Adipose tissue depot specific expression and regulation of fibrosis-related genes and proteins in experimental obesity.

Transforming growth factor beta (Tgfb) is a well-studied pro-fibrotic cytokine, which upregulates cellular communication network factor 2 (Ccn2), collagen, and actin alpha 2, smooth muscle (Acta2) expression. Obesity induces adipose tissue fibrosis, which contributes to metabolic diseases. This work aimed to analyze the expression of Tgfb, Ccn2, collagen1a1 (Col1a1), Acta2 and BMP and activin membrane-bound inhibitor (Bambi), which is a negative regulator of Tgfb signaling, in different adipose tissue depots of mice fed a standard chow, mice fed a high fat diet (HFD) and ob/ob mice. Principally, these genes were low expressed in brown adipose tissues and this difference was less evident for the ob/ob mice. Ccn2 and Bambi protein as well as mRNA expression, and collagen1a1 mRNA were not induced in the adipose tissues upon HFD feeding whereas Tgfb and Acta2 mRNA increased in the white fat depots. Immunoblot analysis showed that Acta2 protein was higher in subcutaneous and perirenal fat of these mice. In the ob/ob mice, Ccn2 mRNA and Ccn2 protein were upregulated in the fat depots. Here, Tgfb, Acta2 and Col1a1 mRNA levels and serum Tgfb protein were increased. Acta2 protein was, however, not higher in subcutaneous and perirenal fat of these mice. Col6a1 mRNA was shown before to be higher in obese fat tissues. Current analysis proved the Col6a1 protein was induced in subcutaneous fat of HFD fed mice. Notably, Col6a1 was reduced in perirenal fat of ob/ob mice in comparison to the respective controls. 3T3-L1 cells express Ccn2 and Bambi protein, whose levels were not changed by fatty acids, leptin, lipopolysaccharide, tumor necrosis factor and interleukin-6. All of these factors led to higher Tgfb in 3T3-L1 adipocyte media but did not increase its mRNA levels. Free fatty acids induced necrosis whereas apoptosis did not occur in any of the in vitro incubations excluding cell death as a main reason for higher Tgfb in cell media. In summary, Tgfb mRNA is consistently induced in white fat tissues in obesity but this is not paralleled by a clear increase of its target genes. Moreover, discrepancies between mRNA and protein expression of Acta2 were observed. Adipocytes seemingly do not contribute to higher Tgfb mRNA levels in obesity. These cells release more Tgfb protein when challenged with obesity-related metabolites connecting metabolic dysfunction and fibrosis.

Lower adiposity does not protect beta-2 syntrophin null mice from hepatic steatosis and inflammation in experimental non-alcoholic steatohepatitis.

Visceral adiposity is strongly associated with liver steatosis, which predisposes to the development of non-alcoholic steatohepatitis (NASH). Mice with loss of the molecular adapter protein beta-2 syntrophin (SNTB2) have greatly reduced intra-abdominal fat mass. Hepatic expression of proteins with a role in fatty acid metabolism such as fatty acid synthase was nevertheless normal. This was also the case for proteins regulating cholesterol synthesis and uptake. Yet, a slight induction of hepatic cholesterol was noticed in the mutant mice. When mice were fed a methionine choline deficient (MCD) diet to induce NASH, liver cholesteryl ester content was induced in the wild type but not the mutant mice. Serum cholesterol of the mice fed a MCD diet declined and this was significant for the SNTB2 null mice. Though the mutant mice lost less fat mass than the wild type animals, hepatic triglyceride levels were similar between the groups. Proteins involved in fatty acid or cholesterol metabolism such as fatty acid synthase, apolipoprotein E and low-density lipoprotein receptor did not differ between the genotypes. Hepatic oxidative stress and liver inflammation of mutant and wild type mice were comparable. Mutant mice had lower hepatic levels of secondary bile acids and higher cholesterol storage in epididymal fat, and this may partly prevent hepatic cholesterol deposition. In summary, the current study shows that SNTB2 null mice have low intra-abdominal fat mass and do not accumulate hepatic cholesteryl esters when fed a MCD diet. Nevertheless, the SNTB2 null mice develop a similar NASH pathology as wild type mice suggesting a minor role of intra-abdominal fat and liver cholesteryl esters in this model.

Targeting of Hmo1 to subcompartments of the budding yeast nucleolus.

The nucleolus is a multilayered, membraneless organelle made up of liquidlike biogenesis compartments surrounding an array of ribosomal RNA genes (rDNA). Biogenesis factors accumulate in the outer compartments through RNA binding and phase separation promoted by intrinsically disordered protein regions. In contrast, the nucleolar localization of rDNA-binding proteins, which reside in the central chromatin compartment, is less well characterized. To gain mechanistic insight, we analyzed the localization, mitotic segregation, nucleic acid binding, and nuclear dynamics of the budding yeast rDNA-binding protein Hmo1. Deletion of the main DNA-binding domain, the HMG boxB, compromised Hmo1 transfer to daughter cells in mitosis and transcription-independent rDNA association but still allowed nucleolar localization. The C-terminal lysine-rich region turned out to be a combined nuclear and nucleolar localization sequence (NLS-NoLS). Its integrity was required for maximal enrichment and efficient retention of Hmo1 in the nucleolus and nucleolar localization of the ΔboxB construct. Moreover, the NLS-NoLS region was sufficient to promote nucleolar accumulation and bound nucleic acids in vitro with some preference for RNA. Bleaching experiments indicated mobility of Hmo1 inside the nucleolus but little exchange with the nucleoplasm. Thus, a bilayered targeting mechanism secures proper localization of Hmo1 to the nucleolus.

Compositional reorganization of the nucleolus in budding yeast mitosis.

The nucleolus is a membraneless organelle of the nucleus and the site of rRNA synthesis, maturation, and assembly into preribosomal particles. The nucleolus, organized around arrays of rRNA genes (rDNA), dissolves during prophase of mitosis in metazoans, when rDNA transcription ceases, and reforms in telophase, when rDNA transcription resumes. No such dissolution and reformation cycle exists in budding yeast, and the precise course of nucleolar segregation remains unclear. By quantitative live-cell imaging, we observed that the yeast nucleolus is reorganized in its protein composition during mitosis. Daughter cells received equal shares of preinitiation factors, which bind the RNA polymerase I promoter and the rDNA binding barrier protein Fob1, but only about one-third of RNA polymerase I and the processing factors Nop56 and Nsr1. The distribution bias was diminished in nonpolar chromosome segregation events observable in dyn1 mutants. Unequal distribution, however, was enhanced by defects in RNA polymerase I, suggesting that rDNA transcription supports nucleolar segregation. Indeed, quantification of pre-rRNA levels indicated ongoing rDNA transcription in yeast mitosis. These data, together with photobleaching experiments to measure nucleolar protein dynamics in anaphase, consolidate a model that explains the differential partitioning of nucleolar components in budding yeast mitosis.