Beta TC3 Research Articles

Desensitization of glucagon-like peptide 1 receptors in insulin-secreting beta TC3 cells: role of PKA-independent mechanisms.

1. The cellular processes involved in the desensitization of the glucagon-like peptide 1 receptors were investigated by measurements of the glucagon-like peptide 1(7-36)amide (GLP-1(7-36)amide)-induced increases in intracellular free Ca2+ concentration ([Ca2+]i) in insulin-secreting beta TC3 cells. 2. In the presence of 11.2 mM glucose, stimulation with GLP-1(7-36)amide led to a small membrane depolarization (< 10 mV), induction of electrical activity and a rapid increase in [Ca2+]i. The increase in [Ca2+]i was not observed in the presence of the L-type Ca(2+)-channel antagonist nifedipine. However, nifedipine was ineffective when applied after addition of GLP-1(7-36)amide. 3. The increase in [Ca2+]i evoked by GLP-1-(7-36)amide was transient and even in the continued presence of the agonist, [Ca2+]i returned to the basal value within 4-5 min. The latter process was slowed, but not prevented, by inhibition of protein kinase C (PKC) by staurosporine and Ro31-8220. 4. Short pretreatment of the cells with the phorbol ester, 4-beta-phorbol-12-beta-myristate-13-alpha-acetate (PMA), an activator of PKC, reduced the GLP-1(7-36)amide-evoked increase in [Ca2+]i by 75%. This effect of PMA was fully reversed by staurosporine and Ro31-8220. 5. The ability of GLP-1(7-36)amide to increase [Ca2+]i disappeared upon pre-exposure of the cells to the hormone (desensitization). This process was maximal within 5 min of exposure to the agonist. Following removal of the agonist from the medium, the ability to respond to subsequent stimulation by GLP-1(7-36)amide recovered gradually with time; half and complete recovery requiring > 20 min and 60 min, respectively. The desensitizing action of GLP-1(7-36)amide persisted in the presence of either staurosporine or forskolin and did not require an elevation of [Ca2+]i. 6. Our data suggest that the GLP-1(7-36)amide-evoked increase in [Ca2+]i is initiated by Ca(2+)-influx though voltage-dependent and nifedipine-sensitive L-type Ca2+ channels but depends principally on Ca2+ mobilization from internal stores for its maintenance. The desensitization of the GLP-1 receptors that occurs in the continued presence of the agonist does not result from the activation of protein kinase A or Ca(2+)-dependent kinases/phosphatases. Our data indicate that activation of PKC may contribute to the desensitization of the GLP-1 receptors but that other (PKC-independent) mechanisms also participate in this process.

Cytokine-induced apoptotic cell death in a mouse pancreatic beta-cell line: inhibition by Bcl-2.

Cytokines are thought to contribute to the induction of pancreatic beta-cell destruction in insulin-dependent diabetes mellitus. The molecular mechanisms that underlie beta-cell death were investigated by studying cytokine-induced cell death in beta-cell lines. A combination of three cytokines (interleukin-1 beta, tumour necrosis factor-alpha, and interferon-gamma) induced apoptotic cell death in the mouse pancreatic beta-cell line beta TC1, as judged from the appearance of cells with hypodiploid nuclei and oligonucleosomal DNA fragmentation. The same treatment also induced apoptosis in the mouse pancreatic alpha-cell line alpha TC1 and the NOD/Lt mouse beta-cell line NIT-1, although to a lesser extent than in beta TC1 cells. The abundance of endogenous Bcl-2 in beta TC1 cells was lower than that in the other two cell lines. Overexpression of human Bcl-2 in beta TC1 cells partially protected them from cytokine-induced cell death. These results suggest that apoptosis may be responsible, at least in part, for cytokine-induced beta-cell destruction and that Bcl-2 prevents apoptosis in pancreatic islet cells.

Glucose metabolism and insulin release in mouse beta HC9 cells, as model for wild-type pancreatic beta-cells.

