A. Wendt

Deletion of the G protein-coupled receptor 30 impairs glucose tolerance, reduces bone growth, increases blood pressure, and eliminates estradiol-stimulated insulin release in female mice.

In vitro studies suggest that the G protein-coupled receptor (GPR) 30 is a functional estrogen receptor. However, the physiological role of GPR30 in vivo is unknown, and it remains to be determined whether GPR30 is an estrogen receptor also in vivo. To this end, we studied the effects of disrupting the GPR30 gene in female and male mice. Female GPR30((-/-)) mice had hyperglycemia and impaired glucose tolerance, reduced body growth, increased blood pressure, and reduced serum IGF-I levels. The reduced growth correlated with a proportional decrease in skeletal development. The elevated blood pressure was associated with an increased vascular resistance manifested as an increased media to lumen ratio of the resistance arteries. The hyperglycemia and impaired glucose tolerance in vivo were associated with decreased insulin expression and release in vivo and in vitro in isolated pancreatic islets. GPR30 is expressed in islets, and GPR30 deletion abolished estradiol-stimulated insulin release both in vivo in ovariectomized adult mice and in vitro in isolated islets. Our findings show that GPR30 is important for several metabolic functions in female mice, including estradiol-stimulated insulin release.

Impaired insulin secretion and glucose tolerance in beta cell-selective Ca(v)1.2 Ca2+ channel null mice.

Insulin is secreted from pancreatic β cells in response to an elevation of cytoplasmic Ca2+ resulting from enhanced Ca2+ influx through voltage‐gated Ca2+ channels. Mouse β cells express several types of Ca2+ channel (L‐, R‐ and possibly P/Q‐type). β cell‐selective ablation of the gene encoding the L‐type Ca2+ channel subtype Cav1.2 (βCav1.2−/− mouse) decreased the whole‐cell Ca2+ current by only ∼45%, but almost abolished first‐phase insulin secretion and resulted in systemic glucose intolerance. These effects did not correlate with any major effects on intracellular Ca2+ handling and glucose‐induced electrical activity. However, high‐resolution capacitance measurements of exocytosis in single β cells revealed that the loss of first‐phase insulin secretion in the βCav1.2−/− mouse was associated with the disappearance of a rapid component of exocytosis reflecting fusion of secretory granules physically attached to the Cav1.2 channel. Thus, the conduit of Ca2+ entry determines the ability of the cation to elicit secretion.

Delay between fusion pore opening and peptide release from large dense-core vesicles in neuroendocrine cells.

Peptidergic neurotransmission is slow compared to that mediated by classical neurotransmitters. We have studied exocytotic membrane fusion and cargo release by simultaneous capacitance measurements and confocal imaging of single secretory vesicles in neuroendocrine cells. Depletion of the readily releasable pool (RRP) correlated with exocytosis of 10%-20% of the docked vesicles. Some remaining vesicles became releasable after recovery of RRP. Expansion of the fusion pore, seen as an increase in luminal pH, occurred after approximately 0.3 s, and peptide release was delayed by another 1-10 s. We conclude that (1) RRP refilling involves chemical modification of vesicles already in place, (2) the release of large neuropeptides via the fusion pore is negligible and only proceeds after complete fusion, and (3) sluggish peptidergic transmission reflects the time course of vesicle emptying.

Regulated exocytosis of GABA-containing synaptic-like microvesicles in pancreatic beta-cells.

We have explored whether γ-aminobutyric acid (GABA) is released by regulated exocytosis of GABA-containing synaptic-like microvesicles (SLMVs) in insulin-releasing rat pancreatic β-cells. To this end, β-cells were engineered to express GABAA-receptor Cl−-channels at high density using adenoviral infection. Electron microscopy indicated that the average diameter of the SLMVs is 90 nm, that every β-cell contains ∼3,500 such vesicles, and that insulin-containing large dense core vesicles exclude GABA. Quantal release of GABA, seen as rapidly activating and deactivating Cl−-currents, was observed during membrane depolarizations from −70 mV to voltages beyond −40 mV or when Ca2+ was dialysed into the cell interior. Depolarization-evoked GABA release was suppressed when Ca2+ entry was inhibited using Cd2+. Analysis of the kinetics of GABA release revealed that GABA-containing vesicles can be divided into a readily releasable pool and a reserve pool. Simultaneous measurements of GABA release and cell capacitance indicated that exocytosis of SLMVs contributes ∼1% of the capacitance signal. Mathematical analysis of the release events suggests that every SLMV contains 0.36 amol of GABA. We conclude that there are two parallel pathways of exocytosis in pancreatic β-cells and that release of GABA may accordingly be temporally and spatially separated from insulin secretion. This provides a basis for paracrine GABAergic signaling within the islet.

