Gene symbol: IPF1. Disease: MODY 4.
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Amino Acid Substitution↗
Biomedical subjects
K Docherty
Publications and source records attributed to K Docherty.
Enhanced expression of a furin-cleavable proinsulin.
Cell engineering or gene therapy may represent an alternative to current methods of treating diabetes mellitus. Cells could be engineered to secrete insulin ex vivo for transplantation or the insulin gene could be administered directly by injection into muscle. A problem has been that non-neuroendocrine cells lack the endoproteases (PC3/1 and PC2) that are responsible for the processing of proinsulin to insulin. This can be surmounted by engineering the paired basic amino acid processing sites within proinsulin to sites that would be recognized by the ubiquitously expressed protease, furin. However, in every study to date, the expression of the furin-cleavable construct was greatly reduced relative to that of the unmodified proinsulin construct. We investigated possible causes for this, including mRNA stability, the presence of additional CpG islands, and the amino acid substitutions within furin-cleavable proinsulin. Several furin-cleavable rat proinsulin I cDNAs were engineered and used to transfect human HEK293, rat L6 and mouse C(2)C(12) cell lines. The stability of wild-type and furin-cleavable proinsulin mRNA in transfected C(2)C(12) cells was measured by RT-PCR. Comparison of the decay rates in the presence of actinomycin D showed no significant difference between the two species of mRNA. A furin-cleavable proinsulin cDNA was created to contain the same distribution of CpG islands as wild-type proinsulin. Comparison of insulin-like immunoreactivity in all three cell lines transfected with either this construct or a widely used furin-cleavable proinsulin containing additional CpG islands showed that the presence of the extra CpG islands had no effect. Studies to examine amino acid substitutions used to create furin consensus sequences showed that the addition of basic residues at the C-peptide/A-chain junction was responsible for the reduced production of furin-cleavable proinsulin. Using this information, we engineered a cDNA for furin-cleavable rat proinsulin I that was efficiently processed to mature insulin and expressed at the same level as wild-type proinsulin.
Amino Acid Sequence↗
The effect of water temperature on the GABAergic and reproductive systems in female and male goldfish (Carassius auratus).
This study investigated the effect of water temperature on the synthesis of the amino acid neurotransmitter gamma-aminobutyric acid (GABA). In goldfish, GABA stimulates the release of pituitary gonadotropin-II (GTH-II), which regulates gonadal function. Fish were maintained in water of 11, 18, or 24 degrees. In the female and male goldfish, GABA synthesis rates estimated following inhibition of GABA catabolism by gamma-vinyl GABA (GVG) in both the telencephalon (TEL) and the hypothalamus (HYP) were increased in fish held at 24 degrees compared to those at either 11 or 18 degrees (P < 0.05). Additionally, GABA synthesis rates in the pituitary increased in a temperature-dependent manner. Glutamate is the precursor for GABA synthesis; however, no consistent pattern was seen between glutamate and GABA synthesis rates, indicating that glutamate is not a limiting factor in GABA synthesis. Both water temperature and GVG administration increased serum GTH-II levels in female goldfish. However, in male goldfish water temperature had no significant effect on serum GTH-II levels, and GVG injection increased serum GTH-II levels only in fish maintained at 24 degrees. The effects of temperature on the levels of mRNA expression of the GABA-synthesizing enzymes glutamate decarboxylase 65 (GAD(65)) and GAD(67) were measured by semiquantitative PCR. In the TEL and HYP of female goldfish, GAD(65) was not affected, whereas temperature change from 11 to 18 degrees increased (P < 0.05) GAD(67) mRNA levels. These results demonstrate that central GABAergic systems in the goldfish are temperature sensitive.
Animals↗
Secretion of bioactive human insulin following plasmid-mediated gene transfer to non-neuroendocrine cell lines, primary cultures and rat skeletal muscle in vivo.