Glucose metabolism and its relationship with glucose-induced insulin release were studied in beta HC9 and beta TC3 cells to identify and characterize key factors controlling the intermediary metabolism of glucose and glucose-induced insulin release. The beta HC9 cell line, derived from pancreatic islets with beta-cell hyperplasia, is characterized by a normal concentration-dependency curve for glucose-stimulated insulin release, whereas the beta TC3 cell line, derived from pancreatic beta-cell tumors, shows a marked leftward shift of this curve. Maximum velocity and the Michaelis-Menten constant of glucose uptake in beta HC9 and beta TC3 cells were similar, even though GLUT-2 expression in these two cell lines differed. In both cell lines, the kinetic characteristics of glucose usage, glucose oxidation, and glucose-induced oxygen consumption were similar to those of glucose phosphorylation, indicating that the kinetics of glucose metabolism from the glucose phosphorylation step in the cytosol to the mitochondrial process of oxidative phosphorylation are determined by the glucose-phosphorylating enzyme, that is, by glucokinase in beta HC9 cells and by hexokinase in beta TC3 cells. Thus beta HC9 cells provide an opportunity for the quantitative analysis of glucose metabolism, the associated generation of coupling factors, and other essential beta-cell functions involved in glucose sensing and insulin secretion.

Expression and Functional Role of Syntaxin 1/HPC-1 in Pancreatic β Cells: SYNTAXIN 1A, BUT NOT 1B, PLAYS A NEGATIVE ROLE IN REGULATORY INSULIN RELEASE PATHWAY

Syntaxin 1/HPC-1 is an integral membrane protein, which is thought to be implicated in the regulation of synaptic neurotransmitter release. We investigated syntaxin 1 expression in pancreatic beta cells and the functional role of syntaxin 1 in the insulin release mechanism. Expression of syntaxin 1A, but not 1B, was detected in mouse isolated islets by the reverse transcriptase-polymerase chain reaction procedure. An immunoprecipitation study of metabolically labeled islets with an anti-rat syntaxin 1/HPC-1 antibody demonstrated syntaxin 1A protein with an apparent molecular mass of approximately 35 kDa. Immunohistochemistry of the mouse pancreas demonstrated that syntaxin 1/HPC-1 was present in the plasma membranes of the islets of Langerhans. In order to determine the functional role of syntaxin 1 in pancreatic beta-cells, rat syntaxin 1A or 1B was overexpressed in mouse beta TC3 cells using the transient transfection procedure. Transfection of beta TC3 cells with either syntaxin 1 resulted in approximately 7-fold increases in their immunodetectable protein levels. Glucose-stimulated insulin release by syntaxin 1A-overexpressing cells was suppressed to about 50% of the level in control cells, whereas insulin release by syntaxin 1B-overexpressing and control cells did not differ. Next, we established stable beta TC3 cell lines that overexpressed syntaxin 1A and used them to evaluate the effect of syntaxin 1A on the regulatory insulin release pathway. Two insulin secretogogues, 4-beta-phorbol 12-myristate 13-acetate or forskolin, increased insulin release by untransfected beta TC3 cells markedly, but their effects were diminished in syntaxin 1A-overexpressing beta TC3 cells. Glucose-unstimulated insulin release and the proinsulin biosynthetic rate were not affected by syntaxin 1A overexpression, indicating a specific role of syntaxin 1A in the regulatory insulin release pathway. Finally, in vitro binding assays showed that syntaxin 1A binds to insulin secretory granules, indicating an inhibitory role of syntaxin 1A in insulin exocytosis via its interaction with vesicular proteins. These results demonstrate that syntaxin 1A is expressed in the islets of Langerhans and functions as a negative regulator in the regulatory insulin release pathway.