Beta-cell specific deletion of Dicer1 leads to defective insulin secretion and diabetes mellitus.

Mature microRNAs (miRNAs), derived through cleavage of pre-miRNAs by the Dicer1 enzyme, regulate protein expression in many cell-types including cells in the pancreatic islets of Langerhans. To investigate the importance of miRNAs in mouse insulin secreting β-cells, we have generated mice with a β-cells specific disruption of the Dicer1 gene using the Cre-lox system controlled by the rat insulin promoter (RIP). In contrast to their normoglycaemic control littermates (RIP-Cre+/− Dicer1 Δ/wt), RIP-Cre+/− Dicer1flox/flox mice (RIP-Cre Dicer1 Δ/Δ) developed progressive hyperglycaemia and full-blown diabetes mellitus in adulthood that recapitulated the natural history of the spontaneous disease in mice. Reduced insulin gene expression and concomitant reduced insulin secretion preceded the hyperglycaemic state and diabetes development. Immunohistochemical, flow cytometric and ultrastructural analyses revealed altered islet morphology, marked decreased β-cell mass, reduced numbers of granules within the β-cells and reduced granule docking in adult RIP-Cre Dicer1 Δ/Δ mice. β-cell specific Dicer1 deletion did not appear to disrupt fetal and neonatal β-cell development as 2-week old RIP-Cre Dicer1 Δ/Δ mice showed ultrastructurally normal β-cells and intact insulin secretion. In conclusion, we have demonstrated that a β-cell specific disruption of the miRNAs network, although allowing for apparently normal β-cell development, leads to progressive impairment of insulin secretion, glucose homeostasis and diabetes development.

Corelease and differential exit via the fusion pore of GABA, serotonin, and ATP from LDCV in rat pancreatic beta cells

The release of γ-aminobutyric acid (GABA) and ATP from rat β cells was monitored using an electrophysiological assay based on overexpression GABAA or P2X2 receptor ion channels. Exocytosis of LDCVs, detected by carbon fiber amperometry of serotonin, correlated strongly (∼80%) with ATP release. The increase in membrane capacitance per ATP release event was 3.4 fF, close to the expected capacitance of an individual LDCV with a diameter of 0.3 μm. ATP and GABA were coreleased with serotonin with the same probability. Immunogold electron microscopy revealed that ∼15% of the LDCVs contain GABA. Prespike “pedestals,” reflecting exit of granule constituents via the fusion pore, were less frequently observed for ATP than for serotonin or GABA and the relative amplitude (amplitude of foot compared to spike) was smaller: in some cases the ATP-dependent pedestal was missing entirely. An inward tonic current, not dependent on glucose and inhibited by the GABAA receptor antagonist SR95531, was observed in β cells in clusters of islet cells. Noise analysis indicated that it was due to the activity of individual channels with a conductance of 30 pS, the same as expected for individual GABAA Cl− channels with the ionic gradients used. We conclude that (a) LDCVs accumulate ATP and serotonin; (b) regulated release of GABA can be accounted for by exocytosis of a subset of insulin-containing LDCVs; (c) the fusion pore of LDCVs exhibits selectivity and compounds are differentially released depending on their chemical properties (including size); and (d) a glucose-independent nonvesicular form of GABA release exists in β cells.

GABAB-receptor activation inhibits exocytosis in rat pancreatic {beta}-cells by G-protein-dependent activation of calcineurin.

We have investigated the regulation of hormone secretion from rat pancreatic islets by the GABAB receptors (GABABRs). Inclusion of the specific GABABR antagonist CGP 55845 in the extracellular medium increased glucose‐stimulated insulin secretion 1.6‐fold but did not affect the release of glucagon and somatostatin. Conversely, addition of the GABABR agonist baclofen inhibited glucose‐stimulated insulin secretion by ∼60%. Using RT‐PCR, transcription of GABABR1a‐c,f and GABABR2 subunits was detected in β‐cells. Measurements of membrane currents and cell capacitance were applied to single β‐cells to investigate the mechanisms by which GABABR activation inhibits insulin secretion. In perforated‐patch measurements, baclofen inhibited exocytosis elicited by 500‐ms voltage‐clamp depolarizations to 0 mV by ≤ 80% and voltage‐gated Ca2+ entry by only ∼30%. Both effects were concentration‐dependent with IC50 values of ∼2 μm. The inhibitory action of baclofen was abolished in the presence of CGP 55845. The ability of baclofen to suppress exocytosis was prevented by pre‐treatment with pertussis toxin and by inclusion of GDPβS in the intracellular medium, and became irreversible in the presence of GTPγS as expected for a process involving inhibitory G‐proteins (Gi/o‐proteins). The inhibitory effect of baclofen resulted from activation of the serine/threonine protein phosphatase calcineurin and pre‐treatment with cyclosporin A or intracellular application of calcineurin autoinhibitory peptide abolished the effect. Addition of baclofen had no effect on [Ca2+]i and electrical activity in glucose‐stimulated β‐cells. These data indicate that GABA released from β‐cells functions as an autocrine inhibitor of insulin secretion in pancreatic islets and that the effect is principally due to direct suppression of exocytosis.

