Lysosomal acid lipase and lipophagy are constitutive negative regulators of glucose-stimulated insulin secretion from pancreatic beta cells

AIMS/HYPOTHESIS: Lipolytic breakdown of endogenous lipid pools in pancreatic beta cells contributes to glucose-stimulated insulin secretion (GSIS) and is thought to be mediated by acute activation of neutral lipases in the amplification pathway. Recently it has been shown in other cell types that endogenous lipid can be metabolised by autophagy, and this lipophagy is catalysed by lysosomal acid lipase (LAL). This study aimed to elucidate a role for LAL and lipophagy in pancreatic beta cells. METHODS: We employed pharmacological and/or genetic inhibition of autophagy and LAL in MIN6 cells and primary islets. Insulin secretion following inhibition was measured using RIA. Lipid accumulation was assessed by MS and confocal microscopy (to visualise lipid droplets) and autophagic flux was analysed by western blot. RESULTS: Insulin secretion was increased following chronic (≥ 8 h) inhibition of LAL. This was more pronounced with glucose than with non-nutrient stimuli and was accompanied by augmentation of neutral lipid species. Similarly, following inhibition of autophagy in MIN6 cells, the number of lipid droplets was increased and GSIS was potentiated. Inhibition of LAL or autophagy in primary islets also increased insulin secretion. This augmentation of GSIS following LAL or autophagy inhibition was dependent on the acute activation of neutral lipases. CONCLUSIONS/INTERPRETATION: Our data suggest that lysosomal lipid degradation, using LAL and potentially lipophagy, contributes to neutral lipid turnover in beta cells. It also serves as a constitutive negative regulator of GSIS by depletion of substrate for the non-lysosomal neutral lipases that are activated acutely by glucose

Substituted hippurates and hippurate analogs as substrates and inhibitors of peptidylglycine alpha-hydroxylating monooxygenase (PHM).

Peptidyl alpha-hydroxylating monooxygenase (PHM) functions in vivo towards the biosynthesis of alpha-amidated peptide hormones in mammals and insects. PHM is a potential target for the development of inhibitors as drugs for the treatment of human disease and as insecticides for the management of insect pests. We show here that relatively simple ground state analogs of the PHM substrate hippuric acid (C(6)H(5)-CO-NH-CH(2)-COOH) inhibit the enzyme with K(i) values as low as 0.5microM. Substitution of sulfur atom(s) into the hippuric acid analog increases the affinity of PHM for the inhibitor. Replacement of the acetylglycine moiety, -CO-NH-CH(2)-COOH with an S-(thioacetyl)thioglycolic acid moiety, -CS-S-CH(2)-COOH, yields compounds with the highest PHM affinity. Both S-(2-phenylthioacetyl)thioglycolate and S-(4-ethylthiobenzoyl)thioglycolic acid inhibit the proliferation of cultured human prostate cancer cells at concentrations \u3e100-fold excess of their respective K(i) values. Comparison of K(i) values between mammalian PHM and insect PHM shows differences in potency suggesting that a PHM-based insecticide with limited human toxicity can be developed

Substituted Hippurates and Hippurate Analogs as Substrates and Inhibitors of Peptidylglycine α-Hydroxylating Monooxygenase (PHM)

Peptidyl α-hydroxylating monooxygenase (PHM) functions in vivo towards the biosynthesis of α-amidated peptide hormones in mammals and insects. PHM is a potential target for the development of inhibitors as drugs for the treatment of human disease and as insecticides for the management of insect pests. We show here that relatively simple ground state analogs of the PHM substrate hippuric acid (C6H5–CO–NH–CH2–COOH) inhibit the enzyme with Ki values as low as 0.5 μM. Substitution of sulfur atom(s) into the hippuric acid analog increases the affinity of PHM for the inhibitor. Replacement of the acetylglycine moiety, –CO–NH–CH2–COOH with an S-(thioacetyl)thioglycolic acid moiety, –CS–S–CH2–COOH, yields compounds with the highest PHM affinity. Both S-(2-phenylthioacetyl)thioglycolate and S-(4-ethylthiobenzoyl)thioglycolic acid inhibit the proliferation of cultured human prostate cancer cells at concentrations \u3e100-fold excess of their respective Ki values. Comparison of Ki values between mammalian PHM and insect PHM shows differences in potency suggesting that a PHM-based insecticide with limited human toxicity can be developed

Identification of Trypanosoma brucei AdoMetDC Inhibitors Using a High-Throughput Mass Spectrometry-Based Assay

Human African trypanosomiasis (HAT) is a fatal infectious disease caused by the eukaryotic pathogen Trypanosoma brucei (<i>Tb</i>). Available treatments are difficult to administer and have significant safety issues. <i>S</i>-Adenosylmethionine decarboxylase (AdoMetDC) is an essential enzyme in the parasite polyamine biosynthetic pathway. Previous attempts to develop <i>Tb</i>AdoMetDC inhibitors into anti-HAT therapies failed due to poor brain exposure. Here, we describe a large screening campaign of two small-molecule libraries (∼400,000 compounds) employing a new high-throughput (∼7 s per sample) mass spectrometry-based assay for AdoMetDC activity. As a result of primary screening, followed by hit confirmation and validation, we identified 13 new classes of reversible <i>Tb</i>AdoMetDC inhibitors with low-micromolar potency (IC₅₀) against both <i>Tb</i>AdoMetDC and <i>T. brucei</i> parasite cells. The majority of these compounds were >10-fold selective against the human enzyme. Importantly, compounds from four classes demonstrated high propensity to cross the blood–brain barrier in a cell monolayer assay. Biochemical analysis demonstrated that compounds from eight classes inhibited intracellular <i>Tb</i>AdoMetDC in the parasite, although evidence for a secondary off-target component was also present. The discovery of several new <i>Tb</i>AdoMetDC inhibitor chemotypes provides new hits for lead optimization programs aimed to deliver a novel treatment for HAT

Substituted hippurates and hippurate analogs as substrates and inhibitors of peptidylglycine a-hydroxylating monooxygenase (PHM)

Peptidyl a-hydroxylating monooxygenase (PHM) functions in vivo towards the biosynthesis of a-amidated peptide hormones in mammals and insects. PHM is a potential target for the development of inhibitors as drugs for the treatment of human disease and as insecticides for the management of insect pests. We show here that relatively simple ground state analogs of the PHM substrate hippuric acid (C6H5-CO-NH-CH2-COOH) inhibit the enzyme with Ki values as low as 0.5 µM. Substitution of sulfur atom(s) into the hippuric acid analog increases the affinity of PHM for the inhibitor. Replacement of the acetylglycine moiety, -CO-NH-CH2-COOH with an S-(thioacetyl)thioglycolic acid moiety, -CS-S-CH2-COOH, yields compounds with the highest PHM affinity. Both S-(2-phenylthioacetyl)thioglycolate and S-(4-ethylthiobenzoyl)thioglycolic acid inhibit the proliferation of cultured human prostate cancer cells at concentrations &gt;100-fold excess of their respective Ki values. Comparison of Ki values between mammalian PHM and insect PHM shows differences in potency suggesting that a PHM-based insecticide with limited human toxicity can be developed. © 2008 Elsevier Ltd. All rights reserved