PubMed Health⌕ Search

Biomedical subjects

M B Wheeler

Publications and source records attributed to M B Wheeler.

At least 55 records · Page 3Linked to original sources
PubMed1999

Overexpression of uncoupling protein 2 inhibits glucose-stimulated insulin secretion from rat islets.

Uncoupling protein 2 (UCP-2) mRNA expression has been shown to be altered by metabolic conditions such as obesity in humans, but its functional significance is unknown. The expression of UCP-2 mRNA and protein in normal rat islets was established by reverse transcriptase-polymerase chain reaction and immunocytochemistry in pancreatic islets and tissue, respectively. Intense immunostaining of UCP-2 correlated with insulin-positive ,-cells. Overexpression of UCP-2 in normal rat islets was accomplished by infection with an adenovirus (AdEGI-UCP-2) containing the full-length human UCP-2 coding sequence. Induction of the AdEGI-UCP-2 gene resulted in severe blunting of glucose-stimulated insulin secretion (GSIS) without affecting islet insulin content or the ability of the calcium ionophore A23187 to increase insulin secretion from AdEGI-UCP-2-expressing islets. Therefore, UCP-2 overexpression affects signal transduction proximal to Ca2+-mediated steps, including exocytosis. Insulin secretion from single beta-cells to 16.5 mmol/l glucose examined by reverse hemolytic plaque assay was nearly ablated if UCP-2 was overexpressed. Thus, a direct, causal relationship between overexpression of UCP-2 and inhibition of GSIS in normal islets has been established. These data suggest that increased expression of UCP-2 has the potential to cause the lack of a glucose effect on insulin secretion in type 2 diabetes.

Adenoviridae↗
PubMed1998

Identification of a novel adenylate cyclase in the ruminal anaerobe, Prevotella ruminicola D31d.

Our previous evaluation of ruminal and other anaerobic bacteria showed only Prevotella ruminicola D31d produced detectable concentrations of cyclic AMP. In order to investigate the synthesis of this important metabolic regulator, the gene for adenylate cyclase (cya), which produces cyclic AMP, was cloned and expressed in a cyaA mutant of Escherichia coli. The cloned P. ruminicola D31d gene was able to complement the cyaA mutation and permitted fermentation of lactose on MacConkey Lactose agar plates. Analysis of the DNA sequence of the 2.5-kilobase pair insert revealed an open reading frame encoding for a 67-kDa protein. This protein was novel in that no amino acid similarity was observed with other procaryotic or eucaryotic adenylate cyclases in the GenBank database. Production of cyclic AMP in the E. coli clone was confirmed with a radioimmunoassay technique. This is the first example of an adenylate cyclase gene identified from an anaerobic bacterium.

Adenylyl Cyclases↗
PubMed1998

In vitro and in vivo evaluation of insulin-producing beta TC6-F7 cells in microcapsules.

In the present study, the insulin secretory capacity of beta TC6-F7 cells in microcapsules was evaluated. The cell mass within capsules was found to expand in a three-dimensional fashion, in contrast to cells seeded on plates that grew as a monolayer. In in vitro studies, both free and encapsulated cells were found to secrete insulin in the absence of glucose, at 13.6 +/- 1.1 and 14.5 +/- 0.9 ng.10(6) cells-1.60 min-1, respectively, with the response rising to a maximum of 26.0 +/- 0.8 and 31 +/- 2.3 ng.10(6) cells-1.60 min-1 in the presence of 16.8 mM glucose. Encapsulated cells were able to produce Ca2+ responses in the presence of KCl (50 mM) and BAY K 8644 (100 microM). In in vivo studies, intraperitoneal transplantation of 3.0 x 10(6) microencapsulated cells into mice (n = 5) with streptozotocin-induced diabetes resulted in the restoration of normoglycemia up to 57 days. Insulin concentrations rose from 0.4 +/- 0.1 ng/ml before the graft administration to 2.2 +/- 0.8 ng/ml after the transplantation in the normoglycemic recipients. An oral glucose challenge in transplant recipients demonstrated a flat glucose response, suggesting extremely high glucose clearance rates. These data demonstrate the potential use of the immunoisolated beta-cell lines for the treatment of diabetes.

Animals↗
PubMed1998

Truncated SNAP-25 (1-197), like botulinum neurotoxin A, can inhibit insulin secretion from HIT-T15 insulinoma cells.

