Alfredo Moreno

A novel regulatory mechanism of the mitochondrial Ca2+ uniporter revealed by the p38 mitogen-activated protein kinase inhibitor SB202190

It is widely acknowledged that mitochondrial Ca2+ uptake modulates the cytosolic [Ca2+] ([Ca2+]c) acting as a transient Ca2+ buffer. In addition, mitochondrial [Ca2+] ([Ca2+]M) regulates the rate of respiration and may trigger opening of the permeability transition pore and start apoptosis. However, no mechanism for the physiological regulation of mitochondrial Ca2+ uptake has been described. We show here that SB202190, an inhibitor of p38 mitogen‐activated protein (MAP) kinase, strongly stimulates ruthenium red‐sensitive mitochondrial Ca2+ uptake, both in intact and in permeabilized HeLa cells. The [Ca2+]M peak induced by agonists was increased about fourfold in the presence of the inhibitor, with a concomitant reduction in the [Ca2+]c peak. The stimulation occurred fast and was rapidly reversible. In addition, experiments in permeabilized cells perfused with controlled [Ca2+] showed that SB202190 stimulated mitochondrial Ca2+ uptake by more than 10‐fold, but only in the physiological [Ca2+]c range (1–4 μM). Other structurally related p38 MAP kinase inhibitors (SB203580, PD169316, or SB220025) produced little or no effect. Our data suggest that in HeLa cells, a protein kinase sensitive to SB202190 tonically inhibits the mitochondrial Ca2+ uniporter. This novel regulatory mechanism may be of paramount importance to modulate mitochondrial Ca2+ uptake under different physiopathological conditions.

Leiurus quinquestriatus venom inhibits different kinds of Ca2+-dependent K+ channels

A minor protein component of Leiurus quinquestriatus venom has been reported to inhibit selectively the apamin-insensitive Ca2+-dependent K+ channels of mammalian skeletal muscle (Miller, C., Moczydlowski, E., Latorre, R. and Phillips, M. (1985) Nature 313, 316-318). We report the effect of the venom on both the apamin-insensitive channels of the human erythrocyte, the Ehrlich cell and the rat thymocyte and the apamin-sensitive channel of the guinea pig hepatocyte. The venom inhibited Ca2+-dependent K+ transport in all the cases with a Ki value within the range of 1 to 10 micrograms/ml, similar to that reported previously in muscle. Valinomycin-induced K+ transport was also antagonized by the venom but its sensitivity was about 1/10 as much as that of the Ca2+-dependent K+ channel.

Regulation and Genetics of Amino Acid Transport

The major transport systems for the uptake of neutral amino acids in mammalian cells have been designated A, ASC, and L.’,’ The transport systems and their regulation have been characterized in Chinese hamster ovary (CHO) System A is sodium-dependent, subject to trans-inhibition, and serves for the uptake of amino acids with short, polar, or linear side chains. System ASC is also sodium dependent and has a strong preference for alanine, serine, and cysteine. In the C H O cell, the ASC system shows a somewhat broader specificity than that found in the Ehrlich cell.* Unlike System A, System ASC does not tolerate N-methylated substrates such as 2-methylaminoisobutyric acid (MeAIB). System L is sodium-independent and serves for the uptake of branched-chain and aromatic amino acids. We operationally define the systems as follows: System A can be represented by the sodium-dependent uptake of 0.2 m M 2-arninoisobutyric acid (AIB) that is inhibited by 25 m M MeAIB; System ASC, the sodium-dependent uptake of 0.2 m M L-alanine that is not inhibited by 25 m M MeAIB; and System L, the sodium-independent uptake of 0.2 m M L-leucine that is inhibited by 10 mM 2-aminobicyclo-[2,2,1]-heptane-2-carboxylic acid (BCH).3 Although the systems have a preferred set of substrates, they do have overlapping specificities. FIGURE 1 shows the contributions of these systems to the uptake of individual amino acids in CHO-K1 cells. This overlap makes the study of transport systems in isolation difficult. The availability of mutations in one or more of the transport systems would greatly facilitate the study of the function and regulation of the transport systems. We are currently combining genetic approaches with kinetic studies using C H O cells because of the relative ease with which mutants can be obtained from these cells. Furthermore, C H O cells can be used to form interspecies hybrids with human cells.’ The hamster-human hybrid cells preferentially segregate the human chromosomes, permitting the assignment of a phenotype to a particular chromosome. In the present study, we have isolated and characterized C H O mutants defective in the regulation of System L6 and mutants with reduced System L transport activity. We have also used hamster-human hybrids to map System L transport activity to human chromosome 20.’

