Lipid droplet (LD) catabolism in hepatocytes is mediated by a combination of lipolysis and a selective autophagic mechanism called lipophagy, but the relative contributions of these seemingly ...distinct pathways remain unclear. We find that inhibition of lipolysis, lipophagy, or both resulted in similar overall LD content but dramatic differences in LD morphology. Inhibition of the lipolysis enzyme adipose triglyceride lipase (ATGL) resulted in large cytoplasmic LDs, whereas lysosomal inhibition caused the accumulation of numerous small LDs within the cytoplasm and degradative acidic vesicles. Combined inhibition of ATGL and LAL resulted in large LDs, suggesting that lipolysis targets these LDs upstream of lipophagy. Consistent with this, ATGL was enriched in larger-sized LDs, whereas lipophagic vesicles were restricted to small LDs as revealed by immunofluorescence, electron microscopy, and Western blot of size-separated LDs. These findings provide new evidence indicating a synergistic relationship whereby lipolysis targets larger-sized LDs to produce both size-reduced and nascently synthesized small LDs that are amenable for lipophagic internalization.
Hepatocytes metabolize energy-rich cytoplasmic lipid droplets (LDs) in the lysosome-directed process of autophagy. An organelleselective form of this process (macrolipophagy) results in the ...engulfment of LDs within double-membrane delimited structures (autophagosomes) before lysosomal fusion. Whether this is an exclusive autophagic mechanism used by hepatocytes to catabolize LDs is unclear. It is also unknown whether lysosomes alonemight be sufficient to mediate LD turnover in the absence of an autophagosomal intermediate. We performed live-cell microscopy of hepatocytes to monitor the dynamic interactions between lysosomes and LDs in real-time. We additionally used a fluorescent variant of the LD-specific protein (PLIN2) that exhibits altered fluorescence in response to LD interactions with the lysosome. We find that mammalian lysosomes and LDs undergo interactions during which proteins and lipids can be transferred from LDs directly into lysosomes. Electron microscopy (EM) of primary hepatocytes or hepatocyte-derived cell lines supports the existence of these interactions. It reveals a dramatic process whereby the lipid contents of the LD can be “extruded” directly into the lysosomal lumen under nutrient-limited conditions. Significantly, these interactions are not affected by perturbations to crucial components of the canonical macroautophagy machinery and can occur in the absence of double-membrane lipoautophagosomes. These findings implicate the existence of an autophagicmechanism used bymammalian cells for the direct transfer of LD components into the lysosome for breakdown. This process further emphasizes the critical role of lysosomes in hepatic LD catabolism and provides insights into the mechanisms underlying lipid homeostasis in the liver.
The objective of this study, which was part of an integrated project to investigate the antimicrobial effects of dietary tannins on native food borne pathogens in beef cattle, was to examine the ...effects of source of tannin (condensed, CT,
vs. hydrolysable, HT) on performance, feed efficiency, ruminal fermentation parameters, and carcass and non-carcass traits in finishing beef steers. Thirty-six crossbred steers averaging 414
±
40
kg BW were stratified by initial BW and randomly assigned to one of three treatments: control (CN), CT, or HT tannins. Commercially available tannin extracts were added to a high-grain diet (ME
=
11.9
MJ/kg DM) at 14.9
g/kg DM. Mimosa and chestnut extracts provided condensed tannin and hydrolysable tannin, respectively. Steers were individually fed using Calan gate feeders a high-grain diet. Rumen fluid was collected on days 0, 21, and 42
via stomach tube and analyzed for VFA and
in vitro methane producing activity. Cattle were harvested at the end of the study and carcass data collected 24-h postharvest. There was no effect (
P>0.05) of tannin supplementation on animal performance, ruminal fermentation parameters,
in vitro methane producing activity, or carcass and non-carcass traits, except for HCW, EBW, and rumen mass and empty GIT (g/kg EBW). Condensed tannin steers had 3.7% lower (
P<0.05) HCW compared to CN with HT steers having intermediate HCW. Hydrolysable tannin treated steers had 2.8% lower (
P<0.05) EBW compared to CN while CT steers had intermediate EBW; CT treated steers also had 15.2% higher (
P<0.05) rumen mass (g/kg EBW) compared to HT with CN steers being intermediate. This resulted in a 10.2% increase (
P<0.05) in total empty GIT (g/kg EBW) for HT steers compared to CT steers with CN steers bring intermediate. There was a treatment
×
day interaction for butyrate concentration. For steers fed CT, there was a linear increase in butyrate while the HT steers remained relatively stable and the control steers had numerically lower butyrate. Despite the significant interaction, treatment means on day 42 were not significantly different. Results indicate that neither source of dietary tannin affected performance and feed efficiency. There were no detrimental effects of tannins on other offal measured indicating that tannins supplementation may be a viable option in finishing beef cattle if bactericidal efficacy is established. More research is needed to further our understanding of how tannin supplementation may fit into real-life feedlot situations.