We and others have previously shown that insulin-secreting cells of the pancreas express high levels of SNAP-25 (synaptosomal-associated protein of 25 kDa), a 206-amino acid t-SNARE (target soluble N-ethylmaleimide-sensitive factor attachment protein receptors) implicated in synaptic vesicle exocytosis. In the present study, we show that SNAP-25 is required for insulin secretion by transient transfection of Botulinum Neurotoxin A (BoNT/A) into insulin-secreting HIT-T15 cells. Transient expression of BoNT/A cleaved the endogenous as well as overexpressed SNAP-25 proteins and caused significant reductions in K+ and glucose-evoked secretion of insulin. To determine whether the inhibition of release was due to the depletion of functional SNAP-25 or the accumulation of proteolytic by-products, we transfected cells with SNAP-25 proteins from which the C-terminal nine amino acids had been deleted to mimic the effects of the toxin. This modified SNAP-25 (amino acids 1-197) remained bound to the plasma membrane but was as effective as the toxin at inhibiting insulin secretion. Microfluorimetry revealed that the inhibition of secretion was due neither to changes in basal cytosolic Ca2+ levels nor in Ca2+ influx evoked by K(+)-mediated plasma membrane depolarization. Electron microscopy revealed that cells transfected with either BoNT/A or truncated SNAP-25 contained significantly higher numbers of insulin granules, many of which clustered close to the plasma membrane. Together, these results demonstrate that functional SNAP-25 proteins are required for insulin secretion and suggest that the inhibitory action of BoNT/A toxin on insulin secretion is in part caused by the production of the plasma membrane-bound cleavage product, which itself interferes with insulin granule docking and fusion.

Botulinum Toxins, Type A↗
PubMed1998

Forms and prevalence of intersexuality and effects of environmental contaminants on sexuality in cricket frogs (Acris crepitans).

Cricket frogs (Acris crepitans) from several different sites in Illinois were collected to assess the effects of environmental contamination on the prevalence of intersex gonads. Of 341 frogs collected in 1993, 1994, and 1995, 2.7% were intersex individuals. There was no statistically significant relationship between the chemical compounds detected and cricket frog intersexuality. However, there was an association approaching significance (p = 0.07) between the detection of atrazine and intersex individuals. A comparison of reference sites with sites that had point polychlorinated biphenyl (PCB) and polychlorinated dibenzofuran (PCDF) contamination revealed a significant relationship between sex-ratio reversal and contamination with PCBs and PCDFs. The sex ratio of juvenile frogs studied from three sites with PCB and PCDF point contamination favored males over females, which was the opposite of the sex ratio in control ponds (p = 0.0007). The statistically significant correlation between organochlorine contamination and sex-ratio reversal suggests PCBs and PCDFs can influence cricket frog sexual differentiation. The current study suggests that in cricket frogs, sex ratios and the prevalence of intersex gonads are altered by environmental contamination.

Aging↗
PubMed1998

Enhanced glucose-dependent insulinotropic polypeptide secretion and insulinotropic action in glucagon-like peptide 1 receptor -/- mice.

Incretins are gastrointestinal hormones that act on the pancreas to potentiate glucose-stimulated insulin secretion. Despite the physiological importance of the enteroinsular axis, disruption of glucagon-like peptide (GLP)-1 action is associated with only modest glucose intolerance in GLP-1 receptor -/- (GLP-1R -/-) mice. We show here that GLP-1R -/- mice exhibit compensatory changes in the enteroinsular axis via increased glucose-dependent insulinotropic polypeptide (GIP) secretion and enhanced GIP action. Serum GIP levels in GLP-1R -/- mice were significantly elevated versus those in +/+ control mice after an oral glucose tolerance test (369 +/- 40 vs. 236 +/- 28 pmol/l; P < or = 0.02). Furthermore, GIP perfusion of mice pancreas and isolated islets in the presence of elevated glucose concentrations elicited a significantly greater insulin response in GLP-1R -/- than in +/+ mice (P < or = 0.02-0.05). In contrast, no significant perturbation in the insulin response to perfused glucagon was detected under conditions of low (4.4 mmol/l) or high (16.6 mmol/l) glucose in GLP-1R -/- mice. Total pancreatic insulin but not glucagon content was significantly reduced in GLP-1R -/- compared with in +/+ mice (77 +/- 9 vs. 121 +/- 10 pmol/mg protein; P < or = 0.005). These observations suggest that upregulation of the GIP component of the enteroinsular axis, at the levels of GIP secretion and action, modifies the phenotype resulting from interruption of the insulinotropic activity of GLP-1 in vivo.

