A comparative study of five centrally acting drugs on the pharmacological treatment of obesity.

CONTEXT: No long-term studies have compared centrally acting drugs for treating obesity. OBJECTIVE: To compare the efficacy and safety of diethylpropion (DEP), fenproporex (FEN), mazindol (MZD), fluoxetine (FXT) and sibutramine (SIB) in promoting weight loss. DESIGN AND SETTING: A prospective, randomized, placebo (PCB)-controlled study conducted at a single academic institution. PATIENTS: A total of 174 obese premenopausal women. INTERVENTION: Participants randomly received DEP 75 mg (n=28), FEN 25 mg (n=29), MZD 2 mg (n=29), SIB 15 mg (n=30), FXT 20 mg (n=29) or PCB (n=29) daily over 52 weeks. Diet and physical activity were encouraged. MAIN OUTCOME MEASURES: The primary endpoints were changes in body weight and the proportion of women who achieved at least 5% weight loss by week 52 in the intent-to-treat population. Other measurements included anthropometry, safety, metabolic and cardiovascular parameters. RESULTS: Weight loss was greater than PCB (-3.1±4.3 kg) with DEP (-10.0±6.4 kg; P<0.001), SIB (-9.5±5.9 kg; P<0.001), FEN (-7.8±6.9 kg; P<0.01) and MZD (-7.4±4.9 kg; P<0.01) but not with FXT (-2.5±4.1 kg). Ten (33.3%) women lost⩾5% of their initial weight with PCB, compared with 20 (71.4%; P<0.001) with DEP, 20 (69%; P<0.02) with FEN, 21 (72.4%; P<0.01) with MZD, 22 (73.3%; P<0.001) with SIB and 10 (35.5%) with FXT. Each medically treated group experienced more adverse events compared with PCB (P<0.001). Compared with PCB, constipation was more prevalent with DEP, SIB and MZD (P<0.01); anxiety was more prevalent with DEP (P=0.01); and irritability occurred more frequently with DEP and FEN (P=0.02). Significant improvements in the depression and anxiety scores, binge-eating episodes and quality of life correlated with weight loss. CONCLUSION: The centrally acting drugs DEP, FEN, MZD and SIB were more effective than PCB in promoting weight loss in obese premenopausal women, with a satisfactory benefit-risk profile.

Proteasome inhibitors block a late step in lysosomal transport of selected membrane but not soluble proteins.

The ubiquitin-proteasome pathway acts as a regulator of the endocytosis of selected membrane proteins. Recent evidence suggests that it may also function in the intracellular trafficking of membrane proteins. In this study, several models were used to address the role of the ubiquitin-proteasome pathway in sorting of internalized proteins to the lysosome. We found that lysosomal degradation of ligands, which remain bound to their receptors within the endocytic pathway, is blocked in the presence of specific proteasome inhibitors. In contrast, a ligand that dissociates from its receptor upon endosome acidification is degraded under the same conditions. Quantitative electron microscopy showed that neither the uptake nor the overall distribution of the endocytic marker bovine serum albumin-gold is substantially altered in the presence of a proteasome inhibitor. The data suggest that the ubiquitin-proteasome pathway is involved in an endosomal sorting step of selected membrane proteins to lysosomes, thereby providing a mechanism for regulated degradation.

Chronic AT1 receptor blockade alters aortic nerve activity in hypertension.

In the chronic phase of coarctation hypertension (CH) we have shown both reduction in baroreceptor sensitivity (Hypertension. 1992;19[suppl II]:II-198-II-201.) and normalization of the depressed baroreceptor reflex control of heart rate, even with the persistence of hypertension in losartan-treated animals (Am J Physiol. 1995;269:H812-H818). In the present study we analyzed the effects of angiotensin II blockade on afferent aortic nerve activity of CH and sham-operated groups treated chronically with vehicle or losartan (10 mg/kg per day p.o.). CH was induced by subdiaphragmatic aortic coarctation, and the treatments lasted 8 days (4 control and 4 experimental days). Aortic pressure (conscious rats) and aortic nerve activity simultaneous to pressure (anesthetized rats) were recorded on the fourth day of the experimental period. Losartan-treated rats showed reduced tail pressure (104+/-3 versus 117+/-3 mm Hg in the vehicle group). In both groups, aortic coarctation caused a significant increase in pressure (25% and 28%, respectively) and a depression of the aortic nerve activity/pressure relationship when compared with sham-operated coarcted animals. In the physiological range of pressure changes, the depression was significantly smaller after losartan treatment (3.30+/-0.33 versus 2.18+/-0.37%/mm Hg in the losartan- and vehicle-treated CH groups, respectively, versus 5.05+/-0.33%/mm Hg in the sham-operated vehicle-treated group). Angiotensin type 1 (AT1) receptor blockade was also accompanied by reduced variability of the afferent discharge. The data suggested that apart from its pressure effect, angiotensin II acts at AT1 receptors to decrease the sensitivity of aortic afferents during physiological (+/-10 mm Hg) increases and decreases in pressure. Thus, angiotensin II may contribute to reductions of baroreceptor gain in chronic hypertension.

