Cold-induced expression of a truncated adenylyl cyclase 3 acts as rheostat to brown fat function.

Promoting brown adipose tissue (BAT) activity innovatively targets obesity and metabolic disease. While thermogenic activation of BAT is well understood, the rheostatic regulation of BAT to avoid excessive energy dissipation remains ill-defined. Here, we demonstrate that adenylyl cyclase 3 (AC3) is key for BAT function. We identified a cold-inducible promoter that generates a 5' truncated AC3 mRNA isoform (Adcy3-at), whose expression is driven by a cold-induced, truncated isoform of PPARGC1A (PPARGC1A-AT). Male mice lacking Adcy3-at display increased energy expenditure and are resistant to obesity and ensuing metabolic imbalances. Mouse and human AC3-AT are retained in the endoplasmic reticulum, unable to translocate to the plasma membrane and lack enzymatic activity. AC3-AT interacts with AC3 and sequesters it in the endoplasmic reticulum, reducing the pool of adenylyl cyclases available for G-protein-mediated cAMP synthesis. Thus, AC3-AT acts as a cold-induced rheostat in BAT, limiting adverse consequences of cAMP activity during chronic BAT activation.

Wt1 haploinsufficiency induces browning of epididymal fat and alleviates metabolic dysfunction in mice on high-fat diet.

AIMS/HYPOTHESIS: Despite a similar fat storing function, visceral (intra-abdominal) white adipose tissue (WAT) is detrimental, whereas subcutaneous WAT is considered to protect against metabolic disease. Recent findings indicate that thermogenic genes, expressed in brown adipose tissue (BAT), can be induced primarily in subcutaneous WAT. Here, we investigate the hypothesis that the Wilms tumour gene product (WT1), which is expressed in intra-abdominal WAT but not in subcutaneous WAT and BAT, suppresses a thermogenic program in white fat cells. METHODS: Heterozygous Wt1 knockout mice and their wild-type littermates were examined in terms of thermogenic and adipocyte-selective gene expression. Glucose tolerance and hepatic lipid accumulation in these mice were assessed under normal chow and high-fat diet conditions. Pre-adipocytes isolated from the stromal vascular fraction of BAT were transduced with Wt1-expressing retrovirus, induced to differentiate and analysed for the expression of thermogenic and adipocyte-selective genes. RESULTS: Expression of the thermogenic genes Cpt1b and Tmem26 was enhanced and transcript levels of Ucp1 were on average more than tenfold higher in epididymal WAT of heterozygous Wt1 knockout mice compared with wild-type mice. Wt1 heterozygosity reduced epididymal WAT mass, improved whole-body glucose tolerance and alleviated severe hepatic steatosis upon diet-induced obesity in mice. Retroviral expression of WT1 in brown pre-adipocytes, which lack endogenous WT1, reduced mRNA levels of Ucp1, Ppargc1a, Cidea, Prdm16 and Cpt1b upon in vitro differentiation by 60-90%. WT1 knockdown in epididymal pre-adipocytes significantly lowered Aldh1a1 and Zfp423 transcripts, two key suppressors of the thermogenic program. Conversely, Aldh1a1 and Zfp423 mRNA levels were increased approximately five- and threefold, respectively, by retroviral expression of WT1 in brown pre-adipocytes. CONCLUSION/INTERPRETATION: WT1 functions as a white adipocyte determination factor in epididymal WAT by suppressing thermogenic genes. Reducing Wt1 expression in this and other intra-abdominal fat depots may represent a novel treatment strategy in metabolic disease.

Loss of the ciliary gene Bbs4 results in defective thermogenesis due to metabolic inefficiency and impaired lipid metabolism.

