Sphk2-/- mice are protected from obesity and insulin resistance.

Sphingosine kinases phosphorylate sphingosine to sphingosine 1­phosphate (S1P), which functions as a signaling molecule. We have previously shown that sphingosine kinase 2 (Sphk2) is important for insulin secretion. To obtain a better understanding of the role of Sphk2 in glucose and lipid metabolism, we have characterized 20- and 52-week old Sphk2-/- mice using glucose and insulin tolerance tests and by analyzing metabolic gene expression in adipose tissue. A detailed metabolic characterization of these mice revealed that aging Sphk2-/- mice are protected from metabolic decline and obesity compared to WT mice. Specifically, we found that 52-week old male Sphk2-/- mice had decreased weight and fat mass, and increased glucose tolerance and insulin sensitivity compared to control mice. Indirect calorimetry studies demonstrated an increased energy expenditure and food intake in 52-week old male Sphk2-/- versus control mice. Furthermore, expression of adiponectin gene in adipose tissue was increased and the plasma levels of adiponectin elevated in aged Sphk2-/- mice compared to WT. Analysis of lipid metabolic gene expression in adipose tissue showed increased expression of the Atgl gene, which was associated with increased Atgl protein levels. Atgl encodes for the adipocyte triglyceride lipase, which catalyzes the rate-limiting step of lipolysis. In summary, these data suggest that mice lacking the Sphk2 gene are protected from obesity and insulin resistance during aging. The beneficial metabolic effects observed in aged Sphk2-/- mice may be in part due to enhanced lipolysis by Atgl and increased levels of adiponectin, which has lipid- and glucose-lowering effects.

Glimepiride Administered in Chow Reversibly Impairs Glucose Tolerance in Mice.

Sulfonylureas are a class of antidiabetes medications prescribed to millions of individuals worldwide. Rodents have been used extensively to study sulfonylureas in the laboratory. Here, we report the results of studies treating mice with a sulfonylurea (glimepiride) in order to understand how the drug affects glucose homeostasis and tolerance. We tested the effect of glimepiride on fasting blood glucose, glucose tolerance, and insulin secretion, using glimepiride sourced from a local pharmacy. We also examined the effect on glucagon, gluconeogenesis, and insulin sensitivity. Unexpectedly, glimepiride exposure in mice was associated with fasting hyperglycemia, glucose intolerance, and decreased insulin. There was no change in circulating glucagon levels or gluconeogenesis. The effect was dose-dependent, took effect by two weeks, and was reversed within three weeks after removal. Glimepiride elicited the same effects in all strains evaluated: four wild-type strains, as well as the transgenic Grn-/- and diabetic db/db mice. Our findings suggest that the use of glimepiride as a hypoglycemic agent in mice should proceed with caution and may have broader implications about mouse models as a proxy to study the human pharmacopeia.

microRNAs in Pancreatic ß-Cell Physiology.

The ß-cells within the pancreas are responsible for production and secretion of insulin. Insulin is released from pancreatic ß-cells in response to increasing blood glucose levels and acts on insulin-sensitive tissues such as skeletal muscle and liver in order to maintain normal glucose homeostasis. Therefore, defects in pancreatic ß-cell function lead to hyperglycemia and diabetes mellitus. A new class of molecules called microRNAs has been recently demonstrated to play a crucial role in regulation of pancreatic ß-cell function under normal and pathophysiological conditions. miRNAs have been shown to regulate endocrine pancreas development, insulin biosynthesis, insulin exocytosis, and ß-cell expansion. Many of the ß-cell enriched miRNAs have multiple functions and regulate pancreas development as well as insulin biosynthesis and exocytosis. Furthermore, several of the ß-cell specific miRNAs have been shown to accumulate in the circulation before the onset of diabetes and may serve as potential biomarkers for prediabetes. This chapter will focus on miRNAs that are enriched in pancreatic ß-cells and play a critical role in modulation of ß-cell physiology and may have clinical significance in the treatment of diabetes.

Transcriptional activity of the islet ß cell factor Pdx1 is augmented by lysine methylation catalyzed by the methyltransferase Set7/9.

