Factors from the mitochondria that induce insulin secretion

Insulin secretion and metabolism

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Factors from the Mitochondria that induce insulin secretion

The main effector from mitochondrial respiration on insulin secretion is ATP. Mitochondrial oxidative metabolism has been estimated to produce 98% of β-cell ATP (Erecinska et al., 1992). The increase in glucose-stimulated ATP production increases the ATP/ADP ratio, closing the ATP-dependent K⁺ channel (K_ATP) channel in the β-cell leading to membrane depolarization (Aguilar-Bryan & Bryan, 1999;Detimary et al., 1996;Civelek et al., 1996;Gembal et al., 1992;Kakei et al., 1986;Newgard & McGarry, 1995). K_ATP channels have been found in pancreatic β-cells and in low levels in pancreatic a-cells (Ashcroft & Rorsman, 1989;Suzuki et al., 1997). Elevated ATP levels displace bound ADP on K_ATP channels resulting in channel closure in normal β-cells (Ashcroft & Rorsman, 1989;Arkhammar et al., 1987). The pancreatic β-cell K_ATP channels consist of four sulphonylurea receptor-1 (SUR1) subunits and four Kir6.2 subunits. The pore of the channel is formed by the inward rectifier potassium channel (Kir6.2) subunits, which allow K⁺ ions to flow through the membrane (Miki et al., 1999). ATP likely acts on the Kir6.2 subunit to inhibit channel activity, whereas, MgADP, sulfonylureas and diazoxide act primarily on the SUR1 subunit (Miki et al., 1999). Kir6.2 knockout mice have shown that this subunit is important for glucose-induced and sulfonylurea-induced insulin secretion (Miki et al., 1997;Miki et al., 1998). SUR1 knockout mouse pancreatic β-cells lack K_ATP channels altogether and have spontaneous Ca²⁺ action potentials equivalent to those seen in patients with persistent hyperinsulinemic hypoglycemia of infancy (PHHI) (Miki et al., 1999).

The exact role of cytosolic or mitochondrial ATP production in K_ATP channel function is controversial, because some investigators have suggested that cytosolic ATP production takes precedence in K_ATP channel regulation in β-cells (Mertz et al., 1996), like that seen in cardiac muscle (Weiss & Lamp, 1989). Support for mitochondrial ATP production in regulating K_ATP channel activity and GSIS in β-cells is that a-ketoisocaproic acid, which is entirely metabolized by mitochondria in the TCA cycle, can inactivate K_ATP channels (Ashcroft & Ashcroft, 1990) and stimulate insulin secretion (Best, 1997). In addition, patients with mitochondrial DNA mutations exhibit impaired b-cell function and overt diabetes (Gerbitz et al., 1996;Maechler & Wollheim, 2001) and blockade of mitochondrial metabolism can inhibit GSIS (Best, 1997). Oxygen consumption is increased when glucose concentrations are elevated (Longo et al., 1991) and insulin secretion is increased by exposing islets to an increase in concentrations of oxygen (Longo et al., 1991;Gerbitz et al., 1996). These pieces of evidence suggest that mitochondrial glucose metabolism is a key player regulating K_ATP channel activity and insulin secretion in pancreatic β-cells.

Closure of the K_ATP channel leads to membrane depolarization and activation of voltage-dependent Ca²⁺channels, increasing the concentration of cytosolic Ca²⁺ (Prentki & Matschinsky, 1987;Ashcroft & Rorsman, 1989;Misler et al., 1992;Henquin, 1987). Elevated cytosolic Ca²⁺ leads to stimulation of exocytosis of insulin (Mariot et al., 1998;Gilon & Henquin, 1992), but the Ca²⁺ signal alone is not sufficient for sustained secretion because under clamped cytosolic Ca²⁺ concentrations, glucose can still elicit insulin secretion (Maechler et al., 1997). This suggests that a mitochondrial messenger must exist which is distinct from ATP that is involved in stimulation of insulin secretion (Maechler et al., 1997;Maechler et al., 1998). These factors include glutamate, malonyl CoA, long-chain acyl CoAs (LC-CoA), and/or NADPH (Figure 3).

Glutamate may be this mitochondrial factor (Maechler & Wollheim, 1999;Maechler et al., 1997). Glutamate is formed in the mitochondria from a-ketoglutarate, a TCA cycle intermediate, by glutamate dehydrogenase (Fisher, 1985). Glutamate has been shown to directly stimulate insulin exocytosis (Maechler & Wollheim, 1999). Insulin secretory granule glutamate uptake is likely involved, as inhibitors of vesicular glutamate transport (Ozkan & Ueda, 1998;Roseth et al., 1995) suppress the glutamate-evoked exocytosis (Maechler & Wollheim, 1999). It has been hypothesized (Maechler & Wollheim, 1999) that glutamate uptake would render the insulin granules secretion competent. Glutamate uptake could possibly reduce granular membrane potential (Maechler & Wollheim, 1999;Maycox et al., 1988), or possibly, promote Ca²⁺ uptake into the granules, as Ca²⁺ depletion of this compartment inhibits insulin exocytosis (Scheenen et al., 1998). Ca²⁺-evoked exocytosis in permeabilized mast cells is strictly dependent on the presence of glutamate (Churcher & Gomperts, 1990). Glutamate could also result in granule swelling, enabling the granule to fuse with the plasma membrane, a phenomenon described in zymogen granules (Jena et al., 1997).

