Charles W. Garner

The insulin-induced down-regulation of IRS-1 in 3T3-L1 adipocytes is mediated by a calcium-dependent thiol protease.

Insulin receptor substrate-1 (IRS-1) is a protein expressed in 3T3-L1 adipocytes that is involved in most, if not all of the biological responses to insulin. Chronic exposure of these cells to insulin down-regulates IRS-1 by stimulating its degradation (Rice, K.M., Turnbow, M.A. and Garner, C.W. (1993) Biochem. Biophys. Res. Commun. 190, 961-967). This insulin-induced down-regulation of IRS-1 was totally abolished by BAPTA-AM (cell-permeable calcium chelator), E-64d (cell-permeable thiol protease inhibitor), Cbz-Leu-Nleu-H and Cbz-Leu-Leu-Tyr-CHN2 (selective cell-permeable calpain inhibitor peptides). Calpastatin (specific calpain inhibitor protein) also inhibited the insulin-induced down-regulation of IRS-1 in transiently permeabilized cells. In addition, 3T3-L1 adipocytes express endogenous calpain which can degrade IRS-1 in cell-free extracts. These results suggest that the insulin-induced down-regulation of IRS-1 in 3T3-L1 adipocytes is mediated by a calcium-dependent thiol protease which is sensitive to inhibition by calpain inhibitors.

Mechanisms of Action of Hormone-Sensitive Lipase in Mouse Leydig Cells: Its Role in the Regulation of the Steroidogenic Acute Regulatory Protein

Background: Hormone-sensitive lipase (HSL) is a multifunctional enzyme that is critically involved in regulating energy homeostasis. Results: HSL catalyzes the hydrolysis of cholesteryl esters and plays an indispensable role in the regulation of Bt2cAMP-induced steroidogenic acute regulatory protein (StAR) expression. Conclusion: The mode of action of HSL in cAMP/PKA-mediated regulation of steroidogenesis involves multiple signaling, including the LXR-regulatory pathway. Significance: The present findings can be useful for better understanding a number of physiopathological processes. Hormone-sensitive lipase (HSL) catalyzes the hydrolysis of cholesteryl esters in steroidogenic tissues and, thus, facilitates cholesterol availability for steroidogenesis. The steroidogenic acute regulatory protein (StAR) controls the rate-limiting step in steroid biosynthesis. However, the modes of action of HSL in the regulation of StAR expression remain obscure. We demonstrate in MA-10 mouse Leydig cells that activation of the protein kinase A (PKA) pathway, by a cAMP analog Bt2cAMP, enhanced expression of HSL and its phosphorylation (P) at Ser-660 and Ser-563, but not at Ser-565, concomitant with increased HSL activity. Phosphorylation and activation of HSL coincided with increases in StAR, P-StAR (Ser-194), and progesterone levels. Inhibition of HSL activity by CAY10499 effectively suppressed Bt2cAMP-induced StAR expression and progesterone synthesis. Targeted silencing of endogenous HSL, with siRNAs, resulted in increased cholesteryl ester levels and decreased cholesterol content in MA-10 cells. Depletion of HSL affected lipoprotein-derived cellular cholesterol influx, diminished the supply of cholesterol to the mitochondria, and resulted in the repression of StAR and P-StAR levels. Cells overexpressing HSL increased the efficacy of liver X receptor (LXR) ligands on StAR expression and steroid synthesis, suggesting HSL-mediated steroidogenesis entails enhanced oxysterol production. Conversely, cells deficient in LXRs exhibited decreased HSL responsiveness. Furthermore, an increase in HSL was correlated with the LXR target genes, steroid receptor element-binding protein 1c and ATP binding cassette transporter A1, demonstrating HSL-dependent regulation of steroidogenesis predominantly involves LXR signaling. LXRs interact/cooperate with RXRs and result in the activation of StAR gene transcription. These findings provide novel insight and demonstrate the molecular events by which HSL acts to drive cAMP/PKA-mediated regulation of StAR expression and steroidogenesis in mouse Leydig cells.

A simple rapid biuret method for the estimation of protein in samples containing thiols

Abstract Interference in the Lowry protein determination by thiol compounds is now well known (1–3). We have found that the estimation of protein by the biuret reaction is also subject to interference when the protein sample contains various thiols. We wish to report that this interference can be prevented in most cases by using a biuret reagent which is chelated with ethylenediaminetetraacetate (EDTA). In samples containing dithiothreitol (DTT) it is also necessary to add iodoacetamide prior to the addition of the biuret reagent. The use of iodoacetate to eliminate thiol interference in the Lowry procedure has been reported previously (3). This report details the extent of interference of dithiothreitol, β-mercaptoethanol, β-mercaptoethylamine, and glutathione, and illustrates the extent of neutralization which is attained in each case. We have also introduced modifications which permit the development of a stable color in only 5 min.