The objective of these studies was to evaluate human insulin gene expression following intramuscular plasmid injection in non-diabetic rats as a potential approach to gene therapy for diabetes mellitus avoiding the need for immunosuppression. A wild-type human preproinsulin construct and a mutant construct in which PC2/PC3 sites were engineered to form furin consensus sites were evaluated in in vitro transfections of hepatocyte (HepG2) and myoblast (C2C12/L6) cell lines, primary rat myoblasts, and dermal fibroblasts. In vivo gene transfer by percutaneous plasmid injection of soleus muscle +/- prior notexin-induced myolysis was assessed in rats. In vitro transfection of non-neuroendocrine cell lines and primary cultures with wild-type human preproinsulin resulted in secretion of predominantly unprocessed proinsulin. Employing the mutant construct, there was significant processing to mature insulin (HepG2, 95%; C2C12, 75%; L6, 65%; primary myoblasts, 48%; neonatal fibroblasts, 56%; adult fibroblasts, 87%). In rats aged 5 weeks, circulating human (pro)insulin was detected from 1 to 37 days following plasmid injection and the potential of augmenting transfection efficiency by prior notexin injection was demonstrated (wild-type processing, 87%; mutant, 90%). Relative hypoglycaemia was confirmed by HbA1C (saline, 5.5%; wild type, 5.1%; mutant, 5.1% (P<0.05)). Human (pro)insulin levels and processing (wild-type, 8%; mutant, 53%) were lower in rats aged 9 months but relative hypoglycaemia was confirmed by serum glucose at 10 days (saline, 6.4 mmol/l; wild-type, 6.0 mmol/l; mutant, 5.4 mmol/l). In conclusion, prolonged constitutive systemic secretion of bioactive human (pro)insulin has been attained in non-neuroendocrine cells in vitro and in growing and mature rats following intramuscular plasmid injection.
Animals↗
Pancreatic duodenal homeobox-1, PDX-1, a major regulator of beta cell identity and function.
Pancreatic duodenal homeobox -1 is a transcription factor that is expressed in beta and delta cells of the islets of Langerhans and in dispersed endocrine cells of the duodenum. It is involved in regulating the expression of a number of key beta-cell genes as well as somatostatin. It also plays a pivotal part in the development of the pancreas and islet cell ontogeny. Thus homozygous disruption of the gene in mice and humans results in pancreatic agenesis. Heterozygous mutations in the gene result in impaired glucose tolerance and symptoms of diabetes as seen in MODY4 and late-onset Type II (non-insulin-dependent) diabetes mellitus. In adults pancreatic duodenal homeobox-1 expression is increased in duct cells of the pancreas that have been induced to proliferate and differentiate to form new islets. Defects in pancreatic duodenal homeobox-1 could therefore contribute to Type II diabetes by affecting compensatory mechanisms that increase the rate of beta-cell neogenesis to meet the increased insulin secretory demand. It could also be a pharmacological target for beta-cell defects in Type II diabetes, while its role as a regulator of islet stem cell activity is being exploited to produce a replenishable source of islet tissue for transplantation in Type I (insulin-dependent) diabetes mellitus.
Animals↗
Growth and development of the islets of Langerhans: implications for the treatment of diabetes mellitus.
In the past couple of years a number of major breakthroughs have been made in understanding the developmental biology of the islets of Langerhans. These include the involvement of the hedgehog signalling pathway in defining the region of the gut endoderm that will develop into the pancreas; the discovery that the transcription factor neurogenin3 and the Delta/Notch signalling pathway control endocrine cell differentiation through a lateral inhibition mechanism; and that alpha and beta cells are derived from an islet progenitor cell and follow independent lineage pathways rather than arising from a common mutihormonal progenitor cell as previously thought. This knowledge had been used in strategies to provide a replenishable supply of insulin-secreting cells for the treatment of diabetes mellitus. Thus, islet progenitor cells in adult pancreatic ducts or in isolated islets of Langerhans have been induced to grow in culture and their endocrine-like properties have been characterised. A proliferating beta-like cell line has been derived from tissue removed from a child with persistent hyperinsulinaemic hypoglycaemia of infancy and been engineered in culture to secrete insulin in response to glucose. And finally, embryonic stem cells have been shown to adopt islet-like characteristics under defined culture conditions.
Animals↗
Sex steroid regulation of glutamate decarboxylase mRNA expression in goldfish brain is sexually dimorphic.
