Nascent shifts in renal cellular metabolism, structure, and function due to chronic empagliflozin in prediabetic mice.

Sodium-glucose cotransporter, type 2 inhibitors (SGLT2i) are emerging as the gold standard for treatment of type 2 diabetes (T2D) with renal protective benefits independent of glucose lowering. We took a high-level approach to evaluate the effects of the SGLT2i, empagliflozin (EMPA) on renal metabolism and function in a prediabetic model of metabolic syndrome. Male and female 12-wk-old TallyHo (TH) mice, and their closest genetic lean strain (Swiss-Webster, SW) were treated with a high-milk-fat diet (HMFD) plus/minus EMPA (@0.01%) for 12-wk. Kidney weights and glomerular filtration rate were slightly increased by EMPA in the TH mice. Glomerular feature analysis by unsupervised clustering revealed sexually dimorphic clustering, and one unique cluster relating to EMPA. Periodic acid Schiff (PAS) positive areas, reflecting basement membranes and mesangium were slightly reduced by EMPA. Phasor-fluorescent life-time imaging (FLIM) of free-to-protein bound NADH in cortex showed a marginally greater reliance on oxidative phosphorylation with EMPA. Overall, net urine sodium, glucose, and albumin were slightly increased by EMPA. In TH, EMPA reduced the sodium phosphate cotransporter, type 2 (NaPi-2), but increased sodium hydrogen exchanger, type 3 (NHE3). These changes were absent or blunted in SW. EMPA led to changes in urine exosomal microRNA profile including, in females, enhanced levels of miRs 27a-3p, 190a-5p, and 196b-5p. Network analysis revealed "cancer pathways" and "FOXO signaling" as the major regulated pathways. Overall, EMPA treatment to prediabetic mice with limited renal disease resulted in modifications in renal metabolism, structure, and transport, which may preclude and underlie protection against kidney disease with developing T2D.NEW & NOTEWORTHY Renal protection afforded by sodium glucose transporter, type 2 inhibitors (SGLT2i), e.g., empagliflozin (EMPA) involves complex intertwined mechanisms. Using a novel mouse model of obesity with insulin resistance, the TallyHo/Jng (TH) mouse on a high-milk-fat diet (HMFD), we found subtle changes in metabolism including altered regulation of sodium transporters that line the renal tubule. New potential epigenetic determinants of metabolic changes relating to FOXO and cancer signaling pathways were elucidated from an altered urine exosomal microRNA signature.

The role of FXR and TGR5 in reversing and preventing progression of Western diet-induced hepatic steatosis, inflammation, and fibrosis in mice.

Nonalcoholic steatohepatitis (NASH) is the most common chronic liver disease in the US, partly due to the increasing incidence of metabolic syndrome, obesity, and type 2 diabetes. The roles of bile acids and their receptors, such as the nuclear receptor farnesoid X receptor (FXR) and the G protein-coupled receptor TGR5, on the development of NASH are not fully clear. C57BL/6J male mice fed a Western diet (WD) develop characteristics of NASH, allowing determination of the effects of FXR and TGR5 agonists on this disease. Here we show that the FXR-TGR5 dual agonist INT-767 prevents progression of WD-induced hepatic steatosis, inflammation, and fibrosis, as determined by histological and biochemical assays and novel label-free microscopy imaging techniques, including third harmonic generation, second harmonic generation, and fluorescence lifetime imaging microscopy. Furthermore, we show INT-767 decreases liver fatty acid synthesis and fatty acid and cholesterol uptake, as well as liver inflammation. INT-767 markedly changed bile acid composition in the liver and intestine, leading to notable decreases in the hydrophobicity index of bile acids, known to limit cholesterol and lipid absorption. In addition, INT-767 upregulated expression of liver p-AMPK, SIRT1, PGC-1α, and SIRT3, which are master regulators of mitochondrial function. Finally, we found INT-767 treatment reduced WD-induced dysbiosis of gut microbiota. Interestingly, the effects of INT-767 in attenuating NASH were absent in FXR-null mice, but still present in TGR5-null mice. Our findings support treatment and prevention protocols with the dual FXR-TGR5 agonist INT-767 arrest progression of WD-induced NASH in mice mediated by FXR-dependent, TGR5-independent mechanisms.

