Wendell E. Nicholson

Assessment of Human Pancreatic Islet Architecture and Composition by Laser Scanning Confocal Microscopy

The recent success of pancreatic islet transplantation has generated considerable enthusiasm. To better understand the quality and characteristics of human islets used for transplantation, we performed detailed analysis of islet architecture and composition using confocal laser scanning microscopy. Human islets from six separate isolations provided by three different islet isolation centers were compared with isolated mouse and non-human primate islets. As expected from histological sections of murine pancreas, in isolated murine islets α and δ cells resided at the periphery of the β-cell core. However, human islets were markedly different in that α, β, and δ cells were dispersed throughout the islet. This pattern of cell distribution was present in all human islet preparations and islets of various sizes and was also seen in histological sections of human pancreas. The architecture of isolated non-human primate islets was very similar to that of human islets. Using an image analysis program, we calculated the volume of α, β, and δ cells. In contrast to murine islets, we found that populations of islet cell types varied considerably in human islets. The results indicate that human islets not only are quite heterogeneous in terms of cell composition but also have a substantially different architecture from widely studied murine islets.

Reduction in Pancreatic Transcription Factor PDX-1 Impairs Glucose-stimulated Insulin Secretion

Complete lack of transcription factor PDX-1 leads to pancreatic agenesis, whereas heterozygosity for PDX-1 mutations has been recently noted in some individuals with maturity-onset diabetes of the young (MODY) and in some individuals with type 2 diabetes. To determine how alterations in PDX-1 affect islet function, we examined insulin secretion and islet physiology in mice with one PDX-1 allele inactivated. PDX-1+/− mice had a normal fasting blood glucose and pancreatic insulin content but had impaired glucose tolerance and secreted less insulin during glucose tolerance testing. The expression of PDX-1 and glucose transporter 2 in islets from PDX-1+/−mice was reduced to 68 and 55%, respectively, whereas glucokinase expression was not significantly altered. NAD(P)H generation in response to glucose was reduced by 30% in PDX-1+/− mice. The in situ perfused pancreas of PDX-1+/− mice secreted about 45% less insulin when stimulated with 16.7 mm glucose. The Km for insulin release was similar in wild type and PDX-1+/− mice. Insulin secretion in response to 20 mm arginine was unchanged; the response to 10 nm glucagon-like peptide-1 was slightly increased. However, insulin secretory responses to 10 mm 2-ketoisocaproate and 20 mm KCl were significantly reduced (by 61 and 66%, respectively). These results indicate that a modest reduction in PDX-1 impairs several events in glucose-stimulated insulin secretion (such as NAD(P)H generation, mitochondrial function, and/or mobilization of intracellular Ca2+) and that PDX-1 is important for normal function of adult pancreatic islets.

Pancreatic Islet Production of Vascular Endothelial Growth Factor-A Is Essential for Islet Vascularization, Revascularization, and Function

To investigate molecular mechanisms controlling islet vascularization and revascularization after transplantation, we examined pancreatic expression of three families of angiogenic factors and their receptors in differentiating endocrine cells and adult islets. Using intravital lectin labeling, we demonstrated that development of islet microvasculature and establishment of islet blood flow occur concomitantly with islet morphogenesis. Our genetic data indicate that vascular endothelial growth factor (VEGF)-A is a major regulator of islet vascularization and revascularization of transplanted islets. In spite of normal pancreatic insulin content and β-cell mass, mice with β-cell–reduced VEGF-A expression had impaired glucose-stimulated insulin secretion. By vascular or diffusion delivery of β-cell secretagogues to islets, we showed that reduced insulin output is not a result of β-cell dysfunction but rather caused by vascular alterations in islets. Taken together, our data indicate that the microvasculature plays an integral role in islet function. Factors modulating VEGF-A expression may influence islet vascularity and, consequently, the amount of insulin delivered into the systemic circulation.

Dopamine Inhibits Angiotensin-Stimulated Aldosterone Biosynthesis in Bovine Adrenal Cells

The possibility that dopamine may play a role in the in vivo control of aldosterone production in man was suggested to us by reports from others; (a) that bromocriptine, a dopaminergic agonist, inhibits the aldosterone response to diuresis and to the infusion of angiotensin or ACTH; and (b) that metaclopramide, a dopamine blocking agent, causes elevations in plasma aldosterone levels. To determine whether such effects were direct or indirect, we examined the action of dopamine on aldosterone biosynthesis in isolated, bovine adrenal cells. Dopamine significantly inhibits the aldosterone response to angiotensin (P < 0.001), but does not influence basal aldosterone biosynthesis. It has previously been reported that angiotensin stimulates both the early and late phases of aldosterone biosynthesis. The present experiments demonstrated that the enhancing effect of angiotensin on the conversion of deoxycorticosterone to aldosterone (late phase of aldosterone biosynthesis) was almost completely inhibited by dopamine (P < 0.001). A significant inhibitory effect of dopamine (10 nM) was seen even when aldosterone biosynthesis was stimulated by a grossly supraphysiological concentration of angiotensin II (10 muM). However, these studies did not demonstrate any direct effect of dopamine on the early phase of aldosterone biosynthesis (cholesterol to pregnenolone) basally or when stimulated, or on the late phase of aldosterone biosynthesis under basal conditions. These in vitro studies suggest a direct inhibitory role for dopamine on the late phase of aldosterone biosynthesis, which may account for the in vivo inhibition of the aldosterone response to angiotensin in subjects treated with a dopaminergic agent.

