The fasting-induced adipose factor (FIAF, ANGPTL4, PGAR, HFARP) was previously identified as a novel adipocytokine that was up-regulated by fasting, by peroxisome proliferator-activated receptor agonists, and by hypoxia. To further characterize FIAF, we studied regulation of FIAF mRNA and protein in liver and adipose cell lines as well as in human and mouse plasma. Expression of FIAF mRNA was up-regulated by peroxisome proliferator-activated receptor ␣ (PPAR␣) and PPAR␤/␦ agonists in rat and human hepatoma cell lines and by PPAR␥ and PPAR␤/␦ agonists in mouse and human adipocytes. Transactivation, chromatin immunoprecipitation, and gel shift experiments identified a functional PPAR response element within intron 3 of the FIAF gene. At the protein level, in human and mouse blood plasma, FIAF was found to be present both as the native protein and in a truncated form. Differentiation of mouse 3T3-L1 adipocytes was associated with the production of truncated FIAF, whereas in human white adipose tissue and SGBS adipocytes, only native FIAF could be detected. Interestingly, truncated FIAF was produced by human liver. Treatment with fenofibrate, a potent PPAR␣ agonist, markedly increased plasma levels of truncated FIAF, but not native FIAF, in humans. Levels of both truncated and native FIAF showed marked interindividual variation but were not associated with body mass index and were not influenced by prolonged semistarvation. Together, these data suggest that FIAF, similar to other adipocytokines such as adiponectin, may partially exert its function via a truncated form.

Both apolipoprotein E (apoE) and 24(S)-hydroxycholesterol are involved in the pathogenesis of Alzheimer disease (AD). It has beenhypothesized that apoE affects AD development via isoform-specific effects on lipid trafficking between astrocytes and neurons. However, the regulation of the cholesterol supply of neurons via apoE-containing high density lipoproteins remains to be clarified. We show for the first time that the brain-specific metabolite of cholesterol produced by neurons, i.e. 24(S)-hydroxycholesterol, induces apoE transcription, protein synthesis, and secretion in a dose-and time-dependent manner in cells of astrocytic but not of neuronal origin. Moreover, 24(S)-hydroxycholesterol primes astrocytoma, but not neuroblastoma cells, to mediate cholesterol efflux to apoE. Similar results were obtained using the synthetic liver X receptor (LXR) agonist GW683965A, suggesting involvement of an LXR-controlled signaling pathway. A 10 -20-fold higher basal LXR␣ and -␤ expression level in astrocytoma compared with neuroblastoma cells may underlie these differential effects. Furthermore, apoE-mediated cholesterol efflux from astrocytoma cells may be controlled by the ATP binding cassette transporters ABCA1 and ABCG1, since their expression was also up-regulated by both compounds. In contrast, ABCG4 seems not to be involved, because its expression was induced only in neuronal cells. The expression of sterol regulatory element-binding protein (SREBP-2), low density lipoprotein receptor, 3-hydroxy-3-methylglutaryl-CoA reductase, and SREBP-1c was transiently up-regulated by GW683965A in astrocytes but down-regulated by 24(S)-hydroxycholesterol, suggesting that cholesterol efflux and synthesis are regulated independently. In conclusion, evidence is provided that 24(S)-hydroxycholesterol induces apoE-mediated efflux of cholesterol in astrocytes via an LXR-controlled pathway, which may be relevant for chronic and acute neurological diseases.

OBJECTIVE:To test the hypothesis that consumption of a high-fat diet leads to an increase in UCP mRNA expression in human skeletal muscle. In a group of endurance athletes, with a range in ®ber type distribution, we hypothesized that the effect of the high-fat diet on UCP2 and UCP3 mRNA expression is more pronounced in muscle ®bers which are known to have a high capacity to shift from carbohydrate to fat oxidation (type IIA ®bers). DESIGN: Ten healthy trained athletes (®ve males, ®ve females) consumed a low-fat diet (17 AE 0.9 en% of fat) and high-fat diet (41.4 AE 1.4 en% fat) for 4 weeks, separated by a 4 week wash-out period. Muscle biopsies were collected at the end of both dietary periods. MEASUREMENTS: Using RT-PCR, levels of UCP2 and UCP3 mRNA expression were measured and the percentage of type I, IIA and IIB ®bers were determined using the myo®brillar ATPase method in all subjects. RESULTS: UCP3L mRNA expression tended to be higher on the high-fat diet, an effect which reached signi®cance when only males were considered (P 0.037). Furthermore, diet-induced change in mRNA expression of UCP3T (r: 0.66, P 0.037), UCP3L (r: 0.61, P 0.06) and UCP2 (r: 0.70, P 0.025), but not UCP3S, correlated signi®cantly with percentage dietary fat on the high-fat diet. Plasma FFA levels were not different during the two diets. Finally, the percentage of type IIA ®bers was positively correlated with the diet-induced change in mRNA expression for UCP2 (r: 0.7, P 0.03), UCP3L (r: 0.73, P 0.016) and UCP3T (r: 0.68, P 0.03) but not with UCP3S (r: 0.06, NS). CONCLUSION: UCP2 and UCP3 mRNAs are upregulated by a high-fat diet. This upregulation is more pronounced in humans with high proportions of type IIA ®bers, suggesting a role for UCPs in lipid utilization.

