FFA cause hepatic insulin resistance by inhibiting insulin suppression of glycogenolysis

Free fatty acids (FFA) have been shown to inhibit insulin suppression of endogenous glucose production (EGP). To determine whether this is the result of stimulation by FFA of gluconeogenesis (GNG) or glycogenolysis (GL) or a combination of both, we have determined rates of GNG and GL (with (2)H(2)O) and EGP in 16 healthy nondiabetic volunteers (11 males, 5 females) during euglycemic-hyperinsulinemic (~450 pM) clamping performed either with or without simultaneous intravenous infusion of lipid plus heparin. During insulin infusion, FFA decreased from 571 to 30 micromol/l (P < 0.001), EGP from 15.7 to 2.0 micromol x kg(-1) x min(-1) (P < 0.01), GNG from 8.2 to 3.7 micromol x kg(-1). min(-1) (P < 0.05), and GL from 7.4 to -1.7 micromol x kg(-1). min(-1) (P < 0.02). During insulin plus lipid/heparin infusion, FFA increased from 499 to 1,247 micromol/l (P < 0.001). EGP decreased 64% less than during insulin alone (-5.1 +/- 0.7 vs. -13.7 +/- 3.4 micromol x kg(-1). min(-1)). The decrease in GNG was not significantly different from the decrease of GNG during insulin alone (-2.6 vs. -4.5 micromol x kg(-1). min(-1), not significant). In contrast, GL decreased 66% less than during insulin alone (-3.1 vs. -9.2 micromol x kg(-1). min(-1), P < 0.05). We conclude that insulin suppressed EGP by inhibiting GL more than GNG and that elevated plasma FFA levels attenuated the suppression of EGP by interfering with insulin suppression of GL.     

Chk2 is a tumor suppressor that regulates apoptosis in both an ataxia telangiectasia mutated (ATM)-dependent and an ATM-independent manner

In response to ionizing radiation (IR), the tumor suppressor p53 is stabilized and promotes either cell cycle arrest or apoptosis. Chk2 activated by IR contributes to this stabilization, possibly by direct phosphorylation. Like p53, Chk2 is mutated in patients with Li-Fraumeni syndrome. Since the ataxia telangiectasia mutated (ATM) gene is required for IR-induced activation of Chk2, it has been assumed that ATM and Chk2 act in a linear pathway leading to p53 activation. To clarify the role of Chk2 in tumorigenesis, we generated gene-targeted Chk2-deficient mice. Unlike ATM(-/-) and p53(-/-) mice, Chk2(-/-) mice do not spontaneously develop tumors, although Chk2 does suppress 7,12-dimethylbenzanthracene-induced skin tumors. Tissues from Chk2(-/-) mice, including those from the thymus, central nervous system, fibroblasts, epidermis, and hair follicles, show significant defects in IR-induced apoptosis or impaired G(1)/S arrest. Quantitative comparison of the G(1)/S checkpoint, apoptosis, and expression of p53 proteins in Chk2(-/-) versus ATM(-/-) thymocytes suggested that Chk2 can regulate p53-dependent apoptosis in an ATM-independent manner. IR-induced apoptosis was restored in Chk2(-/-) thymocytes by reintroduction of the wild-type Chk2 gene but not by a Chk2 gene in which the sites phosphorylated by ATM and ataxia telangiectasia and rad3(+) related (ATR) were mutated to alanine. ATR may thus selectively contribute to p53-mediated apoptosis. These data indicate that distinct pathways regulate the activation of p53 leading to cell cycle arrest or apoptosis.     

Differential responses of temporal and nasal retinal neurites to regional-specific cues in the mouse retinofugal pathway

Retinal explants of mouse embryos were cultured together with explants of different regions in the retinofugal pathway in order to investigate whether ventral temporal (VT) and dorsal nasal (DN) retinal neurites showed differential responses to regional-specific cues in the pathway. In the presence of the chiasm, biased outgrowth of retinal neurites was found in explants of both retinal regions, which was accompanied by a reduction in total neurite growth in the VT but not the DN retina. Such differential responses to the diffusible negative influence were also observed when explants of two retinal origins were cocultured with the ventral diencephalon, but were not found with the dorsal diencephalon that contains targets of the optic axons. Indeed, extensive neurite invasion was found in the dorsal diencephalic explants and this ingrowth was more prominent for VT than DN neurites, showing a difference in axons from a distinct position in the retina to contact-mediated stimulatory activity within the target nuclei. We conclude that neurites from different regions of the retina show differential responses to the regional-specific cues in the diencephalon. These cues exist in both diffusible and contact-mediated forms that may shape the characteristic course and organization of retinal axons in decision regions of the optic pathway and the visual targets.     

