Susanne G. Straub

Glucose-stimulated signaling pathways in biphasic insulin secretion.

Glucose‐stimulated biphasic insulin secretion involves at least two signaling pathways, the KATP channel‐dependent and KATP channel‐independent pathways, respectively. In the former, enhanced glucose metabolism increases the cellular adenosine triphosphate/adenosine diphosphate (ATP/ADP) ratio, closes KATP channels and depolarizes the cell. Activation of voltage‐dependent Ca2+ channels increases Ca2+ entry and [Ca2+]i and stimulates insulin release. The KATP channel‐independent pathways augment the response to increased [Ca2+]i by mechanisms that are currently unknown. However, they affect different pools of insulin‐containing granules in a highly coordinated manner. The β‐cell granule pools can be minimally described as reserve, morphologically docked, readily and immediately releasable. Activation of the KATP channel‐dependent pathway results in exocytosis of an immediately releasable pool that is responsible for the first phase of glucose‐stimulated insulin release. Following glucose metabolism, the rate‐limiting step for the first phase lies in the rate of signal transduction between sensing the rise in [Ca2+]i and exocytosis of the immediately releasable granules. The immediately releasable pool of granules can be enlarged by previous exposure to glucose (by time‐dependent potentiation, TDP), and by second messengers such as cyclic adenosine monophosphate (cyclic AMP) and diacylglycerol (DAG). The second phase of glucose‐stimulated insulin secretion is due mainly to the KATP channel‐independent pathways acting in synergy with the KATP channel‐dependent pathway. The rate‐limiting step here is the conversion of readily releasable granules to the state of immediate releasability, following which, in an activated cell they will undergo exocytosis. In the rat and human β‐cell the KATP channel‐independent pathways induce a time‐dependent increase in the rate of this step that results in the typical rising second‐phase response. In the mouse β‐cell the rate appears not to be changed much by glucose. Potential intermediates involved in controlling the rate‐limiting step include increases in cytosolic long‐chain acyl‐CoA levels, adenosine triphosphate (ATP) and guanosine triphosphate (GTP), DAG binding proteins, including some isoforms of protein kinase (PKC), and protein acyl transferases. Agonists that can change the rate‐limiting steps for both phases of insulin release include those like glucagon‐like peptide 1 (GLP‐1) that raise cyclic AMP levels and those like acetylcholine that act via DAG. Copyright

A Wortmannin-sensitive Signal Transduction Pathway Is Involved in the Stimulation of Insulin Release by Vasoactive Intestinal Polypeptide and Pituitary Adenylate Cyclase-activating Polypeptide

Vasoactive intestinal polypeptide (VIP), pituitary adenylate cyclase-activating polypeptide-27 (PACAP-27), and PACAP-38 stimulated insulin release with EC values of 0.15, 0.15, and 0.06 nM respectively, as expected for the VIP2/PACAP3 receptor subtype. Secretion was stimulated promptly and peaked at 6-10 min. At 30 min, the secretion rate was still 2-3-fold higher than the control rate. The peptides increased cyclic AMP and [Ca] transiently so that at 30 min they had returned to control values. Therefore, an additional signal is required to explain the prolonged stimulation of release. The prolonged effects, but not the acute effects of VIP and PACAP on insulin release were inhibited by low concentrations of wortmannin, a phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor. While wortmannin inhibited PI 3-kinase activity in cell lysates, no activation by the peptides was seen. Therefore, the wortmannin-sensitive pathway is either dependent on basal PI 3-kinase activity, or another target for wortmannin is responsible for inhibition of the peptide-stimulated secretion. It is concluded that the acute stimulation of insulin release by VIP and PACAP is mediated by increased cyclic AMP and [Ca], whereas the sustained release is mediated by a novel wortmannin-sensitive pathway.

Augmentation of Insulin Release by Glucose in the Absence of Extracellular Ca2+: New Insights Into Stimulus-Secretion Coupling

Glucose stimulates insulin secretion in the pancreatic β-cell by means of a synergistic interaction between at least two signaling pathways. One, the KATP channel-dependent pathway, increases the entry of Ca2+ through voltage-gated channels by closure of the KATP channels and depolarization of the β-cell membrane. The resulting increase in [Ca2+]i stimulates insulin exocytosis. The other, a KATP channel-independent pathway, requires that [Ca2+]i be elevated and augments the Ca2+-stimulated release. These mechanisms are in accord with the belief that glucose-stimulated insulin secretion has an essential requirement for extracellular Ca2+ and increased [Ca2+]i. However, when protein kinases A and C are activated simultaneously, a large effect of glucose to augment insulin release can be seen in the absence of extracellular Ca2+, under conditions in which [Ca2+]i is not increased, and even when [Ca2+]i is decreased to low levels by intracellular chelation with BAPTA. In the presence or absence of Ca2+, there are similarities in the characteristics of augmentation of insulin release that suggest that only one augmentation mechanism may be involved. These similarities include time course, glucose dose-responses, augmentation by nutrients other than glucose such as α-ketoisocaproate (α-KIC), and augmentation by the fatty acids palmitate and myristate. However, augmentation in the presence and absence of Ca2+ is distinctly different in GTP dependency. Therefore, exocytosis under these two conditions appears to be triggered differently—one by Ca2+ and the other by GTP or a GTPdependent mechanism. The augmentation pathways are likely responsible for time-dependent potentiation of secretion and for the second phase of glucose-stimulated insulin release.

