Can myoglobin expression in pancreatic beta cells improve insulin secretion under hypoxia? An exploratory study with transgenic porcine islets.
ABSTRACT The feasibility of myoglobin (Mb)-facilitated oxygen transport in improving porcine islet survival under hypoxia was investigated. Discrete groups of islets were transfected with replication-defective adenoviral vector Ad5 respiratory syncitial virus (RSV) to induce expression of Mb or green fluorescent protein (GFP). Native islets served as the controls. In vitro studies at 37 degrees C assessed islet insulin secretion efficacy: (i) to a glucose challenge from 30 to 300 mg/dL at fixed pO2; and (ii) at variable oxygen tensions ranging from 5 to 40 mm Hg over 12 h. The transfection was effective in initiating islet expression of Mb or GFP. Low Mb-expression levels equivalent to 2% the Mb concentration in a muscle cell (0.25 ng of Mb per islet) were documented, with no statistical improvement in insulin secretion. A surprising side note is that insulin secretion was impaired in islets expressing GFP. Improved Mb expression is essential to determine the feasibility of enhancing islet survival under hypoxia.
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ABSTRACT: The purpose of the present study was to visualize myoglobin-facilitated oxygen delivery to mitochondria at a critical mitochondrial oxygen supply in single isolated cardiomyocytes of rats. Using the autofluorescence of mitochondrial reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H), the mitochondrial oxygen supply was imaged from approximately 1.4 microm inside the cell surface at a subcellular spatial resolution. Significant radial gradients of intracellular oxygenation were produced by superfusing the cell suspension with a mixed gas containing 2-4% oxygen while stimulating mitochondrial respiration with an uncoupler of oxidative phosphorylation. Augmentation of the NAD(P)H fluorescence started from the core of the cell (anoxic core) and progressively expanded toward the plasma membrane, as the extracellular Po(2) was lowered. Inactivation of cytosolic myoglobin by 5 mM NaNO(2) significantly enlarged such anoxic regions. Nitrite affected neither mitochondrial respiration in uncoupled cells nor the relationship between Po(2) and the NAD(P)H fluorescence in coupled cells. Thus we conclude that myoglobin significantly facilitates intracellular oxygen transport at a critical level of mitochondrial oxygen supply in single cardiomyocytes.Biophysical Journal 07/2000; 78(6):3252-9. · 3.67 Impact Factor
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ABSTRACT: Of many obstacles involved in developing a bioartificial pancreas, which consists of encapsulated and physically immunoprotected islets, for long-term implantation in insulin-dependent diabetic patients, the impaired functionality and decreasing viability of encapsulated islets over time are critical factors in determining the size and longevity of the implant. These factors are closely associated with short oxygen supply to the encaged islets from the implant site. To facilitate oxygen transport to islets in the capsules, we coencapsulated hemoglobin cross-linked with difunctional polyethylene glycol (Hb-conjugate, Hb-C) which is large in size (>100 kDa), thus preventing diffusional loss through the immunoprotecting membrane. The coencapsulation of Hb-C with islets in alginate-poly-L-lysine microcapsules by dissolving Hb-C in an islet-suspended alginate solution at a concentration of 0.25 mM improved the insulin secretion and viability of the islets. At week 0, the islets, coencapsulated with Hb-C, cultured at P(O2) = 40 mmHg (assumed oxygen partial pressure in the most common implant site, the peritoneal cavity), secreted 200% more insulin compared with the control islets without Hb-C at glucose concentrations of both 100 and 300 mg/dL. The Hb-C effect became more significant with time at higher glucose concentrations. After culturing the islets for 8 weeks at 40 mmHg, the insulin secretion was enhanced 200 and 550% at glucose concentrations of 100 and 300 mg/dL as compared with the control, respectively. The results were closely associated with improved viability and suggest that the introduction of Hb-C is an effective approach to maintaining the oxygen supply to encapsulated islets. In addition, Hb-C coencapsulation with pancreatic islets may (1) provide a partial clue to reducing the large size of the biohybrid artificial pancreas, (2) lead to a reduced need for pancreas donation, and (3) prolong the longevity of the biohybrid artificial pancreas in the body.Tissue Engineering 07/2002; 8(3):379-94. · 4.07 Impact Factor
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ABSTRACT: We recently reported a transgenic [mouse insulin promoter (MIP)-green fluorescent protein (GFP)] mouse in which GFP expression is targeted to the pancreatic islet beta-cells to enable convenient identification of beta-cells as green cells. The GFP-expressing beta-cells of the MIP-GFP mouse were functionally indistinguishable from beta-cells of normal mice. Here we characterized the ionic channel properties and exocytosis of MIP-GFP mouse islet beta- and alpha-cells. Beta-cells displayed delayed rectifying K+ and high-voltage-activated Ca2+ channels and exhibited Na+ currents only at hyperpolarized holding potential. Alpha-cells were nongreen and had both A-type and delayed rectifier K+ channels, both low-voltage-activated and high-voltage-activated Ca2+ channels, and displayed Na+ currents readily at -70 mV holding potential. Alpha-cells had ATP-sensitive K+ channel (KATP) channel density as high as that in beta-cells, and, surprisingly, alpha-cell KATP channels were more sensitive to ATP inhibition (IC50=0.16+/-0.03 mM) than beta-cell KATP channels (IC50=0.86+/-0.10 mM). Whereas alpha-cells were rather uniform in size [2-4.5 picofarad (pF)], beta-cells varied vastly in size (2-12 pF). Of note, small beta-cells (<4.5 pF) showed little exocytosis, whereas medium beta-cells (5-8 pF) exhibited vigorous exocytosis, but large beta-cells (>8 pF) had weaker exocytosis. We found no correlation between beta-cell size and their Ca2+ channel density, suggesting that Ca2+ influx may not be the cause of the heterogeneity in exocytotic responses. The MIP-GFP mouse therefore offers potential to further explore the functional heterogeneity in beta-cells of different sizes. The MIP-GFP mouse islet is therefore a reliable model to efficiently examine alpha-cell and beta-cell physiology and should greatly facilitate examination of their pathophysiology when the MIP-GFP mice are crossed with diabetic models.Endocrinology 11/2005; 146(11):4766-75. · 4.72 Impact Factor