Animals↗
PubMed1998

Production of bovine alpha-lactalbumin in the milk of transgenic pigs.

High production of milk and its components are necessary to allow maximal growth of developing pigs. In this study, transgenic pigs were produced containing the alpha-lactalbumin gene, whose product is a potential limiting component in the production of milk. Two lines of transgenic pigs were produced to analyze the effects that overproduction of the milk protein alpha-lactalbumin may have on milk production and piglet growth. Transgenic pigs were produced through microinjection of the bovine alpha-lactalbumin gene. The gene construct contained 2.0 kb of 5' flanking region, the 2.0 kb coding region, and 329 bp of 3' flanking region. Sows hemizygous for the transgene produced as much as .9 g of bovine alpha-lactalbumin per liter of pig milk. The production of the bovine protein caused approximately a 50% increase in the total alpha-lactalbumin concentration of pig milk throughout a lactation. The concentration of bovine alpha-lactalbumin was highest on d 0 and 5 of lactation and decreased as lactation progressed. The ratio of bovine to porcine alpha-lactalbumin changed during the sow's lactation. This ratio was 4.3 to 1 on d 0 of lactation, but by d 20 of lactation the ratio was .43 to 1. This suggested that the bovine transgene and the endogenous porcine gene are under slightly different control mechanisms. The higher level of total alpha-lactalbumin present on d 0 of lactation was correlated with higher lactose percentage on d 0 in transgenic sows (3.8%), compared with controls (2.6%) (P < .01). Although there was also a trend for higher lactose percentage in transgenic sows on d 5 and 10 of lactation, no significant differences were observed. These data suggest that alpha-lactalbumin is limiting early in lactation of swine. Furthermore, higher concentrations of alpha-lactalbumin early in lactation may boost milk output.

Animals↗
PubMed1998

Genetic engineering of glucose-stimulated insulin secretion in Chinese hamster ovary cells.

To engineer an a non-islet cell capable of glucose-stimulated insulin secretion, a chinese hamster ovary cell line (CHO) was transfected with a mammalian expression vector carrying the human insulin cDNA (pCB/hINS). More proinsulin than insulin was released daily by the stably transformed cell line (CHO-INS). Examination of acid-ethanol extracts confirmed that both insulin and proinsulin were stored. Immunohistochemical analysis of the cells also showed that (pro)insulin was stored. Unlike beta cells, CHO-INS cells did not secrete insulin in response to glucose. To investigate this lack of effect, we examined whether transfection of GLUT2 cDNA, which is ordinarily not expressed in CHO-INS cells, would confer glucose-stimulated insulin secretion. Consequently, we have demonstrated that glucose regulated insulin release occurs in the CHO-INS-GLUT2 cell line and that glucose potentiates the insulin secretory response to non-glucose secretagogues.

Animals↗
PubMed1997

The Xenopus proglucagon gene encodes novel GLP-1-like peptides with insulinotropic properties.

The proglucagon gene encodes several hormones that have key roles in the regulation of metabolism. In particular, glucagon-like peptide (GLP-1), a potent stimulus of insulin secretion, is being developed as a therapy for the treatment of non-insulin-dependent diabetes mellitus. To define structural moieties of the molecule that convey its insulinotropic activity, we have cloned and characterized the proglucagon gene from the amphibian, Xenopus laevis. Unexpectedly, these cDNAs were found to encode three unique glucagon-like-1 peptides, termed xenGLP-1A, xenGLP-1B, and xenGLP-1C in addition to the typical proglucagon-derived hormones glucagon and GLP-2. xenGLP-1A, -1B, and -1C were synthesized and tested for their ability to bind and activate the human GLP-1 receptor (hGLP-1R), and to stimulate insulin release from rat pancreas. All three Xenopus GLP-1-like peptides bind effectively to the hGLP-1R and stimulate cAMP production. Surprisingly, xenGLP-1B(1-30) demonstrated higher affinity for the hGLP-1R than hGLP-1 (IC50 of 1.1 +/- 0.4 nM vs. 4.4 +/- 1.0 nM, respectively, P < 0.02) and was equipotent to hGLP-1 in stimulating cAMP production (EC50 of 0.17 +/- 0.02 nM vs. 0.6 +/- 0. 2 nM, respectively, P > 0.05). Further studies demonstrated that hGLP-1, xenGLP-1A, -1B, and -1C stimulate comparable insulin release from the pancreas. These results demonstrate that despite an average of nine amino acid differences between the predicted Xenopus GLPs and hGLP-1, all act as hGLP-1R agonists.