Testosterone and oestradiol can modulate GABA synthesis in sexually regressed goldfish. Here we investigated their effects on the mRNA expression of two isoforms of the GABA synthesizing enzyme glutamate decarboxylase (GAD(65) and GAD(67), EC 4.1.1.15). Full-length GAD clones were isolated from a goldfish cDNA library and sequenced. Goldfish GAD(65) encodes a polypeptide of 583 amino acid residues, which is 77% identical to human GAD(65). Goldfish GAD(67) encodes a polypeptide of 587 amino acid residues and is 82% identical to human GAD(67). Goldfish GAD(65) and GAD(67) are 63% identical. Sexually regressed male and female goldfish were implanted with solid silastic pellets containing testosterone, oestradiol or no steroid. Semiquantitative PCR analysis showed that oestradiol significantly increased GAD(65) mRNA expression in female hypothalamus and telencephalon, while testosterone resulted in a significant increase only in telencephalon. GAD(67) mRNA levels were not affected by steroids in females. In contrast, both steroids induced significant decreases of GAD(65) and GAD(67) mRNA levels in male hypothalamus, but had no effect on GAD mRNA expression in male telencephalon. Our results indicate that modulation of GAD mRNA expression is a possible mechanism for steroid action on GABA synthesis, which may have opposite effects in males and females.
Amino Acid Sequence↗
Association of prohormone convertase 3 with membrane lipid rafts.
Prohormone convertase 3 (PC3) is a neuroendocrine-specific member of the subtilisin-kexin family, involved in the intracellular processing and maturation of prohormones and proneuropeptides. PC3 is synthesised as a proprotein that undergoes two different cleavages resulting in the mature PC3 and the enzymatically active PC3DeltaC. In vitro translated proPC3 and proPC3DeltaC bind to trans-Golgi network (TGN)/granule-enriched membranes from the AtT20 neuroendocrine cell line in a pH-dependent manner suggesting both a dominant role for the pro-region in membrane association and that the C-terminal region is not essential. However, while PC3 bound to membranes the majority of PC3DeltaC did not, suggesting that either the pro-region or the C-terminal region of PC3 is required for membrane association. Removal of peripheral membrane proteins did not affect the binding properties of any of the in vitro translated proteins. Chromaffin granule membranes (CGMs) were used to study the binding characteristics of endogenous PC3 and its active C-terminal truncated counterpart (PC3DeltaC). Incubation of CGMs with Triton X-100 did not completely solubilise either of these forms of PC3. Moreover, both PC3 and PC3DeltaC remained associated with detergent-resistant membrane microdomains, termed lipid rafts, purified from CGMs. The data raise the possibility that PC3 and PC3DeltaC are sorted to the regulated secretory pathway via their association with membrane lipid rafts.
Aspartic Acid Endopeptidases↗
Phosphorylation-dependent nucleocytoplasmic shuttling of pancreatic duodenal homeobox-1.
Pancreatic duodenal homeobox-1 (PDX-1) is a homeodomain protein that plays an important role in the development of the pancreas and in maintaining the identity and function of the islets of Langerhans. It also regulates the expression of the insulin gene in response to changes in glucose and insulin concentrations. Glucose and insulin regulate PDX-1 by way of a signaling pathway involving phosphatidylinositol 3-kinase (PI 3-kinase) and SAPK2/p38. Activation of this pathway leads to phosphorylation of PDX-1 and its movement into the nucleus. To investigate the intracellular trafficking of PDX-1, immunocytochemistry was used to localize PDX-1 in the human beta-cell line NesPDX-1, in which PDX-1 is overexpressed, and in MIN6 beta-cells. In low-glucose conditions, PDX-1 localized predominantly to the nuclear periphery, with some staining in the cytoplasm. After stimulation with glucose, PDX-1 was present in the nucleoplasm. The translocation of PDX-1 to the nucleoplasm was complete within 15 min and occurred in 5-10 mmol/l glucose. Insulin and sodium arsenite, an activator of the stress-activated pathway, also stimulated PDX-1 movement from the nuclear periphery to the nucleoplasm. When cells were transferred between high glucose- and low glucose-containing medium, PDX-1 rapidly shuttled between the nuclear periphery and the nucleoplasm. Glucose- and insulin-stimulated translocation of PDX-1 to the nucleoplasm was inhibited by wortmannin and SB 203580, indicating that a pathway involving PI 3-kinase and SAPK2/p38 was involved; translocation was unaffected by PD 098959 and rapamycin, suggesting that neither mitogen-activated protein kinase nor p70(s6k) were involved. Arsenite-stimulated import of PDX-1 into the nucleus was inhibited by SB 203580 but not by wortmannin. Export from the nucleoplasm to the nuclear periphery was inhibited by calyculin A and okadaic acid, suggesting that dephosphorylation of PDX-1 was involved. These results demonstrated that PDX-1 shuttles between the nuclear periphery and nucleoplasm in response to changes in glucose and insulin concentrations and that these events are dependent on PI 3-kinase, SAPK2/p38, and a nuclear phosphatase(s).
