Anthony J. Weinhaus

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.

Regulation of glucokinase in pancreatic islets by prolactin: a mechanism for increasing glucose-stimulated insulin secretion during pregnancy

Glucokinase activity is increased in pancreatic islets during pregnancy and in vitro by prolactin (PRL). The underlying mechanisms that lead to increased glucokinase have not been resolved. Since glucose itself regulates glucokinase activity in beta-cells, it was unclear whether the lactogen effects are direct or occur through changes in glucose metabolism. To clarify the roles of glucose metabolism in this process, we examined the interactions between glucose and PRL on glucose metabolism, insulin secretion, and glucokinase expression in insulin 1 (INS-1) cells and rat islets. Although the PRL-induced changes were more pronounced after culture at higher glucose concentrations, an increase in glucose metabolism, insulin secretion, and glucokinase expression occurred even in the absence of glucose. The presence of comparable levels of insulin secretion at similar rates of glucose metabolism from both control and PRL-treated INS-1 cells suggests the PRL-induced increase in glucose metabolism is responsible for the increase in insulin secretion. Similarly, increases in other known PRL responsive genes (e.g. the PRL receptor, glucose transporter-2, and insulin) were also detected after culture without glucose. We show that the upstream glucokinase promoter contains multiple STAT5 binding sequences with increased binding in response to PRL. Corresponding increases in glucokinase mRNA and protein synthesis were also detected. This suggests the PRL-induced increase in glucokinase mRNA and its translation are sufficient to account for the elevated glucokinase activity in beta-cells with lactogens. Importantly, the increase in islet glucokinase observed with PRL is in line with that observed in islets during pregnancy.

Mechanisms of arginine-induced increase in cytosolic calcium concentration in the beta-cell line NIT-1

Summary The effects of l-arginine and its analogues NG-nitro-l-arginine, NG-methyl-l-arginine, l-homoarginine and d-arginine on cytosolic calcium concentration were investigated to characterise the mechanisms of arginine-induced stimulation and to determine if nitric oxide production played a role in this stimulation. NIT-1 cells, a transgenic beta-cell line, were used for this purpose since they release insulin in response to typical beta-cell stimuli. Our data demonstrate that the arginine-induced increase in cytosolic calcium concentration was completely dependent on the influx of extracellular Ca2 + via verapamil-sensitive voltage-activated Ca2 + channels and that arginine stimulation requires the presence of a nutrient in order to cause an increase in cytosolic calcium concentration. The nutrient likely acted by closing the K +ATP channels, since its effect could be inhibited by activation of these channels with diazoxide. l-arginine, as well as nitro-arginine and methyl-arginine which are not substrates for the production of nitric oxide, caused similar increases in cytosolic calcium concentration. Non-metabolisable arginine analogues homoarginine and d-arginine also caused increases in the cytosolic calcium concentration although not to the same extent. Insulin secretion was enhanced to the same extent by all analogues of arginine. It can be concluded that the arginine-induced increase in cytosolic calcium concentration in NIT-1 cells is attributable to an electrogenic effect following the transport of arginine leading to depolarisation of the plasma membrane potential, although metabolism of the amino acid itself may also partially contribute to the response. [Diabetologia (1997) 40: 374–382]

Chiari's network: review of the literature

The Chiari network, present in approximately 2% of the population, and is a reticulated network of fibers originating from the Eustachian connecting to different parts of the right atrium. Its presence results from incomplete reabsorption of the right valve of the sinus venosus. Chiari’s network is often clinically insignificant. However, it has been reported to be involved in the pathogenesis of thromboembolic disease, endocarditis, arrhythmias, and entrapment of catheters upon percutaneous intervention. While initially discovered and researched using autopsy dissections, Chiari’s network is often found as an incidental finding on diagnostic imaging studies, thus providing new methods for studying its incidence and clinical significance.

Insulin Secretagogues, But Not Glucose, Stimulate an Increase in [Ca2+]i in the Fetal Rat β-cell