Amino Acid Sequence↗
PubMed1997

GIP(6-30amide) contains the high affinity binding region of GIP and is a potent inhibitor of GIP1-42 action in vitro.

GIP (Glucose-dependent Insulinotropic Polypeptide) is an important regulator of insulin secretion. The effects of truncated forms of the peptide, GIP(10-30), GIP(6-30amide) and GIP(7-30), on binding of 125I-GIP(1-42) to GIP receptors in transfected CHO-KI cells, and on cyclic AMP responses to GIP(1-42), have been studied with a view to defining further the receptor binding region of GIP, and to establish whether such truncated peptides exhibit agonist or antagonist activity. All three peptides were found to be receptor antagonists, however GIP(6-30amide) exhibited receptor binding affinity equivalent to that of GIP(1-42) in competitive binding studies (IC50 = 3.08+/-0.57 nM). GIP(6-30amide) inhibited GIP(1-42)-induced cAMP production by 58% at a concentration of 100 nM, whereas GIP(10-30) and GIP(7-30), inhibited only in the microM range. GIP(6-30amide) therefore contains the high affinity binding region of GIP and is a potent inhibitor of GIP(1-42) action in vitro.

Animals↗
PubMed1997

Localization of the domains involved in ligand binding and activation of the glucose-dependent insulinotropic polypeptide receptor.

The receptors for the two structurally related insulinotropic hormones Glucose-dependent Insulinotropic Polypeptide (GIP) and Glucagon-Like Peptide-1 (GLP-1) share approximately 40% sequence identity and demonstrate complete specificity for their endogenous ligands, while utilizing similar second messenger pathways. In the current study chimeric GIP-GLP-1 receptors were prepared, and the effect of domain-exchange on ligand binding and adenylyl cyclase activation examined. A chimera (CH-2) consisting of the first 132 amino acids of the external N-terminal (NT) domain bound 125I-GIP with high affinity (27.77 +/- 11.85 nM). However, for receptor coupling to cAMP production it was necessary to extend the NT into the first transmembrane (TM-1) region (CH-3: IC50 = 9.04 +/- 1.07 nM; EC50 = 17.1 +/- 3.5 nM). A chimera which included part of TM-3 (CH-4) demonstrated binding and signalling (IC50 = 8.33 +/- 0.14 nM; EC50 = 467.5 +/- 173.6 pM) similar to the wild type receptor (IC50 = 1.33 +/- 0.19 nM; EC50 = 497.9 +/- 211.7 pM). Surprisingly constructs CH-2 and CH-3, while devoid of detectable 125I-GLP-1 binding, were capable of eliciting GLP-1-specific cAMP production (EC50s CH-2 = 81.4 +/- 19.6 nM; CH-3 = 5.99 +/- 0.68 nM) suggesting that receptor activation is not completely dependent on high affinity receptor binding. These data clearly demonstrate that the NT domain of the GIP receptor acts as the ligand-specific binding domain and that the first transmembrane domain is important for receptor activation.

Adenylyl Cyclases↗
PubMed1997

Scanning of the glucagon-like peptide-1 receptor localizes G protein-activating determinants primarily to the N terminus of the third intracellular loop.

