, and -429-3p) were confirmed to be associated with and predictive of (pro-)insulin transcript levels. Use of doxycycline-inducible microRNA-overexpressing human pancreatic duct cell lines confirmed the regulatory roles of these microRNAs in (pro-)endocrine gene expression. Knockdown of these microRNAs in human islet cells reduced (pro-)insulin transcript abundance. Our data provide specific microRNAs to further study microRNA-mRNA interactions in regulating insulin transcription., Graphical abstract, Highlights • Unbiased machine learning workflow ranks miRNAs associated with insulin transcription • Forced expression of top-ranked miRNAs drives pro-endocrine program in progenitor cells • Knockdown of top-ranked miRNAs retards insulin gene transcription in human islets • Insulin transcript-associated miRNAs are reduced in islets of donors with type 2 diabetes, Pathophysiology; Computational Bioinformatics; Transcriptomics

Type 1 diabetes (T1D) is an autoimmune disease, where insulin-producing β-cells in the pancreas are inappropriately recognized and destroyed by immune cells. Islet transplantation is the most successful cell-based therapy for T1D individuals who experience frequent and severe life-threatening hypoglycemia. However, this therapy is extremely restricted owing to the limited availability of donor pancreas. In recent years, significant progress has been made in generating β-cells from stem/progenitor cells using different approaches of in vitro differentiation. The insulin production from such in vitro generated β-cells is still far less than that observed in islet β-cells. We employed a novel strategy to improve the efficiency of progenitor cell differentiation by performing partial mouse pancreas resection after transplanting in vitro generated insulin-producing cells under the kidney capsule of these mice. Pancreas resection (pancreatectomy) has been shown to induce regenerative pathways, leading to regeneration of almost the entire resected pancreas over 3-5 weeks in mice. We found that in our method, regenerating mouse pancreas promotes better graft differentiation/maturation and insulin production from transplanted cells. In this chapter, we detail the protocols used for transplantation of in vitro differentiated cells in immunocompromised mice, partial pancreatectomy in host (NOD scid) mice, and assessment of graft function. We believe that our protocols provide a solid platform for further studies aimed at understanding growth/differentiation molecules secreted from regenerating pancreas that promote graft maturation.

Type 1 diabetes (T1D) is an autoimmune disease, where insulin-producing β-cells in the pancreas are inappropriately recognized and destroyed by immune cells. Islet transplantation is the most successful cell-based therapy for T1D individuals who experience frequent and severe life-threatening hypoglycemia. However, this therapy is extremely restricted owing to the limited availability of donor pancreas. In recent years, significant progress has been made in generating β-cells from stem/progenitor cells using different approaches of in vitro differentiation. The insulin production from such in vitro generated β-cells is still far less than that observed in islet β-cells. We employed a novel strategy to improve the efficiency of progenitor cell differentiation by performing partial mouse pancreas resection after transplanting in vitro generated insulin-producing cells under the kidney capsule of these mice. Pancreas resection (pancreatectomy) has been shown to induce regenerative pathways, leading to regeneration of almost the entire resected pancreas over 3-5 weeks in mice. We found that in our method, regenerating mouse pancreas promotes better graft differentiation/maturation and insulin production from transplanted cells. In this chapter, we detail the protocols used for transplantation of in vitro differentiated cells in immunocompromised mice, partial pancreatectomy in host (NOD scid) mice, and assessment of graft function. We believe that our protocols provide a solid platform for further studies aimed at understanding growth/differentiation molecules secreted from regenerating pancreas that promote graft maturation.

