Robert L. Sorenson

Multicolor laser scanning confocal immunofluorescence microscopy: practical application and limitations.

Publisher Summary Advances in fluorescent probe chemistry, economical laser availability, and confocal microscope instrumentation are making the enormous potential of multicolor laser scanning confocal microscopy (LSCM) available to a wider range of biologists. This chapter outlines the requirements for performing multicolor immunofluorescence studies with LSCM. The principles of immunofluorescence histochemistry necessary for the preparation of multilabeled specimens are summarized. The chapter examines the technical aspects of confocal microscope instrumentation that affect its application to multicolor studies. The practical application and limitations of this technology are demonstrated for multicolor LSCM, using the inexpensive, air-cooled argon ion and krypton-argon ion lasers as light sources are presented and various aspects of confocal microscopy that affect the comparison of images acquired with different excitation and/or emission wavelengths are examined. The chapter provides a better understanding of the compromises needed to effect the accurate imaging of specimens stained with multiple fluorophores. The chapter also discusses the applications and limitations of multicolor laser scanning confocal immunofluorescence microscopy.

Immunohistochemical Localization of Aldose Reductase: II. Rat Eye and Kidney

Aldose reductase (AR) was purified from rat seminal vesicles. Specific antibodies to this enzyme were prepared in rabbits and were used in the unlabeled antibody-enzyme (PAP) technique to localize AR in a number of tissues known to be sites of diabetic lesions. AR was localized in the following structures in the eye: lens epithelial lining and cortical lenticular fibers; corneal endothelium; the inner, nonpigmented layer of ciliary body epithelium and its extension as the posterior surface of the iris; and neuroglial (Muller) cells in the retina. Retinal capillary endothelium did not contain immunoreactive AR. In the kidney, staining was intense in the inner medulla. Specific structures included thin limbs of the loop of Henle, collecting tubules deep in the inner medulla, and transitional epithelial cells lining the pelvic space: structures in the cortex included gtomerular podocytes and distal convoluted tubules. Collecting tubules in the outer medulla and cortex, as well as proximal convoluted tubules and glomerular capillary endothelium did not contain immunoreactive AR.

Structural and Functional Considerations of GABA in Islets of Langerhans: β-Cells and Nerves

γ-Aminobutyric acid (GABA), a prominent inhibitory neurotransmitter, is present in high concentrations in β-cells of islets of Langerhans. The GABA shunt enzymes, glutamate decarboxylase (GAD) and GABA transaminase (GABA-T), have also been localized in islet β-cells. With the recent demonstration that the 64,000-Mr antigen associated with insulin-dependent diabetes mellitus is GAD, there isincreased interest in understanding the role of GABA in islet function. Only a small component of β-cell GABA is contained in insulin secretory granules, making it unlikely that GABA, co-released with insulin, is physiologically significant. Our immunohistochemical study of GABA in β-cells of intact islets indicates that GABA is associated with a vesicular compartment distinctly different from insulin secretory granules. Whether this compartment represents a releasable pool of GABA has yet to be determined. GAD in β-cells is associated with a vesicular compartment, similar to the GABA vesicles. In addition, GAD is found in a unique extensive tubular cisternal complex (GADcomplex). It islikely that the GABA-GAD vesicles are derived from this GAD-containing complex. Physiological studies on the effect of extracellular GABA on islet hormonal secretion have had variable results. Effects of GABA on insulin, glucagon, and somatostatin secretion have been proposed. The most compelling evidence for GABA regulation of islet hormone secretion comes from studies on somatostatin secretion, where it has an inhibitory effect. We present new evidence demonstrating the presence of GABAergic nerve cell bodies at the periphery of islets with numerous GABA-containing processes extending into the islet mantle. This close association between GABAergic neurons and islet α- and δ-cells strongly suggests that GABA inhibition of somatostatin and glucagon secretionis mediated by these neurons. Intracellular β-cell GABAA and its metabolismmay have a role in β-cell function. New evidence indicates that GABA shunt activity isinvolved in regulation of insulin secretion. In addition, GABA or its metabolites may regulate proinsulin synthesis. These new observations provide insight into the complex nature of GABAergic neurons and β-cell GABA in regulation of islet function.

Glucose- and GTP-dependent stimulation of the carboxyl methylation of CDC42 in rodent and human pancreatic islets and pure beta cells. Evidence for an essential role of GTP-binding proteins in nutrient-induced insulin secretion.