We describe a new principle for assessment of the activity of proteolytic enzymes of all classes and show the application of this principle for the quantitative assay of bacterial collagenase and human matrix metalloproteinases (MMPs). Central to this new principle is the presence of a proenzyme that can be activated into an active enzyme by a single proteolytic event. The regular activation sequence in the proenzyme is replaced using protein engineering by an artificial sequence recognized by the proteinase to be determined. The latter can act as an activator for the newly engineered proenzyme. In the present paper a simple colorimetric assay for the determination for MMPs is described based on this principle. With the aid of protein engineering, a modified pro-urokinase has been prepared in which the activation sequence normally recognized by plasmin (Pro-Arg-Phe-Lys upward arrowIle-Ile-Gly-Gly) has been replaced by a sequence expected to be recognized and hydrolysed by many MMPs (Arg-Pro-Leu-Gly upward arrowIle-Ile-Gly-Gly). The active urokinase resulting from activation of the modified pro-urokinase by a MMP could be measured either directly, using a specific chromogenic peptide substrate for urokinase, or indirectly via urokinase-catalysed plasminogen activation. The response of the assay to equal molar quantities of active MMPs decreases in the order MMP-2>MMP-9>MMP-1>MMP-3>MMP-7. The detection limit for MMP-9 was below 15 pM, corresponding to 3. 75x10(-15) mol per assay. Using the assay, increased MMP activity was detected in synovial tissue extracts from rheumatoid arthritis patients compared with those from osteoarthritis patients, and in stomach tumour extracts as compared with normal stomach tissue extracts.

SUMMARY:The present study provides evidence that chronic intake of a high-fat diet induces a dramatic extravasation of immunoglobulins, indicating alterations in blood-brain barrier (BBB) functioning, in the brains of apolipoprotein E (apoE)-knockout mice, but not of C57Bl/6 control mice. Using sodium fluorescein as a marker for the permeability of the BBB, we found additional support for age-related disturbances of BBB function in apoE-knockout mice. Behavioral analysis of apoE-knockout mice compared with C57Bl/6 mice indicated that they were also less efficient in acquiring the spatial Morris water maze task. Furthermore, apoE-knockout mice are known to develop severe atherosclerosis, which is exacerbated with a high-fat diet. We therefore compared the apoE-knockout mice with the apoE3-Leiden transgenic mice, which are known to develop atherosclerosis. However, apoE3-Leiden mice that were kept on a high-fat, high-cholesterol diet and that developed atherosclerosis to an extent similar to the apoE-knockout mice, showed no signs of BBB disturbances. These results indicate for the first time that apoE plays an essential role in the maintenance of the integrity of the BBB during aging and that it protects the brain from neuropathology induced by a high-fat diet. We therefore hypothesize that the role of apoE in the maintenance of the integrity of the BBB may be the mechanism by which apoE affects the progression of neurodegeneration, as seen in Alzheimer's disease. (Lab Invest 2001, 81:953-960).A polipoprotein E (apoE) is synthesized in almost all cell types and throughout the body. Within the brain, astrocytes are the predominant source of apoE (Pitas et al, 1987). It is thought that apoE functions by mediating neuronal repair and/or remodeling during development and during regeneration after injury to the central nervous system by mediating the distribution of lipids, predominantly cholesterol and phospholipids (Nathan et al, 1994).Three common human isoforms of apoE exist: E2, E3, and E4. ApoE4 is now a well-established risk factor for the development of Alzheimer's disease (AD) (Corder et al, 1993;Saunders et al, 1993). ApoE4 has also been associated with other neurological diseases, including vascular dementia and a poor clinical outcome in patients after stroke and head injury (Nicoll et al, 1995;Slooter et al, 1997). The mechanism by which apoE4 affects the process of neurodegeneration, however, is largely unknown.Mice deficient in apoE display disturbances in learning and memory function as well as in long-term potentiation (Krugers et al, 1997;Krzywkowski et al, 1999;Oitzl et al, 1997;Veinbergs and Masliah, 1999). They display several neuropathologic alterations, including a loss of synapses with increasing age, cytoskeletal alterations (Masliah et al, 1995), and a reduced activity of choline acetyltransferase (Gordon et al, 1995). In addition, they show an impaired response to ischemic, traumatic, and excitotoxic lesions (Chen et al, 1997;Laskowitz et al, 1997). On the contrary, a few studies report neit...