Purification and properties of human intestine alanine aminopeptidase

Human intestinal alanine aminopeptidase has been purified to greater than 90% homogeneity. The enzyme was released from mucosal cell membranes by Triton X-100 treatment. The native enzyme had a molecular weight of 206,000 in dilute buffer and 108,000 in the presence of sodium dodecyl sulfate. The enzyme was inhibited by chelators suggesting the presence of a metal ion in the enzyme. The most potent chelator inhibitor tested, o-phenanthroline, gave mixed kinetics (Ki = 67 micro M). Activity was restored by removal of the chelator. The enzyme was inhibited competitively by amino acids having hydrophobic side chains such as L-phenylalanine (Ki = 0.67 mM). Puromycin and methicillin also inhibited the enzyme in the competitive (Ki = 12.5 micro M) and noncompetitive (Ki = 4.6 mM) manner, respectively. Kinetic analysis of several amino acid beta-naphthylamides as substrates demonstrated the preference for substrates having hydrophobic or basic amino terminal residues with no beta-branching. L-Methionyl-beta-naphthylamide was the most tightly bound with L-alanyl-beta-naphthylamide was the most rapidly hydrolyzed.

Wortmannin and LY294002 inhibit the insulin-induced down-regulation of IRS-1 in 3T3-L1 adipocytes.

The insulin receptor substrate-1 (IRS-1) is expressed in 3T3-L1 adipocytes and is involved in at least some insulin responses, notably mitogenesis. Chronic exposure to insulin down regulates IRS-1 in these cells by stimulating its degradation (Rice, K.M., Turnbow, M.A. and Garner, C.W. (1993) Biochem. Biophys. Res. Commun. 190, 961-967). This insulin response was completely inhibited by wortmannin and LY294002 (2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one), two inhibitors of phosphatidylinositol 3-kinase (PI 3-kinase). Neither wortmannin nor LY294002 had any effect on the calcium-dependent degradation of IRS-1 in vitro nor did they inhibit the phosphorylation of IRS-1 in vitro. In addition, neomycin, a cationic aminoglycoside antibiotic that binds to phosphoinositides, inhibited the insulin-induced down-regulation of IRS-1 in 3T3-L1 adipocytes and, also, the C8-PIP3-stimulated degradation of IRS-1 in vitro. These results suggest that PI 3-kinase and its 3-phosphoinositide products mediate the insulin-induced down-regulation of IRS-1 in 3T3-L1 adipocytes.

Stimulation of DNA synthesis in rat uterine cells by growth factors and uterine extracts

Replicative DNA synthesis, as measured by thymidine incorporation, has been measured in rat uterine cells in primary culture in response to growth factors. Insulin, insulin-like growth factor-I (IGF-I), multiplication-stimulating activity (MSA) and platelet-derived growth factor (PDGF) stimulated DNA synthesis, while estradiol, epidermal growth factor (EGF), transforming growth factor-beta (TGF-beta), basic fibroblast growth factor (bFGF) and relaxin did not stimulate or did so weakly and only at very high concentrations. Uterine acid extracts also stimulated DNA synthesis. IGF-I stimulated at concentrations consistent with its acting through the IGF-I receptor; however, insulin stimulated at concentrations higher than expected for its acting through its receptor and this its action may be mediated through the IGF-I receptor. IGF-I was found in uterine tissue by radioimmunoassay (RIA). There was a 5- to 10-fold increase in IGF-I in the uteri from ovariectomized rats that had been treated with estradiol 24 h earlier. This is analogous to the increase in growth factor activity found previously in rat uterus after 24-h estradiol treatment (Beck, C.A. and Garner, C.W. (1989) Mol. Cell. Endocrinol. 63, 93-101). These data are consistent with the hypothesis that estradiol effects in the uterus are in part mediated through IGF-I.