Adipose tissue is central to the regulation of energy balance. While white adipose tissue (WAT) is responsible for triglyceride storage, brown adipose tissue specializes in energy expenditure. Deterioration of brown adipocyte function contributes to the development of metabolic complications like obesity and diabetes. These disorders are also leading symptoms of the Bardet-Biedl syndrome (BBS), a hereditary disorder in humans which is caused by dysfunctions of the primary cilium and which therefore belongs to the group of ciliopathies. The cilium is a hair-like organelle involved in cellular signal transduction. The BBSome, a supercomplex of several Bbs gene products, localizes to the basal body of cilia and is thought to be involved in protein sorting to and from the ciliary membrane. The effects of a functional BBSome on energy metabolism and lipid mobilization in brown and white adipocytes were tested in whole-body Bbs4 knockout mice that were subjected to metabolic challenges. Chronic cold exposure reveals cold-intolerance of knockout mice but also ameliorates the markers of metabolic pathology detected in knockouts prior to cold. Hepatic triglyceride content is markedly reduced in knockout mice while circulating lipids are elevated, altogether suggesting that defective lipid metabolism in adipose tissue creates increased demand for systemic lipid mobilization to meet energetic demands of reduced body temperatures. These findings taken together suggest that Bbs4 is essential for the regulation of adipose tissue lipid metabolism, representing a potential target to treat metabolic disorders.

Adipocyte Accumulation in the Bone Marrow during Obesity and Aging Impairs Stem Cell-Based Hematopoietic and Bone Regeneration.

Aging and obesity induce ectopic adipocyte accumulation in bone marrow cavities. This process is thought to impair osteogenic and hematopoietic regeneration. Here we specify the cellular identities of the adipogenic and osteogenic lineages of the bone. While aging impairs the osteogenic lineage, high-fat diet feeding activates expansion of the adipogenic lineage, an effect that is significantly enhanced in aged animals. We further describe a mesenchymal sub-population with stem cell-like characteristics that gives rise to both lineages and, at the same time, acts as a principal component of the hematopoietic niche by promoting competitive repopulation following lethal irradiation. Conversely, bone-resident cells committed to the adipocytic lineage inhibit hematopoiesis and bone healing, potentially by producing excessive amounts of Dipeptidyl peptidase-4, a protease that is a target of diabetes therapies. These studies delineate the molecular identity of the bone-resident adipocytic lineage, and they establish its involvement in age-dependent dysfunction of bone and hematopoietic regeneration.

Adult human liver contains intermediate-type proteasomes with different enzymatic properties.

BACKGROUND: The 20S proteasome is the proteolytic core of the major intracellular protein degradative system, the ubiquitin-proteasome system. Since little is known about proteasomes of human liver, we have investigated the proteasome spectrum in adult human liver. MATERIAL AND METHODS: 20S proteasomes were chromatographically purified from adult human liver and from HuH7 cells. They were divided into subpopulations and subtypes and characterized with regard to their proteolytic activities using short fluorogenic oligo- and long poly-peptide substrates. Their subunit composition was studied by immunoblotting. RESULTS: Proteasomes from adult human liver tissue can be separated into three subpopulations (I, II, III), each of which is composed of several subtypes, which total to a spectrum of 14 different subtypes. Two minor subtypes contain only the immuno-subunits ß1i and ß5i but not their standard counterparts; all others are intermediate subtypes containing ß1 and ß5 standard- and ß1i and ß5i immuno-subunits in various compositions. With regard to the proteolytic activities we observed that a decreasing content of subunit ß1i in the subtypes goes along with a decreasing ratio of chymotrypsin-like/caspase-like activity, whereas the degradation rate of a 30 mer polypeptide substrate increased with decreasing ß1i content. By comparison, 20S proteasomes from HuH7 cells do not contain immuno-subunits but are pure standard proteasomes, which can be separated into three subtypes. CONCLUSION: These findings suggest that adult human liver contains a spectrum of 14 different 20S proteasome subtypes with different enzymatic properties reflecting most probably an adaptive response of liver cell functions to challenging factors during lifetime.

Molecular alterations in proteasomes of rat liver during aging result in altered proteolytic activities.

