Post-click labeling enables highly accurate single cell analyses of glucose uptake ex vivo and in vivo.

Cellular glucose uptake is a key feature reflecting metabolic demand of cells in physiopathological conditions. Fluorophore-conjugated sugar derivatives are widely used for monitoring glucose transporter (GLUT) activity at the single-cell level, but have limitations in in vivo applications. Here, we develop a click chemistry-based post-labeling method for flow cytometric measurement of glucose uptake with low background adsorption. This strategy relies on GLUT-mediated uptake of azide-tagged sugars, and subsequent intracellular labeling with a cell-permeable fluorescent reagent via a copper-free click reaction. Screening a library of azide-substituted monosaccharides, we discover 6-azido-6-deoxy-D-galactose (6AzGal) as a suitable substrate of GLUTs. 6AzGal displays glucose-like physicochemical properties and reproduces in vivo dynamics similar to 18F-FDG. Combining this method with multi-parametric immunophenotyping, we demonstrate the ability to precisely resolve metabolically-activated cells with various GLUT activities in ex vivo and in vivo models. Overall, this method provides opportunities to dissect the heterogenous metabolic landscape in complex tissue environments.

Pten regulates endocytic trafficking of cell adhesion and Wnt signaling molecules to pattern the retina.

The retina is exquisitely patterned, with neuronal somata positioned at regular intervals to completely sample the visual field. Here, we show that phosphatase and tensin homolog (Pten) controls starburst amacrine cell spacing by modulating vesicular trafficking of cell adhesion molecules and Wnt proteins. Single-cell transcriptomics and double-mutant analyses revealed that Pten and Down syndrome cell adhesion molecule Dscam) are co-expressed and function additively to pattern starburst amacrine cell mosaics. Mechanistically, Pten loss accelerates the endocytic trafficking of DSCAM, FAT3, and MEGF10 off the cell membrane and into endocytic vesicles in amacrine cells. Accordingly, the vesicular proteome, a molecular signature of the cell of origin, is enriched in exocytosis, vesicle-mediated transport, and receptor internalization proteins in Pten conditional knockout (PtencKO) retinas. Wnt signaling molecules are also enriched in PtencKO retinal vesicles, and the genetic or pharmacological disruption of Wnt signaling phenocopies amacrine cell patterning defects. Pten thus controls vesicular trafficking of cell adhesion and signaling molecules to establish retinal amacrine cell mosaics.

Progenitor division and cell autonomous neurosecretion are required for rod photoreceptor sublaminar positioning.

Migration is essential for the laminar stratification and connectivity of neurons in the central nervous system. In the retina, photoreceptors (PRs) migrate to positions according to birthdate, with early-born cells localizing to the basal-most side of the outer nuclear layer. It was proposed that apical progenitor mitoses physically drive these basal translocations non-cell autonomously, but direct evidence is lacking, and whether other mechanisms participate is unknown. Here, combining loss- or gain-of-function assays to manipulate cell cycle regulators (Sonic hedgehog, Cdkn1a/p21) with an in vivo lentiviral labelling strategy, we demonstrate that progenitor division is one of two forces driving basal translocation of rod soma. Indeed, replacing Shh activity rescues abnormal rod translocation in retinal explants. Unexpectedly, we show that rod differentiation also promotes rod soma translocation. While outer segment function or formation is dispensable, Crx and SNARE-dependent synaptic function are essential. Thus, both non-cell and cell autonomous mechanisms underpin PR soma sublaminar positioning in the mammalian retina.

Flow cytometric analysis of phosphatidylcholine metabolism using organelle-selective click labeling.

Here, we present a protocol to analyze phosphatidylcholine (PC) metabolism in mammalian cells using organelle-selective click labeling coupled with flow cytometry (O-ClickFC). We describe steps for the metabolic incorporation of azide-choline into PC. We then detail fluorescent labeling of the azide-modified PC with organelle-targeting clickable dyes in the ER-Golgi, plasma membrane, and mitochondria, and by flow cytometry. This protocol is optimized for flow cytometric quantification of the labeled PC at the organelle level within single live cells. For complete details on the use and execution of this protocol, please refer to Tsuchiya et al. (2023).1.

Organelle-selective click labeling coupled with flow cytometry allows pooled CRISPR screening of genes involved in phosphatidylcholine metabolism.