Screening for inborn errors of metabolism in high-risk children from Rio de Janeiro, Brazil.

From 1988 to 1995, our laboratory at the Institute of Chemistry of the Federal University of Rio de Janeiro, in Rio de Janeiro, screened 2650 samples from 2000 high-risk patients (mostly children) for Inborn Errors of Metabolism (IEM). Chemical tests, various chromatographic techniques and enzyme assays were performed on urine, plasma and in some cases, cerebrospinal fluid (CSF). A total of 145 cases of IEM (7.2%) was identified. These were related to: the metabolism of amino acids (41) and carbohydrates (17), organic acids (7), lysosomal enzymes (61), membrane transport system (16), metals (2), intestinal disaccharidases (1) and porphyrin metabolism (3). Furthermore, a relevant number of patients with abnormal findings is still under investigation. Biochemical results and clinical symptoms are presented and the importance of reference laboratories for the detection of IEM is stressed.

Growth hormone receptor ubiquitination, endocytosis, and degradation are independent of signal transduction via Janus kinase 2.

The ubiquitin-proteasome system is required in growth hormone receptor (GHR) endocytosis. For cytokine receptors, which lack intrinsic tyrosine kinase activity, signal transduction is initiated by the activation of a member of the Janus kinase (JAK) family. Previously, we have shown that GHR and JAK2 tyrosine (de) phosphorylation are regulated via the ubiquitin system. In this study, we examined the role of JAK2-mediated signal transduction in GHR internalization and down-regulation. Mutation of the attachment site for JAK2, box-1, in the GHR cytoplasmic tail resulted in the complete absence of GHR and JAK2 phosphorylation. This modification did not alter the rate and extent of receptor-bound growth hormone internalization as compared with a functional GHR, nor did it change its turnover and transport to the plasma membrane. In addition, the receptor was still normally ubiquitinated and remained dependent on both an intact ubiquitin system and proteasomal action for its internalization. Thus, GHR ubiquitination, endocytosis, and degradation occur independently of GHR signal transduction via JAK2. We conclude that whereas endocytosis and degradation require the ubiquitin system, they are independent of GHR signal transduction.

Endocytosis and degradation of the growth hormone receptor are proteasome-dependent.

The ubiquitin conjugation system is involved in ligand-induced endocytosis of the growth hormone receptor (GHR) via a cytosolic 10-amino acid ubiquitin-dependent endocytosis motif. Herein, we demonstrate that the proteasome is also involved in growth hormone receptor down-regulation. Ligand-induced degradation was blocked in the presence of specific proteasomal inhibitors. In addition, growth hormone (GH) internalization was inhibited, whereas the transferrin receptor cycle remained unaffected. A truncated GHR entered the cells independent of proteasome action. In addition, we show that GH internalization is independent of the presence of lysine residues in the cytosolic domain of the receptor, whereas its internalization can still be inhibited by proteasomal inhibitors. Thus, GHR internalization requires proteasome action in addition to an active ubiquitin conjugation system, but ubiquitination of the GHR itself seems not to be required.

The signal transduction of the growth hormone receptor is regulated by the ubiquitin/proteasome system and continues after endocytosis.