Several GTP-binding proteins (G-proteins) undergo post-translational modifications (isoprenylation and carboxyl methylation) in pancreatic beta cells. Herein, two of these were identified as CDC42 and rap 1, using Western blotting and immunoprecipitation. Confocal microscopic data indicated that CDC42 is localized only in islet endocrine cells but not in acinar cells of the pancreas. CDC42 undergoes a guanine nucleotide-specific membrane association and carboxyl methylation in normal rat islets, human islets, and pure beta (HIT or INS-1) cells. GTPgammaS-dependent carboxyl methylation of a 23-kD protein was also demonstrable in secretory granule fractions from normal islets or beta cells. AFC (a specific inhibitor of prenyl-cysteine carboxyl methyl transferases) blocked the carboxyl methylation of CDC42 in five types of insulin-secreting cells, without blocking GTPgammaS-induced translocation, implying that methylation is a consequence (not a cause) of transfer to membrane sites. High glucose (but not a depolarizing concentration of K+) induced the carboxyl methylation of CDC42 in intact cells, as assessed after specific immunoprecipitation. This effect was abrogated by GTP depletion using mycophenolic acid and was restored upon GTP repletion by coprovision of guanosine. In contrast, although rap 1 was also carboxyl methylated, it was not translocated to the particulate fraction by GTPgammaS; furthermore, its methylation was also stimulated by 40 mM K+ (suggesting a role which is not specific to nutrient stimulation). AFC also impeded nutrient-induced (but not K+-induced) insulin secretion from islets and beta cells under static or perifusion conditions, whereas an inactive structural analogue of AFC failed to inhibit insulin release. These effects were reproduced not only by S-adenosylhomocysteine (another methylation inhibitor), but also by GTP depletion. Thus, the glucose- and GTP-dependent carboxyl methylation of G-proteins such as CDC42 is an obligate step in the stimulus-secretion coupling of nutrient-induced insulin secretion, but not in the exocytotic event itself. Furthermore, AFC blocked glucose-activated phosphoinositide turnover, which may provide a partial biochemical explanation for its effect on secretion, and implies that certain G-proteins must be carboxyl methylated for their interaction with signaling effector molecules, a step which can be regulated by intracellular availability of GTP.

Further Studies of an Inhibitor of the Two Antibody Immunoassay System

Previous studies with the two antibody immunoassay method have demonstrated the presence of an “inhibitor” in fresh plasma which interferes with the precipitating step of the two antibody system. Immunoelectrophoretic studies described here demonstrated no cross-reactivity of rat or human gamma globulin with anti-guinea pig plasma (rabbit source), which had been suggested by other workers. Concomitant inactivation of complement and “inhibitor” by heat, antigen-antibody complex, EDTA, or ammonia indicates that complement is the inhibitor of the two antibody immunoassay system. EDTA (0.01 M) inactivates the plasma “inhibitor,” permitting assay of undiluted plasma by the two antibody method.

Immunohistochemical Localization of Aldose Reductase: I. Enzyme Purification and Antibody Preparation—Localization in Peripheral Nerve, Artery, and Testis

Aldose reductase (AR) was purified from rat and bovine seminal vesicles using DEAE-cellulose, hydroxylapatite, and Sephadex-gel column chromatography. The purification resulted in the obtention of an AR pool and a contaminating pool. Antibodies were raised in rabbits against both enzymes by subcutaneous injection of the AR pool. The antisera was judged to be specific for AR by immunoprecipitation of AR activity and by Ouchterlony double immunodiffusion and immunoelectrophoretic methods. Antibodies against rat AR were used in the unlabeled antibody-enzyme (PAP) technique to demonstrate the cellular location of the enzyme in a number of tissues known to be sites of diabetic lesions. Antibodies against bovine AR were not cross reactive with the rat enzyme, as determined by Ouchterlony and competitive protein-binding studies. AR was localized in rat tissues to the Schwann cell sheath of peripheral nerve, arterial endothelium, and the sustentacular (Sertoli) cells and mature spermatids of the testis.