Linear Combination Properties of the Phasor Space in Fluorescence Imaging.

The phasor approach to fluorescence lifetime imaging, and more recently hyperspectral fluorescence imaging, has increased the use of these techniques, and improved the ease and intuitiveness of the data analysis. The fit-free nature of the phasor plots increases the speed of the analysis and reduces the dimensionality, optimization of data handling and storage. The reciprocity principle between the real and imaginary space-where the phasor and the pixel that the phasor originated from are linked and can be converted from one another-has helped the expansion of this method. The phasor coordinates calculated from a pixel, where multiple fluorescent species are present, depends on the phasor positions of those components. The relative positions are governed by the linear combination properties of the phasor space. According to this principle, the phasor position of a pixel with multiple components lies inside the polygon whose vertices are occupied by the phasor positions of these individual components and the distance between the image phasor to any of the vertices is inversely proportional to the fractional intensity contribution of that component to the total fluorescence from that image pixel. The higher the fractional intensity contribution of a vertex, the closer is the resultant phasor. The linear additivity in the phasor space can be exploited to obtain the fractional intensity contribution from multiple species and quantify their contribution. This review details the various mathematical models that can be used to obtain two/three/four components from phasor space with known phasor signatures and then how to obtain both the fractional intensities and phasor positions without any prior knowledge of either, assuming they are mono-exponential in nature. We note that other than for blind components, there are no restrictions on the type of the decay or their phasor positions for linear combinations to be valid-and they are applicable to complicated fluorescence lifetime decays from components with intensity decays described by multi-exponentials.

Empagliflozin Treatment Attenuates Hepatic Steatosis by Promoting White Adipose Expansion in Obese TallyHo Mice.

Sodium-glucose co-transporters (SGLTs) serve to reabsorb glucose in the kidney. Recently, these transporters, mainly SGLT2, have emerged as new therapeutic targets for patients with diabetes and kidney disease; by inhibiting glucose reabsorption, they promote glycosuria, weight loss, and improve glucose tolerance. They have also been linked to cardiac protection and mitigation of liver injury. However, to date, the mechanism(s) by which SGLT2 inhibition promotes systemic improvements is not fully appreciated. Using an obese TallyHo mouse model which recapitulates the human condition of diabetes and nonalcoholic fatty liver disease (NAFLD), we sought to determine how modulation of renal glucose handling impacts liver structure and function. Apart from an attenuation of hyperglycemia, Empagliflozin was found to decrease circulating triglycerides and lipid accumulation in the liver in male TallyHo mice. This correlated with lowered hepatic cholesterol esters. Using in vivo MRI analysis, we further determined that the reduction in hepatic steatosis in male TallyHo mice was associated with an increase in nuchal white fat indicative of "healthy adipose expansion". Notably, this whitening of the adipose came at the expense of brown adipose tissue. Collectively, these data indicate that the modulation of renal glucose handling has systemic effects and may be useful as a treatment option for NAFLD and steatohepatitis.

Bile acid sequestration reverses liver injury and prevents progression of nonalcoholic steatohepatitis in Western diet-fed mice.

Nonalcoholic fatty liver disease is a rapidly rising problem in the 21st century and is a leading cause of chronic liver disease that can lead to end-stage liver diseases, including cirrhosis and hepatocellular cancer. Despite this rising epidemic, no pharmacological treatment has yet been established to treat this disease. The rapidly increasing prevalence of nonalcoholic fatty liver disease and its aggressive form, nonalcoholic steatohepatitis (NASH), requires novel therapeutic approaches to prevent disease progression. Alterations in microbiome dynamics and dysbiosis play an important role in liver disease and may represent targetable pathways to treat liver disorders. Improving microbiome properties or restoring normal bile acid metabolism may prevent or slow the progression of liver diseases such as NASH. Importantly, aberrant systemic circulation of bile acids can greatly disrupt metabolic homeostasis. Bile acid sequestrants are orally administered polymers that bind bile acids in the intestine, forming nonabsorbable complexes. Bile acid sequestrants interrupt intestinal reabsorption of bile acids, decreasing their circulating levels. We determined that treatment with the bile acid sequestrant sevelamer reversed the liver injury and prevented the progression of NASH, including steatosis, inflammation, and fibrosis in a Western diet-induced NASH mouse model. Metabolomics and microbiome analysis revealed that this beneficial effect is associated with changes in the microbiota population and bile acid composition, including reversing microbiota complexity in cecum by increasing Lactobacillus and decreased Desulfovibrio The net effect of these changes was improvement in liver function and markers of liver injury and the positive effects of reversal of insulin resistance.