Glucose Modulates γ-Aminobutyric Acid Release from the Pancreatic βTC6 Cell Line

To determine if endogenous gamma-aminobutyric acid (GABA) is secreted by a pancreatic beta-cell-derived cell line and to determine the effects of glucose on GABA release, beta TC6 cultures were incubated in the presence of 1 or 10 mmol/l glucose for 12 h and then subjected to a 2-h secretion test in Krebs-Ringer buffer containing 1 or 10 mmol/l glucose. beta TC6-conditioned medium was collected at 15, 30, 60, and 120 min after glucose stimulation for GABA analysis by high pressure liquid chromatography-electrochemical detection. After 30 min, medium GABA concentrations were significantly higher (p < 0.05) in cultures that were exposed to high glucose during both the 12-h incubation period and the 2-h secretion test than in the remaining three glucose combinations. To address possible roles of beta-cell-derived GABA, the effect of GABA on glucagon secretion from pancreatic alpha TC6 cells was tested at concentrations released from beta TC6 cells. Inhibition of glucagon secretion by alpha TC6 cells was observed in the presence of GABA at concentrations equivalent to concentrations secreted by beta TC6 cells. The inhibitory effects of GABA on glucagon secretion by alpha TC6 cells were blocked by the GABAA receptor antagonist bicuculline and were dissociated from the inhibitory effects of glucose. Together, these results provide the first documentation that endogenous GABA is released from a highly differentiated beta-cell line and that glucose and GABA independently attenuate glucagon secretion by a pancreatic alpha-cell line.

Regulated secretion of prolactin by the mouse insulinoma cell line beta TC-3.

Our aim is to use cultured cells capable of regulated protein secretion for the production of recombinant proteins that require particular types of post-translational modifications. Here we have generated a stable transfected beta TC-3 cell line, beta TC-IPR9, that secretes high levels of recombinant prolactin. Transfected cells synthesize both the 27 kDa glycosylated and a 23 kDa nonglycosylated prolactin; the 23 kDa nonglycosylated species was secreted preferentially when cells were placed in secretion medium containing isobutylmethylxanthine (IBMX) and high concentrations of glucose, K+, and Ca2+. When the cells were cultured in medium containing low concentrations of glucose, K+, and Ca2+, most of the prolactin and insulin were not secreted; much of the prolactin was proteolytically converted to a 16 kDa form. Within the first 30 minutes after transferring the cells to medium containing secretagogues there was a 20-fold increase in the rate of secretion of prolactin; all of the 16 kDa species was secreted. The recombinant cells could be cycled several times between medium in which prolactin was biosynthesized and medium in which it was secreted. Preferential secretion of proteolytically processed prolactin in a medium without contaminating proteins offers an example of the advantage of this technology for production of other recombinant proteins.

Differential Processing of Proglucagon by the Subtilisin-like Prohormone Convertases PC2 and PC3 to Generate either Glucagon or Glucagon-like Peptide

Proglucagon is processed differently in the islet alpha cells and the intestinal endocrine L cells to release either glucagon or glucagon-like peptide 1-(7-37) (GLP1-(7-37)), peptide hormones with opposing actions in vivo. In previous studies with a transformed alpha cell line (alpha TC1-6) we demonstrated that the kexin/subtilisin-like prohormone convertase, PC2 (SPC2), is responsible for generating the typical alpha cell pattern of proglucagon processing, giving rise to glucagon and leaving unprocessed the entire C-terminal half-molecule known as major proglucagon fragment or MPGF (Rouillé, Y., Westermark, G., Martin, S. K., Steiner. D. F. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 3242-3246). Here we present evidence, using mouse pituitary AtT-20 cells infected with a vaccinia viral vector encoding proglucagon, that PC3 (SPC3), the major neuroendocrine prohormone convertase in these cells, reproduces the intestinal L cell processing phenotype, in which MPGF is processed to release two glucagon-related peptides, GLP1 and GLP2, while the glucagon-containing N-terminal half-molecule (glicentin) is only partially processed to oxyntomodulin and small amounts of glucagon. Moreover, in AtT-20 cells stably transfected with PC2 (AtT-20/PC2 cells), glicentin was efficiently processed to glucagon, providing further support for the conclusion that PC2 is the enzyme responsible for the alpha cell processing phenotype. In other cell lines expressing both PC2 and PC3 (STC-1 and beta TC-3), proglucagon was also processed extensively to both glucagon and GLP1-(7-37), although STC-1 cells express lower levels of PC2 and processed the N-terminal domain to glucagon less efficiently. In contrast, GH4C1 and COS 7 cells, which express very little or no PC2 or PC3, failed to process proglucagon, aside from a low level of interdomain cleavage which occurred only in the GH4C1 cells. In vitro PC3 did not cleave at the single Arg residue in GLP1 to generate GLP1-(7-37), its truncated biologically active form, indicating the likelihood that another convertase is required for this cleavage.

