Before considering the mechanisms of action underlying incretin-induced insulin secretion, it is important to understand the basic cellular physiology underlying glucose-induced insulin secretion in β-cells. The details of the regulation of insulin secretion by glucose. Briefly, glucose enters β-cells via facilitated transport (Glut transporters), where it is metabolized, and adenosine triphosphate (ATP) is generated. The ensuing increase in the intracellular ATP/adenosine diphosphate (ADP) ratio causes inhibition of ATP-sensitive potassium channels (KATP).
Potassium efflux through KATP channels normally keeps the β-cell membrane polarized (negative resting voltage); thus, when KATP channels are inhibited, the cell membrane is depolarized (moves toward a neutral or positive resting voltage) in the immediate vicinity of the KATP channels. This depolarization activates voltage-dependent calcium channels (VDCCs), allowing calcium to enter the cell. Calcium entry leads to insulin secretory vesicle exocytosis and insulin release. Under normal circumstances, delayed rectifier voltagedependent potassium channels (Kv) then open, allowing potassium to leave the cell. This efflux repolarizes the cell membrane and halts the insulin release.
The cellular and molecular mechanisms by which GLP-1 and GIP elicit insulin secretion overlap considerably and include (see Fig. 4.2) the following:
KATP Channel Modulation. Both GLP-1 and GIP bind to G protein-coupled receptors and activate adenylate cyclase, which catalyzes the conversion of ATP to the cellular second messenger 3′–5′ cyclic adenosine monophosphate (cAMP). These initial steps begin a series of cellular actions by which GLP-1 and GIP are thought to exert insulinotropic effects. The first downstream mechanism involves modulation of KATP channels. A number of groups have shown that both GLP-1 and GIP cause closure of KATP channels. As described above, inhibition of KATP channels facilitates membrane depolarization which induces downstream insulin release. The mechanism underlying the effect on KATP channels is thought to involve. cAMP-dependent protein kinase (PKA); inhibition of PKA reverses the effects of both incretin hormones on KATP channels (but see also Suga and colleagues). Furthermore, in mouse models with a genetic mutation causing an absence of KATP channels, GLP-1- and GIP-induced insulin secretion is diminished. These results provide further evidence that KATP channel modulation represents an important component of incretin-induced insulin secretion. Interestingly, GLP-1 action at the KATP channel may play an important role in the glucose-dependence of GLP-1-dependent insulin secretion. In the absence of elevated glucose concentrations, GLP-1 cannot inhibit KATP channels enough to affect exocytosis. However, when GLP-1 is administered with a sulfonylurea, which directly inhibits KATP channels in a glucose-independent manner, GLP-1 dependent insulin secretion is augmented. This effect – uncoupling the glucose dependence of GLP-1 – has consequences in GLP- 1-based therapy.
- Calcium Efflux Through VDCCs. Gromada and colleagues have demonstrated that GLP-1 and GIP administration also increase VDCC activity, leading to increased calcium entry into β-cells and insulin exocytosis. As with KATP channel effects, PKA activation appears to underlie the effects on VDCC current changes.
- Kv Channel Modulation. As described above, Kv channels are integral in restoring cell membrane potential following depolarization and thereby limiting calcium entry and further exocytosis of insulin-containing granules. GLP-1 receptor activation has been shown to inhibit Kv channel currents by approximately 40% in rat pancreatic β-cells. GIP has been reported to have similar effects on Kv channel currents. Thus, inhibiting Kv channel currents may lead to prolonged exocytosis. The effects on Kv channels appear to be dependent on PKA signaling as well as the phosphatidylinositol kinase pathway. In addition to effects on Kv channel currents, GIP has also been reported to affect cell surface expression and modulation of Kv channels.
- Intracellular Calcium Stores. In addition to the direct and indirect effects that GLP-1 and GIP have on calcium entry into the cell through VDCCs, additional calcium is released from intracellular stores such as the endoplasmic reticulum (ER). This process is thought to be dependent on converging intracellular signals. For example, GLP-1-stimulated PKA and cAMP-regulated guanine nucleotide exchange factor-II (Epac, also termed cAMP-GEFII) sensitize calcium channels in the ER. Intracellular calcium release is then initiated by the transient increase in calcium entering the cell through VDCCs, the net result being a further increase in intracellular calcium as well as a wider spatial distribution of intracellular calcium. GIP has been reported to have similar effects. The entire process, termed “calcium-induced calcium release,” is thought to contribute to exocytosis of insulin granules located in subcellular regions not located in the immediate vicinity of the VDCCs.7,106,107 Thus, calcium-induced calcium release may play a prominent role in the postprandial state, allowing for an even greater incretin-induced insulin secretory response.
- Mitochondrial ATP synthesis. In addition to stimulating exocytosis, the increase in calcium-induced intracellular calcium release described above has also been shown to affect mitochondrial ATP production. Amplified ATP production may lead to further effects on KATP channels in a feed-forward manner.
- cAMP-associated Insulin Granule Mobilization. The insulinotropic activity of GLP-1 results in part from calcium influx through VDCCs (described above). However, only a small fraction of insulin-containing granules (less than 1%109) belong to what is termed the “readily releasable pool,” meaning that they are located close enough to VDCCs that they undergo exocytosis soon after VDCC opening. The remaining insulin-containing granules must be “primed” by series of cellular steps involving cAMP, calcium, and both PKA and Epac. These steps involve granule mobilization (via PKA) and increases in the size of granules (via Epac2), both processes which are influenced by GLP-1 and GIP signaling. The increased availability of insulin-containing granules for exocytosis has been estimated to account for as much as 70% of the insulinotropic activity of GLP-1 and GIP.
- Insulin Biosynthesis. In addition to effecting acute changes in insulin release, both GLP-1 and GIP simulate
insulin synthesis and gene transcription in β-cells. This process ensures that adequate insulin remains
available for secretion. Moreover, GLP-1 has been shown to upregulate the transcription of genes involved
in insulin secretion.
Fig. 4.2 Schematic of intracellular mechanisms of action underlying incretin-induced insulin secretion. GLP-1 receptor activation leads cAMP generation and PKA activation, leading to (1) inhibition of KATP channels, which depolarizes β-cells leading to increased excitability and downstream insulin release; (2) increased VDCC activity, leading to an increase in intracellular calcium; (3) inhibition of Kv channels, delaying repolarization and extending β-cell excitability. (4) Additional calcium is released from intracellular stores via PKA, EPAC2, and calcium entry through VDCCs. (5) Intracellular calcium increases stimulate mitochondrial ATP production, increasing the ATP/ADP ratio, and leading to additional effects on KATP channels. (6) Multiple intracellular steps involving PKA, EPAC2, calcium, and cAMP lead to the priming and mobilization of insulin granules for release. (7) Receptor activation leads to new insulin synthesis as well as increases in the transcription of genes involved in insulin synthesis
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