The growth hormone receptor (GHR) intracellular domain contains all of the information required for signal transduction as well as for endocytosis. Previously, we showed that the proteasome mediates the clathrin-mediated endocytosis of the GHR. Here, we present evidence that the proteasomal inhibitor MG132 prolongs the GH-induced activity of both GHR and JAK2, presumably through stabilization of GHR and JAK2 tyrosine phosphorylation. If proteasomal inhibitor was combined with ligand in an endocytosis-deficient GHR mutant, the same phenomenon occurred indicating that proteasomal action on tyrosine dephosphorylation is independent of endocytosis. Experiments with a GHR-truncated tail mutant (GHR-(1-369)) led to a prolonged JAK2 phosphorylation caused by the loss of a phosphatase-binding site. This raised the question of what happens to the signal transduction of the GHR after its internalization. Co-immunoprecipitation of GH.GHR complexes before and after endocytosis showed that JAK2 as well as other activated proteins are bound to the GHR not only at the cell surface but also intracellularly, suggesting that the GHR signal transduction continues in endosomes. Additionally, these results provide evidence that GHR is present in endosomes both in its full-length and truncated form, indicating that the receptor is down-regulated by the proteasome.

Chronic AT(1) receptor blockade alters autonomic balance and sympathetic responses in hypertension.

In the coarctation hypertension model, we have shown that chronic treatment with losartan causes both normalization of impaired reflex control of heart rate and partial correction of the depressed aortic nerve activity/pressure relationship, even with the persistence of hypertension. In the present study, we analyzed the effects of angiotensin II blockade on the efferent pathways of coarcted and sham-operated groups treated chronically with vehicle or losartan (10 mg/kg per day PO). Hypertension was induced by subdiaphragmatic aortic coarctation, and the treatments lasted 9 days (4 control and 5 experimental days). On day 5, autoregressive power spectral analysis was performed on heart rate recordings made in conscious rats. Other groups were used for sympathetic splanchnic nerve activity recordings made simultaneously with pressure (anesthetized rats) at basal condition and during loading/unloading of baroreceptors. Losartan treatment induced a significant reduction in basal pressure but did not interfere with the development of hypertension (similar pressure increases of 24% and 28% over control values in losartan and vehicle groups, respectively). In vehicle-treated rats, establishment of hypertension was accompanied by a marked change in power spectral density from high- (1.19+/-0.06 Hz, 33+/-6%) to low-frequency components (0,42+/-0.03 Hz, 54+/-6%), with increased low-frequency-to-high-frequency ratio. When compared with sham-operated vehicle-treated rats, there was also increase in the gain of sympathetic activity/pressure relationship, with displacement of lower plateau toward high levels of sympathetic activity. No changes in the power spectral density and sympathetic activity/pressure relationship were observed when hypertension developed in the presence of chronic angiotensin type 1 (AT(1)) receptor blockade. The data suggest that angiotensin II, activated during the establishment of coarctation hypertension, acts via AT(1) receptors to alter sympathovagal balance, facilitating the sympathetic outflow to heart and peripheral circulation during baroreceptors unloading. Data also indicate that the observed effects are not conditioned by preexisting pressure levels.

Identification of a calcium binding site in Staphylococcus hyicus lipase: generation of calcium-independent variants.

In this study we have identified the presence of a high-affinity binding site for calcium in the lipase from Staphylococcus hyicus. By means of isothermal titration calorimetry we showed that the enzyme binds one calcium per molecule of enzyme with a dissociation constant of 55 microM. The residual activity of the apoenzyme compared to the activity in the presence of calcium ions varies from 65% at 10 degreesC to nearly zero at 40 degreesC. On the basis of primary sequence alignment with other staphylococcal lipases and the lipases from Bacillus thermocatenulatus and from Pseudomonas glumae in combination with site-directed mutagenesis, aspartates 354 and 357 could be identified as calcium ligands. Kinetic measurements with the D357E variant showed that replacement of Asp357 by a glutamate decreased the affinity for calcium ions 30-fold. Introduction of a lysine, an asparagine, or an alanine at position 357 and of a lysine or an asparagine at position 354 resulted in calcium-independent variants. Isothermal titration calorimetry confirmed the loss of calcium binding. Although the D357K, D357N, and D357A variants did not bind calcium, at room temperature they were nearly as active as wild-type lipase in the presence of calcium, but at elevated temperatures these calcium-independent lipases showed a reduced activity. Over the whole temperature range the activities of the D354K and D354N variants are significantly lower than wild-type enzyme in the presence of calcium and are comparable to the activity of the wild-type apoenzyme. Our results show that binding of calcium is important for the structural stabilization of staphylococcal lipases (and possibly other lipases) and that it is possible to engineer calcium-independent variants on the basis of limited structural homology with another lipase.