Aging induces alterations of tissue protein homoeostasis. To investigate one of the major systems catalysing intracellular protein degradation we have purified 20S proteasomes from rat liver of young (2 months) and aged (23 months) animals and separated them into three subpopulations containing different types of intermediate proteasomes with standard- and immuno-subunits. The smallest subpopulation ΙΙΙ and the major subpopulation Ι comprised proteasomes containing immuno-subunits ß1i and ß5i beside small amounts of standard-subunits, whereas proteasomes of subpopulation ΙΙ contained only ß5i beside standard-subunits. In favour of a relative increase of the major subpopulation Ι, subpopulation ΙΙ and ΙΙΙ were reduced for about 55 % and 80 %, respectively, in aged rats. Furthermore, in all three 20S proteasome subpopulations from aged animals standard-active site subunits were replaced by immuno-subunits. Overall, this transformation resulted in a relative increase of immuno-subunit-containing proteasomes, paralleled by reduced activity towards short fluorogenic peptide substrates. However, depending on the substrate their hydrolysing activity of long polypeptide substrates was significantly higher or unchanged. Furthermore, our data revealed an altered MHC class I antigen-processing efficiency of 20S proteasomes from liver of aged rats. We therefore suggest that the age-related intramolecular alteration of hepatic proteasomes modifies its cleavage preferences without a general decrease of its activity. Such modifications could have implications on protein homeostasis as well as on MHC class I antigen presentation as part of the immunosenescence process.

Driving forces of proteasome-catalyzed peptide splicing in yeast and humans.

Proteasome-catalyzed peptide splicing (PCPS) represents an additional activity of mammalian 20S proteasomes recently identified in connection with antigen presentation. We show here that PCPS is not restricted to mammalians but that it is also a feature of yeast 20S proteasomes catalyzed by all three active site ß subunits. No major differences in splicing efficiency exist between human 20S standard- and immuno-proteasome or yeast 20S proteasome. Using H(2)(18)O to monitor the splicing reaction we also demonstrate that PCPS occurs via direct transpeptidation that slightly favors the generation of peptides spliced in cis over peptides spliced in trans. Splicing efficiency itself is shown to be controlled by proteasomal cleavage site preference as well as by the sequence characteristics of the spliced peptides. By use of kinetic data and quantitative analyses of PCPS obtained by mass spectrometry we developed a structural model with two PCPS binding sites in the neighborhood of the active Thr1.

A tandem sequence motif acts as a distance-dependent enhancer in a set of genes involved in translation by binding the proteins NonO and SFPQ.

BACKGROUND: Bioinformatic analyses of expression control sequences in promoters of co-expressed or functionally related genes enable the discovery of common regulatory sequence motifs that might be involved in co-ordinated gene expression. By studying promoter sequences of the human ribosomal protein genes we recently identified a novel highly specific Localized Tandem Sequence Motif (LTSM). In this work we sought to identify additional genes and LTSM-binding proteins to elucidate potential regulatory mechanisms. RESULTS: Genome-wide analyses allowed finding a considerable number of additional LTSM-positive genes, the products of which are involved in translation, among them, translation initiation and elongation factors, and 5S rRNA. Electromobility shift assays then showed specific signals demonstrating the binding of protein complexes to LTSM in ribosomal protein gene promoters. Pull-down assays with LTSM-containing oligonucleotides and subsequent mass spectrometric analysis identified the related multifunctional nucleotide binding proteins NonO and SFPQ in the binding complex. Functional characterization then revealed that LTSM enhances the transcriptional activity of the promoters in dependency of the distance from the transcription start site. CONCLUSIONS: Our data demonstrate the power of bioinformatic analyses for the identification of biologically relevant sequence motifs. LTSM and the here found LTSM-binding proteins NonO and SFPQ were discovered through a synergistic combination of bioinformatic and biochemical methods and are regulators of the expression of a set of genes of the translational apparatus in a distance-dependent manner.