Nutrient-stimulated insulin secretion is dependent upon the generation of metabolic coupling factors in the mitochondria of the pancreatic B cell.

In the pancreatic beta-cell, insulin secretion is stimulated by glucose metabolism resulting in membrane potential-dependent elevation of cytosolic Ca2+ ([Ca2+]c). This cascade involves the mitochondrial membrane potential (delta psi[m]) hyperpolarization and elevation of mitochondrial Ca2+ ([Ca2+]m) which activates the Ca(2+)-sensitive NADH-generating dehydrogenases. Metabolism-secretion coupling requires unidentified signals, other than [Ca2+]c, possibly generated by the mitochondria through the rise in [Ca2+]m. To test this paradigm, we have established an alpha-toxin permeabilized cell preparation permitting the simultaneous monitoring of [Ca2+] with mitochondrially targeted aequorin and insulin secretion under conditions of saturating [ATP] (10 mM) and of clamped [Ca2+]c at substimulatory levels (500 nM). The tricarboxylic acid (TCA) cycle intermediate succinate hyperpolarized delta psi(m), raised [Ca2+]m up to 1.5 microM and stimulated insulin secretion 20-fold, without changing [Ca2+]c. Blockade of the uniporter-mediated Ca2+ influx into the mitochondria abolished the secretory response. Moreover, glycerophosphate, which raises [Ca2+]m by hyperpolarizing delta psi(m) without supplying carbons to the TCA cycle, failed to stimulate exocytosis. Activation of the TCA cycle with citrate evoked secretion only when combined with glycerophosphate. Thus, mitochondrially driven insulin secretion at permissive [Ca2+]c requires both a substrate for the TCA cycle and a rise in [Ca2+]m. Therefore, mitochondrial metabolism generates factors distinct from Ca2+ and ATP capable of inducing insulin exocytosis.

Mitochondrial dysfunction due to alterations in the mitochondrial genome (mtDNA) has recently attracted much attention, with the finding that mutations in the mitochondrially encoded proteins perturb cell function. Several disorders have been linked to such genetic changes, including a specific diabetic phenotype. Using ethidium bromide (EtBr) that intercalates into mtDNA, we have effectively eliminated functions under the control of mtDNA from the highly differentiated INS-1 insulin-secreting cell line. We have investigated the consequences on insulin secretion, mitochondrial enzyme activity, organelle structure, and membrane polarization in such cells (INS-1 rho0). Under these conditions, the mitochondrial membrane potential fails to hyperpolarize in response to either glucose or methylsuccinate. In agreement with this finding, the morphology of the mitochondria is altered in the presence of EtBr, sharing similarities with mitochondria in which the membrane potential has been collapsed with the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). In addition, there is no effect of either nutrient secretagogue at the level of the plasma membrane potential, although the effect of the depolarizing agent KCl on membrane depolarization is completely preserved. Similarly, glucose and methylsuccinate fail to increase insulin secretion, whereas KCl is still effective. To test further the effects of mtDNA depletion on exocytosis, we permeabilized INS-1 cells with Staphylococcus aureus alpha-toxin, which forms small holes in the plasma membrane. In contrast to control cells, mitochondrial substrates were incapable of stimulating insulin secretion in mtDNA-deficient cells, emphasizing that the defect in secretion lies at the level of mitochondrial function rather than in the exocytotic process. The results indicate the paramount importance of the mitochondria in the downstream effects elicited by exposure to elevated concentrations of nutrient secretagogue.

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The ability of lithium to interfere with phosphoinositide metabolism in rat cerebral cortex slices has been examined by monitoring the accumulation of CMP-phosphatidate (CMP-PtdOH) and the reduction in Ins(1,4,5)P3 and Ins(1,3,4,5)P4 levels. A small accumulation of [14C]CMP-PtdOH was seen in slices prelabelled with [14C]cytidine and stimulated with carbachol (1 mM) or Li+ (1 mM). However, simultaneous addition of both agents for 30 min produced a 22-fold accumulation, with Li+ producing a half-maximal effect at a concentration of 0.61 +/- 0.19 mM. Kinetic studies revealed that the effects of carbachol and Li+ on CMP-PtdOH accumulation occurred with no initial lag apparent under these conditions and that preincubation with myo-inositol (10 or 30 mM) dramatically attenuated CMP-PtdOH accumulation. myo-Inositol could also attenuate the rate of accumulation of CMP-PtdOH when added 20 min after carbachol and Li+; these effects were not observed when equimolar concentrations of scyllo-inositol were added. Use of specific radioreceptor assays allowed the mass accumulations of Ins(1,4,5)P3 and Ins(1,3,4,5)P4 to be monitored. Following a lag of 5-10 min, Li+ resulted in a marked reduction in the accumulation of both inositol polyphosphates resulting from muscarinic-cholinergic stimulation. Preincubation of cerebral cortex slices with myo- (but not scyllo-) inositol delayed, but did not prevent, the reduction in the accumulation of Ins(1,4,5)P3 or Ins(1,3,4,5)P4. The results suggest that cerebral cortex, at least in vitro, is very sensitive to myo-inositol depletion under conditions of muscarinic receptor stimulation. The relationship of such depletion to the generation of inositol polyphosphate second messengers is discussed.