Regulation of Islet β-Cell Proliferation by Prolactin in Rat Islets

This study examined the effects of prolactin on β-cell proliferation in pancreatic islet of Langerhans. Insulin secretion and β-cell proliferation were significantly increased from neonatal rat islets cultured for 4 days in the presence of either 500 ng/ml ovine prolactin (oPRL) or rat prolactin (rPRL). These effects could be prevented by including anti-oPRL serum in the culture media. Although insulin secretion and β-cell proliferation were slightly higher during the first 24 h of exposure to rPRL, maximal response was observed after 4 days for insulin secretion and 6–10 days for β-cell proliferation. The initial mitogenic response of β-cell to rPRL occurred by the limited recruitment of nondividing β-cells into the cell cycle and by most daughter cells proceeding directly into additional cell division cycles. Subsequently, the maximal effect of rPRL on β-cell proliferation was maintained by a higher rate of recruitment of previously nondividing β-cells into cell cycle with only one fourth of the daughter cells continuing to divide. These observations are difficult to reconcile with the proposal that a limited pool of β-cells capable of undergoing cell division exists in islets. Instead, these observations suggest that individual β-cells are transiently re-entering the cell cycle and dividing infrequently in response to rPRL. In this case, the majority of the β-cells would not be expected to be in an irreversible Go phase. We also demonstrated that the effects of rPRL on β-cell proliferation occur at normal serum glucose concentrations and are affected by inhibitors of polyamine metabolism. Additional studies on the effects of rPRL on β-cells should provide important information on the regulation of β-cell proliferation during conditions of increased insulin demand.

Reversal of New-Onset Diabetes through Modulating Inflammation and Stimulating β-Cell Replication in Nonobese Diabetic Mice by a Dipeptidyl Peptidase IV Inhibitor

Inhibition of dipeptidyl peptidase IV (DPP-IV) activity by NVP-DPP728, a DPP-IV inhibitor, improves the therapeutic efficacy of glucagon-like peptide-1 (GLP-1). CD26 is a membrane-associated glycoprotein with DPP-IV activity and is expressed on lymphocytes. We investigated the effect of NVP-DPP728 on reversing new-onset diabetes in nonobese diabetic (NOD) mice and modulating the inflammatory response and stimulating beta-cell regeneration. New-onset diabetic NOD mice were treated with NVP-DPP728 for 2, 4, and 6 wk. Blood glucose level was monitored. Regulatory T cells in thymus and secondary lymph nodes, TGF-beta1 and GLP-1 in plasma, and the insulin content in the pancreas were measured. Immunostaining for insulin and bromodeoxyuridine (BrdU) were performed. The correlation of beta-cell replication with inflammation was determined. In NVP-DPP728-treated NOD mice, diabetes could be reversed in 57, 74, and 73% of mice after 2, 4, and 6 wk treatment, respectively. Insulitis was reduced and the percentage of CD4(+)CD25(+)FoxP3(+) regulatory T cells was increased in treated NOD mice with remission. Plasma TGF-beta1 and GLP-1, the insulin content, and both insulin(+) and BrdU(+) beta-cells in pancreas were also significantly increased. No significant correlations were found between numbers of both insulin(+) and BrdU(+) beta-cells in islets and beta-cell area or islets with different insulitis score in NOD mice with remission of diabetes. In conclusion, NVP-DPP728 treatment can reverse new-onset diabetes in NOD mice by reducing insulitis, increasing CD4(+)CD25(+)FoxP3(+) regulatory T cells, and stimulating beta-cell replication. beta-Cell replication is not associated with the degree of inflammation in NVP-DPP728-treated NOD mice.

Three-Dimensional Imaging of Intact Isolated Islets of Langerhans With Confocal Microscopy

We present a new technique for analyzing the three-dimensional structure of intact isolated islets of Langerhans. Adult rat and human islets were stained with whole-mount immunofluorescence techniques and optically sectioned with a confocal microscope. This has several advantages over traditional methods: 1) the technical difficulties in serial sectioning and handling the large numbers of sections are avoided, 2) optical sectioning by confocal microscopy gives improved resolution and strongly suppresses light from out-of-focus structures, and 3) entire islets can be rapidly imaged for the presence of positive staining. This new technique should facilitate the study of the three-dimensional structure of islets of Langerhans.

Studies of an Inhibitor of the Two Antibody Immunoassay System

A heat labile plasma inhibitor of the two antibody insulin immunoassay system has been investigated by means of paper electrophoresis, ultracentrifugation, and DEAE cellulose anion exchange resin. These studies indicate that the inhibitor does not interfere with the binding of insulin to its specific antibody (first antibody reaction); but that the inhibitor interferes with the reaction of anti-guinea pig serum (rabbit source) with the insulin-anti-insulin soluble complex (second antibody reaction). When plasma samples are appropriately diluted the effect of the inhibitor is minimized.