Phasor approach to autofluorescence lifetime imaging FLIM can be a quantitative biomarker of chronic renal parenchymal injury.

Diabetic kidney disease continues to be the leading cause of chronic kidney disease, often advancing to end stage kidney disease. In addition to the well characterized glomerular alterations including mesangial expansion, podocyte injury, and glomerulosclerosis, tubulointerstitial fibrosis is also an important component of diabetic kidney injury. Similarly, tubulointerstitial fibrosis is a critical component of any chronic kidney injury. Therefore, sensitive and quantitative identification of tubulointerstitial fibrosis is critical for the assessment of long-term prognosis of kidney disease. Here, we employed phasor approach to fluorescence lifetime imaging, commonly known as FLIM, to understand tissue heterogeneity and calculate changes in the tissue autofluorescence lifetime signatures due to diabetic kidney disease. FLIM imaging was performed on cryostat sections of snap-frozen biopsy material of patients with diabetic nephropathy. There was an overall increase in phase lifetime (τphase) with increased disease severity. Multicomponent phasor analysis shows the distinctive differences between the different disease states. Thus, phasor autofluorescence lifetime imaging, which does not involve any staining, can be used to understand and evaluate the severity of kidney disease.

Multicomponent Analysis of Phasor Plot in a Single Pixel to Calculate Changes of Metabolic Trajectory in Biological Systems.

Phasor FLIM in cells undergoing oxidative stress and in mice liver sections have shown the presence of a third autofluorescent component indicative of lipid droplets along with free and enzyme-bound NADH with similar emissions. This third component affects the position and shape of the phasor distribution, pushing it away from the metabolic trajectory. Phasor rule of addition is still valid and was exploited here to create a multicomponent analysis where the phasor distribution can be reassigned to the metabolic trajectory and changes in metabolism can be detected independently of the intensity of this third component. Calculation of multiple components from FLIM imaging data of biological systems is a difficult process, especially if different fluorescent species are present at the same pixel. This paper describes the methodology that can be used to separate these multiple components when they are present in the phasor signature acquired in a single pixel of an image.

Spatial dynamics of SIRT1 and the subnuclear distribution of NADH species.

Sirtuin 1 (SIRT1) is an NAD+-dependent deacetylase that functions as metabolic sensor of cellular energy and modulates biochemical pathways in the adaptation to changes in the environment. SIRT1 substrates include histones and proteins related to enhancement of mitochondrial function as well as antioxidant protection. Fluctuations in intracellular NAD+ levels regulate SIRT1 activity, but how SIRT1 enzymatic activity impacts on NAD+ levels and its intracellular distribution remains unclear. Here, we show that SIRT1 determines the nuclear organization of protein-bound NADH. Using multiphoton microscopy in live cells, we show that free and bound NADH are compartmentalized inside of the nucleus, and its subnuclear distribution depends on SIRT1. Importantly, SIRT6, a chromatin-bound deacetylase of the same class, does not influence NADH nuclear localization. In addition, using fluorescence fluctuation spectroscopy in single living cells, we reveal that NAD+ metabolism in the nucleus is linked to subnuclear dynamics of active SIRT1. These results reveal a connection between NAD+ metabolism, NADH distribution, and SIRT1 activity in the nucleus of live cells and pave the way to decipher links between nuclear organization and metabolism.

FXR/TGR5 Dual Agonist Prevents Progression of Nephropathy in Diabetes and Obesity.