Background Previous studies indicate that low birth weight and exposure to maternal stress during pregnancy may result in shortened telomeres in infants. Shorter telomere length has in turn been linked with accelerated ageing and with age-related diseases. This study aimed to investigate the association between pregnancy and birth factors and relative telomere length in offspring at 11 years of age. Methods Participants were aged 11 years enrolled in the Auckland Birthweight Collaborative Study at birth (n = 380). Half of the children were born small for gestational age (SGA = birthweight ≤ 10th percentile) and half were appropriate for gestational age (AGA = birthweight > 10th percentile). Maternal stress during pregnancy was assessed using the Perceived Stress Scale. Relative leukocyte telomere length (RTL) in leukocytes was measured at 11 years of age using quantitative real-time PCR. Results RTL was normally distributed (mean = 3.78, SD = 1.05). There were no significant associations between RTL at age 11 years and birthweight, sex, maternal smoking, maternal stress during pregnancy or maternal pre-pregnancy body mass index. Conclusion At age 11 years, RTL did not differ between children by birthweight or pregnancy-related stressors. Further telomere-related studies in newborns, children and adolescents are merited to increase knowledge of potential telomere modulating factors.

Supplementary_Table_1_new for Epigenetic and Transcriptome Profiling Identifies a Population of Visceral Adipose-Derived Progenitor Cells with the Potential to Differentiate into an Endocrine Pancreatic Lineage by Michael D. Williams, Mugdha V. Joglekar, Sarang N. Satoor, Wilson Wong, Effie Keramidaris, Amanda Rixon, Philip O’Connell, Wayne J. Hawthorne, Geraldine M. Mitchell, and Anandwardhan A. Hardikar in Cell Transplantation

SummaryPeople in developing countries have faced multigenerational undernutrition and are currently undergoing major lifestyle changes, contributing to an epidemic of metabolic diseases, though the underlying mechanisms remain unclear. Using a Wistar rat model of undernutrition over 50 generations, we show that Undernourished rats exhibit low birth-weight, high visceral adiposity (DXA/MRI), and insulin resistance (hyperinsulinemic-euglycemic clamps), compared to age-/gender-matched control rats. Undernourished rats also have higher circulating insulin, homocysteine, endotoxin and leptin levels, lower adiponectin, vitamin B12 and folate levels, and an 8-fold increased susceptibility to Streptozotocin-induced diabetes compared to control rats. Importantly, these metabolic abnormalities are not reversed after two generations of unrestricted access to commercial chow (nutrient recuperation). Altered epigenetic signatures in insulin-2 gene promoter region of Undernourished rats are not reversed by nutrient recuperation, and may contribute to the persistent detrimental metabolic profiles in similar multigenerational undernourished human populations.

It is well known that human cells are diverse with respect to their epigenome, transcriptome, and proteome. In the context of regenerative medicine, it is important for the transplanted cells or tissues to faithfully recapitulate their intended tissue type in each of these respects. Whether the cells chosen for such an application are embryonic, postnatal, or induced pluripotent stem cells, the transplanted product must behave in a predictable and reliable manner to be a safe and effective treatment option. Irrespective of the choice of cells used in such an application, the characterization and understanding of the developmental cues responsible for establishing and maintaining the desired cell phenotype are essential.Animal models are extremely important in understanding the development of a specific tissue, which can then be subsequently extrapolated to human studies. Generation of transgenic animal models with whole-body gene knockout, conditional knockout, constitutive fluorescent gene reporters, and Cre-Lox-based conditional and lineage reporters has revolutionized the field of developmental biology. An intrinsically complex network of the actions and interactions of the multitude of different signalling cascades is required for development. A thorough understanding of such networks, gained through studies on transgenic animal models, is essential for the development of the techniques necessary to reliably differentiate a given stem or progenitor cell population into a specific cell type, such as an islet-like, insulin-producing cell aggregate.In this chapter, we describe the use of GFP (green fluorescent protein)-based reporter mice for isolation of cells of choice, analyzing gene expression in those cells as well as their use for screening signalling molecules to understand their effect on differentiation.