The role of the insulin control element and RIPE3b1 activators in glucose-stimulated transcription of the insulin gene.

The most important regulator of insulin expression in islet beta-cells is glucose, which stimulates insulin gene transcription, protein synthesis, and secretion. Glucose-induced insulin gene transcription is regulated by cis-acting elements found within the 5'-flanking region of the insulin gene. We previously demonstrated that the insulin control element (ICE, -100 to -91) and RIPE3b1 (-115 to -107) elements mediated this response in the HIT T-15 beta-cell line. In this study, we examined more closely how these insulin gene control elements regulate glucose-induced transcription. RIPE3b1 element binding was shown to be induced by glucose in both mouse beta TC-6 and beta TC-3 cell lines, although higher glucose concentrations were necessary in the beta-cells (beta TC-6) that responded to physiological glucose concentrations. RIPE3b1 binding was also regulated in glucose-stimulated beta- cells by various effectors of this response. The RIPE3b1 or ICE elements were shown to independently direct glucose-stimulated expression from minimal heterologous promoter constructs. We conclude that the RIPE3b1 and ICE elements are the principal mediators of glucose-stimulated transcription of the insulin gene.

Glucose transporter expression and functional role of hexokinase in insulin biosynthesis in mouse beta TC3 cells.

It was previously reported that insulin biosynthesis in mouse beta TC3 cells was regulated by glucose (Nagamatsu, S., and D. F. Steiner. Endocrinology 130: 748-754, 1992). In the present study, we examined the effect of glucose on the glucose transporter expression and hexokinase activities and determined the relationship between them and glucose-stimulated insulin biosynthesis in beta TC3 cells. Reverse transcriptase-polymerase chain reaction and Northern blot analysis revealed that beta TC3 cells expressed GLUT-1 and GLUT-3 glucose transporter mRNAs, but not GLUT-2. The levels of GLUT-1 and GLUT-3 mRNAs were not affected by glucose (0 or 11 mM glucose) over a period of 48 h. Immunoprecipitation of metabolically labeled beta TC3 cells with specific antibodies against GLUT-1 or GLUT-3 proteins revealed no effect of glucose on the biosynthesis of glucose transporters. Hexokinase [low Michaelis constant (Km) hexokinase] activity from cells incubated in 11 mM glucose for 48 h increased nearly twofold compared with cells maintained in 0 mM glucose, although the amount of cellular hexokinase protein detected by immunoblot analysis was unchanged between 0 and 11 mM glucose conditions. Glucokinase (high Km hexokinase) activity, in contrast, was not affected by glucose. Preincubation of beta TC3 cells with 2-deoxyglucose to inhibit hexokinase, thereby inhibiting all glycolysis, resulted in the decrease of glucose-stimulated insulin biosynthesis. Thus, in mouse beta TC3 cells that do not express GLUT-2, there is a close relationship between hexokinase activity and glucose-stimulated insulin biosynthesis, but not between the glucose transporter and glucose-stimulated insulin biosynthesis.