Cellular lipid synthesis and transport are governed by intricate protein networks. Although genetic screening should contribute to deciphering the regulatory networks of lipid metabolism, technical challenges remain-especially for high-throughput readouts of lipid phenotypes. Here, we coupled organelle-selective click labeling of phosphatidylcholine (PC) with flow cytometry-based CRISPR screening technologies to convert organellar PC phenotypes into a simple fluorescence readout for genome-wide screening. This technique, named O-ClickFC, was successfully applied in genome-scale CRISPR-knockout screens to identify previously reported genes associated with PC synthesis (PCYT1A, ACACA), vesicular membrane trafficking (SEC23B, RAB5C), and non-vesicular transport (PITPNB, STARD7). Moreover, we revealed previously uncharacterized roles of FLVCR1 as a choline uptake facilitator, CHEK1 as a post-translational regulator of the PC-synthetic pathway, and CDC50A as responsible for the translocation of PC to the outside of the plasma membrane bilayer. These findings demonstrate the versatility of O-ClickFC as an unprecedented platform for genetic dissection of cellular lipid metabolism.

Three dimensional reconstruction of the mouse cerebellum in Hedgehog-driven medulloblastoma models to identify Norrin-dependent effects on preneoplasia.

Spontaneous mouse models of medulloblastoma (MB) offer a tractable system to study malignant progression in the brain. Mouse Sonic Hedgehog (Shh)-MB tumours first appear at postnatal stages as preneoplastic changes on the surface of the cerebellum, the external granule layer (EGL). Here we compared traditional histology and 3DISCO tissue clearing in combination with light sheet fluorescence microscopy (LSFM) to identify and quantify preneoplastic changes induced by disrupting stromal Norrin/Frizzled 4 (Fzd4) signalling, a potent tumour inhibitory signal in two mouse models of spontaneous Shh-MB. We show that 3DISCO-LSFM is as accurate as traditional histology for detecting Norrin/Fzd4-associated changes in PNL formation in Ptch+/- mice and EGL hyperplasia in Neurod2-SmoA1+/- mice. Moreover, we show that the anti-tumour effect of Norrin/Fzd4 signalling is restricted to the posterior region of the cerebellum and is characterized by defective neural progenitor migration away from the EGL. In conclusion, 3DISCO-LSFM is a valid way to monitor tumour initiation events in mouse MB models and reveals an unanticipated regional restriction of stromal signalling in constraining tumour initiation.

Soy Phospholipids Exert a Renoprotective Effect by Inhibiting the Nuclear Factor Kappa B Pathway in Macrophages.

Complications associated with chronic kidney disease (CKD), which involves kidney inflammation, are a major health problem. Soy protein isolate (SPI) reportedly inhibits CKD exacerbation; however, its detailed action mechanism remains obscure. Therefore, the role of the polar lipid component of SPI in suppressing inflammation was investigated. Zucker fatty rats were divided into three groups and fed a diet containing casein, SPI, or casein + SPI ethanol extract (SPIEE) for 16 weeks. The isoflavones and phospholipids of SPIEE were evaluated for their anti-inflammatory effects. Rats in the SPI and casein + SPIEE groups showed reduced levels of the urinary N-acetyl-ß-d-glucosaminidase and renal IL-1ß mRNA (an inflammatory marker) compared with those in the casein group. In proximal tubular cells, genistein significantly inhibited monocyte chemoattractant protein-1 (MCP-1) expression induced by an IL-1ß stimulus. In macrophages, soybean phospholipids suppressed lipopolysaccharide-induced IL-1ß gene expression by inhibiting the phosphorylation of inhibitor κB and p65. Phosphatidylinositol (PI) was found to be essential for inhibition of IL-1ß expression. SPIEE inhibited the exacerbation of kidney disease. Genistein and soybean phospholipids, especially soybean-specific phospholipids containing PI, effectively inhibited the inflammatory spiral in vitro. Hence, daily soybean intake may be effective for inhibiting chronic inflammation and slowing kidney disease progression.

Intake of Docosahexaenoic Acid-Enriched Milk Beverage Prevents Age-Related Cognitive Decline and Decreases Serum Bone Resorption Marker Levels.

The pathogenic mechanism of dementia is still unknown, and the fundamental treatment remains to be established. Thus, there is growing interest in preventing dementia through diet. One of the functional ingredients attracting attention is docosahexaenoic acid. We conducted a 12-month, randomized, double-blind, placebo-controlled clinical trial in healthy elderly Japanese individuals with a Mini-Mental State Examination score of 28 or higher at baseline using a docosahexaenoic acid-enriched milk beverage containing 297 mg docosahexaenoic acid and 137 mg eicosapentaenoic acid. Consumption of a docosahexaenoic acid-enriched milk beverage increased the fatty acid levels of docosahexaenoic acid and eicosapentaenoic acid in erythrocyte membranes, which was the primary outcome of this study. Moreover, intake of this beverage prevented age-related cognitive decline and decreased serum bone resorption marker levels. Our data demonstrate that, even at a low dose, long-term daily intake of docosahexaenoic acid prevents dementia and may show beneficial effect on bone health.