An Evaluation of Some Theories of the Mechanism of Aging

Two theories of aging are considered in this review. Although there exists substantial experimental evidence in support of the somatic mutation and error catastrophe hypotheses, several experiments have been published which are extremely difficult to reconcile with these models, at least in their simplest forms. These include the observation that biochemical and morphological degenerative changes observed in fibroblasts aged in vitro do not resemble alterations observed in cells obtained from aged donors, and the fact that tissues transplanted serially through different hosts do not decline in vigor in the manner predicted by the somatic mutation theory. Although biochemical and mutational alterations appear to accumulate in fibroblasts aged in vitro (in support of the error catastrophe model), there are substantial problems with the interpretation of such experiments, and some observations (such as the lack of increase in translational error in hemoglboin synthesis as a function of age) seem to argue directly against the error catastrophe theory. Some alternative theoretical and experimental possibilities are discussed, including the concept of programmed aging as the cause of senescence.

Peroxidation of free and esterified fatty acids by horseradish peroxidase

Linoleic and arachidonic acids and unsaturated esterified fatty acids of soybean lecithin liposomes were oxidized by horseradish peroxidase in the presence of hydrogen peroxide. The major products formed in the presence of oxygen were fatty acid hydroperoxides. In the absence of oxygen, other unidentified products were formed. Diene conjugation was about 5 times faster in oxygen than in nitrogen. Malondialdehyde was formed only in the presence of oxygen. Superoxide, singlet oxygen and hydroxyl radical may have been involved in the free fatty acid oxidation system but not in the liposome system. Replacement of hydrogen peroxide with the hydrogen peroxide (and superoxide) generators xanthine oxidase or galactose oxidase caused a more efficient oxidation in the presence of peroxidase than in its absence, suggesting that the in vivo toxicity of hydrogen peroxide and superoxide may be greatly increased in the presence of peroxidase.

Stimulation of glucose transport in cultured uterine cells by rat and rabbit uterine extracts

Estradiol-17 beta was previously shown to stimulate glucose transport (as measured by phosphorylation of 2-deoxyglucose) in rat uterine tissue in vivo (Meier, D.A. and Garner, C.W. (1987) Endocrinology 121, 1366-1374) but attempts to demonstrate this in uterine organ strips in vitro, in uterine tumor cell lines or in uterine cells in primary culture have been unsuccessful. However, aqueous uterine extracts and uterine luminal fluid did stimulate glucose transport in uterine tumor cells and uterine cells in primary culture. Estradiol in vivo and uterine extracts in vitro each increased the initial rate of glucose transport 1.5- to 3-fold. In each case, 2-3 h were required for the stimulation to be fully expressed. The stimulation was not inhibited by cycloheximide suggesting that protein synthesis was not required. Uteri from ovariectomized rats injected daily for 4 days with 10 micrograms estradiol contained 4-fold more activity than uteri from saline-injected control animals. The activity was acid- and heat-stable, inactivated by trypsin treatment but not removed by dextran-coated charcoal treatment, suggesting that the activity is (or is associated with) a protein. The activity eluted in the 6-12 kDa range upon chromatography on Sephadex G-50. Insulin (1-1000 ng/ml) and epidermal growth factor (1-100 ng/ml) stimulated glucose transport, but only less than 50% of the stimulation by extracts. The substance(s) present in the extracts, possibly a known growth factor, may be involved in the estradiol stimulation of glucose transport and other estradiol actions in vivo.

Hydrophobic binding sites of human liver alanine aminopeptidase.

Abstract Fatty acids, alkyl amines, and amides of α-amino fatty acids inhibit human liver alanine aminopeptidase apparently by binding to residue binding site 1 of the active center, i.e., the N-terminal binding site. The pKi values of the acids, amines, and amides increase until the overall chain length reaches eight carbons. The pKi values are the same for members of the series with chain lengths longer than eight carbon atoms. Assuming an extended structure of the inhibitors, this site will accommodate amino acid side chains of not longer than 11.7 A from the α-carbon to the end of the chain. Long chain amino acids inhibit by binding apparently at residue site 3. The pKi values of dl -α-amino acids from α-aminobutyric acid to α-aminodecanoic acid increase with the addition of each methylene unit. Thus, site 3 will accommodate amino acid side chains which are at least 13.0 A from the α-carbon to the end of the chain. Methanol and other organic solvents reversibly inhibit the binding of substrates at pH 6.9 without affecting the maximum rate of catalysis. At lower pH values, the maximum rate of catalysis is lowered. Sodium chloride also inhibits substrate hydrolysis at pH 6.9 but does not affect the maximum rate of catalysis. The pKi values of fatty acids, alkyl amines, and amino acids are strongly decreased by methanol and slightly increased by sodium chloride. These data indicate that a major portion of the interactions of the enzyme with fatty acids, amines, and amino acids is of a hydrophobic nature.