Functional Expression of an Epitope-tagged G protein-coupled K+ Channel (GIRK1)

An epitope-tagged form of an inwardly rectifying and G protein-coupled K+ channel (GIRK1-cp) was expressed at high levels in transfected mammalian cells. Immunoblot analysis of transfected human embryonic kidney cells (HEK293) and mouse insulinoma cells (beta TC3) revealed several GIRK1-cp polypeptides, including the major 59-kDa band, corresponding to the predicted mass of the GIRK1 polypeptide plus the epitope tag. Immunohistochemical staining using two anti-tag antibodies showed abundant immunoreactive material, which was predominantly concentrated in the perinuclear area in both transfected cell types. While functional GIRK1-cp message was present in poly(A)+ RNA prepared from HEK293 cells expressing GIRK1-cp protein, appropriate K+ currents could not be detected. In contrast, whole cell recordings made directly from transfected beta TC3 cells expressing GIRK1-cp revealed inwardly rectifying, pertussis toxin-sensitive currents activated by norepinephrine and galanin. Single channel recordings in excised patches of beta TC3 cells expressing GIRK1-cp showed rectifying K+ currents when activated by 50 microM guanosine 5'-O-(thiotriphosphate), with a slope conductance of 39.1 +/- 1.0 picosiemens. This is the first report of stable heterologous expression of a functional G protein-coupled K+ channel in mammalian cells. The activity of an epitope-tagged channel in insulinoma cells demonstrates the utility of this system for further biochemical and biophysical analyses of G protein-K+ channel interactions.

Evidence that Rap1 carboxylmethylation is involved in regulated insulin secretion.

Protein carboxylmethylation is a reversible posttranslational modification that regulates protein function. We examined the carboxylmethylation of small GTP-binding proteins in a pancreatic beta-cell line (beta TC cells). In vitro assays showed that carboxylmethylation of a membrane 23-kDa protein was induced by guanine nucleotides, best demonstrated by the nonhydrolyzable GTP analog, guanosine 5'-(3-O-thio)triphosphate (GTP gamma S). GTP gamma S also induced translocation of this 23-kilodalton (kDa) protein from cytosol to particulate fractions. Immunoblotting with antiserum sc-65 raised against Rap1 identified the carboxyl-methylated 23-kDa protein as Rap1. 1) The 23-kDa carboxyl-methylated protein separated by two-dimensional electrophoresis overlapped with the 23-kDa protein detected by immunoblotting. 2) GTP gamma S, in the presence of cytosol, increased the amount of detectable membrane-associated Rap1. Studies in intact beta TC cells demonstrated the carboxylmethylation of the 23-kDa protein in response to glucose and depolarizing concentrations of potassium, an effect that was abolished by the calcium channel inhibitor, D600. Similarly, N-acetyl-S-trans,trans-farnesyl-L-cysteine, an inhibitor of in vivo carboxylmethylation at COOH-terminal S-farnesylcysteine by methyltransferase, inhibited carboxylmethylation of the 23-kDa protein in intact cells and reduced insulin secretion in response to glucose and potassium. These data establish a correlation between insulin secretion and carboxylmethylation of a 23-kDa protein that comigrates with Rap1.

Enzymatic characterization of immunopurified prohormone convertase 2: potent inhibition by a 7B2 peptide fragment.