Bile acids are ligands for the nuclear hormone receptor farnesoid X receptor (FXR) and the G protein-coupled receptor TGR5. We have shown that FXR and TGR5 have renoprotective roles in diabetes- and obesity-related kidney disease. Here, we determined whether these effects are mediated through differential or synergistic signaling pathways. We administered the FXR/TGR5 dual agonist INT-767 to DBA/2J mice with streptozotocin-induced diabetes, db/db mice with type 2 diabetes, and C57BL/6J mice with high-fat diet-induced obesity. We also examined the individual effects of the selective FXR agonist obeticholic acid (OCA) and the TGR5 agonist INT-777 in diabetic mice. The FXR agonist OCA and the TGR5 agonist INT-777 modulated distinct renal signaling pathways involved in the pathogenesis and treatment of diabetic nephropathy. Treatment of diabetic DBA/2J and db/db mice with the dual FXR/TGR5 agonist INT-767 improved proteinuria and prevented podocyte injury, mesangial expansion, and tubulointerstitial fibrosis. INT-767 exerted coordinated effects on multiple pathways, including stimulation of a signaling cascade involving AMP-activated protein kinase, sirtuin 1, PGC-1α, sirtuin 3, estrogen-related receptor-α, and Nrf-1; inhibition of endoplasmic reticulum stress; and inhibition of enhanced renal fatty acid and cholesterol metabolism. Additionally, in mice with diet-induced obesity, INT-767 prevented mitochondrial dysfunction and oxidative stress determined by fluorescence lifetime imaging of NADH and kidney fibrosis determined by second harmonic imaging microscopy. These results identify the renal signaling pathways regulated by FXR and TGR5, which may be promising targets for the treatment of nephropathy in diabetes and obesity.

Reversing the direction of galvanotaxis with controlled increases in boundary layer viscosity.

Weak external electric fields (EFs) polarize cellular structure and direct most migrating cells (galvanotaxis) toward the cathode, making it a useful tool during tissue engineering and for healing epidermal wounds. However, the biophysical mechanisms for sensing weak EFs remain elusive. We have reinvestigated the mechanism of cathode-directed water flow (electro-osmosis) in the boundary layer of cells, by reducing it with neutral, viscous polymers. We report that increasing viscosity with low molecular weight polymers decreases cathodal migration and promotes anodal migration in a concentration dependent manner. In contrast, increased viscosity with high molecular weight polymers does not affect directionality. We explain the contradictory results in terms of porosity and hydraulic permeability between the polymers rather than in terms of bulk viscosity. These results provide the first evidence for controlled reversal of galvanotaxis using viscous agents and position the field closer to identifying the putative electric field receptor, a fundamental, outside-in signaling receptor that controls cellular polarity for different cell types.

Label-free fluorescence lifetime and second harmonic generation imaging microscopy improves quantification of experimental renal fibrosis.

All forms of progressive renal diseases develop a final pathway of tubulointerstitial fibrosis and glomerulosclerosis. Renal fibrosis is usually quantified using histological staining, a process that is time-consuming and pathologist dependent. Here we develop a fast and operator-independent method to measure fibrosis utilizing the murine unilateral ureteral obstruction model which manifests a time-dependent fibrotic increase in obstructed kidneys while the contralateral kidneys are used as controls. After ureteral obstruction, kidneys were analyzed at 7, 14, and 21 days. Fibrosis was quantified using fluorescence lifetime imaging (FLIM) and second harmonic generation (SHG) in a Deep Imaging via Enhanced photon Recovery deep tissue imaging microscope. This microscope was developed for deep tissue along with second and third harmonic generation imaging and has extraordinary sensitivity toward harmonic generation. SHG data suggest the presence of more fibrillar collagen in the obstructed kidneys. The combination of short-wavelength FLIM and SHG analysis results in a robust assessment procedure independent of observer interpretation and let us create criteria to quantify the extent of fibrosis directly from the image. Thus, the FLIM-SHG technique shows remarkable improvement in quantification of renal fibrosis compared to standard histological techniques.

Intrinsic stability and oligomerization dynamics of DNA processivity clamps.