The role of mitochondria in the desensitization of insulin secretion was investigated. In rat pancreatic beta cells, both insulin secretion and mitochondrial [Ca 2؉ ] increases were desensitized following two challenges with the mitochondrial substrate methyl succinate. In the beta cell line INS-1, similar results were observed when a 5-min interval separated two 5-min pulses. In contrast, ATP generation monitored in luciferase-expressing INS-1 cells was stimulated to the same extent during both exposures to methyl succinate. Succinate, like ␣-glycerophosphate, activates the electron transport chain at complex II. As a consequence, the mitochondrial membrane hyperpolarizes, promoting ATP synthesis and Ca 2؉ influx into the mitochondria through the uniporter. The mitochondrial desensitization was further studied in permeabilized INS-1 cells. Increasing extramitochondrial [Ca 2؉ ] from 100 to 500 nM enhanced succinate oxidation 4-fold. At 500 nM Ca 2؉ , 1 mM succinate caused a blunted mitochondrial [Ca 2؉ ] increase upon the second, compared with the first, stimulation. These effects were mimicked by ␣-glycerophosphate, and there was cross-desensitization between the two compounds. Succinate hyperpolarized the mitochondrial membrane during both the first and second applications. This suggests that the uniporter itself, rather than the respiratory chain, is desensitized. These results emphasize the key role of the mitochondria not only in the stimulation of insulin secretion, but also in its desensitization.Desensitization is a common feature of cell biology in general and of insulin secretion in particular. However, the molecular mechanism of desensitization toward nutrient stimuli is poorly understood. Nesher and Cerasi (1) first observed that successive short stimuli with glucose or arginine in the isolated perfused rat pancreas resulted in the inhibition of the insulin secretory response to the second stimulus. Insensitivity of the pancreatic beta cell to glucose was reported in pancreata taken from hyperglycemic rats (2) and is found in several diabetic animal models (3). A reduced responsiveness of the pancreatic beta cell to glucose has also been described after prolonged exposure of beta cells to hexose in vitro (4, 5) or in human subjects (6). This desensitization phenomenon is distinguished from glucose toxicity, the latter being irreversible, whereas the former implies a reversible state of cellular refractoriness due to repeated exposures to an agonist (7). Desensitization can occur at any of the multiple steps coupling glucose recognition to insulin secretion, including the exocytotic process itself, as shown in permeabilized cells exposed to repeated Ca 2ϩ pulses (8).In the pancreatic beta cell, mitochondrial metabolism plays a pivotal role in the generation of signals coupling glucose recognition to insulin secretion (9 -13). (22,23). On the other hand, the hyperpolarization of the mitochondrial membrane exerts a negative feedback by lowering the oxygen consumption and the rate of H ϩ cycling (24, ...

The ability of lithium to interfere with the metabolism of inositol phosphates in brain may underlie its therapeutic action in manic-depressive illness. In these experiments, lithium, at therapeutic concentrations, enhanced the accumulation of [3H]inositol monophosphate but suppressed the accumulation of the putative second messengers [3H]inositol 1,4,5-trisphosphate [( 3H]Ins(1,4,5)P3) and [3H]inositol 1,3,4,5-tetrakisphosphate following stimulation of cerebral cortex slices with carbachol. Mass measurements of Ins(1,4,5)P3 showed similar inhibitory effects, which could be prevented by preincubation with myo-inositol. These data may reveal the mechanism by which lithium can reduce polyphosphoinositide-mediated neurotransmission in brain.

In 1321N1 astrocytoma cells, thrombin, but not carbachol, induces AP-1-mediated gene expression and DNA synthesis. To understand the divergent effects of these G proteincoupled receptor agonists on cellular responses, we examined Gq-dependent signaling events induced by thrombin receptor and muscarinic acetylcholine receptor stimulation. Thrombin and carbachol induce comparable changes in phosphoinositide and phosphatidylcholine hydrolysis, mobilization of intracellular Ca2', diglyceride generation, and redistribution of protein kinase C; thus, activation of these Gq-signaling pathways appears to be insufficient for gene expression and mitogenesis. Thrombin increases Ras and mitogen-activated protein kinase activation to a greater extent than carbachol in 1321N1 cells. The effects of thrombin are not mediated through Gi, since ribosylation of Gi/Go proteins by pertussis toxin does not prevent thrombin-induced gene expression or thrombin-stimulated DNA synthesis. We recently reported that the pertussis toxininsensitive G12 protein is required for thrombin-induced DNA synthesis. We demonstrate here, using transfection of receptors and G proteins in COS-7 cells, that Ga12 selectively couples the thrombin receptor to AP-1-mediated gene expression. This does not appear to result from increased mitogen-activated protein kinase activity but may reflect activation of a tyrosine kinase pathway. We suggest that preferential coupling of the thrombin receptor to G12 accounts for the selective ability of thrombin to stimulate Ras, mitogen-activated protein kinase, gene expression, and mitogenesis in 1321N1 cells.