CO2/HCO3−- and calcium-regulated soluble adenylyl cyclase as a physiological ATP sensor.

Background: The affinity of soluble adenylyl cyclase (sAC) for its substrate ATP suggested that it might be sensitive to fluctuations in ATP. Results: In sAC-overexpressing and glucose-responsive cells, sAC-generated cAMP reflects intracellular ATP levels. Conclusion: sAC can be an ATP sensor inside cells. Significance: sAC serves as a metabolic sensor via regulation by three cellular metabolites: ATP, bicarbonate, and calcium. The second messenger molecule cAMP is integral for many physiological processes. In mammalian cells, cAMP can be generated from hormone- and G protein-regulated transmembrane adenylyl cyclases or via the widely expressed and structurally and biochemically distinct enzyme soluble adenylyl cyclase (sAC). sAC activity is uniquely stimulated by bicarbonate ions, and in cells, sAC functions as a physiological carbon dioxide, bicarbonate, and pH sensor. sAC activity is also stimulated by calcium, and its affinity for its substrate ATP suggests that it may be sensitive to physiologically relevant fluctuations in intracellular ATP. We demonstrate here that sAC can function as a cellular ATP sensor. In cells, sAC-generated cAMP reflects alterations in intracellular ATP that do not affect transmembrane AC-generated cAMP. In β cells of the pancreas, glucose metabolism generates ATP, which corresponds to an increase in cAMP, and we show here that sAC is responsible for an ATP-dependent cAMP increase. Glucose metabolism also elicits insulin secretion, and we further show that sAC is necessary for normal glucose-stimulated insulin secretion in vitro and in vivo.

Time-resolved metabolomics analysis of β-cells implicates the pentose phosphate pathway in the control of insulin release

Insulin secretion is coupled with changes in β-cell metabolism. To define this process, 195 putative metabolites, mitochondrial respiration, NADP+, NADPH and insulin secretion were measured within 15 min of stimulation of clonal INS-1 832/13 β-cells with glucose. Rapid responses in the major metabolic pathways of glucose occurred, involving several previously suggested metabolic coupling factors. The complexity of metabolite changes observed disagreed with the concept of one single metabolite controlling insulin secretion. The complex alterations in metabolite levels suggest that a coupling signal should reflect large parts of the β-cell metabolic response. This was fulfilled by the NADPH/NADP+ ratio, which was elevated (8-fold; P<0.01) at 6 min after glucose stimulation. The NADPH/NADP+ ratio paralleled an increase in ribose 5-phosphate (>2.5-fold; P<0.001). Inhibition of the pentose phosphate pathway by trans-dehydroepiandrosterone (DHEA) suppressed ribose 5-phosphate levels and production of reduced glutathione, as well as insulin secretion in INS-1 832/13 β-cells and rat islets without affecting ATP production. Metabolite profiling of rat islets confirmed the glucose-induced rise in ribose 5-phosphate, which was prevented by DHEA. These findings implicate the pentose phosphate pathway, and support a role for NADPH and glutathione, in β-cell stimulus-secretion coupling.

Noradrenaline inhibits exocytosis via the G protein βγ subunit and refilling of the readily releasable granule pool via the αi1/2 subunit

The molecular mechanisms responsible for the ‘distal’ effect by which noradrenaline (NA) blocks exocytosis in the β‐cell were examined by whole‐cell and cell‐attached patch clamp capacitance measurements in INS 832/13 β‐cells. NA inhibited Ca2+‐evoked exocytosis by reducing the number of exocytotic events, without modifying vesicle size. Fusion pore properties also were unaffected. NA‐induced inhibition of exocytosis was abolished by a high level of Ca2+ influx, by intracellular application of antibodies against the G protein subunit Gβ and was mimicked by the myristoylated βγ‐binding/activating peptide mSIRK. NA‐induced inhibition was also abolished by treatment with BoNT/A, which cleaves the C‐terminal nine amino acids of SNAP‐25, and also by a SNAP‐25 C‐terminal‐blocking peptide containing the BoNT/A cleavage site. These data indicate that inhibition of exocytosis by NA is downstream of increased [Ca2+]i and is mediated by an interaction between Gβγ and the C‐terminus of SNAP‐25, as is the case for inhibition of neurotransmitter release. Remarkably, in the course of this work, a novel effect of NA was discovered. NA induced a marked retardation of the rate of refilling of the readily releasable pool (RRP) of secretory granules. This retardation was specifically abolished by a Gαi1/2 blocking peptide demonstrating that the effect is mediated via activation of Gαi1 and/or Gαi2.