CORRELATION OF PLASMA ACTH CONCENTRATION WITH ADRENOCORTICAL RESPONSE IN NORMAL HUMAN SUBJECTS, SURGICAL PATIENTS, AND PATIENTS WITH CUSHING'S DISEASE.

The role of ACTH in various clinical disorders has been difficult to ascertain because the available assay methods have lacked the sensitivity necessary for valid quantitation of the hormone in the plasma of normal subjects (1-4). Even the method of Lipscomb and Nelson (5), the most sensitive practical bioassay procedure now available, usually requires the injection of at least 0.05 mUof ACTHper rat, if responses are to be elicited that will be statistically significant without the use of a prohibitive number of animals. It is usually impractical to inject more than 5 ml of crude human plasma into a single rat. Therefore, in order to be accurately measurable by this procedure, the concentration of ACTH in the plasma must be at least 0.05 mUper 5 ml, or 1 mUper 100 ml. Numerous studies indicate that normal plasma levels of ACTHare well below this concentration. Byr the adrenal ascorbic acid depletion assay method, Sydnor, Sayers, Brown, and Tyler (1) were unable to detect ACTH in plasma of normal subjects, even after attempting to extract the hormone with oxvcellulose in preparation for the bioassay. These workers concluded that blood ACTH concentrations of normal human subjects were less than 0.5 mUper 100 ml. Using a similar procedure, Fujita (3) estimated the normal level of ACTHto be about 1 mUper L, i.e., 0.1 mUper 100 ml. Cooper and Nelson (6) were able to detect ACTH in the plasma of only 3 of 10 patients before surgery, by a method that they

Oxidative stress is a mediator of glucose toxicity in insulin-secreting pancreatic islet cell lines.

Pancreatic β cells secrete insulin in response to changes in the extracellular glucose. However, prolonged exposure to elevated glucose exerts toxic effects on β cells and results in β cell dysfunction and ultimately β cell death (glucose toxicity). To investigate the mechanism of how increased extracellular glucose is toxic to β cells, we used two model systems where glucose metabolism was increased in β cell lines by enhancing glucokinase (GK) activity and exposing cells to physiologically relevant increases in extracellular glucose (3.3–20 mm). Exposure of cells with enhanced GK activity to 20 mm glucose accelerated glycolysis, but reduced cellular NAD(P)H and ATP, caused accumulation of intracellular reactive oxygen species (ROS) and oxidative damage to mitochondria and DNA, and promoted apoptotic cell death. These changes required both enhanced GK activity and exposure to elevated extracellular glucose. A ROS scavenger partially prevented the toxic effects of increased glucose metabolism. These results indicate that increased glucose metabolism in β cells generates oxidative stress and impairs cell function and survival; this may be a mechanism of glucose toxicity in β cells. The level of β cell GK may also be critical in this process.

Normal and abnormal regulation of β-MSH in man

A B S T R A C T The regulation of plasma 8-melanocyte-stimulating hormone (,8-MSH) in man has been studied utilizing a radioimmunoassay previously described (1). In normal subjects plasma p-MSH values ranged from 20 to 110 pg/ml. Metyrapone increased and dexamethasone decreased plasma P-MSH levels. Surgical stress stimulated f-MSH secretion. Plasma P-MSH levels were elevated in patients with untreated Addisons disease and untreated congenital adrenal hyperplasia, and these levels fell to normal during glucocorticoid therapy. In patients with Cushings syndrome due to pituitary adrenocorticotropic hormone (ACTH) excess, plasma fi-MSH was slightly elevated before treatment. In those patients who developed pituitary tumors and hyperpigmentation after bilateral adrenalectomy, plasma P-MSH was greatly elevated. In patients with Cushings syndrome due to adrenal tumor, plasma j9-MSH was subnormal. In patients with the ectopic ACTH syndrome, the levels of plasma f8-MSH were high. Plasma fi-MSH had a diurnal variation in normal subjects, patients with Addisons disease, and patients with congenital adrenal hyperplasia; but the normal diurnal variation was lost in patients with Cushings disease. In patients with high plasma 8-MSH, simultaneous determinations of plasma ACTH showed close correlation between the degree of elevation of ACTH and that of 8-MSH. In extracts of tumors from patients with the ectopic ACTH-MSH syndrome the quantities of the two hormones were roughly equivalent. In patients with hyperpigmentation due to a variety of disorders other than pituitary-adrenal abnormalities, plasma 8-MSH was normal. It is concluded that the secretion of P-MSH is regulated by the same factors that regulate ACTH.