The transcription factor Pdx1 is crucial to islet ß cell function and regulates target genes in part through interaction with coregulatory factors. Set7/9 is a Lys methyltransferase that interacts with Pdx1. Here we tested the hypothesis that Lys methylation of Pdx1 by Set7/9 augments Pdx1 transcriptional activity. Using mass spectrometry and mutational analysis of purified proteins, we found that Set7/9 methylates the N-terminal residues Lys-123 and Lys-131 of Pdx1. Methylation of these residues occurred only in the context of intact, full-length Pdx1, suggesting a specific requirement of secondary and/or tertiary structural elements for catalysis by Set7/9. Immunoprecipitation assays and mass spectrometric analysis using ß cells verified Lys methylation of endogenous Pdx1. Cell-based luciferase reporter assays using wild-type and mutant transgenes revealed a requirement of Pdx1 residue Lys-131, but not Lys-123, for transcriptional augmentation by Set7/9. Lys-131 was not required for high-affinity interactions with DNA in vitro, suggesting that its methylation likely enhances post-DNA binding events. To define the role of Set7/9 in ß cell function, we generated mutant mice in which the gene encoding Set7/9 was conditionally deleted in ß cells (Set(Δ)ß). Set(Δ)ß mice exhibited glucose intolerance similar to Pdx1-deficient mice, and their isolated islets showed impaired glucose-stimulated insulin secretion with reductions in expression of Pdx1 target genes. Our results suggest a previously unappreciated role for Set7/9-mediated methylation in the maintenance of Pdx1 activity and ß cell function.

Minireview: microRNA function in pancreatic ß cells.

MicroRNAs are small noncoding ribonucleotides that regulate mRNA translation or degradation and have major roles in cellular function. MicroRNA (miRNA) levels are deregulated or altered in many diseases. There is overwhelming evidence that miRNAs also play an important role in the regulation of glucose homeostasis and thereby may contribute to the establishment of diabetes. MiRNAs have been shown to affect insulin levels by regulating insulin production, insulin exocytosis, and endocrine pancreas development. Although a large number of miRNAs have been identified from pancreatic ß-cells using various screens, functional studies that link most of the identified miRNAs to regulation of pancreatic ß-cell function are lacking. This review focuses on miRNAs with important roles in regulation of insulin production, insulin secretion, and ß-cell development, and will discuss only miRNAs with established roles in ß-cell function.

Obesity and diabetes cause cognitive dysfunction in the absence of accelerated ß-amyloid deposition in a novel murine model of mixed or vascular dementia.

Mid-life obesity and type 2 diabetes mellitus (T2DM) confer a modest, increased risk for Alzheimer's disease (AD), though the underlying mechanisms are unknown. We have created a novel mouse model that recapitulates features of T2DM and AD by crossing morbidly obese and diabetic db/db mice with APPΔNL/ΔNLx PS1P264L/P264L knock-in mice. These mice (db/AD) retain many features of the parental lines (e.g. extreme obesity, diabetes, and parenchymal deposition of ß-amyloid (Aß)). The combination of the two diseases led to additional pathologies-perhaps most striking of which was the presence of severe cerebrovascular pathology, including aneurysms and small strokes. Cortical Aß deposition was not significantly increased in the diabetic mice, though overall expression of presenilin was elevated. Surprisingly, Aß was not deposited in the vasculature or removed to the plasma, and there was no stimulation of activity or expression of major Aß-clearing enzymes (neprilysin, insulin degrading enzyme, or endothelin-converting enzyme). The db/AD mice displayed marked cognitive impairment in the Morris Water Maze, compared to either db/db or APPΔNLx PS1P264L mice. We conclude that the diabetes and/or obesity in these mice leads to a destabilization of the vasculature, leading to strokes and that this, in turn, leads to a profound cognitive impairment and that this is unlikely to be directly dependent on Aß deposition. This model of mixed or vascular dementia provides an exciting new avenue of research into the mechanisms underlying the obesity-related risk for age-related dementia, and will provide a useful tool for the future development of therapeutics.

MicroRNA-30d induces insulin transcription factor MafA and insulin production by targeting mitogen-activated protein 4 kinase 4 (MAP4K4) in pancreatic ß-cells.