Indazole inhibition of cystic fibrosis transmembrane conductance regulator Cl(-) channels in rat epididymal epithelial cells

Previous studies have shown that two indazole compounds, lonidamine [1-(2,4-dichlorobenzyl)-indazole-3-carboxylic acid] and its analogue AF2785 [(1-(2,4-dichlorobenzyl)-indazol-3-acrylic acid], suppress fertility in male rats. We also found that these compounds inhibit the cystic fibrosis transmembrane conductance regulator chloride (CFTR-Cl(-)) current in epididymal epithelial cells. To further investigate how lonidamine and AF2785 inhibit the current, we used a spectral analysis protocol to study whole-cell CFTR current variance. Application of lonidamine or AF2785 to the extracellular membrane of rat epididymal epithelial cells introduced a new component to the whole-cell current variance. Spectral analysis of this variance suggested a block at a rate of 3.68 micro mol(-1)/sec(-1) and an off rate of 69.01 sec(-1) for lonidamine, and an on rate of 3.27 micro mol(-1)/sec(-1) and an off rate of 108 sec(-1) for AF2785. Single CFTR-Cl(-) channel activity using excised inside-out membrane patches from rat epididymal epithelial cells revealed that addition of lonidamine to the intracellular solution caused a flickery block (a reduction in channel-open time) at lower concentration (10 micro M) without any effect on open channel probability or single-channel current amplitude. At higher concentrations (50 and 100 micro M), lonidamine showed a flickery block and a decrease in open-channel probability. The flickery block by lonidamine was both voltage-dependent and concentration-dependent. These results suggest that lonidamine and AF2785, which are open-channel blockers of CFTR at low concentrations, also affect CFTR gating at high concentrations. We conclude that these indazole compounds provide new pharmacological tools for the investigation of CFTR. By virtue of their interference with reproductive processes, these drugs have the potential for being developed into novel male contraceptives.     

Crystallization of membrane proteins from media composed of connected-bilayer gels

The use of hydrated-lipid gels in which the bilayer is an infinitely periodic (or at least continuous), three-dimensional structure offers a relatively new approach for the crystallization of membrane proteins. While excellent crystals of the Halobacterial rhodopsins have been obtained with such media, success remains poor in extending their use to other membrane proteins. Experience with crystallization of bacteriorhodopsin has led us to recognize a number of improvements that can be made in the use of such hydrated-gel media, which may now prove to be of general value for the crystallization of other membrane proteins.     

Roles of specific extracellular domains of the glucagon receptor in ligand binding and signaling

To identify structural determinants of ligand binding in the glucagon receptor, eight receptor chimeras and additional receptor point mutants were prepared and studied. Amino acid residues 103-117 and 126-137 in the extracellular N-terminal tail and residues 206-219 and 220-231 in the first extracellular loop of the glucagon receptor were replaced with the corresponding segments of the glucagon-like peptide-1 receptor or the secretin receptor. Specific segments of both the N-terminal tail and the first extracellular loop of the glucagon receptor are required for hormone binding. The 206-219 segment of the first loop appears to be important for both glucagon binding and receptor activation. Functional studies with a synthetic chimeric peptide consisting of the N-terminal 14 residues of glucagon and the C-terminal 17 residues of glucagon-like peptide 1 suggest that hormone binding specificity may involve this segment of the first loop. The binding selectivity may arise in part from aspartic acid residues in this segment. Mutation of R-202 located at the junction between the second transmembrane helix and the first loop resulted in a mutant receptor that failed to bind glucagon or signal. We conclude that high-affinity glucagon binding requires multiple contacts with residues in the N-terminal tail and first extracellular loop domain of the glucagon receptor, with hormone specificity arising primarily from the amino acid 206-219 segment. The data suggest a model whereby glucagon first interacts with the N-terminal domain of the receptor followed by more specific interactions between the N-terminal half of the peptide and the first extracellular loop of the receptor, leading to activation.     

Measuring kinesin's first step

A variety of models have recently emerged to explain how the molecular motor kinesin is able to maintain processive movement for over 100 steps. Although these models differ in significant features, they all predict that kinesin's catalytic domains intermittently separate from each other as the motor takes 8-nm steps along the microtubule. Furthermore, at some point in this process, one molecule of ATP is hydrolyzed per step. However, exactly when hydrolysis and product release occur in relation to this forward step have not been established. Furthermore, the rate at which this separation occurs as well as the speed of motor stepping onto and release from the microtubule have not been measured. In the absence of this information, it is difficult to critically evaluate competing models of kinesin function. We have addressed this issue by developing spectroscopic probes whose fluorescence is sensitive to motor-motor separation or microtubule binding. The kinetics of these fluorescence changes allow us to directly measure how fast kinesin steps onto and releases from the microtubule and provide insight into how processive movement is maintained by this motor.     

Cloning and characterization of the human neural cell adhesion molecule, CNTN4 (alias BIG-2)

We report the isolation and characterization of human contactin 4 (CNTN4), a brain-derived, immunoglobulin superfamily molecule-2 (alias BIG-2) as a candidate gene responsible for the differentiation potential of human neuroblastoma cells. Northern blot analysis showed highest CNTN4 expression in testes, thyroid, small intestine, uterus and brain. Induction of CNTN4 mRNA expression in human neuroblastoma tumor cells treated with retinoic acid correlated with a block in retinoid-induced neuritogenesis. Our findings suggest a role for human contactin 4 protein in the response of neuroblastoma cells to differentiating agents.