Cannabinoid type 1 receptor blockade induces transdifferentiation towards a brown fat phenotype in white adipocytes

Aim: The endocannabinoid (EC) system is a major component in the control of energy homeostasis. It mediates a positive energy balance via central and peripheral pathways. Blockade of the cannabinoid type 1 receptor induces weight reduction and improves cardiovascular risk factors in overweight patients. Cannabinoid receptor type 1 (CB1R)‐deficient mice are resistant to diet‐induced obesity. The mechanisms responsible for these effects remain only partially elucidated. We hypothesized peripheral effects via direct modulation of adipocyte function to be an integral part of EC action on energy metabolism and insulin sensitivity. Methods: SV40 immortalized murine white and brown adipocytes were used for all experiments. We investigated the effect of CB1R blockade by stimulating the cells acutely and chronically with rimonabant, a selective antagonist for the CB1R, or by knocking down the receptor with small interfering RNA (siRNA). Changes in thermogenic mRNA and protein expression as well as mitochondrial biogenesis and function were assessed by real‐time RT‐PCR, immunoblotting, fluorescent staining techniques, electron microscopy and by measuring oxygen consumption. Results: Acute and chronic blockade of the CB1R with the selective antagonist rimonabant or by siRNA in murine white adipocytes strongly induced the thermogenic uncoupling protein‐1 (UCP‐1). UCP‐1 expression was increased in a time‐ and dose‐dependent manner both at the RNA and protein level. Furthermore, this effect was paralleled by enhanced peroxisome proliferator‐activated receptor γ coactivator 1α (PGC‐1α) expression. In accordance with these findings, AMP‐activated protein kinase (AMPK) phosphorylation was also increased after rimonabant treatment. Mitochondria‐specific fluorescent staining demonstrated an augmentation in the number of mitochondria. This was confirmed by electron microscopy images. Moreover, rimonabant treatment enhanced the cytochrome c oxidase activity and increased cellular oxygen consumption. Conclusions: Taken together, our data demonstrate that inhibition of peripheral CB1R action in adipocytes directly promotes transdifferentiation of white adipocytes into a mitochondria‐rich, thermogenic brown fat phenotype. Enhanced thermogenesis and insulin sensitivity may represent a peripheral mechanism contributing to weight loss and improved glucose homeostasis in rimonabant‐treated patients.     

"自杀性传输"及相关毒素在疼痛治疗中的应用

某些毒素具有逆向轴浆运输作用,可造成神经系统的选择性损伤,即“自杀性传输”,并被命名为“分子神经外科”。近年来此技术应用于伤害性感受研究领域,证明为分析伤害性感受系统的一种有用工具,并有希望界定特殊类型神经元在疼痛感受和发现疼痛治疗新方法中的作用。     

A gene involved in the biogenesis of cytochromes is co-transcribed with a ribosomal protein gene in wheat mitochondria

Sequence analysis of a transcribed region of the wheat mitochondrial (mt) genome revealed two open reading frames (orfs) coding for proteins of 589 and 174 amino acids. Both genes are co-transcribed in a 2.6-kb RNA. The largest orf codes for a hydrophobic protein which bears similarity to a bacterial protein involved in the biogenesis of c-type cytochromes. Its corresponding RNA sequence is fully edited at 34 positions. The second orf encodes a protein homologous to the amino-terminal third of E. coli ribosomal protein S1, corresponding to the ribosome-binding domain of this protein. Its RNA sequence is edited at four positions, one of the edits creating a stop codon. The presence of both proteins in wheat mitochondria was demonstrated using specific antibodies raised against fusion proteins obtained in E. coli from the corresponding cDNAs.     

Changes in content of cytochromes c and a (a3) in liver mitochondria of hyperthyroid rats

Male Wistar rats aged 1, 3, 12, and 24 months were given thyroxine in a dose of 250 g/100 g body weight daily. After 9 days the content of cytochromes c and a (a₃) was considerably increased. An appreciable increase in the cytochrome c content was observed as early as after 24 h, whereas the content of cytochromes a (a₃) did not exceed normal even after 2 days. The content of cytochromes a (a₃) in rats aged 3, 12, and 24 months was little lower after 24 h than normally. A significant temporary increase in the ratio c/a (a₃) after 1–2 days was observed only in rats aged 12 and 24 months. An increase in the ratio c/a (a₃) with age also was demonstrated. The prospects for the use of thyroid hormones in the study of regulation of biogenesis of the mitochondria are suggested.Department of Biochemistry of Ontogeny, Research Institute of Biology, Khar'kov University. (Presented by Academician of the Academy of Medical Sciences of the USSR S. E. Severin.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 89, No. 2, pp. 174–176, February, 1980.     

