Poul Jacobsen

Adipose Triglyceride Lipase and Hormone-sensitive Lipase Are the Major Enzymes in Adipose Tissue Triacylglycerol Catabolism

The mobilization of free fatty acids from adipose triacylglycerol (TG) stores requires the activities of triacylglycerol lipases. In this study, we demonstrate that adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) are the major enzymes contributing to TG breakdown in in vitro assays and in organ cultures of murine white adipose tissue (WAT). To differentiate between ATGL- and HSL-specific activities in cytosolic preparations of WAT and to determine the relative contribution of these TG hydrolases to the lipolytic catabolism of fat, mutant mouse models lacking ATGL or HSL and a mono-specific, small molecule inhibitor for HSL (76-0079) were used. We show that 76-0079 had no effect on TG catabolism in HSL-deficient WAT but, in contrast, essentially abolished free fatty acid mobilization in ATGL-deficient fat. CGI-58, a recently identified coactivator of ATGL, stimulates TG hydrolase activity in wild-type and HSL-deficient WAT but not in ATGL-deficient WAT, suggesting that ATGL is the sole target for CGI-58-mediated activation of adipose lipolysis. Together, ATGL and HSL are responsible for more than 95% of the TG hydrolase activity present in murine WAT. Additional known or unknown lipases appear to play only a quantitatively minor role in fat cell lipolysis.

Adipose triglyceride lipase in human skeletal muscle is upregulated by exercise training

Mobilization of fatty acids from stored triacylglycerol (TG) in adipose tissue and skeletal muscle [intramyocellular triacylglycerol (IMTG)] requires activity of lipases. Although exercise training increases the lipolytic capacity of skeletal muscle, the expression of hormone-sensitive lipase (HSL) is not changed. Recently, adipose triglyceride lipase (ATGL) was identified as a TG-specific lipase in various rodent tissues. To investigate whether human skeletal muscle ATGL protein is regulated by endurance exercise training, 10 healthy young men completed 8 wk of supervised endurance exercise training. Western blotting analysis on lysates of skeletal muscle biopsy samples revealed that exercise training induced a twofold increase in skeletal muscle ATGL protein content. In contrast to ATGL, expression of comparative gene identification 58 (CGI-58), the activating protein of ATGL, and HSL protein was not significantly changed after the training period. The IMTG concentration was significantly decreased by 28% at termination of the training program compared with before. HSL-phoshorylation at Ser(660) was increased, HSL-Ser(659) phosporylation was unchanged, and HSL-phoshorylation at Ser(565) was decreased altogether, indicating an enhanced basal activity of this lipase. No change was found in the expression of diacylglycerol acyl transferase 1 (DGAT1) after training. Inhibition of HSL with a monospecific, small molecule inhibitor (76-0079) and stimulation of ATGL with CGI-58 revealed that significant ATGL activity is present in human skeletal muscle. These results suggest that ATGL in addition to HSL may be important for human skeletal muscle lipolysis.

Protection against post-ischemic behavioral pathology by the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) antagonist 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(f)quinoxaline (NBQX) in the gerbil

The neuroprotective effects of NBQX (2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(f)quinoxaline) were assessed on hippocampal CA1 neuronal loss and locomotor hyperactivity following transient bilateral carotid artery occlusion (BCAO) in the gerbil. NBQX, a selective blocker of the AMPA glutamate receptor subtype, was injected 1 h after 5 or 10 min BCAO, or sham surgery. Both 5 and 10 min ischemia produced equivalent hyperactivity 3 days post ischemia and CA1 neuronal loss on Day 4, while activity was unchanged in the sham-operated group. NBQX protected from both hippocampal damage and post-ischemic hyperactivity. These results demonstrate that NBQX can protect from behavioral pathology induced by global cerebral ischemia.

The Critical Role of Neutral Cholesterol Ester Hydrolase 1 in Cholesterol Removal From Human Macrophages

Rationale: Hydrolysis of intracellular cholesterol ester (CE) is the key step in the reverse cholesterol transport in macrophage foam cells. We have recently shown that neutral cholesterol ester hydrolase (Nceh)1 and hormone-sensitive lipase (Lipe) are key regulators of this process in mouse macrophages. However, it remains unknown which enzyme is critical in human macrophages and atherosclerosis. Objective: We aimed to identify the enzyme responsible for the CE hydrolysis in human macrophages and to determine its expression in human atherosclerosis. Methods and Results: We compared the expression of NCEH1, LIPE, and cholesterol ester hydrolase (CES1) in human monocyte-derived macrophages (HMMs) and examined the effects of inhibition or overexpression of each enzyme in the cholesterol trafficking. The pattern of expression of NCEH1 was similar to that of neutral CE hydrolase activity during the differentiation of HMMs. Overexpression of human NCEH1 increased the hydrolysis of CE, thereby stimulating cholesterol mobilization from THP-1 macrophages. Knockdown of NCEH1 specifically reduced the neutral CE hydrolase activity. Pharmacological inhibition of NCEH1 also increased the cellular CE in HMMs. In contrast, LIPE was barely detectable in HMMs, and its inhibition did not decrease neutral CE hydrolase activity. Neither overexpression nor knockdown of CES1 affected the neutral CE hydrolase activity. NCEH1 was expressed in CD68-positive macrophage foam cells of human atherosclerotic lesions. Conclusions: NCEH1 is expressed in human atheromatous lesions, where it plays a critical role in the hydrolysis of CE in human macrophage foam cells, thereby contributing to the initial part of reverse cholesterol transport in human atherosclerosis.