Sliding clamps are ring-shaped oligomeric proteins that are essential for processive deoxyribonucleic acid replication. Although crystallographic structures of several clamps have been determined, much less is known about clamp structure and dynamics in solution. Here, we characterized the intrinsic solution stability and oligomerization dynamics of the homodimeric Escherichia coli ß and the homotrimeric Saccharomyces cerevisiae proliferating cell nuclear antigen (PCNA) clamps using single-molecule approaches. We show that E. coli ß is stable in solution as a closed ring at concentrations three orders of magnitude lower than PCNA. The trimeric structure of PCNA results in slow subunit association rates and is largely responsible for the lower solution stability. Despite this large difference, the intrinsic lifetimes of the rings differ by only one order of magnitude. Our results show that the longer lifetime of the E. coli ß dimer is due to more prominent electrostatic interactions that stabilize the subunit interfaces.

3D fluorescence anisotropy imaging using selective plane illumination microscopy.

Fluorescence anisotropy imaging is a popular method to visualize changes in organization and conformation of biomolecules within cells and tissues. In such an experiment, depolarization effects resulting from differences in orientation, proximity and rotational mobility of fluorescently labeled molecules are probed with high spatial resolution. Fluorescence anisotropy is typically imaged using laser scanning and epifluorescence-based approaches. Unfortunately, those techniques are limited in either axial resolution, image acquisition speed, or by photobleaching. In the last decade, however, selective plane illumination microscopy has emerged as the preferred choice for three-dimensional time lapse imaging combining axial sectioning capability with fast, camera-based image acquisition, and minimal light exposure. We demonstrate how selective plane illumination microscopy can be utilized for three-dimensional fluorescence anisotropy imaging of live cells. We further examined the formation of focal adhesions by three-dimensional time lapse anisotropy imaging of CHO-K1 cells expressing an EGFP-paxillin fusion protein.

Mapping diffusion in a living cell via the phasor approach.

Diffusion of a fluorescent protein within a cell has been measured using either fluctuation-based techniques (fluorescence correlation spectroscopy (FCS) or raster-scan image correlation spectroscopy) or particle tracking. However, none of these methods enables us to measure the diffusion of the fluorescent particle at each pixel of the image. Measurement using conventional single-point FCS at every individual pixel results in continuous long exposure of the cell to the laser and eventual bleaching of the sample. To overcome this limitation, we have developed what we believe to be a new method of scanning with simultaneous construction of a fluorescent image of the cell. In this believed new method of modified raster scanning, as it acquires the image, the laser scans each individual line multiple times before moving to the next line. This continues until the entire area is scanned. This is different from the original raster-scan image correlation spectroscopy approach, where data are acquired by scanning each frame once and then scanning the image multiple times. The total time of data acquisition needed for this method is much shorter than the time required for traditional FCS analysis at each pixel. However, at a single pixel, the acquired intensity time sequence is short; requiring nonconventional analysis of the correlation function to extract information about the diffusion. These correlation data have been analyzed using the phasor approach, a fit-free method that was originally developed for analysis of FLIM images. Analysis using this method results in an estimation of the average diffusion coefficient of the fluorescent species at each pixel of an image, and thus, a detailed diffusion map of the cell can be created.

Cyanine dyes in biophysical research: the photophysics of polymethine fluorescent dyes in biomolecular environments.