Prohormone convertase (PCs) are thought to mediate the controlled proteolysis of prohormones and neuropeptide precursors. While recombinant PC1 and furin are currently available, thus far it has not been possible to produce recombinant PC2. We have used conditioned medium obtained from the mouse insulinoma cell line beta TC3 to generate a working preparation of enzymatically active PC2 through immunopurification. Immunopurified PC2 cleaved the fluorogenic substrate Cbz-Arg-Ser-Lys-Arg-AMC in a time- and calcium-dependent manner. It was half-maximally stimulated at 75 microM Ca2+, had an optimum pH of 5, and exhibited PCMS and EDTA sensitivity similar to that reported for furin and PC1. The tight-binding inhibitor 27 kDa 7B2 was used to calculate the Kd for this inhibitor and the active enzyme concentration. The Kd was 7.3 +/- 1.7 nM, and the turnover rate of PC2 was 5.2 molecules substrate per enzyme molecule per minute. The specific activity was 4.9 nmol/micrograms/h (assuming a molecular mass for PC2 of 64 kDa). The enzyme preparation was able to cleave recombinant proenkephalin at at least four of the expected paired basic sites in the absence, but not in the presence, of 27 kDa 7B2. Since 21 kDa 7B2 is functionally inactive as a proteinase inhibitor, we examined the inhibitory activity of the carboxy-terminal portion of 27 kDa 7B2 (7B2 CT-peptide). Synthetic peptides were used to demonstrate that the 7B2 CT-peptide (a) represents a potent inhibitor of PC2 (Ki = 57 nM), (b) can block the conversion of proPC2 to PC2, and (c) can block the PC2-mediated conversion of proenkephalin to smaller peptide fragments.(ABSTRACT TRUNCATED AT 250 WORDS)

Mouse insulinoma beta TC3 cells express prodynorphin messenger ribonucleic acid and derived peptides: a unique cellular model for the study of prodynorphin biosynthesis and processing

The tumor cell line beta TC3 has been established from insulinomas derived from transgenic mice carrying a hybrid insulin promoter-simian virus-40 tumor antigen gene. The beta TC3 cells express high steady state levels of proinsulin messenger RNA (mRNA). In this same cell line, we describe in the present study high expression levels of prodynorphin (pro-Dyn) mRNA and its derived peptides. By Northern blot analysis, the screening of 23 cell lines of endocrine (n = 10) and of nonendocrine (n = 13) origin revealed the presence of high levels of the 2.6-kilobase pro-Dyn transcript only in beta TC3 cells. The beta TC3 cells expressed levels of pro-Dyn mRNA comparable to those in rat tissues expressing pro-Dyn. Chromatographic and radioimmunological studies showed that pro-Dyn mRNA was translated and fully processed into opioid peptides with leucine-enkephalin (Leu-Enk)-extended sequences [dynorphin-A-(1-8), dynorphin-B-(1-13), and alpha-neo-endorphin]. The expression of the prohormone convertases was also examined in beta TC3 cells by Northern blot analysis. In addition to the ubiquitously expressed furin, beta TC3 cells have abundant levels of prohormone convertase-1 (PC1) and PC2 mRNAs, but undetectable levels of PACE4 or PC5 mRNAs. Incubation of beta TC3 cells with 8-bromo-cAMP for 24 h stimulated 3-fold both the pro-Dyn mRNA levels and the secretion of opioid peptides. In contrast to pro-Dyn mRNA, furin, PC1, and PC2 mRNA levels were not affected by 8-bromo-cAMP. The beta TC3 cells constitute a unique model to elucidate the biosynthetic pathway of pro-Dyn processing, to identify the proteolytic enzymes responsible for the production of pro-Dyn end products, and to assess the potential role of opioid peptides in the regulation of pancreatic function.

Mouse insulinoma beta TC3 cells express prodynorphin messenger ribonucleic acid and derived peptides: a unique cellular model for the study of prodynorphin biosynthesis and processing.