Radioimmunoassay of β-MSH in Human Plasma and Tissues

: A radioimmunoassay method for beta-melanocyte-stimulating hormone (beta-MSH) has been developed and utilized in the identification and quantification of this hormone in human plasma and tissues. The concentration of beta-MSH in two human pituitary glands was found to be approximately 350 mug/g. beta-MSH was identified in the tumor tissue of all 11 patients with the ectopic ACTH syndrome who were studied; concentrations in individual cases ranged from 3 to 1600 ng/g. In plasma of chronically hyperpigmented patients with Addisons disease, Cushings disease (after bilateral adrenalectomy), and the ectopic ACTH syndrome, beta-MSH concentrations of 0.5-6 ng/ml were found. The degree of clinical hyperpigmentation was well correlated with the quantity of beta-MSH in the plasma. beta-MSH concentrations in the plasma of normal subjects were less than 0.09 ng/ml. In all of these circumstances, bioassays for MSH were also performed, and it was found that most of the biologic MSH activity of the plasma and tissues could be accounted for by beta-MSH.

Assessment of pancreatic islet mass after islet transplantation using in vivo bioluminescence imaging.

Background. Pancreatic islet transplantation is an emerging therapy for type 1 diabetes, but it is difficult to assess islets after transplantation and thus to design interventions to improve islet survival. Methods. To image and quantify islets, the authors transplanted luciferase-expressing murine or human islets (by adenovirus-mediated gene transfer) into the liver or beneath the renal capsule of immunodeficient mice and quantified the in vivo bioluminescence imaging (BLI) of mice using a cooled charge-coupled device camera and digital photon-counting image analysis. To account for variables that are independent of islet mass such as transplant site, animal positioning, and wound healing, the BLI of transplanted islets was calibrated against measurement of luminescence of an implanted bead emitting a constant light intensity. Results. BLI of mice bearing islet transplants was seen in the expected anatomic location, was stable for more than 8 weeks after transplantation, and correlated with the number of islets transplanted into the liver or kidney. BLI of the luminescent bead and of transplanted islets in the kidney was approximately four times greater than when transplanted in the liver, indicating that photon emission is dependent on optical absorption of generated light and thus light source location. Conclusion. In vivo BLI allows for quantitative, serial measurements of pancreatic islet mass after transplantation and should be useful in assessing interventions to sustain or increase islet survival of transplanted islets.

Biologic and Immunologic Characterization and Physical Separation of ACTH and ACTH Fragments in the Ectopic ACTH Syndrome

Extracts of tumors from 32 patients with the ectopic ACTH syndrome were subjected to simultaneous bioassay and radioimmunoassays for ACTH. Radioimmunoassays were performed using three antisera, one of which reacts with the extreme N-terminal 1-13 amino acid sequence of ACTH, the second with the N-terminal 1-23 sequence of the ACTH molecule, and the third with the C-terminal 25-39 amino acid sequence of ACTH. There was, in general, good correlation between bioactivity and N-terminal ACTH immunoreactivity. However, there were large excesses of both extreme N-terminal and C-terminal immunoreactive materials in most tumor extracts, which were not found in extracts of three human pituitaries. Three tumor extracts were subjected to molecular sieve chromatography on Sephadex G-50 fine resin. The bioactive ACTH eluted in the same fractions as pituitary ACTH (mol wt approximately 4,500 daltons) and reacted equally in all three ACTH radioimmunoassay systems. The bioactive tumor ACTH was neutralized by incubation with the C-terminal antiserum, indicating it has an intact C-terminal sequence of amino acids. The next several fractions from the Sephadex column contained a material, mol wt approximately 3,100, which was biologically inactive and had C-terminal immunoreactivity but no N-terminal or extreme N-terminal immunoreactivity. Incubation with the N-terminal 1-23 ACTH antiserum did not adsorb these C-terminal fragments, indicating they lacked an intact sequence of amino acids in this region. A smaller ACTH fragment (mol wt approximately 1,800 daltons) eluted in still later fractions and reacted with the extreme N-terminal antiserum but not with the N-terminal or C-terminal antisera. It had no steroidogenic activity, but appeared to have significant melanocyte-stimulating activity. It is concluded that, in addition to an ACTH similar, if not identical, to pituitary ACTH, tumors of patients with the ectopic ACTH syndrome contain both N-terminal and C-terminal ACTH fragments.