Sphingomyelin synthase-related protein SMSr controls ceramide homeostasis in the ER

Ceramides are central intermediates of sphingolipid metabolism with critical functions in cell organization and survival. They are synthesized on the cytosolic surface of the endoplasmic reticulum (ER) and transported by ceramide transfer protein to the Golgi for conversion to sphingomyelin (SM) by SM synthase SMS1. In this study, we report the identification of an SMS1-related (SMSr) enzyme, which catalyses the synthesis of the SM analogue ceramide phosphoethanolamine (CPE) in the ER lumen. Strikingly, SMSr produces only trace amounts of CPE, i.e., 300-fold less than SMS1-derived SM. Nevertheless, blocking its catalytic activity causes a substantial rise in ER ceramide levels and a structural collapse of the early secretory pathway. We find that the latter phenotype is not caused by depletion of CPE but rather a consequence of ceramide accumulation in the ER. Our results establish SMSr as a key regulator of ceramide homeostasis that seems to operate as a sensor rather than a converter of ceramides in the ER

Design and baseline characteristics of the finerenone in reducing cardiovascular mortality and morbidity in diabetic kidney disease trial

Background: Among people with diabetes, those with kidney disease have exceptionally high rates of cardiovascular (CV) morbidity and mortality and progression of their underlying kidney disease. Finerenone is a novel, nonsteroidal, selective mineralocorticoid receptor antagonist that has shown to reduce albuminuria in type 2 diabetes (T2D) patients with chronic kidney disease (CKD) while revealing only a low risk of hyperkalemia. However, the effect of finerenone on CV and renal outcomes has not yet been investigated in long-term trials. Patients and Methods: The Finerenone in Reducing CV Mortality and Morbidity in Diabetic Kidney Disease (FIGARO-DKD) trial aims to assess the efficacy and safety of finerenone compared to placebo at reducing clinically important CV and renal outcomes in T2D patients with CKD. FIGARO-DKD is a randomized, double-blind, placebo-controlled, parallel-group, event-driven trial running in 47 countries with an expected duration of approximately 6 years. FIGARO-DKD randomized 7,437 patients with an estimated glomerular filtration rate >= 25 mL/min/1.73 m(2) and albuminuria (urinary albumin-to-creatinine ratio >= 30 to <= 5,000 mg/g). The study has at least 90% power to detect a 20% reduction in the risk of the primary outcome (overall two-sided significance level alpha = 0.05), the composite of time to first occurrence of CV death, nonfatal myocardial infarction, nonfatal stroke, or hospitalization for heart failure. Conclusions: FIGARO-DKD will determine whether an optimally treated cohort of T2D patients with CKD at high risk of CV and renal events will experience cardiorenal benefits with the addition of finerenone to their treatment regimen. Trial Registration: EudraCT number: 2015-000950-39; ClinicalTrials.gov identifier: NCT02545049

Ethanol metabolism and oxidative stress are required for unfolded protein response activation and steatosis in zebrafish with alcoholic liver disease

