adipocyte ͉ Cide ͉ diabetes ͉ fat droplet ͉ fat metabolism

Fat-specific protein (FSP)27/Cidec is most highly expressed in white and brown adipose tissues and increases in abundance by over 50-fold during adipogenesis. However, its function in adipocytes has remained elusive since its discovery over 15 years ago. Here we demonstrate that FSP27/Cidec localizes to lipid droplets in cultured adipocytes and functions to promote lipid accumulation. Ectopically expressed FSP27-GFP surrounds lipid droplets in 3T3-L1 adipocytes and colocalizes with the known lipid droplet protein perilipin. Immunostaining of endogenous FSP27 in 3T3-L1 adipocytes also confirmed its presence on lipid droplets. FSP27-GFP expression also markedly increases lipid droplet size and enhances accumulation of total neutral lipids in 3T3-L1 preadipocytes as well as other cell types such as COS cells. Conversely, RNA interference-based FSP27/Cidec depletion in mature adipocytes significantly stimulates lipolysis and reduces the size of lipid droplets. These data reveal FSP27/Cidec as a novel adipocyte lipid droplet protein that negatively regulates lipolysis and promotes triglyceride accumulation.Adipose tissue is a major determinant of whole body glucose homeostasis and insulin sensitivity, as evidenced by its ability to secrete bioactive peptides and control lipid storage (1-6). The nuclear receptor peroxisome proliferator-activated receptor-␥ promotes adipogenesis and enhances these functions, acting in mice and humans to increase insulin signaling and glucose tolerance (7-12). Several proteins that are highly and selectively expressed in adipocytes, such as the secreted proteins adiponectin and leptin as well as adipsin, are under the control of peroxisome proliferator-activated receptor-␥ (3-5, 13, 14). These and other adipocyte proteins are highly up-regulated during adipogenesis and confer unique characteristics to these cells, including high capacity to store triglyceride and release fatty acids.Fat-specific protein (FSP)27, also denoted as CIDEC for the human homolog (15), was discovered over 15 years ago to be strikingly up-regulated during adipogenesis and is highly expressed in both white and brown adipose tissues (15-18). However, the question of FSP27/Cidec function has remained unsolved. The results we present here demonstrate FSP27/Cidec to be a novel lipid droplet protein that shares many features characteristic of the lipid droplet protein perilipin (19,20), including the ability to enhance neutral lipid accumulation when expressed in 3T3-L1 preadipocytes or even COS cells. Our data also reveal that a mechanism whereby FSP27/Cidec functions to promote triglyceride deposition in adiocytes is by inhibiting lipolysis. FSP27 is thus a major new modulator of lipid droplet function that is required for optimal storage of triglycerides by adipocytes. EXPERIMENTAL PROCEDURESMaterials-C57BL/6J (male, 10 weeks old) mice were obtained from The Jackson Laboratory. Human insulin was obtained from Lilly. Fetal bovine serum was purchased from Atlanta Biologicals, Inc. (Lawrenceville, GA). Other reagents ...

Storing excess energy for future use during starvation is critical for the survival of mammals. Much of this energy is stored in the form of triacylglycerol (TAG) within lipid droplets, which are present most abundantly in adipocytes and which, in turn, accumulate in depots such as subcutaneous and visceral adipose tissue in mice and humans. TAG in lipid droplets is mobilized during starvation by lipase-catalyzed hydrolysis (lipolysis) to release energy in the forms of glycerol and free fatty acids, providing fuel to other cell types such as muscle and liver. Previous work investigating formation of lipid droplets and regulation of lipolysis has elucidated the importance of lipid dropletassociated proteins for these processes ( 1, 2 ). Based on shared sequence homology, one set of lipid droplet proteins is grouped as the perilipin-adipophilin-tail interacting protein 47 (PAT/TIP47) family of proteins ( 3 ). PAT-related proteins are functionally conserved from mammals to lower organisms such as Drosophila and Dictyostelium spp ( 4 ). In Drosophila , two PAT domain proteins are encoded by the Lsdp1 and Lsd2 genes. Drosophila loss-of-function Lsd2 mutants are lean, whereas Lsd2 overexpression causes obesity ( 5 ). In mammals, PAT proteins can be divided into exchangeable TAG-associated PAT proteins (EPATs) or constitutively TAG-associated PAT proteins (CPATs). EPATs include the TIP47/perilipin-3 (PLIN3), S3-12/PLIN4, and Abstract The lipid droplet-associated fat specifi c protein 27 (FSP27) suppresses lipolysis and thereby enhances triglyceride accumulation in adipocytes. We and others have recently found FSP27 to be a remarkably short-lived protein (half-life, 15 min) due to its rapid ubiquitination and proteasomal degradation. Thus, we tested the hypothesis that lipolytic agents such as tumor necrosis factor-␣ (TNF-␣ ) and isoproterenol modulate FSP27 levels to regulate FFA release. Consistent with this concept, we showed that the lipolytic actions of TNF-␣ , interleukin-1 ␤ (IL-1 ␤ ), and IFN-␥ are accompanied by marked decreases in FSP27 expression and lipid droplet size in mouse adipocytes. Similar depletion of FSP27 using short interfering RNA (siRNA) mimicked the lipolysis-enhancing effect of TNF-␣ , while maintaining stable FSP27 levels using expression of hemagglutinin epitopetagged FSP27 blocked TNF-␣ -mediated lipolysis. In contrast, we show the robust lipolytic action of isoproterenol is paradoxically associated with increases in FSP27 levels and a delayed degradation rate corresponding to decreased ubiquitination. This catecholamine-mediated increase in FSP27 abundance, probably a feedback mechanism for restraining excessive lipolysis by catecholamines, is mimicked by forskolin or 8-bromo-cAMP treatment and is prevented by the protein kinase A (PKA) inhibitor KT5720 or by PKA depletion using siRNA. Taken together, these data identify the regulation of FSP27 as an important intermediate in the mechanism of lipolysis in adipocytes in response to TNF-␣ and isoproterenol. -Ranjit, S