MicroRNAs (miRNAs) represent small noncoding RNAs that play a role in many diseases, including diabetes. miRNAs target genes important for pancreas development, ß-cell proliferation, insulin secretion, and exocytosis. Previously, we documented that microRNA-30d (miR-30d), one of miRNAs up-regulated by glucose, induces insulin gene expression in pancreatic ß-cells. Here, we found that the induction of insulin production by overexpression of miR-30d is associated with increased expression of MafA, a ß-cell-specific transcription factor. Of interest, overexpression of miR-30d prevented the reduction in both MafA and insulin receptor substrate 2 (IRS2) with TNF-α exposure. Moreover, we identified that mitogen-activated protein 4 kinase 4 (MAP4K4), a TNF-α-activated kinase, is a direct target of miR-30d. Overexpression of miR-30d protected ß-cells against TNF-α suppression on both insulin transcription and insulin secretion through the down-regulation of MAP4K4 by the miR-30d. A decrease of miR-30d expression was observed in the islets of diabetic db/db mice, in which MAP4K4 expression level was elevated. Our data support the notion that miR-30d plays multiple roles in activating insulin transcription and protecting ß-cell functions from impaired by proinflammatory cytokines and underscore the concept that miR-30d may represent a novel pharmacological target for diabetes intervention.

Sphingosine 1-phosphate (S1P) regulates glucose-stimulated insulin secretion in pancreatic beta cells.

Recent studies suggest that sphingolipid metabolism is altered during type 2 diabetes. Increased levels of the sphingolipid ceramide are associated with insulin resistance. However, a role for sphingolipids in pancreatic beta cell function, or insulin production, and release remains to be established. Our studies in MIN6 cells and mouse pancreatic islets demonstrate that glucose stimulates an intracellular rise in the sphingolipid, sphingosine 1-phosphate (S1P), whereas the levels of ceramide and sphingomyelin remain unchanged. The increase in S1P levels by glucose is due to activation of sphingosine kinase 2 (SphK2). Interestingly, rises in S1P correlate with increased glucose-stimulated insulin secretion (GSIS). Decreasing S1P levels by treatment of MIN6 cells or primary islets with the sphingosine kinase inhibitor reduces GSIS. Moreover, knockdown of SphK2 alone results in decreased GSIS, whereas knockdown of the S1P phosphatase, Sgpp1, leads to a rise in GSIS. Treatment of mice with the sphingosine kinase inhibitor impairs glucose disposal due to decreased plasma insulin levels. Altogether, our data suggest that glucose activates SphK2 in pancreatic beta cells leading to a rise in S1P levels, which is important for GSIS.

Regulation of insulin gene transcription by multiple histone acetyltransferases.

Glucose-stimulated insulin gene transcription is mainly regulated by a 340-bp promoter region upstream of the transcription start site by beta-cell-enriched transcription factors Pdx-1, MafA, and NeuroD1. Previous studies have shown that histone H4 hyperacetylation is important for acute up-regulation of insulin gene transcription. Until now, only the histone acetyltransferase (HAT) protein p300 has been shown to be involved in this histone H4 acetylation event. In this report we investigated the role of the additional HAT proteins CREB binding protein (CBP), p300/CBP-associated factor (PCAF), and general control of amino-acid synthesis 5 (GCN5) in regulation of glucose-stimulated insulin gene transcription. Utilizing quantitative chromatin immunoprecipitation analysis, we demonstrate that glucose regulates the binding of p300, CBP, PCAF, and GCN5 to the proximal insulin promoter. siRNA-mediated knockdown of each of these HAT proteins revealed that depletion of p300 and CBP leads to a drastic decrease in histone H4 acetylation at the insulin promoter and in insulin gene expression, whereas knockdown of PCAF and GCN5 leads to a more moderate decrease in histone H4 acetylation and insulin gene expression. These data suggest that high glucose mediates the recruitment of p300, CBP, PCAF, and GCN5 to the insulin promoter and that all four HATs are important for insulin gene expression.

Modulation of transcription factor function by O-GlcNAc modification.