SUMMARY Secretory pathway dysfunction and lipid accumulation (steatosis) are the two most common responses of hepatocytes to ethanol exposure and are major factors in the pathophysiology of alcoholic liver disease (ALD). However, the mechanisms by which ethanol elicits these cellular responses are not fully understood. Recent data indicates that activation of the unfolded protein response (UPR) in response to secretory pathway dysfunction can cause steatosis. Here, we examined the relationship between alcohol metabolism, oxidative stress, secretory pathway stress and steatosis using zebrafish larvae. We found that ethanol was immediately internalized and metabolized by larvae, such that the internal ethanol concentration in 4-day-old larvae equilibrated to 160 mM after 1 hour of exposure to 350 mM ethanol, with an average ethanol metabolism rate of 56 μmol/larva/hour over 32 hours. Blocking alcohol dehydrogenase 1 (Adh1) and cytochrome P450 2E1 (Cyp2e1), the major enzymes that metabolize ethanol, prevented alcohol-induced steatosis and reduced induction of the UPR in the liver. Thus, we conclude that ethanol metabolism causes ALD in zebrafish. Oxidative stress generated by Cyp2e1-mediated ethanol metabolism is proposed to be a major culprit in ALD pathology. We found that production of reactive oxygen species (ROS) increased in larvae exposed to ethanol, whereas inhibition of the zebrafish CYP2E1 homolog or administration of antioxidants reduced ROS levels. Importantly, these treatments also blocked ethanol-induced steatosis and reduced UPR activation, whereas hydrogen peroxide (H2O2) acted as a pro-oxidant that synergized with low doses of ethanol to induce the UPR. Collectively, these data demonstrate that ethanol metabolism and oxidative stress are conserved mechanisms required for the development of steatosis and hepatic dysfunction in ALD, and that these processes contribute to ethanol-induced UPR activation and secretory pathway stress in hepatocytes

The Cx43-like Connexin Protein Cx40.8 Is Differentially Localized during Fin Ontogeny and Fin Regeneration

Connexins (Cx) are the subunits of gap junctions, membraneous protein channels that permit the exchange of small molecules between adjacent cells. Cx43 is required for cell proliferation in the zebrafish caudal fin. Previously, we found that a Cx43-like connexin, cx40.8, is co-expressed with cx43 in the population of proliferating cells during fin regeneration. Here we demonstrate that Cx40.8 exhibits novel differential subcellular localization in vivo, depending on the growth status of the fin. During fin ontogeny, Cx40.8 is found at the plasma membrane, but Cx40.8 is retained in the Golgi apparatus during regeneration. We next identified a 30 amino acid domain of Cx40.8 responsible for its dynamic localization. One possible explanation for the differential localization is that Cx40.8 contributes to the regulation of Cx43 in vivo, perhaps modifying channel activity during ontogenetic growth. However, we find that the voltage-gating properties of Cx40.8 are similar to Cx43. Together our findings reveal that Cx40.8 exhibits differential subcellular localization in vivo, dependent on a discrete domain in its carboxy terminus. We suggest that the dynamic localization of Cx40.8 differentially influences Cx43-dependent cell proliferation during ontogeny and regeneration

Molecularly defined unfolded protein response subclasses have distinct correlations with fatty liver disease in zebrafish

The unfolded protein response (UPR) is a complex network of sensors and target genes that ensure efficient folding of secretory proteins in the endoplasmic reticulum (ER). UPR activation is mediated by three main sensors, which regulate the expression of hundreds of targets. UPR activation can result in outcomes ranging from enhanced cellular function to cell dysfunction and cell death. How this pathway causes such different outcomes is unknown. Fatty liver disease (steatosis) is associated with markers of UPR activation and robust UPR induction can cause steatosis; however, in other cases, UPR activation can protect against this disease. By assessing the magnitude of activation of UPR sensors and target genes in the liver of zebrafish larvae exposed to three commonly used ER stressors (tunicamycin, thapsigargin and Brefeldin A), we have identified distinct combinations of UPR sensors and targets (i.e. subclasses) activated by each stressor. We found that only the UPR subclass characterized by maximal induction of UPR target genes, which we term a stressed-UPR, induced steatosis. Principal component analysis demonstrated a significant positive association between UPR target gene induction and steatosis. The same principal component analysis showed significant correlation with steatosis in samples from patients with fatty liver disease. We demonstrate that an adaptive UPR induced by a short exposure to thapsigargin prior to challenging with tunicamycin reduced both the induction of a stressed UPR and steatosis incidence. We conclude that a stressed UPR causes steatosis and an adaptive UPR prevents it, demonstrating that this pathway plays dichotomous roles in fatty liver disease

Activating Transcription Factor 6 Is Necessary and Sufficient for Alcoholic Fatty Liver Disease in Zebrafish

