The molecular and functional heterogeneity of pancreatic β-cells is well recognized, but the underlying mechanisms remain unclear. Pancreatic islets harbor a subset of β-cells that co-express Tyrosine Hydroxylase (TH), an enzyme involved in synthesis of catecholamines that repress insulin secretion. Restriction of the TH+ β-cells within islets is essential for appropriate function in mice, such that higher proportion of these cells corresponds to reduced insulin secretion. Here, we use these cells as a model to dissect the developmental control of β-cell heterogeneity. We define the specific molecular and metabolic characteristics of TH+ β-cells, and show differences in their developmental restriction in mice and humans. We show that TH expression in β-cells is restricted by DNA methylation during β-cell differentiation. Ablation of de novo DNA methyltransferase Dnmt3a in the embryonic progenitors results in a dramatic increase in the proportion of TH+ β-cells, while β-cell specific ablation of Dnmt3a does not. We demonstrate that maintenance of Th promoter methylation is essential for its continued restriction in postnatal β-cells. Loss of Th promoter methylation in response to chronic overnutrition increases the number of TH+ β-cells, corresponding to impaired β-cell function. These results reveal a regulatory role of DNA methylation in determining β-cell heterogeneity.

Increasing evidence of new-onset diabetes during the COVID19 pandemic indicates that the SARS-CoV2 virus may drive beta-cell dysfunction leading to diabetes, but it is unclear if it is a primary or secondary effect. Here, we present evidence of SARS-CoV-2 infection of pancreatic beta cells in vivo using a robust and reproducible non-human primates model of mild to moderate COVID19 pathogenesis. Pancreas from SARS-CoV-2 infected subjects were positive for the SARS-CoV2 spike protein by immunohistochemistry and structures indicative of viral replication were evident by electron microscopy. Total beta cell area was decreased in SARS-CoV-2-infected pancreas, attributable to beta cell atrophy. Beta cell granularity was decreased. These histologic phenotypes persisted beyond the duration of the clinical disease course. Detailed electron microscopy of SARS-CoV-2 infected beta-cells revealed ultrastructural hallmarks of beta cell stress that are seen in islets of patients with Type 2 diabetes, including disrupted mitochondria and dilated endoplasmic reticulum. To assess the metabolic status of beta cells from SARS-CoV-2-infected subjects, we used fluorescence life-time imaging to measure the ratio of free and bound NADH as a surrogate of glycolytic and oxidative metabolism. We report an increase in free NADH levels, suggesting that beta cells from SARS-CoV-2-infected subjects adopt a more glycolytic metabolic profile. Taken together, we conclude that SARS-CoV-2 infection induces beta cell stress that may compromise beta-cell function beyond the duration of the disease course. This raises the possibility that the beta cell stress and injury may have clinical implications of the long-term future health of patients that have recovered from COVID19.

<p>The molecular and functional heterogeneity of pancreatic b-cells is well recognized, but the underlying mechanisms remain unclear. Pancreatic islets harbor a subset of b-cells that co-express Tyrosine Hydroxylase (TH), an enzyme involved in synthesis of catecholamines that repress insulin secretion. Restriction of the TH+ b-cells within islets is essential for appropriate function in mice, such that higher proportion of these cells corresponds to reduced insulin secretion. Here, we use these cells as a model to dissect the developmental control of b-cell heterogeneity. We define the specific molecular and metabolic characteristics of TH+ b-cells, and show differences in their developmental restriction in mice and humans. We show that TH expression in b-cells is restricted by DNA methylation during b-cell differentiation. Ablation of <em>de novo</em> DNA methyltransferase Dnmt3a in the embryonic progenitors results in a dramatic increase in the proportion of TH+ b-cells, while b-cell specific ablation of Dnmt3a does not. We demonstrate that maintenance of <em>Th</em> promoter methylation is essential for its continued restriction in postnatal b-cells. Loss of <em>Th</em> promoter methylation in response to chronic overnutrition increases the number of TH+ b-cells, corresponding to impaired b-cell function. These results reveal a regulatory role of DNA methylation in determining b-cell heterogeneity.</p>

We set out to determine whether the C-terminus (amino acids 481–798) of peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α, UniProt Q9UBK2), a regulatory metabolic protein involved in mitochondrial biogenesis, and respiration, is an arginine methyltransferase substrate. Arginine methylation by protein arginine methyltransferases (PRMTs) alters protein function and thus contributes to various cellular processes. In addition to confirming methylation of the C-terminus by PRMT1 as described in the literature, we have identified methylation by another member of the PRMT family, PRMT7. We performed in vitro methylation reactions using recombinant mammalian PRMT7 and PRMT1 at 37, 30, 21, 18, and 4 °C. Various fragments of PGC-1α corresponding to the C-terminus were used as substrates, and the methylation reactions were analyzed by fluorography and mass spectrometry to determine the extent of methylation throughout the substrates, the location of the methylated PGC-1α arginine residues, and finally, whether temperature affects the deposition of methyl groups. We also employed two prediction programs, PRmePRed and MePred-RF, to search for putative methyltransferase sites. Methylation reactions show that arginine residues R548 and R753 in PGC-1α are methylated at or below 30 °C by PRMT7, while methylation by PRMT1 was detected at these same residues at 30 °C. Computational approaches yielded additional putative methylarginine sites, indicating that since PGC-1α is an intrinsically disordered protein, additional methylated arginine residues have yet to be experimentally verified. We conclude that temperature affects the extent of arginine methylation, with more methylation by PRMT7 occurring below physiological temperature, uncovering an additional control point for PGC-1α.

The molecular and functional heterogeneity of pancreatic β-cells is well recognized. Pancreatic islets harbor a small subset of β-cells that co-express Tyrosine Hydroxylase (TH), an enzyme involved in synthesis of catecholamines that repress insulin secretion. Restriction of this sub-population within islets is essential for appropriate insulin secretion. However, the distinguishing characteristics of this subpopulation and the mechanisms that restrict TH expression in β-cells are not known. Here, we define the specific molecular and metabolic characteristics of the TH+ β-cells and show that TH expression in β-cells is restricted by DNA methylation patterning during β-cell lineage specification. Ablation of de novo DNA methyltransferase Dnmt3a in the pancreatic- and endocrine-progenitor lineages results in a dramatic increase in the proportion of TH+ β-cells, while β-cell specific ablation of Dnmt3a has no effect on this sub-population. We demonstrate that maintenance of Th promoter DNA methylation patterns is essential for its continued restriction in postnatal β-cells, and that loss of DNA methylation dysregulates TH expression in β-cells in response to chronic overnutrition, contributing to impairment of β-cell identity. These data highlight the essential requirement of DNA methylation patterning in regulating endocrine cell fates, and reveal a novel role of DNA methylation in β-cell heterogeneity.