Pleiotropic Effects of GIP on Islet Function Involve Osteopontin

OBJECTIVE The incretin hormone GIP (glucose-dependent insulinotropic polypeptide) promotes pancreatic β-cell function by potentiating insulin secretion and β-cell proliferation. Recently, a combined analysis of several genome-wide association studies (Meta-analysis of Glucose and Insulin-Related Traits Consortium [MAGIC]) showed association to postprandial insulin at the GIP receptor (GIPR) locus. Here we explored mechanisms that could explain the protective effects of GIP on islet function. RESEARCH DESIGN AND METHODS Associations of GIPR rs10423928 with metabolic and anthropometric phenotypes in both nondiabetic (N = 53,730) and type 2 diabetic individuals (N = 2,731) were explored by combining data from 11 studies. Insulin secretion was measured both in vivo in nondiabetic subjects and in vitro in islets from cadaver donors. Insulin secretion was also measured in response to exogenous GIP. The in vitro measurements included protein and gene expression as well as measurements of β-cell viability and proliferation. RESULTS The A allele of GIPR rs10423928 was associated with impaired glucose- and GIP-stimulated insulin secretion and a decrease in BMI, lean body mass, and waist circumference. The decrease in BMI almost completely neutralized the effect of impaired insulin secretion on risk of type 2 diabetes. Expression of GIPR mRNA was decreased in human islets from carriers of the A allele or patients with type 2 diabetes. GIP stimulated osteopontin (OPN) mRNA and protein expression. OPN expression was lower in carriers of the A allele. Both GIP and OPN prevented cytokine-induced reduction in cell viability (apoptosis). In addition, OPN stimulated cell proliferation in insulin-secreting cells. CONCLUSIONS These findings support β-cell proliferative and antiapoptotic roles for GIP in addition to its action as an incretin hormone. Identification of a link between GIP and OPN may shed new light on the role of GIP in preservation of functional β-cell mass in humans.

Antiquity of the South Atlantic Anomaly and evidence for top-down control on the geodynamo

The dramatic decay of dipole geomagnetic field intensity during the last 160 years coincides with changes in Southern Hemisphere (SH) field morphology and has motivated speculation of an impending reversal. Understanding these changes, however, has been limited by the lack of longer-term SH observations. Here we report the first archaeomagnetic curve from southern Africa (ca. 1000–1600 AD). Directions change relatively rapidly at ca. 1300 AD, whereas intensities drop sharply, at a rate greater than modern field changes in southern Africa, and to lower values. We propose that the recurrence of low field strengths reflects core flux expulsion promoted by the unusual core–mantle boundary (CMB) composition and structure beneath southern Africa defined by the African large low shear velocity province (LLSVP). Because the African LLSVP and CMB structure are ancient, this region may have been a steady site for flux expulsion, and triggering of geomagnetic reversals, for millions of years.

Regulation of Pancreatic Beta Cell Stimulus-Secretion Coupling by microRNAs

Increased blood glucose after a meal is countered by the subsequent increased release of the hypoglycemic hormone insulin from the pancreatic beta cells. The cascade of molecular events encompassing the initial sensing and transport of glucose into the beta cell, culminating with the exocytosis of the insulin large dense core granules (LDCVs) is termed “stimulus-secretion coupling.” Impairment in any of the relevant processes leads to insufficient insulin release, which contributes to the development of type 2 diabetes (T2D). The fate of the beta cell, when exposed to environmental triggers of the disease, is determined by the possibility to adapt to the new situation by regulation of gene expression. As established factors of post-transcriptional regulation, microRNAs (miRNAs) are well-recognized mediators of beta cell plasticity and adaptation. Here, we put focus on the importance of comprehending the transcriptional regulation of miRNAs, and how miRNAs are implicated in stimulus-secretion coupling, specifically those influencing the late stages of insulin secretion. We suggest that efficient beta cell adaptation requires an optimal balance between transcriptional regulation of miRNAs themselves, and miRNA-dependent gene regulation. The increased knowledge of the beta cell transcriptional network inclusive of non-coding RNAs such as miRNAs is essential in identifying novel targets for the treatment of T2D.