The large GTPase Dynamin 2 (Dyn2) is markedly upregulated in pancreatic cancer, is a potent activator of metastatic migration, and is required for Rac1-mediated formation of lamellipodia. Here we ...demonstrate an unexpected mechanism of Dyn2 action in these contexts via direct binding to the Rac1 guanine nucleotide exchange factor (GEF) Vav1. Surprisingly, disruption of the Dyn2-Vav1 interaction targets Vav1 to the lysosome for degradation via an interaction with the cytoplasmic chaperone Hsc70, resulting in a dramatic reduction of Vav1 protein stability. Importantly, a specific mutation in Vav1 near its Dyn2-binding C-terminal Src homology 3 (SH3) domain prevents Hsc70 binding, resulting in a stabilization of Vav1 levels. Dyn2 binding regulates the interaction of Vav1 with Hsc70 to control the stability and subsequent activity of this oncogenic GEF. These findings elucidate how Dyn2 activates Rac1, lamellipod protrusion, and invasive cellular migration and provide insight into how this specific Vav is ectopically expressed in pancreatic tumors.
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► Dyn2 promotes lamellipodia formation and pancreatic tumor cell migration ► Direct binding between Dyn2 and Vav1 promotes Rac1 activation and migration ► Dyn2 binding protects Vav1 from degradation by the lysosome ► Vav1 is targeted for degradation through an interaction with Hsc70
The large GTPase Dynamin 2 (Dyn2) is upregulated in pancreatic tumors and potentiates invasive migration. Razidlo et al. show that Dyn2 promotes Rac1-mediated migration through an interaction with, and stabilization of, the Rac1 activator Vav1. Unexpectedly, in the absence of Dyn2, Vav1binds Hsc70 and is targeted for lysosomal degradation.
Lipid droplets (LDs) are lipid storage organelles that in hepatocytes may be catabolized by autophagy for use as an energy source, but the membrane-trafficking machinery regulating such a process is ...poorly characterized. We hypothesized that the large GTPase dynamin 2 (Dyn2), well known for its involvement in membrane deformation and cellular protein trafficking, could orchestrate autophagy-mediated LD breakdown. Accordingly, depletion or pharmacologic inhibition of Dyn2 led to a substantial accumulation of LDs in hepatocytes. Strikingly, the targeted disruption of Dyn2 induced a dramatic four- to fivefold increase in the size of autolysosomes. Chronic or acute Dyn2 inhibition combined with nutrient deprivation stimulated the excessive tubulation of these autolysosomal compartments. Importantly, Dyn2 associated with these tubules along their length, and the tubules vesiculated and fragmented in the presence of functional Dyn2. These findings provide new evidence for the participation of the autolysosome in LD metabolism and demonstrate a novel role for dynamin in the function and maturation of an autophagic compartment.
The process by which tumor cells mechanically invade through surrounding stroma into peripheral tissues is an essential component of metastatic dissemination. The directed recruitment of the ...metalloproteinase MT1-MMP to invadopodia plays a critical role in this invasive process. Here, we provide mechanistic insight into MT1-MMP cytoplasmic tail binding protein 1 (MTCBP-1) with respect to invadopodia formation, matrix remodeling, and invasion by pancreatic tumor cells. MTCBP-1 localizes to invadopodia and interacts with MT1-MMP. We find that this interaction displaces MT1-MMP from invadopodia, thereby attenuating their number and function and reducing the capacity of tumor cells to degrade matrix. Further, we observe an inverse correlation between MTCBP-1 and MT1-MMP expression both in cultured cell lines and human pancreatic tumors. Consistently, MTCBP-1-expressing cells show decreased ability to invade in vitro and metastasize in vivo. These findings implicate MTCBP-1 as an inhibitor of the metastatic process.
Selective digestive decontamination (SDD) and selective oropharyngeal decontamination (SOD) improved intensive care unit (ICU), hospital and 28-day survival in ICUs with low levels of antibiotic ...resistance. Yet it is unclear whether the effect differs between medical and surgical ICU patients.
In an individual patient data meta-analysis, we systematically searched PubMed and included all randomized controlled studies published since 2000. We performed a two-stage meta-analysis with separate logistic regression models per study and per outcome (hospital survival and ICU survival) and subsequent pooling of main and interaction effects.
Six studies, all performed in countries with low levels of antibiotic resistance, yielded 16 528 hospital admissions and 17 884 ICU admissions for complete case analysis. Compared to standard care or placebo, the pooled adjusted odds ratios for hospital mortality was 0.82 (95% confidence interval (CI) 0.72–0.93) for SDD and 0.84 (95% CI 0.73–0.97) for SOD. Compared to SOD, the adjusted odds ratio for hospital mortality was 0.90 (95% CI 0.82–0.97) for SDD. The effects on hospital mortality were not modified by type of ICU admission (p values for interaction terms were 0.66 for SDD and control, 0.87 for SOD and control and 0.47 for SDD and SOD). Similar results were found for ICU mortality.
In ICUs with low levels of antibiotic resistance, the effectiveness of SDD and SOD was not modified by type of ICU admission. SDD and SOD improved hospital and ICU survival compared to standard care in both patient populations, with SDD being more effective than SOD.
The membrane deforming dynamin family members MxA and MxB are large GTPases that convey resistance to a variety of infectious viruses. During viral infection, Mx proteins are known to show markedly ...increased expression via an interferon-responsive promoter to associate with nuclear pores. In this study we report that MxB is an inner mitochondrial membrane GTPase that plays an important role in the morphology and function of this organelle. Expression of mutant MxB or siRNA knockdown of MxB leads to fragmented mitochondria with disrupted inner membranes that are unable to maintain a proton gradient, while expelling their nucleoid-based genome into the cytoplasm. These findings implicate a dynamin family member in mitochondrial-based changes frequently observed during an interferon-based, anti-viral response.
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