The mammalian 5-AMP-activated protein kinase (AMPK) is related to a growing family of protein kinases in yeast and plants that are regulated by nutritional stress. We find the most prominent expressed form of the hepatic AMPK catalytic subunit (␣ 1 ) is distinct from the previously cloned kinase subunit (␣ 2 ). The ␣ 1 (548 residues) and ␣ 2 (552 residues) isoforms have 90% amino acid sequence identity within the catalytic core but only 61% identity elsewhere. The tissue distribution of the AMPK activity most closely parallels the low abundance 6-kilobase ␣ 1 mRNA distribution and the ␣ 1 immunoreactivity rather than ␣ 2 , with substantial amounts in kidney, liver, lung, heart, and brain. Both ␣ 1 and ␣ 2 isoforms are stimulated by AMP and contain noncatalytic ␤ and ␥ subunits. The liver ␣ 1 isoform accounts for approximately 94% of the enzyme activity measured using the SAMS peptide substrate. The tissue distribution of the ␣ 2 immunoreactivity parallels the ␣ 2 8.5-kilobase mRNA and is most prominent in skeletal muscle, heart, and liver. Isoforms of the ␤ and ␥ subunits present in the human genome sequence reveal that the AMPK consists of a family of isoenzymes.The 5Ј-AMP-activated protein kinase (AMPK) 1 was initially identified as a protein kinase regulating hydroxymethylglutaryl-CoA reductase (1). Subsequently, the AMPK was shown to phosphorylate hepatic acetyl-CoA carboxylase (2) and adipose hormone-sensitive lipase (3). The AMPK appears to act as a metabolic stress-sensing protein kinase switching off biosynthetic pathways when cellular ATP levels are depleted and when 5Ј-AMP rises in response to fuel limitation and/or hypoxia (4). Partial amino acid sequencing of hepatic AMPK (5, 6) revealed that it consists of 3 subunits, the catalytic subunit ␣ (63 kDa), and two noncatalytic subunits, ␤ (40 kDa) and ␥ (38 kDa).The AMPK is a member of the yeast SNF1 protein kinase subfamily that includes protein kinases present in plants, nematodes, and humans (5-9). The AMPK catalytic subunit, ␣, has strong sequence identity to the catalytic domain of the yeast protein kinase SNF1, which is involved in the induction of invertase (SUC2) under conditions of nutritional stress due to glucose starvation (10). Both Snf1p and the AMPK require phosphorylation by an activating protein kinase for full activity (11). The sequence relationship between Snf1p and AMPK led to the finding that these enzymes share functional similarities, both phosphorylating and inactivating yeast acetyl-CoA carboxylase (5,11,12). Nevertheless, the AMPK does not complement SNF1 in yeast (11), indicating that their full range of functions are not identical. The noncatalytic ␤ and ␥ subunits of AMPK are also related to proteins that interact with Snf1p; the ␤ subunit is related to the SIP1/SIP2/GAL83 family of transcription regulators and the ␥ subunit to SNF4 (also called CAT3) (6, 13). EXPERIMENTAL PROCEDURESPeptide Sequencing-Peptides were derived from rat and porcine ␣ 1 subunit of the AMPK, by in situ proteolysis (5), and sequenced on either an Ap...

No abstract

No abstract

No abstract

It has been shown that treatment with miglustat (Zavesca, N-butyldeoxynojirimycin, OGT 918) improves key clinical features of type I Gaucher disease after 1 year of treatment. This study reports longer-term efficacy and safety data. Patients who had completed 12 months of treatment with open-label miglustat (100-300 mg three times daily) were enrolled to continue with therapy in an extension study. Data are presented up to month 36. Liver and spleen volumes measured by CT or MRI were scheduled every 6 months. Biochemical and haematological parameters, including chitotriosidase activity (a sensitive marker of Gaucher disease activity) were monitored every 3 months. Safety data were also collected every 3 months. Eighteen of 22 eligible patients at four centres entered the extension phase and 14 of these completed 36 months of treatment with miglustat. After 36 months, there were statistically significant improvements in all major efficacy endpoints. Liver and spleen organ volumes were reduced by 18% and 30%, respectively. In patients whose haemoglobin value had been below 11.5 g/dl at baseline, mean haemoglobin increased progressively from baseline by 0.55 g/dl at month 12 (NS), 1.28 g/dl at month 24 (p =0.007), and 1.30 g/dl at month 36 (p =0.013). The mean platelet count at month 36 increased from baseline by 22 x 10(9)/L. No new cases of peripheral neuropathy occurred since previously reported. Diarrhoea and weight loss, which were frequently reported during the initial 12-month study, decreased in magnitude and prevalence during the second and third years. Patients treated with miglustat for 3 years show significant improvements in organ volumes and haematological parameters. In conclusion, miglustat was increasingly effective over time and showed acceptable tolerability in patients who continued with treatment for 3 years.

No abstract

Gaucher's disease is characterized by hepatosplenomegaly, bone-marrow infiltration, osteonecrosis and bone thinning, associated with the presence of pathological macrophages that contain undegraded glycosphingolipids. To investigate the possible role of cytokines in the systemic and local manifestations of established Gaucher's disease, interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6), tumour necrosis factor-alpha (TNF alpha) and interleukin-10 (IL-10) were measured in freshly-separated serum. Samples from eight male and 14 female patients with type 1 Gaucher's disease were compared with sera from 22 healthy age- and sex-matched controls. Concentrations of IL-6 and IL-10 were significantly elevated in sera from patients with Gaucher's disease (11.9 +/- 1.8 (SEM) pg/ml and 5.4 +/- 0.5 (SEM) pg/ml, respectively) compared with those of controls (4.1 +/- 0.9 (SEM) and 0.8 +/- 0.3 (SEM) pg/ml, p < 0.0001). No significant differences in concentrations of TNF alpha or IL-1 beta were identified. IL-6 has been implicated in the development of localized osteolysis in multiple myeloma and in the development of post-menopausal osteoporosis. High concentrations of IL-6 in the serum of patients with Gaucher's disease may thus reflect the development of the bone lesions commonly associated with this disorder. Since IL-6 and IL-10 are important regulators of lymphocyte growth and differentiation, and IL-6 concentrations were significantly raised in patients with oligo- or polyclonal increases in serum immunoglobulins, enhanced release of these cytokines from pathological macrophages provides a pathological link between Gaucher's disease and associated lympho-proliferative disorders.

Gaucher's disease (GD), an inherited metabolic disorder caused by mutations in the glucocerebrosidase gene (GBA), is the most common lysosomal storage disease. Heterozygous mutations in GBA are a major risk factor for Parkinson's disease. GD is divided into three clinical subtypes based on the absence (type 1) or presence (types 2 and 3) of neurological signs. Type 1 GD was the first lysosomal storage disease (LSD) for which enzyme therapy became available, and although infusions of recombinant glucocerebrosidase (GCase) ameliorate the systemic effects of GD, the lack of efficacy for the neurological manifestations, along with the considerable expense and inconvenience of enzyme therapy for patients, renders the search for alternative or complementary therapies paramount. Glucosylceramide and glucosylsphingosine accumulation in the brain leads to massive neuronal loss in patients with neuronopathic GD (nGD) and in nGD mouse models. However, the mode of neuronal death is not known. Here, we show that modulating the receptor-interacting protein kinase-3 (Ripk3) pathway markedly improves neurological and systemic disease in a mouse model of GD. Notably, Ripk3 deficiency substantially improved the clinical course of GD mice, with increased survival and motor coordination and salutary effects on cerebral as well as hepatic injury.