The breakthroughs in single molecule spectroscopy of the last decade and the recent advances in super resolution microscopy have boosted the popularity of cyanine dyes in biophysical research. These applications have motivated the investigation of the reactions and relaxation processes that cyanines undergo in their electronically excited states. Studies show that the triplet state is a key intermediate in the photochemical reactions that limit the photostability of cyanine dyes. The removal of oxygen greatly reduces photobleaching, but induces rapid intensity fluctuations (blinking). The existence of non-fluorescent states lasting from milliseconds to seconds was early identified as a limitation in single-molecule spectroscopy and a potential source of artifacts. Recent studies demonstrate that a combination of oxidizing and reducing agents is the most efficient way of guaranteeing that the ground state is recovered rapidly and efficiently. Thiol-containing reducing agents have been identified as the source of long-lived dark states in some cyanines that can be photochemically switched back to the emissive state. The mechanism of this process is the reversible addition of the thiol-containing compound to a double bond in the polymethine chain resulting in a non-fluorescent molecule. This process can be reverted by irradiation at shorter wavelengths. Another mechanism that leads to non-fluorescent states in cyanine dyes is cis-trans isomerization from the singlet-excited state. This process, which competes with fluorescence, involves the rotation of one-half of the molecule with respect to the other with an efficiency that depends strongly on steric effects. The efficiency of fluorescence of most cyanine dyes has been shown to depend dramatically on their molecular environment within the biomolecule. For example, the fluorescence quantum yield of Cy3 linked covalently to DNA depends on the type of linkage used for attachment, DNA sequence and secondary structure. Cyanines linked to the DNA termini have been shown to be mostly stacked at the end of the helix, while cyanines linked to the DNA internally are believed to partially bind to the minor or major grooves. These interactions not only affect the photophysical properties of the probes but also create a large uncertainty in their orientation.

Visualizing orthogonal RNAs simultaneously in live mammalian cells by fluorescence lifetime imaging microscopy (FLIM).

Visualization of RNAs in live cells is critical to understand biology of RNA dynamics and function in the complex cellular environment. Detection of RNAs with a fluorescent marker frequently involves genetically fusing an RNA aptamer tag to the RNA of interest, which binds to small molecules that are added to live cells and have fluorescent properties. Engineering efforts aim to improve performance and add versatile features. Current efforts focus on adding multiplexing capabilities to tag and visualize multiple RNAs simultaneously in the same cell. Here, we present the fluorescence lifetime-based platform Riboglow-FLIM. Our system requires a smaller tag and has superior cell contrast when compared with intensity-based detection. Because our RNA tags are derived from a large bacterial riboswitch sequence family, the riboswitch variants add versatility for using multiple tags simultaneously. Indeed, we demonstrate visualization of two RNAs simultaneously with orthogonal lifetime-based tags.

Fibrosis quantification using multiphoton excitation imaging of picrosirius red stained cardiac tissue.

Traditional methodologies for fibrosis quantification involve histological measurements, staining with Masson's trichrome and picrosirius red (PSR), and label-free imaging using second harmonic generation (SHG). The difficulty of label-free cardiac SHG imaging is that both collagen (i.e., collagen 1 fibrils) and myosin are harmonophores that generate SHG signals, and specific identification of either collagen or myosin is difficult to achieve. Here we present an alternate method of quantifying cardiac fibrosis by using PSR staining followed by multiphoton excitation fluorescence imaging. Our data from the deoxycorticosterone model of cardiac fibrosis shows that this imaging method and downstream analyses, including background correction, are robust and easy to perform. These advantages are due to the high signal-to-noise ratio provided by PSR in areas of collagen fibers. Furthermore, the hyperspectral and fluorescence lifetime information of PSR-stained area of fibrosis shows better quantification can eventually be obtained using more complex instrumentation.

Establishing Riboglow-FLIM to visualize noncoding RNAs inside live zebrafish embryos.

The central role of RNAs in health and disease calls for robust tools to visualize RNAs in living systems through fluorescence microscopy. Live zebrafish embryos are a popular system to investigate multicellular complexity as disease models. However, RNA visualization approaches in whole organisms are notably underdeveloped. Here, we establish our RNA tagging and imaging platform Riboglow-FLIM for complex cellular imaging applications by systematically evaluating FLIM capabilities. We use adherent mammalian cells as models for RNA visualization. Additional complexity of analyzing RNAs in whole mammalian animals is achieved by injecting these cells into a zebrafish embryo system for cell-by-cell analysis in this model of multicellularity. We first evaluate all variable elements of Riboglow-FLIM quantitatively before assessing optimal use in whole animals. In this way, we demonstrate that a model noncoding RNA can be detected robustly and quantitatively inside live zebrafish embryos using a far-red Cy5-based variant of the Riboglow platform. We can clearly resolve cell-to-cell heterogeneity of different RNA populations by this methodology, promising applicability in diverse fields.