Progesterone inhibits insulin secretion by a membrane delimited, non-genomic action.

In rat islets, progesterone caused a prompt concentration-dependent inhibition of glucose-stimulated insulin release with an IC50 of 10 μM at 8.4 mM glucose. The inhibition was specific since both testosterone and 17β-estradiol had no such effect. The degree of inhibition was similar in islets from male and female rats. The inhibition was not blocked in PTX-treated islets thus ruling out the Gi/Go proteins as mediators of the inhibition. Progesterone inhibited both glucose- and BayK-8644-stimulated insulin secretion in HIT-T15 cells and the IC50 vs. 10 mM glucose was also 10 μM. There was no effect on intracellular cyclic AMP concentration in the presence 0.2 and 10 mM glucose. Progesterone decreased [Ca2+]i under all conditions tested. The decrease in [Ca2+]i was due to blockade of the L-type voltage-dependent Ca2+ channels. Under Ca2+-free conditions, progesterone did not inhibit the stimulation of insulin release due to the combination of glucose, phorbol ester and forskolin. Thus blockade of Ca2+ entry appears to be the sole mechanism by which progesterone inhibits insulin release. As progesterone covalently linked to albumin had a similar inhibitory effect as progesterone itself, it is concluded that the steroid acts at the outer surface of the β-cell plasma membrane. These effects would be classified as either AI or AIIb in the Mannheim classification of nongenomically initiated steroid actions.

Both Gi and Go Heterotrimeric G Proteins Are Required to Exert the Full Effect of Norepinephrine on the β-Cell KATP Channel

The effects of norepinephrine (NE), an inhibitor of insulin secretion, were examined on membrane potential and the ATP-sensitive K+ channel (KATP) in INS 832/13 cells. Membrane potential was monitored under the whole cell current clamp mode. NE hyperpolarized the cell membrane, an effect that was abolished by tolbutamide. The effect of NE on KATP channels was investigated in parallel using outside-out single channel recording. This revealed that NE enhanced the open activities of the KATP channels ∼2-fold without changing the single channel conductance, demonstrating that NE-induced hyperpolarization was mediated by activation of the KATP channels. The NE effect was abolished in cells preincubated with pertussis toxin, indicating coupling to heterotrimeric Gi/Go proteins. To identify the G proteins involved, antisera raised against α and β subunits (anti-Gαcommon, anti-Gβ, anti-Gαi1/2/3, and anti-Gαo) were used. Anti-Gαcommon totally blocked the effects of NE on membrane potential and KATP channels. Individually, anti-Gαi1/2/3 and anti-Gαo only partially inhibited the action of NE on KATP channels. However, the combination of both completely eliminated the action. Antibodies against Gβ had no effects. To confirm these results and to further identify the G protein subunits involved, the blocking effects of peptides containing the sequence of 11 amino acids at the C termini of the α subunits were used. The data obtained were similar to those derived from the antibody work with the additional information that Gαi3 and Gαo1 were not involved. In conclusion, both Gi and Go proteins are required for the full effect of norepinephrine to activate the KATP channel.

Mechanisms of Action of VIP and PACAP in the Stimulation of Insulin Release

VIP and PACAP stimulate insulin release by interaction with the VIP-2/PACAP-3 receptor on the beta cell. Activation of the receptor results in Gs-mediated stimulation of adenylyl cyclase and increased cellular cyclic AMP levels. Increased cyclic AMP results in a small and transient increase in [Ca2+]i, which is likely to have only a small and transient effect on the secretion rate. Cyclic AMP also potentiates insulin secretion by an as yet unknown action at a distal site. A third action of VIP and PACAP is responsible for the continued stimulation of insulin secretion after the levels of cyclic AMP and [Ca2+]i have returned to basal values. This third pathway, which is identified at present only by its sensitivity to low concentrations of wortmannin, plays a major role in the prolonged stimulation of insulin release by VIP and PACAP.

Inhibition of insulin secretion by cerulenin might be due to impaired glucose metabolism

Cerulenin, an inhibitor of protein acylation, has been used as a tool to study the potential role of protein acylation in a variety of activities in different cells, and in stimulus‐secretion coupling in pancreatic islets and clonal β‐cells.