Androstadienes↗
Involvement of the membrane lipid bilayer in sorting prohormone convertase 2 into the regulated secretory pathway.
Prohormone convertase 2 (PC2) is a neuroendocrine-specific protease involved in the intracellular maturation of prohormones and proneuropeptides. PC2 is synthesised as a proprotein (proPC2) that undergoes proteolysis, aggregation and membrane association during its transit through the regulated secretory pathway. We have previously shown that the pro region of proPC2 plays a key role in its aggregation and membrane association. To investigate this further, we determined the binding properties of a peptide containing amino acids 45-84 of proPC2 (proPC2(45-84)) to trans-Golgi network/granule-enriched membranes from the AtT20 cell line. Removal of peripheral membrane proteins or hydrolysis of integral membrane proteins did not affect the binding properties of proPC2(45-84). Rather, proPC2(45-84) was shown to bind to protein-free liposomes in a pH- and Ca(2+)-dependent manner. To identify the component of the lipid bilayer involved in this membrane association, we used chromaffin-granule membranes and studied the binding properties of the endogenous PC2. Treatment of the membranes with saponin, a cholesterol-depleting detergent, failed to extract PC2 from the membranes, whereas chromogranin A (CgA) was removed. Treatment of the membranes with Triton X-100 yielded a low-density detergent-insoluble fraction enriched in PC2, but not CgA. The detergent-insoluble fraction also contained glycoprotein III, known to be part of the lipid rafts (membrane microdomains rich in sphingolipids). Finally, sphingolipid depletion of AtT20 cells resulted in the mis-sorting of PC2, suggestive of a link between the association of PC2 with lipid rafts in the membrane and its sorting into the regulated secretory pathway.
Animals↗
Glucose regulates islet amyloid polypeptide gene transcription in a PDX1- and calcium-dependent manner.
Islet amyloid polypeptide (IAPP) and insulin are expressed in the beta-cells of the islets of Langerhans. They are co-secreted in response to changes in glucose concentration, and their mRNA levels are also regulated by glucose. The promoters of both genes share similar cis-acting sequence elements, and both bind the homeodomain transcription factor PDX1, which plays an important role in the regulation of the insulin promoter and insulin mRNA levels by glucose. Here we examine the role of PDX1 in the regulation of the human IAPP promoter by glucose. The experiments were facilitated by the availability of a human beta-cell line (NES2Y) that lacks PDX1. NES2Y cells also lack operational K(ATP) channels, resulting in a loss of control of calcium signaling. We have previously used these cells to show that glucose regulation of the insulin gene is dependent on PDX1, but not calcium. In the mouse beta-cell line Min6, glucose (16 mm) stimulated a 3.5-4-fold increase in the activity of a -222 to +450 IAPP promoter construct compared with values observed in 0.5 mm glucose. In NES2Y cells, glucose failed to stimulate transcriptional activation of the IAPP promoter. Overexpression of PDX1 in NES2Y cells failed to reinstate glucose-responsive control of the IAPP promoter. Glucose effects on the IAPP promoter were observed only in the presence of PDX1 when normal calcium signaling was restored by overexpression of the two K(ATP) channel subunits SUR1 and Kir6.2. The importance of calcium was further emphasized by an experiment in which glucose-stimulated IAPP promoter activity was inhibited by the calcium channel blocker verapamil (50 microm). Verapamil was further shown to inhibit the stimulatory effect of glucose on IAPP mRNA levels. These results demonstrate that like the insulin promoter, glucose regulation of the IAPP promoter is dependent on the activity of PDX1, but unlike the insulin promoter, it additionally requires the activity of another, as yet uncharacterized factor(s), the activity of which is calcium-dependent.