O-linked beta-N-acetylglucosamine (O-GlcNAc) modification of nuclear and cytoplasmic proteins is important for many cellular processes, and the number of proteins that contain this modification is steadily increasing. This modification is dynamic and reversible, and in some cases competes for phosphorylation of the same residues. O-GlcNAc modification of proteins is regulated by cell cycle, nutrient metabolism, and other extracellular signals. Compared to protein phosphorylation, which is mediated by a large number of kinases, O-GlcNAc modification is catalyzed only by one enzyme called O-linked N-acetylglucosaminyl transferase or OGT. Removal of O-GlcNAc from proteins is catalyzed by the enzyme beta-N-acetylglucosaminidase (O-GlcNAcase or OGA). Altered O-linked GlcNAc modification levels contribute to the establishment of many diseases, such as cancer, diabetes, cardiovascular disease, and neurodegeneration. Many transcription factors have been shown to be modified by O-linked GlcNAc modification, which can influence their transcriptional activity, DNA binding, localization, stability, and interaction with other co-factors. This review focuses on modulation of transcription factor function by O-linked GlcNAc modification.

Adenoviral gene transfer into isolated pancreatic islets.

The beta cells within the pancreatic islets are responsible for production of insulin, a peptide hormone required for maintaining normoglycemia. The establishment of efficient gene transfer into pancreatic islets is very important for studies of insulin and glucagon production and secretion, as well as for gene therapy purposes for the treatment of diabetes. We describe here in detail a protocol for adenoviral gene transfer into isolated mouse islets of the pancreas. Effective gene transfer into pancreatic islets using recombinant adenoviruses can be achieved with a multiplicity of infection (MOI) of 10. However, if the islets are not dispersed, adenoviral gene transfer is limited only to the cells on the periphery of the islets, which represent the glucagon-producing alpha cells in rodents. Dispersion of pancreatic islets with EGTA increases the efficiency of gene transfer into the cells within the core of the islets, which consist of insulin-producing beta cells.

The stability and transactivation potential of the mammalian MafA transcription factor are regulated by serine 65 phosphorylation.

The level of the MafA transcription factor is regulated by a variety of effectors of beta cell function, including glucose, fatty acids, and insulin. Here, we show that phosphorylation at Ser(65) of mammalian MafA influences both protein stability and transactivation potential. Replacement of Ser(65) with Glu to mimic phosphorylation produced a protein that was as unstable as the wild type, whereas Asp or Ala mutation blocked degradation. Analysis of MafA chimeric and deletion constructs suggests that protein phosphorylation at Ser(65) alone represents the initial degradation signal, with ubiquitinylation occurring within the C terminus (amino acids 234-359). Although only wild type MafA and S65E were polyubiquitinylated, both S65D and S65E potently stimulated transactivation compared with S65A. Phosphorylation at Ser(14) also enhanced activation, although it had no impact on protein turnover. The mobility of MafA S65A was profoundly affected upon SDS-PAGE, with the S65E and S65D mutants influenced less due to their ability to serve as substrates for glycogen synthase kinase 3, which acts at neighboring N-terminal residues after Ser(65) phosphorylation. Our observations not only illustrate the sensitivity of the cellular transcriptional and degradation machinery to phosphomimetic mutants at Ser(65), but also demonstrate the singular importance of phosphorylation at this amino acid in regulating MafA activity.

Identification of glucose-regulated miRNAs from pancreatic {beta} cells reveals a role for miR-30d in insulin transcription.

MicroRNAs (miRNAs) are small noncoding ribonucleotides that bind mRNAs and function mainly as translational repressors in mammals. MicroRNAs have been implicated to play a role in many diseases, including diabetes. Several reports indicate an important function for miRNAs in insulin production as well as insulin secretion. We have recently carried out a screen in the pancreatic beta-cell line MIN6 to identify miRNAs with altered abundance in response to changes in glucose concentrations. This screen resulted in identification of 61 glucose-regulated miRNAs from a total of 108 miRNAs detectable in MIN6 cells. Many of the identified miRNAs, including miR-124a, miR-107, and miR-30d were up-regulated in the presence of high glucose. Only a few of the miRNAs, including miR-296, miR-484, and miR-690 were significantly down-regulated by high glucose treatment. Interestingly, we found that overexpression of miR-30d, one of the miRNAs up-regulated by glucose, increased insulin gene expression, while inhibition of miR-30d abolished glucose-stimulated insulin gene transcription. Overexpression or inhibition of miR-30d did not have any effect on insulin secretion. These data suggest that the putative target genes of miR-30d may be negative regulators of insulin gene expression.

MiR-30 family and EMT in human fetal pancreatic islets.