GABA‐Mimetic Activity and Effects on Diazepam Binding of Aminosulphonic Acids Structurally Related to Piperidine‐4‐Sulphonic Acid

The relationship between structure, in vivo activity, and in vitro activity of some analogues of the γ‐aminobutyric acid (GABA) agonist piperidine‐4‐sulphonic acid (P4S) was studied. The syntheses of 1,2,3,6‐tetrahydropyridine‐4‐sulphonic acid (DH‐P4S) and (RS)‐pyrrolidin‐3‐yl‐methanesulphonamide (PMSA‐amide) are described. Like P4S, its unsaturated analogue DH‐P4S and the five‐ring isomer (RS)‐pyrrolidin‐3‐yl‐methanesulphonic acid (PMSA) were bicuculline methochloride (BMC)‐sensitive inhibitors of the firing of neurones in the cat spinal cord. Whereas isonipecotic acid was less potent than its unsaturated analogue isoguvacine as a GABA‐mimetic and as an inhibitor of GABA binding, the opposite relative potencies of P4S and DH‐P4S were observed, P4S being proportionally more potent than DH‐P4S. In contrast with P4S and DH‐P4S, PMSA, which is an analogue of the potent GABA uptake inhibitor and BMC‐sensitive GABA‐mimetic homo‐β‐proline, was a relatively weak inhibitor of GABA uptake in vitro. PMSA‐amide was more than two orders of magnitude weaker than PMSA as an inhibitor of GABA binding and did not significantly affect GABA uptake in vitro. The effects of 3‐aminopropanesulphonic acid (3‐APS), PMSA, P4S, and DH‐P4S on the binding of [3H]diazepam in vitro at 30°C, in the presence or absence of chloride ions, were studied and compared with those of the structurally related amino acids GABA, homo‐β‐proline, isonipecotic acid, and isoguvacine. Under these conditions the aminosulphonic acids were weaker than the respective amino acids in enhancing [3H]diazepam binding, the difference being more pronounced in the absence of chloride.

Synthesis and pharmacology of a novel pyrrolo[2,1,5-cd] indolizine (NNC 45-0095), a high affinity non-steroidal agonist for the estrogen receptor.

1-Ethyl-2-(4-hydroxyphenyl)pyrrolo[2,1,5-cd]indolizine (NNC 45-0095) is a novel compound which represents the parent pharmacophore structure of a series of pyrrolo[2,1,5-cd]indolizine derivatives with mixed estrogen agonist/antagonist properties. NNC 45-0095 binds with high affinity to the estrogen receptor (IC50=9.5 nM) and exhibits full protection of bone loss in the ovariectomized mouse model for post-menopausal osteoporosis.

Inhibition of cisplatin-induced emesis in ferrets by the non-NMDA receptor antagonists NBQX and CNQX.

The excitatory amino acid (EAA) receptor antagonists, 2,3-dihydroxy-6-nitro-7-sulphamoylbenzo(f)quinoxaline (NBQX) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), which preferentially block non-N-methyl-D-aspartate (non-NMDA) subtypes of EAA receptors, effectively inhibit cisplatin-induced emesis in ferrets. A high dose of cisplatin (10 mg/kg i.v.) was used which induced emesis in all saline-treated control ferrets. At 10 mg/kg i.v., NBQX totally prevented cisplatin-induced emesis in 5 of 6 ferrets and CNQX totally prevented emesis in 3 of 5 ferrets. By comparison, each of the 5-HT3 inhibitors, zacopride and ondansetron, at 1.0 mg/kg i.v. (a dose considered in the high therapeutic range for controlling emesis by these compounds), totally prevented emesis in 2 of 5 ferrets. It is concluded that non-NMDA antagonists effectively inhibit cisplatin-induced emesis. They are potential antiemetic compounds, alone or in combination with 5-HT3 antagonists or other more conventional drugs of choice.

Synthesis and estrogen receptor binding affinities of novel pyrrolo[2,1,5-cd]indolizine derivatives

A series of pyrrolo[2,1,5-cd]indolizine derivatives has been synthesized and evaluated as ligands for the estrogen receptor. Properly substituted mono- and di-hydroxy derivatives showed binding in the low nanomolar range in accordance with their structural resemblance to estrogen.

Synthesis and pharmacological evaluation of novel cis-3,4-diaryl-hydroxychromanes as high affinity partial agonists for the estrogen receptor.

The syntheses and in vitro pharmacological evaluation of a number of cis-3,4-diaryl-hydroxy-chromanes are reported, along with the results of a thorough in vivo profiling of the tissue-selective estrogen partial-agonist NNC 45-0781 [3, (-)-(3S,4R)-7-hydroxy-3-phenyl-4-(4-(2-pyrrolidinoethoxy)phenyl)chromane]. These studies showed that NNC 45-0781 is a very promising candidate for the prevention of post-menopausal osteoporosis, and the treatment of other health issues related to the loss of endogenous estrogen production.

4,5,6,7-Tetrahydroisothiazolo[5,4-c]pyridin-3-ol and Related Analogues of THIP. Synthesis and Biological Activity.

Dargestellt werden die Verbindungen (IV) bis (VI), die auf γ-Aminobuttersaure (GABA)-agonistische und antagonistische Wirkung untersucht werden.