Riluzole Suppresses Growth and Enhances Response to Endocrine Therapy in ER+ Breast Cancer.

Background: Resistance to endocrine therapy in estrogen receptor-positive (ER+) breast cancer remains a significant clinical problem. Riluzole is FDA-approved for the treatment of amyotrophic lateral sclerosis. A benzothiazole-based glutamate release inhibitor with several context-dependent mechanism(s) of action, riluzole has shown antitumor activity in multiple malignancies, including melanoma, glioblastoma, and breast cancer. We previously reported that the acquisition of tamoxifen resistance in a cellular model of invasive lobular breast cancer is accompanied by the upregulation of GRM mRNA expression and growth inhibition by riluzole. Methods: We tested the ability of riluzole to reduce cell growth, alone and in combination with endocrine therapy, in a diverse set of ER+ invasive ductal and lobular breast cancer-derived cell lines, primary breast tumor explant cultures, and the estrogen-independent, ESR1-mutated invasive lobular breast cancer patient-derived xenograft model HCI-013EI. Results: Single-agent riluzole suppressed the growth of ER+ invasive ductal and lobular breast cancer cell lines in vitro, inducing a histologic subtype-associated cell cycle arrest (G0-G1 for ductal, G2-M for lobular). Riluzole induced apoptosis and ferroptosis and reduced phosphorylation of multiple prosurvival signaling molecules, including Akt/mTOR, CREB, and Fak/Src family kinases. Riluzole, in combination with either fulvestrant or 4-hydroxytamoxifen, additively suppressed ER+ breast cancer cell growth in vitro. Single-agent riluzole significantly inhibited HCI-013EI patient-derived xenograft growth in vivo, and the combination of riluzole plus fulvestrant significantly reduced proliferation in ex vivo primary breast tumor explant cultures. Conclusion: Riluzole may offer therapeutic benefits in diverse ER+ breast cancers, including lobular breast cancer.

Sphingosine kinase 1 mediates sexual dimorphism in fibrosis in a mouse model of NASH.

OBJECTIVE: Men with non-alcoholic fatty liver disease (NAFLD) are more likely to progress to non-alcoholic steatohepatitis (NASH) and liver fibrosis than women. However, the underlying molecular mechanisms of this dimorphism is unclear. We have previously shown that mice with global deletion of SphK1, the enzyme that produces the bioactive sphingolipid metabolite sphingosine 1-phosphate (S1P), were protected from development of NASH. The aim of this study was to elucidate the role of hepatocyte-specific SphK1 in development of NASH and to compare its contribution to hepatosteatosis in male and female mice. METHODS: We assessed mouse livers in early-stage fibrosis induced by high fat feeding, using single harmonic generation microscopy, LC-MS/MS analysis of hydroxyproline levels, and expression of fibrosis markers. We identified an antifibrotic intercellular signaling mechanism by culturing primary mouse hepatocytes alongside, and in co-culture with, LX2 hepatic stellate cells. RESULTS: We generated hepatocyte-specific SphK1 knockout mice (SphK1-hKO). Unlike the global knockout, SphK1-hKO male mice were not protected from diet-induced steatosis, inflammation, or fibrogenesis. In contrast, female SphK1-hKO mice were protected from inflammation. Surprisingly, however, in these female mice, there was a ∼10-fold increase in the fibrosis markers Col1α1 and 2-3 fold induction of alpha smooth muscle actin and the pro-fibrotic chemokine CCL5. Because increased fibrosis in female SphK1-hKO mice occurred despite an attenuated inflammatory response, we investigated the crosstalk between hepatocytes and hepatic stellate cells, central players in fibrosis. We found that estrogen stimulated release of S1P from female hepatocytes preventing TGFß-induced expression of Col1α1 in HSCs via S1PR3. CONCLUSIONS: The results revealed a novel pathway of estrogen-mediated cross-talk between hepatocytes and HSCs that may contribute to sex differences in NAFLD through an anti-fibrogenic function of the S1P/S1PR3 axis. This pathway is susceptible to pharmacologic manipulation, which may lead to novel therapeutic strategies.