Small, non-coding ribonucleotides called micro RNAs (miRNAs) have recently emerged as important regulators of cell function. MiRNAs regulate gene expression mainly by binding to the 3' UTR of mRNAs and thereby inhibit the translation of target mRNAs. The abundance of many miRNAs is altered under physiological and pathophysiological conditions, such as cancer and neurodegeneration. Recent data suggest that miRNAs also have an important role in regulation of insulin gene transcription, insulin secretion, and pancreatic islet cell function. An interesting study by Joglekar et al. published in this issue of ISLETS suggests that the miR-30 family of miRNAs may control the epithelial-to-mesenchymal transition of primary cultures of human pancreatic epithelial cells by negatively regulating the translation of mesenchymal gene transcripts such as vimentin. These findings are in agreement with previous data that suggest an important role for one of the miR-30 family members (miR-30d) in insulin gene expression in pancreatic beta cells. In summary, the report by Joglekar et al provides a significant advancement in understanding the role of miRNAs in pancreatic islet development and suggests that manipulation of miR-30 family of miRNA expression may provide an important means to generate and expand pancreatic beta cells from human fetal pancreatic progenitors.

Multiple kinases regulate mafA expression in the pancreatic beta cell line MIN6.

MafA is a basic leucine zipper transcription factor expressed within the beta cells of the pancreas and is required to maintain normal glucose homeostasis as it is involved in various aspects of beta cell biology. MafA protein levels are known to increase in response to high glucose through mechanisms that have yet to be fully characterized. We investigated whether discrete intracellular signaling events control mafA expression. We found that the general kinase inhibitor staurosporine induces mafA expression without altering the stability of the protein. Inhibition of the MAP-kinase JNK mimics the effects of staurosporine on the expression of mafA. Calmodulin kinase and calcium signaling are also important in stimulating mafA expression by high glucose. However, staurosporine, JNK, and calmodulin kinase have different effects on the induction of insulin expression. These data reveal that MafA levels are tightly controlled by the coordinated action of multiple kinase pathways.

Glucose regulation of insulin gene expression in pancreatic beta-cells.

Production and secretion of insulin from the beta-cells of the pancreas is very crucial in maintaining normoglycaemia. This is achieved by tight regulation of insulin synthesis and exocytosis from the beta-cells in response to changes in blood glucose levels. The synthesis of insulin is regulated by blood glucose levels at the transcriptional and post-transcriptional levels. Although many transcription factors have been implicated in the regulation of insulin gene transcription, three beta-cell-specific transcriptional regulators, Pdx-1 (pancreatic and duodenal homeobox-1), NeuroD1 (neurogenic differentiation 1) and MafA (V-maf musculoaponeurotic fibrosarcoma oncogene homologue A), have been demonstrated to play a crucial role in glucose induction of insulin gene transcription and pancreatic beta-cell function. These three transcription factors activate insulin gene expression in a co-ordinated and synergistic manner in response to increasing glucose levels. It has been shown that changes in glucose concentrations modulate the function of these beta-cell transcription factors at multiple levels. These include changes in expression levels, subcellular localization, DNA-binding activity, transactivation capability and interaction with other proteins. Furthermore, all three transcription factors are able to induce insulin gene expression when expressed in non-beta-cells, including liver and intestinal cells. The present review summarizes the recent findings on how glucose modulates the function of the beta-cell transcription factors Pdx-1, NeuroD1 and MafA, and thereby tightly regulates insulin synthesis in accordance with blood glucose levels.

Role of microRNAs in diabetes.

Diabetes is one of the most common chronic diseases in the world. Multiple and complex factors including various genetic and physiological changes can lead to type 1 and type 2 diabetes. However, the major mechanisms underlying the pathogenesis of diabetes remain obscure. With the recent discovery of microRNAs (miRNAs), these small ribonucleotides have been implicated as new players in the pathogenesis of diabetes and diabetes-associated complications. MiRNAs have been shown to regulate insulin production, insulin secretion, and insulin action. This review summarizes the recent progress in the cutting-edge research of miRNAs involved in diabetes and diabetes related complications.

Induction of insulin secretion in engineered liver cells by nitric oxide.

