N-butyldeoxynojirimycin causes weight loss as a result of appetite suppression in lean and obese mice.

AIM: To determine the mechanism of weight loss caused by high doses of N-butyldeoxynojirimycin (NB-DNJ) in healthy lean and leptin-deficient obese (ob/ob) mice. METHODS: Healthy lean and obese mice were treated with NB-DNJ by the following methods: admixed with their diet, delivered by subcutaneously implanted mini-pumps or by intraperitoneal or intracerebroventricular (ICV) injection. Daily changes in body weight and food intake were recorded during the experimental period. The effect of NB-DNJ treatment on subcutaneous adipose tissue and on epididymal fat pads was measured. RESULTS: Lean mice treated with NB-DNJ, admixed with their diet, lost weight in the form of adipose tissue. This resulted in a 40% reduction in skin thickness (control, 358 +/- 11 microm; NB-DNJ treated 203 +/- 6 microm) and a reduction in epididymal fat pad weights after 5 weeks of treatment at 2400 mg/kg/day (control, 0.0154 +/- 0.001; NB-DNJ treated, 0.0026 +/- 0.0005 as ratios of fat pad weight to total body weight). Following the depletion of adipose tissue mass, the mice grew normally and did not have any reduction in lean mass. Obese mice treated with NB-DNJ also lost weight or gained weight at a greatly reduced rate compared with non-treated controls. Body weights at 6 months of age were: lean control, 29.10 +/- 1.15 g; lean NB-DNJ treated, 22.73 +/- 0.29 g; obese control, 63.25 +/- 1.5 g; obese NB-DNJ treated from 5 weeks of age, 35.30 +/- 1.68 g; obese NB-DNJ treated from 12 weeks of age, 38.84 +/- 1.26 g. Both the lean and obese groups of mice treated with NB-DNJ ate up to 30% less than untreated controls. Daily food intake (powder diet) were: lean control, 4.15 +/- 0.54 g; obese control, 4.14 +/- 0.2 g; lean NB-DNJ treated 2.9 +/- 0.37 g; obese NB-DNJ treated, 2.88 +/- 0.47 g. Mice treated with the N-substituted galactose imino sugar analogue, N-butyldeoxygalactonojirimycin (NB-DGJ) did not lose weight. Mice experienced similar weight loss or lack of weight gain when fed a restricted diet that mimics the drug-induced level of food consumption. Delivery of 2 nmol NB-DNJ by ICV injection into lean mice also caused similar reductions in food intake. Food intake: saline vehicle, 4.30 +/- 0.12 g; NB-DNJ, 3.37 +/- 0.19 g; NB-DGJ, 4.03 +/- 0.16 g; 2-deoxyglucose, 4.7 +/- 0.15 g. CONCLUSION: NB-DNJ causes weight loss as a result of reduced food consumption due to central appetite suppression.

Central nervous system inflammation is a hallmark of pathogenesis in mouse models of GM1 and GM2 gangliosidosis.

Mouse models of the GM2 gangliosidoses [Tay-Sachs, late onset Tay-Sachs (LOTS), Sandhoff] and GM1 gangliosidosis have been studied to determine whether there is a common neuro-inflammatory component to these disorders. During the disease course, we have: (i) examined the expression of a number of inflammatory markers in the CNS, including MHC class II, CD68, CD11b (CR3), 7/4, F4/80, nitrotyrosine, CD4 and CD8; (ii) profiled cytokine production [tumour necrosis factor alpha (TNF alpha), transforming growth factor (TGF beta 1) and interleukin 1 beta (IL1 beta)]; and (iii) studied blood-brain barrier (BBB) integrity. The kinetics of apoptosis and the expression of Fas and TNF-R1 were also assessed. In all symptomatic mouse models, a progressive increase in local microglial activation/expansion and infiltration of inflammatory cells was noted. Altered BBB permeability was evident in Sandhoff and GM1 mice, but absent in LOTS mice. Progressive CNS inflammation coincided with the onset of clinical signs in these mouse models. Substrate reduction therapy in the Sandhoff mouse model slowed the rate of accumulation of glycosphingolipids in the CNS, thus delaying the onset of the inflammatory process and disease pathogenesis. These data suggest that inflammation may play an important role in the pathogenesis of the gangliosidoses.

Alpha 1----3-galactosyltransferase: the use of recombinant enzyme for the synthesis of alpha-galactosylated glycoconjugates.

We have reported the isolation and characterization of a bovine cDNA clone containing the complete coding sequence for UDP-Gal:Gal beta 1----4GlcNAc alpha 1----3-galactosyltransferase [Joziasse, D. H., Shaper, J. H., Van den Eijnden, D. H., Van Tunen, A. J. & Shaper, N. L. (1989) J. Biol. Chem. 264, 14290-14297]. Insertion of this cDNA clone into the genome of Autographa californica nuclear polyhedrosis virus (AcNPV) and subsequent infection of Spodoptera frugiperda (Sf9) insect cells with recombinant virus, resulted in high-level expression of enzymatically active alpha 1----3-galactosyltransferase. The expressed enzyme accounted for about 2% of the cellular protein; the corresponding specific enzyme activity was 1000-fold higher than observed in calf thymus, the tissue with the highest specific enzyme activity reported to date. The recombinant alpha 1----3-galactosyltransferase could be readily detergent-solubilized and subsequently purified by affinity chromatography on UDP-hexanolamine-Sepharose. The recombinant alpha 1----3-galactosyltransferase showed the expected preference for the acceptor substrate N-acetyllactosamine (Gal beta 1----4GlcNAc), and demonstrated enzyme kinetics identical to those previously reported for affinity-purified calf thymus alpha 1----3-galactosyltransferase [Blanken, W. M. & Van den Eijnden, D. H. (1985) J. Biol. Chem. 260, 12927-12934]. In pilot studies, the recombinant enzyme was examined for the ability to synthesize alpha 1----3-galactosylated oligosaccharides, glycolipids and glycoproteins. By a combination of 1H-NMR, methylation analysis, HPLC, and exoglycosidase digestion it was established that, for each of the model compounds, the product of galactose transfer had the anticipated terminal structure, Gal alpha 1----3Gal beta 1----4-R. Our results demonstrate that catalysis by recombinant alpha 1----3-galactosyltransferase can be used to obtain preparative quantities of various alpha 1----3-galactosylated glycoconjugates. Therefore, enzymatic synthesis using the recombinant enzyme is an effective alternative to the chemical synthesis of these biologically relevant compounds.