Photoreceptor nanotubes mediate the in vivo exchange of intracellular material.

Emerging evidence suggests that intracellular molecules and organelles transfer between cells during embryonic development, tissue homeostasis and disease. We and others recently showed that transplanted and host photoreceptors engage in bidirectional transfer of intracellular material in the recipient retina, a process termed material transfer (MT). We used cell transplantation, advanced tissue imaging approaches, genetic and pharmacologic interventions and primary cell culture to characterize and elucidate the mechanism of MT. We show that MT correlates with donor cell persistence and the accumulation of donor-derived proteins, mitochondria and transcripts in acceptor cells in vivo. MT requires cell contact in vitro and is associated with the formation of stable microtubule-containing protrusions, termed photoreceptor nanotubes (Ph NTs), that connect donor and host cells in vivo and in vitro. Ph NTs mediate GFP transfer between connected cells in vitro. Furthermore, interfering with Ph NT outgrowth by targeting Rho GTPase-dependent actin remodelling inhibits MT in vivo. Collectively, our observations provide evidence for horizontal exchange of intracellular material via nanotube-like connections between neurons in vivo.

InVision: An optimized tissue clearing approach for three-dimensional imaging and analysis of intact rodent eyes.

The mouse eye is used to model central nervous system development, pathology, angiogenesis, tumorigenesis, and regenerative therapies. To facilitate the analysis of these processes, we developed an optimized tissue clearing and depigmentation protocol, termed InVision, that permits whole-eye fluorescent marker tissue imaging. We validated this method for the analysis of normal and degenerative retinal architecture, transgenic fluorescent reporter expression, immunostaining and three-dimensional volumetric (3DV) analysis of retinoblastoma and angiogenesis. We also used this method to characterize material transfer (MT), a recently described phenomenon of horizontal protein exchange that occurs between transplanted and recipient photoreceptors. 3D spatial distribution analysis of MT in transplanted retinas suggests that MT of cytoplasmic GFP between photoreceptors is mediated by short-range, proximity-dependent cellular interactions. The InVision protocol will allow investigators working across multiple cell biological disciplines to generate novel insights into the local cellular networks involved in cell biological processes in the eye.

Dietary Sodium Nitrite Causes Similar Modifications to Splenic Inflammatory Gene Expression as a High-Fat Diet.

Sodium nitrite (NaNO2) is a widely used food additive. The present study compared the outcomes from intakes of dietary NaNO2 and a high-fat diet (HFD), and assessed their combined effects on inflammatory gene expression in the immune tissues of the mouse. In experiment I, mice were fed a standard low-fat diet (LFD) without or with NaNO2 (0.02 and 0.08%, w/w) for 11 wk. In experiment II, mice were fed an LFD without or with NaNO2 (0.02%) or HFD without or with NaNO2 (0.02%) for 11 wk. Inflammatory gene expression in the immune tissues was then measured. NaNO2 consumption and HFD feeding each resulted in increased splenic mRNAs for cell markers of neutrophils (Ngp, NE, Ly6g, Mpo) and eosinophils (Epo, Ear6), and an S100 family member (S100A8). In contrast, NaNO2 consumption and HFD feeding each resulted in decreased splenic mRNAs for cell markers of macrophages (Emr1, Itgax, CD68, CD206, Dectin-1, TLRs 4, 6, and 7), T- (CD3, CD4), NK- (CD56) and B-cells (CD20, CD40), pro- and anti-inflammatory cytokines (TNF-α, IL-6, IL-1ß, IFN-γ, IL-18, IL-10, TGF-ß), interleukin receptor antagonists (IL1ra, IL6ra) and cell adhesion molecules (ICAM-1, VCAM-1). However, dietary NaNO2 combined with HFD feeding caused no further decrease in these transcript levels compared with dietary NaNO2 alone. These NaNO2- or HFD-induced modifications were less profound in the liver and abdominal adipose tissues than in the spleen. These findings indicate that dietary NaNO2 has similar modulatory effects to HFD feeding on splenic inflammatory genes.

A Diet Including Red Bell Pepper Juice and Soy Protein Suppress Physiological Markers of Muscle Atrophy in Mice.