Cold-sensitive mutation in Neurospora crassa affecting the production of 17S ribosomal RNA from ribosomal precursor RNA

Summary The cold-sensitive mutant strain of Neurospora crassa, crib-1 (PJ30201) has a conditional defect in ribosome biosynthesis. At 10 °C this mutant underproduces the small (37S) ribosomal subunit. Experiments were done to study rRNA synthesis in crib-1 after a shift from the permissive (25 °C) to the nonpermissive temperature and vice versa. The results showed that the primary cold-sensitive defect in crib-1 is in the function, rather than the synthesis, of a molecule needed for the production of the 17S rRNA component of the 37S subunit. Pulse-labeling experiments showed that at 10 °C crib-1 synthesizes the 2.4-Mdal pre-rRNA molecule which contains the sequences for the 17S, 5.8S and 25S rRNA species, but that processing of this molecule is defective such that relatively few stable 17S rRNA molecules accumulate.     

A single nucleotide substitution at the rib2 locus of the yeast mitochondrial gene for 21S rRNA confers resistance to erythromycin and cold-sensitive ribosome assembly

Summary We have studied a mutation (cs23) in the mitochondrial gene for 21SrRNA that affects the peptidyl transferase center of the ribosome and conditionally blocks the assembly of the 54S ribosomal subunit. Strains carrying this mutation are resistant to erythromycin and cold-sensitive for growth on nonfermentable carbon sources (Singh et al. 1978) Mitochondria isolated from mutant cells grown on glucose at 20°C, the nonpermissive temperature, were depleted of the 54S subunit and instead contained a novel 45S ribosomal particle. After mutant cells were shifted from 20°C to 32°C, 54S subunits were assembled, apparently from the 45S particles and pre-existing ribosomal proteins. DNA sequencing revealed that the mutant phenotype is a consequence of a C to A transversion at position 3993 of the 21SrRNA gene. Previously, C to U and C to G mutations have been identified at the same position in the 21S rRNA sequence. This position corresponds to C-2611 in the E. coli 23S RNA, a nucleotide that appears to be conserved in the large rRNA of all erythromycin-sensitive ribosomes.     

Identification of the molecular defect in patients with peroxisomal mosaicism using a novel method involving culturing of cells at 40 degrees C: implications for other inborn errors of metabolism

The peroxisome biogenesis disorders (PBDs), which comprise Zellweger syndrome (ZS), neonatal adrenoleukodystrophy, and infantile Refsum disease (IRD), represent a spectrum of disease severity, with ZS being the most severe, and IRD the least severe disorder. The PBDs are caused by mutations in one of the at least 12 different PEX genes encoding proteins involved in the biogenesis of peroxisomes. We report the biochemical characteristics and molecular basis of a subset of atypical PBD patients. These patients were characterized by abnormal peroxisomal plasma metabolites, but otherwise normal to very mildly abnormal peroxisomal parameters in cultured skin fibroblasts, including a mosaic catalase immunofluorescence pattern in fibroblasts. Since this latter feature made standard complementation analysis impossible, we developed a novel complementation technique in which fibroblasts were cultured at 40 degrees C, which exacerbates the defect in peroxisome biogenesis. Using this method, we were able to assign eight patients to complementation group 3 (CG3), followed by the identification of a single homozygous c.959C>T (p.S320F) mutation in their PEX12 gene. We also investigated various peroxisomal biochemical parameters in fibroblasts at 30 degrees C, 37 degrees C, and 40 degrees C, and found that all parameters showed a temperature-dependent behavior. The principle of culturing cells at elevated temperatures to exacerbate the defect in peroxisome biogenesis, and thereby preventing certain mutations from being missed, may well have a much wider applicability for a range of different inborn errors of metabolism.     

Genomic structure and identification of 11 novel mutations of the PEX6 (peroxisome assembly factor‐2) gene in patients with peroxisome biogenesis disorders

The PEX6 (peroxisome assembly factor‐2, PAF‐2) gene encodes a member of the AAA protein (ATPases associated with diverse cellular activities) family and restores peroxisome assembly in fibroblasts from peroxisome biogenesis disorder patients belonging to complementation group C (group 4 in the United States). We have now clarified the genomic DNA structure of human PEX6 and identified mutations in patients from various ethnic groups. The human PEX6 gene consists of 17 exons and 16 introns, spanning about 14kb. The largest exon, exon 1, has at least 952 bp nucleotides. Eleven novel mutations (18 alleles) were identified by direct sequencing of the PEX6 cDNA from 10 patients. All these mutations have been confirmed in the corresponding genomic DNA. There was no common mutation, but an exon skip was identified in two unrelated Japanese patients. Most of the mutations led to premature termination or large deletions of the PEX6 protein and resulted in the most severe peroxisome biogenesis disorder phenotype of Zellweger syndrome. A patient with an atypical Zellweger syndrome had a missense mutation that was shown to disrupt the cell's ability to form peroxisomes. This mutation analysis will aid in understanding the functions of the PEX6 protein in peroxisomal biogenesis. Hum Mutat 13:487–496, 1999. © 1999 Wiley‐Liss, Inc.