The mammalian gut is inhabited by a complex and highly diverse population of bacteria. About 100 trillion microbes are present in the human gut, a number ten times more than the total number of cells in an adult human body. These microorganisms play an important role in several fundamental and crucial processes such as immunity, digestion, synthesis of vitamins, and metabolizing bile acids, sterols, and xenobiotics in the host, thereby influencing human health. Identification and manipulation of these metabolic interfaces is therefore critical. Here, we present a set of methods for manipulation and targeting the 16S rRNA based identification of rodent gut microbiota using Sanger's and next-generation sequencing platforms. Novel methods for manipulation of gut microbiota are also presented. In principle, these methods can be easily adapted to most rodent models for successful screening and manipulation of gut microbiome, to generate a better understanding of their role in metabolic disease.

The mammalian gut is inhabited by a complex and highly diverse population of bacteria. About 100 trillion microbes are present in the human gut, a number ten times more than the total number of cells in an adult human body. These microorganisms play an important role in several fundamental and crucial processes such as immunity, digestion, synthesis of vitamins, and metabolizing bile acids, sterols, and xenobiotics in the host, thereby influencing human health. Identification and manipulation of these metabolic interfaces is therefore critical. Here, we present a set of methods for manipulation and targeting the 16S rRNA based identification of rodent gut microbiota using Sanger's and next-generation sequencing platforms. Novel methods for manipulation of gut microbiota are also presented. In principle, these methods can be easily adapted to most rodent models for successful screening and manipulation of gut microbiome, to generate a better understanding of their role in metabolic disease.

It is well known that human cells are diverse with respect to their epigenome, transcriptome, and proteome. In the context of regenerative medicine, it is important for the transplanted cells or tissues to faithfully recapitulate their intended tissue type in each of these respects. Whether the cells chosen for such an application are embryonic, postnatal, or induced pluripotent stem cells, the transplanted product must behave in a predictable and reliable manner to be a safe and effective treatment option. Irrespective of the choice of cells used in such an application, the characterization and understanding of the developmental cues responsible for establishing and maintaining the desired cell phenotype are essential.Animal models are extremely important in understanding the development of a specific tissue, which can then be subsequently extrapolated to human studies. Generation of transgenic animal models with whole-body gene knockout, conditional knockout, constitutive fluorescent gene reporters, and Cre-Lox-based conditional and lineage reporters has revolutionized the field of developmental biology. An intrinsically complex network of the actions and interactions of the multitude of different signalling cascades is required for development. A thorough understanding of such networks, gained through studies on transgenic animal models, is essential for the development of the techniques necessary to reliably differentiate a given stem or progenitor cell population into a specific cell type, such as an islet-like, insulin-producing cell aggregate.In this chapter, we describe the use of GFP (green fluorescent protein)-based reporter mice for isolation of cells of choice, analyzing gene expression in those cells as well as their use for screening signalling molecules to understand their effect on differentiation.

MicroRNAs (miRNAs) are small noncoding RNAs that act as regulators of gene expression by targeting mature messenger RNAs. Following the initial report of the presence of miRNAs in serum and plasma a number of studies have successfully demonstrated the use of these miRNAs as biomarkers of disease. Currently, there are many methods of isolating total RNA from liquid samples. Here, we describe a simple, cost effective method for extraction of RNA from human serum as well as subsequent real time PCR analysis of miRNA levels.

Visceral adiposity is a risk factor for cardiovascular disorders, type 2 diabetes mellitus (T2D) and associated metabolic diseases. Sub-cutaneous fat is believed to be intrinsically different from visceral fat. To understand molecular mechanisms involved in metabolic advantages of fat transplantation, we studied a rat model of diet-induced adiposity. Adipokine genes (Adiponectin, Leptin, Resistin and Visfatin) were expressed at 10,000 to a million-fold lower in visceral fat depot as compared to peripheral (thigh/chest) fat depots. Interestingly, autologous transplantation of visceral fat to subcutaneous sites resulted in increased gene transcript abundance in the grafts by 3 weeks post-transplantation, indicating the impact of local (residence) factors influencing epigenetic memory. We show here that active transcriptional state of adipokine genes is linked with glucose mediated recruitment of enzymes that regulate histone methylation. Adipose depots have “residence memory” and autologous transplantation of visceral fat to sub-cutaneous sites offers metabolic advantage.