Amyloid↗
Evidence for recognition of novel islet T cell antigens by granule-specific T cell lines from new onset type 1 diabetic patients.
Type 1 diabetes is a T cell-mediated autoimmune disease where a number of islet beta-cell target autoantigens have been characterized on the basis of reactivity with autoantibodies. Nevertheless, there remains uncertainty of the nature of another group of autoantigens associated with the secretory granule fraction of islet beta-cells that appear to be targeted predominantly by autoreactive T cells. We have previously characterized CD4+, HLA-DR-restricted T cell lines from new onset type 1 diabetic patients that are specific for the secretory granule fraction of rat tumour insulinoma, RIN. The T cell line from the first patient, HS, proliferates in response to crude microsomal membranes prepared from a recently established, pure human islet beta-cell line NES2Y. In addition, the HS line also responds to secretory granule fractions prepared from a murine tumour insulinoma grown in RIP-Tag mice, showing the recognition of species-conserved antigen(s) in beta-cells. Using partially matched antigen-presenting cells, the HS T cells and another line derived from a second patient, MR, were shown to be restricted by disease-associated DRB1*0101 and DRB1*0404 alleles, respectively. Neither the HS or MR T cell lines proliferate in response to a large panel of candidate islet cell antigens, including insulin, proinsulin, glutamic acid decarboxylase, the protein tyrosine phosphatase IA-2/phogrin, imogen-38, ICA69 or hsp60. Our data provide compelling evidence of the presence of a group of antigens associated with the secretory granule fraction of islet beta-cells recognized by the T cell lines, whose definition may contribute to our knowledge of disease induction as well as to diagnosis.
Animals↗
Processing of synthetic pro-islet amyloid polypeptide (proIAPP) 'amylin' by recombinant prohormone convertase enzymes, PC2 and PC3, in vitro.
Islet amyloid polypeptide (IAPP), amylin, is the constituent peptide of pancreatic islet amyloid deposits which form in islets of Type 2 diabetic subjects. Human IAPP is synthesized as a 67-residue propeptide in islet beta-cells and colocalized with insulin in beta-cell granules. The mature 37-amino acid peptide is produced by proteolysis at pairs of basic residues at the C- and N-termini of the mature peptide. To determine the enzymes responsible for proteolysis and their activity at the potential cleavage sites, synthetic human proIAPP was incubated (0.5-16 h) with recombinant prohormone convertases, PC2 or PC3 at appropriate conditions of calcium and pH. The products were analysed by MS and HPLC. Proinsulin was used as a control and was cleaved by both recombinant enzymes resulting in intermediates. PC3 was active initially at the N-terminal-IAPP junction and later at the C-terminus, whereas initial PC2 activity was at the IAPP-C-terminal junction. Processing at the basic residues within the C-terminal flanking peptide rarely occurred. There was no evidence for substantial competition for the processing enzymes when the combined substrates proinsulin and proIAPP were incubated with both PC2 and PC3. As proinsulin cleavage is sequential in vivo (PC3 active at the B-chain-C-peptide junction, followed by PC2 at A chain-C-peptide junction), these data suggest that proteolysis of proIAPP and proinsulin is coincident in secretory granules and increased proinsulin secretion in diabetes could be accompanied by increased production of proIAPP.
Amyloid↗
Sexually dimorphic expression of glutamate decarboxylase mRNA in the hypothalamus of the deep sea armed grenadier, Coryphaenoides (Nematonurus) armatus.