Activation-induced cytidine deaminase (AID) is essential for class switch recombination (CSR) and somatic hypermutation (SHM) of Ig genes. The AID C terminus is required for CSR but not for S region DNA DSBs during CSR, and it is not required for SHM. AID lacking the C terminus (ΔAID) is a dominant negative (DN) mutant, as human patients heterozygous for this mutant fail to undergo CSR. In agreement, we show that ΔAID is a DN mutant when expressed in AID-sufficient mouse splenic B cells. In order to have DN function,ΔAID must have deaminase activity, suggesting that its ability to induce DSBs is important for the DN function. Supporting this hypothesis, Msh2-Msh6 have previously been shown to contribute to DSB formation in S regions, and here we find that Msh2 is required for the DN activity, as ΔAID is not a DN mutant in msh2−/− cells. Our results suggest that the DNA DSBs induced by ΔAID are unable to participate in CSR, and might interfere with the ability of full-length AID to participate in CSR. We propose thatΔAID is impaired in its ability to recruit non-homologous end joining (NHEJ) repair factors, resulting in accumulation of DSBs that undergo aberrant resection. Supporting this hypothesis, we find that the S-S junctions induced by ΔAID have longer microhomologies than those induced by full-length AID. In addition, our data suggest that AID binds Sµ regions in vivo as a monomer.

Introduction: A systematic approach to analysis of the fluid in conjunction with the clinical presentation allows clinicians to diagnose the cause of an effusion, narrow the differential diagnoses, and design a management plan. However, the number of cases where pleural fluid examination gives no proper diagnosis is depressingly high. This study aims to find out the prevalence of negative pleural fluid cultures among patients with pleural effusion in a tertiary care hospital. Methods: This was a descriptive cross-sectional conducted among 273 patients with pleural effusion admitted to a tertiary care hospital between January, 2019 and February, 2020. Ethical clearance was taken from the Institutional Review Committee (Reference number: 134/20). Convenience sampling was done. All patients whose pleural fluid was sent for analysis during the study period were included in the study. Pleural fluid analysis was done, and data were analysed using Statistical Package for the Social Sciences 25.0. Point estimate was done at a 95% Confidence Interval along with frequency and percentages for binary data. Results: Among 273 pleural fluid cultures from patients with pleural effusion, negative pleural fluid cultures were seen in 269 (98.53%) (97.12-99.96 at a 95% Confidence Interval). Conclusions: Our study reported that the prevalence of negative pleural fluid cultures was higher when compared to similar studies conducted in similar settings. The routine pleural fluid analysis could add a very little to the diagnosis and management of pleural effusion.

Introduction: Bloodstream Infection is one of the leading causes of mortality and morbidity among various groups of patients in a developing country like Nepal. Identification of the bacteria and their susceptibility to commonly used antibiotics is very essential for the treatment of the patients. Aims: This study aims to analyze current trends in bloodstream infection by accessing their bacteriological profile and antibiotic susceptibility. Methods: This is a hospital-based retrospective analysis of blood cultures of patients suspected with bloodstream infection. We conducted a three years (January 2018 to January 2021) retrospective analysis of blood culture reports from patients suspected with bloodstream infection. Data of the laboratory reports were used to determine bloodstream infection, blood culture contamination, pathogen profile and antimicrobial resistance patterns. Results: Out of 12811 blood samples that were sent to microbiology laboratory for culture, 438 were positive cultures. The most common etiological agent causing Bloodstream Infection was found to be Salmonella Typhi with129 isolates (29.6%) followed by Escherichia coli (21.9%). There was a significant rise in resistance to most of the frequently used antibiotics. Conclusion: Salmonella Typhi is the most frequent bacteria to be isolated in Bloodstream Infection. Resistance towards different etiological agents of Bloodstream Infection is alarmingly increasing every year. Resistance to frequently prescribed drugs should be of concern to the clinicians prescribing the drugs.