Wide occurrence of guanosine 5′-tetraphosphate in commercial preparations of GTP

Abstract Guanosine 5′-tetraphosphate is present in commercial preparations of GTP in proportions from 1–4.5%, apparently regardless of the type of salt, method of preparation, age of the sample or commercial source.

Leptolide Improves Insulin Resistance in Diet-Induced Obese Mice

Type 2 diabetes (T2DM) is a complex disease linked to pancreatic beta-cell failure and insulin resistance. Current antidiabetic treatment regimens for T2DM include insulin sensitizers and insulin secretagogues. We have previously demonstrated that leptolide, a member of the furanocembranolides family, promotes pancreatic beta-cell proliferation in mice. Considering the beneficial effects of leptolide in diabetic mice, in this study, we aimed to address the capability of leptolide to improve insulin resistance associated with the pathology of obesity. To this end, we tested the hypothesis that leptolide should protect against fatty acid-induced insulin resistance in hepatocytes. In a time-dependent manner, leptolide (0.1 µM) augmented insulin-stimulated phosphorylation of protein kinase B (PKB) by two-fold above vehicle-treated HepG2 cells. In addition, leptolide (0.1 µM) counteracted palmitate-induced insulin resistance by augmenting by four-fold insulin-stimulated phosphorylation of PKB in HepG2 cells. In vivo, acute intraperitoneal administration of leptolide (0.1 mg/kg and 1 mg/kg) improved glucose tolerance and insulin sensitivity in lean mice. Likewise, prolonged leptolide treatment (0.1 mg/kg) in diet-induced obese mice improved insulin sensitivity. These effects were paralleled with an ~50% increased of insulin-stimulated phosphorylation of PKB in liver and skeletal muscle and reduced circulating pro-inflammatory cytokines in obese mice. We concluded that leptolide significantly improves insulin sensitivity in vitro and in obese mice, suggesting that leptolide may be another potential treatment for T2DM.

Liver-specific ablation of insulin-degrading enzyme causes hepatic insulin resistance and glucose intolerance, without affecting insulin clearance in mice

The role of insulin-degrading enzyme (IDE), a metalloprotease with high affinity for insulin, in insulin clearance remains poorly understood. OBJECTIVE This study aimed to clarify whether IDE is a major mediator of insulin clearance, and to define its role in the etiology of hepatic insulin resistance. METHODS We generated mice with liver-specific deletion of Ide (L-IDE-KO) and assessed insulin clearance and action. RESULTS L-IDE-KO mice exhibited higher (~20%) fasting and non-fasting plasma glucose levels, glucose intolerance and insulin resistance. This phenotype was associated with ~30% lower plasma membrane insulin receptor levels in liver, as well as ~55% reduction in insulin-stimulated phosphorylation of the insulin receptor, and its downstream signaling molecules, AKT1 and AKT2 (reduced by ~40%). In addition, FoxO1 was aberrantly distributed in cellular nuclei, in parallel with up-regulation of the gluconeogenic genes Pck1 and G6pc. Surprisingly, L-IDE-KO mice showed similar plasma insulin levels and hepatic insulin clearance as control mice, despite reduced phosphorylation of the carcinoembryonic antigen-related cell adhesion molecule 1, which upon its insulin-stimulated phosphorylation, promotes receptor-mediated insulin uptake to be degraded. CONCLUSION IDE is not a rate-limiting regulator of plasma insulin levels in vivo.