Function and structure of adult pancreatic islets are determined by early postnatal development, which in rats corresponds to the first month of life. We analyzed changes in blood glucose and hormones during this stage and their association with morphological and functional changes of alpha and beta cell populations during this period. At day 20 (d20), insulin and glucose plasma levels were two- and six-fold higher, respectively, as compared to d6. Interestingly, this period is characterized by physiological hyperglycemia and hyperinsulinemia, where peripheral insulin resistance and a high plasmatic concentration of glucagon are also observed. These functional changes were paralleled by reorganization of islet structure, cell mass and aggregate size of alpha and beta cells. Cultured beta cells from d20 secreted the same amount of insulin in 15.6 mM than in 5.6 mM glucose (basal conditions), and were characterized by a high basal insulin secretion. However, beta cells from d28 were already glucose sensitive. Understanding and establishing morphophysiological relationships in the developing endocrine pancreas may explain how events in early life are important in determining adult islet physiology and metabolism.

Background: Pancreatic islets are not fully developed at birth and it is not clear how they are vascularised and innervated. Nerve Growth Factor (NGF) is required to guide sympathetic neurons that innervate peripheral organs and also in cardiovascular system and ovary angiogenesis. Pancreatic beta cells of a transgenic mouse that over-expressed NGF in attracts sympathetic hyperinnervation towards them. Moreover, we have previously demonstrated that adult beta cells synthesize and secrete NGF; however, we do not know how is NGF secreted during development, nor if it might be trophic for sympathetic innervation and survival in the pancreas.

Transient receptor potential channels have been put forward as regulators of insulin secretion. A role for the TRPV1 ion channel in insulin secretion has been suggested in pancreatic beta cell lines. We explored whether TRPV1 is functionally expressed in RINm5F and primary beta cells from neonate and adult rats. We examined if capsaicin could activate cationic non-selective currents. Our results show that TRPV1 channels are not functional in insulin-secreting cells, since capsaicin did not produce current activation, not even under culture conditions known to induce the expression of other ion channels in these cells. Although TRPV1 channels seem to be irrelevant for the physiology of isolated beta cells, they may play a role in glucose homeostasis acting through the nerve fibers that regulate islet function. At the physiological level, we observed that Trpv1 (-/-) mice presented lower fasting insulin levels than their wild-type littermates, however, we did not find differences between these experimental groups nor in the glucose tolerance test or in the insulin secretion. However, we did find that the Trpv1 (-/-) mice exhibited a higher insulin sensitivity compared to their wild-type counterparts. Our results demonstrate that TRPV1 does not contribute to glucose-induced insulin secretion in beta cells as was previously thought, but it is possible that it may control insulin sensitivity.

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It has been generally assumed that insulin circulates freely in blood. However it can also interact with plasma proteins. Insulin receptors are located in the membrane of target cells and consist of an alpha and beta subunits with a tyrosine kinase cytoplasmic domain. The ectodomain, called soluble insulin receptor (SIR) has been found elevated in patients with diabetes mellitus. We explored if insulin binds to SIRs in circulation under physiological conditions and hypothesize that this SIR may be released by hepatocytes in response to high insulin concentrations. The presence of SIR in rat and human plasmas and the culture medium of hepatocytes was explored using Western blot analysis. A purification protocol was performed to isolated SIR using affinity, gel filtration, and ion exchange chromatographies. A modified reverse hemolytic plaque assay was used to measure SIR release from cultured hepatocytes. Incubation with 1 nmol l−1 insulin induces the release of the insulin receptor ectodomains from normal rat hepatocytes. This effect can be partially prevented by blocking protease activity. Furthermore, plasma levels of SIR were higher in a model of metabolic syndrome, where rats are hyperinsulinemic. We also found increased SIR levels in hyperinsulinemic humans. SIR may be an important regulator of the amount of free insulin in circulation. In hyperinsulinemia, the amount of this soluble receptor increases and this could lead to higher amounts of insulin bound to this receptor, rather than free insulin, which is the biologically active form of the hormone. This observation could enlighten the mechanisms of insulin resistance.

Physalia physalis is a marine cnidarian from which high molecular weight toxins with hemolytic and neurotoxic effects have been isolated. In the present work, two novel toxins, PpV9.4 and PpV19.3 were purified from P. physalis by bioactive guideline isolation. It involved two steps of column chromatography, gel filtration and RP-HPLC. The molecular weights were 550.7 and 4720.9 Da for PpV9.4 and PpV19.3, respectively. In the light of the Edman sequencing results, the structure of these toxins included the presence of modified amino acids. Both toxins increased the percentage of insulin secreting beta-cells and induced cytosolic Ca2+ elevation. To date, this is the first report of low molecular weight toxins increasing insulin secretion purified from cnidarians, by constituting a new approach to the study of beta-cells physiology.

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Glucose-induced insulin secretion by pancreatic beta-cells depends on membrane depolarization and [Ca2+]i increase. We correlated voltage- and current-clamp recordings, [Ca2+]i measurements, and insulin reverse hemolytic plaque assay to analyze the activity of a thapsigargin-sensitive cationic channel that can be important for membrane depolarization in single rat pancreatic beta-cells. We demonstrate the presence of a thapsigargin-sensitive cationic current, which is mainly carried by Na+. Moreover, in basal glucose concentration (5.6 mM), thapsigargin depolarizes the plasma membrane, producing electrical activity and increasing [Ca2+]i. The latter is prevented by nifedipine, indicating that Ca2+ enters the cell through L-type Ca2+ channels, which are activated by membrane depolarization. Thapsigargin also increased insulin secretion by increasing the percentage of cells secreting insulin and amplifying hormone secretion by individual beta-cells. Nifedipine blocked the increase completely in 5.6 mM glucose and partially in 15.6 mM glucose. We conclude that thapsigargin potentiates a cationic current that depolarizes the cell membrane. This, in turn, increases Ca2+ entry through L-type Ca2+ channels promoting insulin secretion.