Lipid droplets are now been characterized as organelles existing in most types of cells and tissues of higher eukaryotes. Storage of energy as triglyceride in large adipose‐specific lipid droplets is a fundamental need in all mammals. Recently we found that the Cide domain‐ containing proteins, Cidea and FSP27 (Cidec), are associated with lipid droplets in adipocytes. Expression of Cidea and FSP27 greatly enhance lipid droplet size and neutral lipids when expressed ectopically in COS cells and 3T3‐L1 fibroblasts. Their expression in adipocytes is differentiation dependent, controlled by PPARγ and their depletion causes fragmentation of lipid droplets accompanied by markedly elevated lipolysis. Our results indicate an important role of Cidea and FSP27 in lipid droplet dynamics. Strikingly, in omental and subcutaneous white adipose tissue (WAT) from BMI‐matched obese humans, expression of Cidea, Cidec and well known lipid droplet protein, perilipin, correlates positively with insulin sensitivity. Thus, Cidea, Cidec and Perilipin define a novel, highly regulated pathway of triglyceride deposition in human WAT. The data support a model whereby failure of this pathway results in ectopic lipid accumulation, insulin resistance and its associated co‐morbidities in humans.

The essential role of glycoprotein hormone erythropoietin (Epo) and its receptor, EpoR, in erythroid development is well established: both the EpoR−/− and Epo−/− mouse embryos die on embryonic day 13 (E13) due to failure of definitive erythropoiesis in fetal liver (Wu et al. 1995). It has been suggested that Epo’s principal role during erythropoiesis is to protect erythroid progenitors from apoptosis (Koury and Bondurant, Science 1990). Bcl-xL, an anti-apoptotic member of the bcl-2 family, is induced by EpoR signaling in erythroid cells via the Jak2/Stat5 pathway (Silva et al., Blood 1996; Socolovsky et al., Cell, 1999). Bcl-xL is essential for erythroid maturation: bcl-xL−/− embryos die in utero at the same stage as as EpoR−/− mice, lacking definitive erythropoiesis (Motoyama et al., Science 1995; J Exp Med, 1999). Recenlty, it has been shown that over-expression of bcl-xL in primary wild-type erythroblasts confers Epo independence on these cells in vitro and allows them to complete their differentiaion into red blood cells (Dolznig et al., Curr Biol, 2002). Here we reasoned that if the principal function of EpoR signaling is suppression of apoptosis via bcl-xL, it should be possible to rescue all aspects of erythroid differentiation in EpoR−/− fetal liver progenitors by retrovirally-transducing these cells with bcl-xL. We infected EpoR−/− fetal liver progenitors with bicistronic retroviral vectors expressing either bcl-xL or EpoR, each linked via an IRES sequence to a GFP reporter. Control EpoR−/− cells were infected with ‘empty’ bicistronic vector. Infection rates were in excess of 30% for all constructs, and transduced cells were identified for further analysis using GFP fluorescence. We examined terminal differentiation of the transduced EpoR−/− cells over the ensuing 48 hours, using several distinct assays, including their expression of the cell-surface differentiation markers CD71 and Ter119 by FACS, their ability to give rise to CFU-e colonies in semi-solid medium, their cell-cycle status using DNA content analysis and BrdU incorporation, and their maturation and hemoglobinization by diaminobenzidine staining and light microscopy. We found that EpoR−/− progenitors transduced with bcl-xL were protected from apoptosis, and underwent morphological changes characteristic of erythroid maturation, including decreasing cell size, nuclear condensation and expulsion, and accumulation of hemoglobin. These cells also upregulated the erythroid-specific cell surface marker Ter119. However, unlike EpoR−/− cells transduced with EpoR, bcl-xL -transduced cells did not express high levels of CD71, and failed to give rise to CFU-e colonies in semi-solid medium. Instead, they gave rise to small colonies of 6 cells or less. Cell cycle analysis showed that, throughout the 48 hours of erythroid terminal differentiation, the population of bcl-xL-transduced EpoR−/− cells had a lower fraction of cells in S-phase than control, EpoR-transduced EpoR−/− cells. The cell-cycle status of control, terminally-differentiating wild-type erythroid fetal liver progenitors was not altered by transduction with bcl-xL, excluding the possibility that it directly inhibits S-phase. Taken together our results indicate that bcl-xL does not rescue all aspects of erythroid differentiation in EpoR−/− erythroid progenitors. Specifically, the proliferative program during erythroid terminal differentiation is directly dependent on EpoR signaling, and is not simply a default pathway secondary to EpoR’s anti-apoptotic effect.