Fetal pancreatic islets release insulin poorly in response to glucose; however, the cellular mechanism for this is controversial. By using fura 2 to measure changes in the cytoplasmic free Ca2+ concentration ([Ca2+]i) in β-cells, we have examined islets from fetal, neonatal, and adult rats to determine the ability of glucose and other secretagogues to cause an increase in [Ca2+]i. The effects of glucose (20 mmol/l), glyceraldehyde (20 mmol/l), leucine (20 mmol/l), arginine (20 mmol/l), and the channel effectors glipizide (50 μmol/l), BAY K8644 (2 μmol/l), diazoxide (300 μmol/l), and verapamil (20 μmol/l) on changes in [Ca2+]i were studied. In both the fetal and the mature islet, glyceraldehyde, leucine, arginine, glipizide, and BAY K8644 caused an increase in [Ca2+]i. In mature islets, glucose also increased [Ca2+]i; however, in the fetal islet, glucose had no effect on [Ca2+]i. The stimulus-induced increases in [Ca2+]i in fetal and adult islets were both significantly inhibited by the addition of either diazoxide or verapamil. Similar results were obtained when insulin secretion was measured. Our data show that various secretagogues are able to stimulate fetal islets and cause an increase in [Ca2+]i. Glucose, however, fails to cause an increase in [Ca2+]i in the fetal islet. Hence, the immature insulin secretory response to glucose by the fetal islet is due to the inability of the fetal β-cell to translate glucose stimulation into the increase in [Ca2+]i required for exocytosis of the insulin granule.

Anatomy of the Human Heart

This chapter covers the internal and external anatomy and function of the heart, as well as its positioning within the thorax. Briefly, the heart is a muscular pump, located in the protective thorax, which serves two functions: (1) collect blood from the tissues of the body and pump it to the lungs; and (2) collect blood from the lungs and pump it to all the tissues of the body. The heart’s two upper chambers (or atria) function primarily as collecting chambers, while two lower chambers (ventricles) are much stronger and function to pump blood. The right atrium and ventricle collect blood from the body and pump it to the lungs, and the left atrium and ventricle collect blood from the lungs and pump it throughout the body. There is a one-way flow of blood through the heart which is maintained by a set of four valves (tricuspid, bicuspid, pulmonary, and aortic). The tissues of the heart are supplied with nourishment and oxygen by a separate vascular supply committed only to the heart; the arterial supply to the heart arises from the base of the aorta as the right and left coronary arteries, and the venous drainage is via cardiac veins that return deoxygenated blood to the right atrium.

Basic Biology of Pig Fetal Pancreas and Its Use as an Allograft

Diabetes is a chronic disorder of metabolism, which has the potential to result in the development of microvascular complications, such as retinopathy and nephropathy, many years after the onset of the disease process. This is particularly relevant to insulin-dependent diabetes, which affects the young, with blindness and renal failure occurring early in life, rather than at an advanced age, which can occur in non-insulin dependent diabetes. Until insulin dependent diabetes can be prevented, or cured by means of gene transfer, pancreatic transplantation is probably the most likely means at our disposal to normalize blood glucose levels, and thus prevent the occurrence of complications.

Islet Transplantation in Spontaneously Diabetic BB/Wor Rats

We investigated the effectiveness of islet transplantation as therapy in an animal model of spontaneous type I (insulin-dependent) diabetes mellitus. Grafting MHC-matched and -mismatched islets with the spontaneously diabetic BB rat as a model has been previously reported to result in recurrence of the disease in the grafted tissue. When transplanted with nonimmunogenic islets isolated by nonenzymatic culture, we found that MHC-matched grafts proved to be susceptible to disease recurrence when allowed to remain in situ until ketosis developed in the host. Conversely, the MHC-mismatched grafts did not succumb to the disease process despite the destruction of the β-cell population of the endogenous pancreas. Four manifestly hyperglycemie BB/Wor rats received sufficient islet mass by allotransplantation to reverse this state. All four animals had ameliorated conditions, and three of the four were restored to a normoglycemic state. Recurrence of diabetes in the BB rat was not observed.

Suitability of the Pericardiophrenic Veins for Phrenic Nerve Stimulation: An Anatomic Study

Objective:  The objective of this study was to assess the potential of the pericardiophrenic veins (PPVs) as conduits for transvenous stimulation of the phrenic nerves. Modulating respiration with transvenous phrenic nerve stimulation via the PPVs might reduce or eliminate the adverse effects of central sleep apnea in heart failure.

The successful allotransplantation of neonatal rat islets across multiple combined major and minor histocompatibility barriers

Cultured neonatal rat islets were transplanted across six strain combinations into nonimmunosuppressed allogeneic recipients. Islets were isolated nonenzymatically by an in vitro method and were cultured at 37°C in 5% CO2 in air for 10 days prior to transplant. Transplants to nondiabetic recipients across four allogeneic barriers resulted in morphologically intact and wellgranulated islet tissue present at the graft site in 54 of 55 cases for periods lasting as long as 445 days (mean day of sacrifice was 163). In trials using diabetic recipients, ACIs receiving WF islets (n = 3) and outbred Holtzmans receiving Holtzman islets (n = 3) were reversed and did not return to the hyperglycemic state for experimental periods of up to 430 days.