BACKGROUND: Type 1 Diabetes Mellitus results from an autoimmune destruction of the pancreatic beta cells, which produce insulin. The lack of insulin leads to chronic hyperglycemia and secondary complications, such as cardiovascular disease. The currently approved clinical treatments for diabetes mellitus often fail to achieve sustained and optimal glycemic control. Therefore, there is a great interest in the development of surrogate beta cells as a treatment for type 1 diabetes. Normally, pancreatic beta cells produce and secrete insulin only in response to increased blood glucose levels. However in many cases, insulin secretion from non-beta cells engineered to produce insulin occurs in a glucose-independent manner. In the present study we engineered liver cells to produce and secrete insulin and insulin secretion can be stimulated via the nitric oxide pathway. RESULTS: Expression of either human insulin or the beta cell specific transcription factors PDX-1, NeuroD1 and MafA in the Hepa1-6 cell line or primary liver cells via adenoviral gene transfer, results in production and secretion of insulin. Although, the secretion of insulin is not significantly increased in response to high glucose, treatment of these engineered liver cells with L-arginine stimulates insulin secretion up to three-fold. This L-arginine-mediated insulin release is dependent on the production of nitric oxide. CONCLUSION: Liver cells can be engineered to produce insulin and insulin secretion can be induced by treatment with L-arginine via the production of nitric oxide.

Glucose mediates the translocation of NeuroD1 by O-linked glycosylation.

O-Linked GlcNAc modification of nuclear and cytosolic proteins has been shown to regulate the function of many cellular proteins. Increased O-linked glycosylation, observed under chronic hyperglycemia conditions, has been implicated in the pathogenesis of diabetes. However, the exact role of O-GlcNAc modification in regulating glucose homeostasis remains to be established. We report here that the subcellular localization of the pancreatic beta cell-specific transcription factor NeuroD1 is regulated by O-linked glycosylation in the mouse insulinoma cell line MIN6. Under low glucose conditions, NeuroD1 is mainly in the cytosol. However, treatment of MIN6 cells with high glucose results in O-linked GlcNAc modification of NeuroD1 and its subsequent translocation into the nucleus. Consistent with these data, treatment of MIN6 cells with O-(2-acetamido-2-deoxy-d-glucopyranosylidene)-amino N-phenylcarbamate, an inhibitor of O-GlcNAcase, causes Neuro-D1 localization to the nucleus and induction of insulin gene expression even on low glucose. Furthermore, we demonstrate that NeuroD1 interacts with the O-GlcNAc transferase, OGT only at high concentrations of glucose and depletion of OGT by using small interfering RNA oligos interferes with the nuclear localization of NeuroD1 on high glucose. On low glucose NeuroD1 interacts with the O-GlcNAcase and becomes deglycosylated, which is likely to be important for export of Neuro-D1 into cytosol in the presence of low glucose. In summary, the presented data suggest that glucose regulates the subcellular localization of NeuroD1 in pancreatic beta cells via O-linked GlcNAc modification of NeuroD1 by OGT.

Glucose induces MafA expression in pancreatic beta cell lines via the hexosamine biosynthetic pathway.

MafA is a basic leucine zipper transcription factor that regulates gene expression in both the neuroretina and pancreas. Within the pancreas, MafA is exclusively expressed in the beta cells and is involved in insulin gene transcription, insulin secretion, and beta cell survival. The expression of the mafA gene within beta cells is known to increase in response to high glucose levels by an unknown mechanism. In this study, we demonstrate that pyruvate, which is produced by glycolysis from glucose, is not sufficient to induce mafA gene expression compared with high glucose. This suggests that the signal for MafA induction is independent of ATP levels and that a metabolic event occurring upstream of pyruvate production leads to the induction of MafA. Furthermore, insulin secretion mediated by high glucose is not important for MafA expression. However, the addition of glucosamine to beta cell lines stimulates MafA expression in the absence of high glucose, and inhibition of the hexosamine biosynthetic pathway in the presence of high glucose abolishes MafA induction. Moreover, we demonstrate that the expression of UDP-N-acetylglucosaminyl transferase, the enzyme mediating O-linked glycosylation of cytosolic and nuclear proteins, is essential for glucose-dependent MafA expression. Consistent with this observation, inhibition of N-acetylglucosaminidase, the enzyme involved in the removal of the O-GlcNAc modification from proteins, with O-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-N-phenylcarbamate stimulates MafA expression under low glucose conditions. The presented data suggest that MafA expression mediated by high glucose requires flux through the hexosamine biosynthetic pathway and the O-linked glycosylation of an unknown protein(s) by UDP-N-acetylglucosaminyl transferase.