Prentki, Corkey and co-workers have proposed that long-chain acyl CoA may be a potential modulator of insulin secretion (Corkey et al., 1989;Prentki et al., 1992). This model holds that GSIS in part via its metabolism to citrate in the mitochondria, which is then transported out and is metabolized to malonyl CoA in the cytosol. Malonyl CoA then inhibits carnitine palmitoyltransferase I (CPT I), a key regulatory enzyme of fatty acid oxidation. Inhibition of CPTI leads to an increase in cytosolic long-chain acyl CoAs (LC-acyl CoA). The elevation of LC-acyl CoAs could potentiate insulin secretion by direct modulation of the K_ATP channel or by their conversion to other bioactive metabolites such as inositol trisphophate (IP₃) or diacylglycerol (DAG), or by direct acylation of regulatory proteins (Deeney et al., 2000). Correlative experiments have provided support for this model (Corkey et al., 1989;Prentki et al., 1992;Deeney et al., 2000;Chen et al., 1994). However this mechanism is still controversial since other studies have shown that there is no alteration in GSIS after the link between glucose and lipid metabolism has been directly perturbed (Antinozzi et al., 1998).

NADPH is another potential signaling molecule derived from glucose metabolism that could be a direct or an indirect mitochondrial-derived factor that modulates insulin secretion. The model was proposed by Newgard’s group (Lu et al., 2002) found that application of ¹³C NMR analysis to newly developed INS-1 derived cell lines and provides an alternate hypothesis in which NADPH modulates insulin secretion (Lu et al., 2002). This hypothesis involves recycling of pyruvate across the mitochondrial inner membrane, which leads to the generation of NADPH. Pyruvate recycling involves one key pathway called the pyruvate-malate shuttle system for NADPH production in the cytosol of cells. The first step of this pathway involves a TCA cycle anaplerotic step where pyruvate is converted to oxaloacetate via the pyruvate carboxylase (PC) reaction. This anaplerotic step is a key step in β-cells since 40% of all pyruvate in mitochondria enters the PC reaction (Khan et al., 1996). Oxaloacetate is part of the TCA cycle and has several potential fates in mitochondria. Oxaloacetate exists in the mitochondria mainly as malate. Oxaloacetate is converted to malate by the TCA cycle enzyme mitochondrial malate dehydrogenase (MDH_m). Oxaloacetate after its conversion to malate, can participate in a number of pathways including the malate-aspartate shuttle (a NADH shuttle) or the pyruvate-malate shuttle (a NADPH production pathway). In the pyruvate-malate shuttle system malate is transported out of the mitochondria via a malate-P_i antiporter and then converted to pyruvate via malic enzyme resulting also in the generation of CO₂ and NADPH. Pyruvate can then be transported back into the mitochondria via a pyruvate-H⁺ symporter. Pyruvate can then reenter the cycle. The pyruvate recycling pathway exists in β-cells (MacDonald, 1995;Lu et al., 2002). An alternate pathway leading to an increase in cytosolic malate concentration is via the malate-aspartate shuttle. MacDonald has provided support for this pathway showing that NADPH could potentially modulate insulin secretion inhibiting voltage-dependent K⁺ channels (MacDonald et al., 2003).

Electrical excitability, exocytosis and insulin secretion

In the absence of stimulatory glucose b-cells are electrically silent (Matthews & Sakamoto, 1975;Meissner & Schmelz, 1974). Inhibition of the K_ATP channel by an increase in the ATP/ADP ratio results in membrane depolarization and initiation of β-cell electrical activity. In mouse and human islets this electrical activity is characterized by slow wave depolarization with superimposed bursts of action potentials (Matthews & Sakamoto, 1975;Dean & Matthews, 1970). Action potentials in the b-cell are composed primarily of Ca²⁺ currents controlled by L-type voltage-dependent Ca²⁺ channels (VDCC).

Exocytosis is highly regulated by the molecular machinery broadly known as soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins (Bratanova-Tochkova et al., 2002). The SNARE complex is composed of two groups of SNARE proteins, those associated with the plasma membrane and those associated with the secretory vesicle (Sollner et al., 1993). SNARE proteins in pancreatic β-cells (Wheeler et al., 1996), both plasma membrane and vesicle bound SNAREs, are involved in docking the secretory vesicle to the plasma membrane (Pasyk et al., 2004). Increased cytosolic Ca²⁺ activates a Ca²⁺-sensitive protein in the SNARE complex called synaptotagmin (Mackler et al., 2002) leading to SNARE-mediated fusion of the secretatory vesicle with the plasma membrane and thus insulin secretion from the β-cell.