The tumor cell line beta TC3 has been established from insulinomas derived from transgenic mice carrying a hybrid insulin promoter-simian virus-40 tumor antigen gene. The beta TC3 cells express high steady state levels of proinsulin messenger RNA (mRNA). In this same cell line, we describe in the present study high expression levels of prodynorphin (pro-Dyn) mRNA and its derived peptides. By Northern blot analysis, the screening of 23 cell lines of endocrine (n = 10) and of nonendocrine (n = 13) origin revealed the presence of high levels of the 2.6-kilobase pro-Dyn transcript only in beta TC3 cells. The beta TC3 cells expressed levels of pro-Dyn mRNA comparable to those in rat tissues expressing pro-Dyn. Chromatographic and radioimmunological studies showed that pro-Dyn mRNA was translated and fully processed into opioid peptides with leucine-enkephalin (Leu-Enk)-extended sequences [dynorphin-A-(1-8), dynorphin-B-(1-13), and alpha-neo-endorphin]. The expression of the prohormone convertases was also examined in beta TC3 cells by Northern blot analysis. In addition to the ubiquitously expressed furin, beta TC3 cells have abundant levels of prohormone convertase-1 (PC1) and PC2 mRNAs, but undetectable levels of PACE4 or PC5 mRNAs. Incubation of beta TC3 cells with 8-bromo-cAMP for 24 h stimulated 3-fold both the pro-Dyn mRNA levels and the secretion of opioid peptides. In contrast to pro-Dyn mRNA, furin, PC1, and PC2 mRNA levels were not affected by 8-bromo-cAMP. The beta TC3 cells constitute a unique model to elucidate the biosynthetic pathway of pro-Dyn processing, to identify the proteolytic enzymes responsible for the production of pro-Dyn end products, and to assess the potential role of opioid peptides in the regulation of pancreatic function.

Tissue engineering of a bioartificial pancreas: modeling the cell environment and device function.

Cell-based implantable artificial tissues are most promising for the long-term treatment of endocrine diseases, such as diabetes. One type of a bioartificial pancreas device consists of calcium alginate microbeads containing insulin-secreting cells and is surrounded by a poly(L-lysine) (PLL) membrane. The membrane is semipermeable, allowing cellular nutrients and metabolites to diffuse through but excluding the antibodies and cytotoxic cells of the host, thus immunoprotecting the cells. The device can be modeled by writing the equations for diffusion of nutrients and metabolites through the polymer and for consumption of the former and production of the latter by the cells. In this paper, we describe the construction and analysis of such a model for alginate/PLL microbeads with insulin-secreting recombinant mouse pituitary AtT-20 and mouse insulinoma beta TC3 cells. Entrapped AtT-20 cells are a simplified model system, whereas microbeads with beta TC3 cells constitute a realistic artificial pancreatic device. Effective diffusivities of key compounds through the polymer with entrapped, inactivated AtT-20 spheroids were measured first. The kinetics of glucose and oxygen consumption and insulin secretion were modeled next, and the equations for diffusion and reaction were then combined to describe the entire system. The model was used to compute nutrient and metabolite concentration profiles in beads and the bead secretory response for different bead sizes and cell loadings. The size and loading necessary for the cells to be well nourished and for the beads to be rapidly responsive to step-ups and step-downs of secretion stimuli were evaluated. It was shown that if the cells are hypersensitive to glucose, i.e., they do not shut off secretion at the physiological glucose threshold but at a lower one, so are the microbeads. This work demonstrates the usefulness of mechanistic models with representative parameter values in optimizing the design of artificial tissues and in characterizing aspects of their behavior that are of importance for restoring in vivo function.

Morphological and functional characterization of beta TC-6 cells--an insulin-secreting cell line derived from transgenic mice

Morphological and functional characterization of beta TC-6 cells--an insulin-secreting cell line derived from transgenic mice

Functional analysis of DNA-elements involved in transcriptional control of the human glucose transporter 2 (GLUT 2) gene in the insulin-producing cell line beta TC-3.

The uptake of glucose into pancreatic beta cells as a 'non-rate-limiting-step' is guaranteed by the expression and action of the high-Km glucose transporter 2 (GLUT 2). This transporter is not saturated by physiological plasma glucose levels and hence functions as a "glucose sensor/glucoreceptor". Here we describe DNA-elements of the human GLUT 2 gene promoter which contribute to transcriptional control in the insulin-producing cell line beta TC-3. Nested 5'-as well as 3'-deletions of a DNA-fragment containing up to 1245 bp of the 5'-flanking region and up to 308 bp of the first exon of the human GLUT 2 gene were investigated for their ability to control the expression of a CAT reporter gene in beta TC-3 cells. For tissue-specific transcriptional control 5'-deletional analysis revealed that the region -220/+309 was sufficient. Truncation from the 3'-end from nucleotide +308 to +204 led to a threefold drop in CAT expression. In vitro DNase I footprinting analysis was performed to delineate cis-elements within the region -220/+1. Five specifically protected areas could be defined.