Glutamate decarboxylase (GAD), is a key enzyme in the central nervous system (CNS) that synthesizes the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) from glutamate. Our previous phylogenetic studies on the evolution of this enzyme indicates that there are at least two distinct forms: GAD65 and GAD67. They are the products of separate genes and probably derive from a common ancestral GAD gene following gene duplication prior to the emergence of the teleosts more than 200 Myr ago. Furthermore, a third GAD-like molecule, GAD3, discovered in the armed grenadier, Coryphaenoides (Nematonurus) armatus, is equally divergent from both GAD65 and GAD67. Specimens of C. (N.) armatus were collected by trawl at a depth of 4,000 m in the Porcupine Seabight (Northeastern Atlantic), and brains dissected and frozen for RNA extraction. All three GAD forms are found in the cerebellum, telencephalon and hypothalamus. Semiquantitative PCR analysis showed that males and females have similar levels of expression of GAD67 and GAD3 in the tissues studied. Independent of the sex examined, the levels of expression of GAD65 and GAD67 in the cerebellum were approximately half that in the telencephalon. GAD3 levels were approximately 30% higher in the cerebellum than in either the telencephalon or hypothalamus. In contrast to GAD67 and GAD3, hypothalamic expression of GAD65 mRNA is 1.8 times higher (p < 0.05) in males than in females. These data indicate that the expression of GAD65, a key enzyme for the synthesis of GABA is sexually dimorphic in females and males of C. (N.) armatus.
Animals↗
Growth and development of the islets of langerhans: implications for the treatment of diabetes. A special section consisting of proceedings from the journal of molecular endocrinology symposium at the 190th meeting of the society for endocrinology, london, 8 november 1999
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Journal Article↗
Glucose modulation of insulin mRNA levels is dependent on transcription factor PDX-1 and occurs independently of changes in intracellular Ca2+.
Glucose regulates insulin production in pancreatic beta-cells in the long term by stimulating insulin gene transcription. These effects are partially mediated through the activity of a homeodomain transcription factor, PDX-1, which binds to four sites within the human insulin gene promoter. The availability of a human beta-like cell line, NES2Y, which lacks PDX-1 but expresses the insulin gene, allowed us to determine whether PDX-1 was essential for the stimulatory effect of glucose on insulin mRNA levels. In NES2Y cells, glucose had no effect on the insulin gene promoter linked to a firefly luciferase reporter or on endogenous insulin mRNA levels. However, in NES2Y cells stably transfected with PDX-1 (NES-PDX-1), glucose exhibited a marked stimulatory effect on both the insulin promoter (5+/-0.2-fold, n = 6) and insulin mRNA levels (4.8+/-0.5-fold, n = 4). NES2Y cells were derived from a patient with persistent hyperinsulinemic hypoglycemia of infancy; the cells therefore lacked operational ATP-sensitive potassium channels, which results in the failure to control depolarization-dependent intracellular Ca2+ signaling. Despite the loss of control of Ca2+ channel activity, NES-PDX-1 cells maintained normal glucose-responsive insulin gene regulation. These results demonstrate that glucose modulation of insulin mRNA levels is dependent on the activity of PDX-1 and that these effects are independent of changes in intracellular Ca2+ concentrations.
Calcium↗
Sulfonylurea receptor 1 and Kir6.2 expression in the novel human insulin-secreting cell line NES2Y.
NES2Y is a proliferating human insulin-secreting cell line that we have derived from a patient with persistent hyperinsulinemic hypoglycemia of infancy. This disease is characterized by unregulated insulin release despite profound hypoglycemia. NES2Y cells, like beta-cells isolated from the patient of origin, lack functional ATP-sensitive potassium channels (KATP) and also carry a defect in the insulin gene-regulatory transcription factor PDX1. Here, we report that the NES2Y beta-cells that are transfected with the genes encoding the components of KATP channels in beta-cells, sulfonylurea receptor (SUR) 1 and Kir6.2, have operational KATP channels and show normal intracellular Ca2+ and secretory responses to glucose. However, these cells, designated NESK beta-cells, have impaired insulin gene transcription responses to glucose. NES2Y beta-cells that are transfected with either Kir6.2 or SUR1 alone do not express functional KATP channels and have impaired intracellular free Ca2+ concentration-signaling responses to depolarization-dependent beta-cell agonists. These findings document that in NES2Y beta-cells, coexpression of both subunits is critically required for fully operational KATP channels and KATP channel-dependent signaling events. This article further characterizes the properties of the novel human beta-cell line, NES2Y, and documents the usefulness of these cells in diabetes-related research.
ATP-Binding Cassette Transporters↗
Missense mutations in the insulin promoter factor-1 gene predispose to type 2 diabetes
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Journal Article↗