<div><p>Fatty liver disease (FLD) is characterized by lipid accumulation in hepatocytes and is accompanied by secretory pathway dysfunction, resulting in induction of the unfolded protein response (UPR). Activating transcription factor 6 (ATF6), one of three main UPR sensors, functions to both promote FLD during acute stress and reduce FLD during chronic stress. There is little mechanistic understanding of how ATF6, or any other UPR factor, regulates hepatic lipid metabolism to cause disease. We addressed this using zebrafish genetics and biochemical analyses and demonstrate that Atf6 is necessary and sufficient for FLD. <i>atf6</i> transcription is significantly upregulated in the liver of zebrafish with alcoholic FLD and morpholino-mediated <i>atf6</i> depletion significantly reduced steatosis incidence caused by alcohol. Moreover, overexpression of active, nuclear Atf6 (nAtf6) in hepatocytes caused FLD in the absence of stress. mRNA-Seq and qPCR analyses of livers from five day old nAtf6 transgenic larvae revealed upregulation of genes promoting glyceroneogenesis and fatty acid elongation, including fatty acid synthase (<i>fasn</i>), and nAtf6 overexpression in both zebrafish larvae and human hepatoma cells increased the incorporation of ¹⁴C-acetate into lipids. Srebp transcription factors are key regulators of lipogenic enzymes, but reducing Srebp activation by <i>scap</i> morpholino injection neither prevented FLD in nAtf6 transgenics nor synergized with <i>atf6</i> knockdown to reduce alcohol-induced FLD. In contrast, <i>fasn</i> morpholino injection reduced FLD in nAtf6 transgenic larvae and synergistically interacted with <i>atf6</i> to reduce alcoholic FLD. Thus, our data demonstrate that Atf6 is required for alcoholic FLD and epistatically interacts with <i>fasn</i> to cause this disease, suggesting triglyceride biogenesis as the mechanism of UPR induced FLD.</p></div

Fasn functions downstream of Atf6 to cause steatosis independent of Srebp activation.

<p><b>A:</b> qPCR analysis of <i>fasn</i> expression in <i>atf6</i> morphants and uninjected larvae treated with EtOH for 32 hours. <b>B:</b> qPCR analysis of <i>fasn</i> expression in nAtf6 TG larvae and HepG2 cells transfected with nATF6. Statistics: unpaired t-test. *, p<0.05. <b>C:</b> Quantification of whole mount oil red O staining of nAtf6 TG larvae injected with <i>scap</i> MO. “Total n” and “clutch n” corresponds to the number of larvae and number of clutches scored, respectively. Statistics: chi-square with Fisher's Exact Test. *, p<0.05. <b>D:</b> Quantification of oil red O staining in nAtf6 TG larvae injected with <i>fasn</i> MO. <b>E:</b> Quantification of oil red O staining in <i>atf6/fasn</i> double morphants treated with 350 mM EtOH for 24 hours. Statistics: chi-square with Fisher's Exact Test. *, p<0.05.</p

Cx43-mApple and Cx40.8-EGFP co-assemble in common gap junction channels.

<p>High resolution fluorescence microscopy was used to provide evidence for co-association of Cx43-mApple and Cx40.8-EGFP in common gap junction channels. Constructs that were co-transfected in HeLa cells are indicated to the left of the panels (A, B) <i>Homo sapiens</i> (Hs) Cx43-mApple + Hs Cx43-EGFP show uniformly yellow plaques, suggesting co-asociation. (C, D) Hs Cx43-mApple + Hs Cx26-EGFP show discrete green and red domains, revealing a lack of co-association. Arrows indicate green Hs Cx26-EGFP localization and arrowheads indicate red Hs Cx43-mApple localization. (E, F) <i>Danio rerio</i> (Dr) Cx43-mApple + Dr Cx40.8-EGFP show uniform yellow distribution.</p