Characterization of GIP(1–30) and GIP(1–42) as stimulators of proinsulin gene transcription

Originally characterized in terms of its gastric acid inhibitory properties, GIP (gastric inhibitory polypeptide) expressed in the upper small intestine, was subsequently shown to exert strong glucose-dependent insulin-releasing properties. This action is generally attributed to GIP(1-42) and, so far, no evidence for the contribution of other relevant GIP forms exists. In this study, we compared the effects of GIP(1-42) and C-terminally truncated GIP(1-30) on cAMP production and proinsulin gene transcription at clonal insulin-secreting cell lines (RIN 1046-38, beta TC-3). Both peptides were equally potent stimulators of cAMP generation in both cell lines. Insulin release from RIN 1046-38 cells stimulated by both GIP forms was identical. In both B-cell lines GIP(1-42) and GIP(1-30) stimulated proinsulin gene expression equipotently. GIP not only enhances insulin secretion but also insulin gene expression and, therefore, it is a true insulinotropic hormone.

Interaction of glucagon-like peptide-I (GLP-I) and galanin in insulin (beta TC-1)- and somatostatin (RIN T3)-secreting cells and evidence that both peptides have no receptors on glucagon (INR1G9)-secreting cells.

The interaction of glucagon-like peptide-I (GLP-I) and galanin in clonal endocrine pancreatic cells was characterized. By Northern blot analysis the presence of GLP-I receptor mRNA was shown in B (beta TC-1 cells) and D (RIN 1048-38) cells but not in A (INR1 G9) cells, thus confirming functional data demonstrating the absence of active GLP-I receptors on glucagon-producing cells. Galanin receptors were detected on B and D cells but not on A cells. In B and D cells galanin inhibited the GLP-I stimulated adenylate cyclase activity. Treatment of insulin- and somatostatin-producing cells with GLP-I increased intracellular cAMP levels, and this was dampened by galanin, GLP-I stimulated the activity of protein kinase A in B and D cells, which was also inhibited by galanin. Galanin alone did not influence B- and D-cell function. These data show that in the endocrine pancreas B and D cells but not A cells express GLP-I and galanin receptors. The interaction of GLP-I and galanin might act in the endocrine pancreas as a physiological inhibitor of the potent incretin hormone GLP-I. Therefore, we suggest galanin is a 'decretin'.

Cellular Distributions of the Prohormone Processing Enzymes PC1 and PC2

The prohormone convertases PC1 (also known as sPC3) and PC2 are known to mediate the proteolytic conversion of inactive neuropeptide and hormone precursors to bioactive peptide products. In this study we have used sucrose density centrifugation to determine the subcellular distributions of the various forms of PC1 and PC2 in three different cell types, AtT-20, beta TC3, and PC12 cells. The former two cell lines naturally express PC enzymes, while PC12 cell clones expressing PCs were obtained by stable transfection. Our data show considerable cell-line specific variation in PC processing, with PC12 cells exhibiting the most complete processing of both enzyme precursors. While in all cell lines mature forms of both enzymes were stored within particles having the same buoyant density as secretory granule markers, in some cell lines substantial amounts of mature PC1 and PC2 were also associated with the Golgi marker. Processing of the two PC precursors was not interdependent since PC12 cells expressing only one of the two PCs were fully capable of enzyme maturation. Interestingly, analysis of intracellular processing of an endogenous peptide precursor, proneurotensin, revealed that transfected PC1, but not PC2, showed enzymatic activity against this precursor.