It is well known that glucagon-like peptide-1 (7-36 amide) (tGLP-1) is a potent insulinotropic hormone with powerful antidiabetogenic effects. In the present study we sought to determine the precise regions of the intracellular domains of the tGLP-1 receptor that are required for its efficient coupling to adenylyl cyclase because cAMP is the primary candidate second messenger coupling tGLP-1 to insulin secretion. Recently, we identified an amino acid within the third intracellular loop, K334, that was required for efficient coupling of tGLP-1 receptor to adenylyl cyclase. A similar mutagenesis-based strategy was employed here to examine the first and second intracellular loops and to further define sequences in the third loop required for the efficient coupling of the receptor to its second messengers. Receptor mutants were expressed in COS-7 cells and examined for tGLP-1 binding and cAMP stimulation. Three alanine substitution mutations, V327A, 1328A, and V331A, resulted in significantly lower tGLP-1-stimulated cAMP production without reductions in receptor expression. Analysis of the first and second intracellular loops revealed only one mutation contained within the first loop, R176A, where a significant reduction in cAMP activation was observed with normal receptor expression. These studies suggest that specific determinants of coupling for tGLP-1 receptor are primarily localized to the predicted junction of the fifth transmembrane helix and the third intracellular loop. We predict that V327, 1328, and V331 form part of a hydrophobic face that directly contacts the G protein.

Amino Acid Sequence↗
PubMed1997

Characterization and partial purification of bovine alpha-lactalbumin and beta-casein produced in milk of transgenic mice.

Bovine alpha-lactalbumin (alpha-LA) and bovine beta-casein (beta-CN), from milk from transgenic mice were characterized and partially purified using electrophoretic, immunoblotting, and chromatographic methods. The transgenically expressed bovine milk proteins were identified using PAGE or by a combination of preparative isoelectrofocusing followed by Western immunoblotting. The heterologous bovine alpha-IA and bovine beta-CN had molecular masses that were identical to those of those of the native proteins. The estimated expression of the proteins was 1.0 mg/ml of milk for alpha-LA and 3.0 mg/ml for beta-CN. The calcium binding of bovine alpha-LA suggested that the protein produced in murine milk has the same electrophoretic shift as native bovine alpha-LA after the removal of calcium. Nitrogen-linked glycosylation of native and murine synthesized bovine alpha-LA was identified by peptide-N-glycosidase F treatment, and the N-terminal amino acid sequence of HPLC-purified bovine alpha-LA from mouse milk was confirmed to be identical to native bovine alpha-LA. In addition, the phosphorylation of the bovine beta-CN expressed in the milk of transgenic mice was the same as that of native bovine beta-CN, as determined by phosphatase digestion.

Amino Acid Sequence↗
PubMed1997

Evaluation of immunoisolated insulin-secreting beta TC6-F7 cells as a bioartificial pancreas.

OBJECTIVE: To evaluate the growth and insulin secretion from microencapsulated beta TC6-F7 cells in vitro and to assess the in vivo function of microencapsulated cells transplanted in rats with steptozotocin (STZ)-induced diabetes. METHOD: Alginate-poly-L-lysine encapsulated beta TC6-F7 cells were exposed to glucose, isobutylmethylxanthine (IBMX) and glucagon-like peptide I (7-36 amide) in a static in vitro challenge. In vivo, 2.5-3.5 x 10(7) encapsulated cells were implanted into diabetic rats. Graft function was evaluated by monitoring blood glucose concentrations and by an intraperitoneal glucose tolerance test. RESULTS: The cell density (number of cells per capsule) of cultured microencapsulated beta TC6-F7 cells increased almost 35-fold over a 55 day observation period to reach a plateau of approximately 3500 cells/capsule. While insulin secretion per capsule remained unchanged over the first 21 days of culture, a 7-fold increase was observed during the last 14 days of the 55 day observation period. Intraperitoneal transplantation of 3.5 x 10(7) encapsulated cells into diabetic rats resulted, within 24 hours, in reversal of hyperglycemia for up to 60 days. Post-transplantation blood glucose concentrations varied between 2 and 4 mM. Glucose clearance rates evaluated by an intraperitoneal glucose tolerance test at 30 days post-transplantation resulted in a markedly flat glucose clearance curve with blood glucose never rising above 4 mM. The glucose challenge of microencapsulated cells recovered 30 days post-transplantation resulted in a 2-fold increase in insulin response at glucose concentrations greater than 5.5 mM as compared to glucose-free media. In addition, immunostaining of recovered grafted tissue for insulin, reveals a strong presence of the peptide within the cell population. CONCLUSIONS: These data demonstrate the potential use of an immunoisolated beta-cell line for the treatment of diabetes.

1-Methyl-3-isobutylxanthine↗