Although muscle atrophy can be caused by disuse and lifestyle-related syndromes, it may be possible to prevent this condition through dietary intervention. We hypothesized that a diet including red bell pepper juice (RBPJ) and soy protein isolate (SPI) would prevent muscle atrophy. Accordingly, an experimental diet containing RBPJ and/or SPI was administered for 18 d to normal C57BL/6J mice. The control group was administered a casein diet. Four days before the end of the test period, denervation-induced muscle atrophy and/or sham operation were performed. Anterior tibialis muscle samples were then obtained to assess muscle degradation and perform metabolome analysis. Under the denervation condition, the 20% SPI diet did not alter the mRNA expression levels of muscle atrophy marker genes compared with the 20% casein group. Although the diet comprising RBPJ and 20% casein did not prevent muscle atrophy compared with the control group, the diet containing RBPJ and 20% SPI did. Metabolome analysis revealed that a diet including RBPJ and SPI induced a greater than 1.5-fold change in the levels of 20 muscle atrophy-related metabolites. In particular, the level of S-adenosylmethionine, which concerned with energy metabolism and lifespan, showed a strong positive correlation with the muscle atrophy marker. These findings suggest that a diet including RBPJ and soy protein suppress gene expressions related with muscle atrophy. Further research in humans is needed to confirm whether a combination of RBPJ and SPI can indeed prevent muscle atrophy.

Soy ß-Conglycinin Peptide Attenuates Obesity and Lipid Abnormalities in Obese Model OLETF Rats.

We previously reported that soy ß-conglycinin (ßCG) improves obesity-induced metabolic abnormalities, but not obesity, in obese model Otsuka Long-Evans Tokushima fatty (OLETF) rats. In the present study, we aimed to investigate the effects of ßCG-derived peptide consumption on obesity and lipid abnormality in OLETF rats. To this end, wild-type Long-Evans Tokushima Otsuka and OLETF rats were provided a normal diet containing 20% casein for four weeks as a control. In addition, we prepared ßCG peptide by enzymatic hydrolysis, and OLETF rats were fed a diet in which half of the casein was replaced by ßCG peptide (ßCG peptide group). Consequently, rats in the ßCG peptide group showed decreased abdominal white adipose tissue weight and lipid content (serum and liver triglycerides, and serum and liver cholesterol) compared to control OLETF rats. Further analysis demonstrated that ßCG peptide consumption decreased lipogenic enzyme activity and increased lipolytic enzyme activity in the liver of OLETF rats. In addition, suppressive effects on both synthesis and absorption of cholesterol were observed in ßCG peptide-fed OLETF rats. These findings suggest that peptidization of ßCG enhanced the anti-obese and hypolipidemic effects of ßCG.

Norrin mediates tumor-promoting and -suppressive effects in glioblastoma via Notch and Wnt.

Glioblastoma multiforme (GBM) contains a subpopulation of cells, GBM stem cells (GSCs), that maintain the bulk tumor and represent a key therapeutic target. Norrin is a Wnt ligand that binds Frizzled class receptor 4 (FZD4) to activate canonical Wnt signaling. Although Norrin, encoded by NDP, has a well-described role in vascular development, its function in human tumorigenesis is largely unexplored. Here, we show that NDP expression is enriched in neurological cancers, including GBM, and its levels positively correlated with survival in a GBM subtype defined by low expression of ASCL1, a proneural factor. We investigated the function of Norrin and FZD4 in GSCs and found that it mediated opposing tumor-suppressive and -promoting effects on ASCL1lo and ASCL1hi GSCs. Consistent with a potential tumor-suppressive effect of Norrin suggested by the tumor outcome data, we found that Norrin signaling through FZD4 inhibited growth in ASCL1lo GSCs. In contrast, in ASCL1hi GSCs Norrin promoted Notch signaling, independently of WNT, to promote tumor progression. Forced ASCL1 expression reversed the tumor-suppressive effects of Norrin in ASCL1lo GSCs. Our results identify Norrin as a modulator of human brain cancer progression and reveal an unanticipated Notch-mediated function of Norrin in regulating cancer stem cell biology. This study identifies an unanticipated role of Norrin in human brain cancer progression. In addition, we provide preclinical evidence suggesting Norrin and canonical Wnt signaling as potential therapeutic targets for GBM subtype-restricted cancer stem cells.

Dietary soybean protein ameliorates high-fat diet-induced obesity by modifying the gut microbiota-dependent biotransformation of bile acids.

The consumption of soybean protein has well-known favorable metabolic effects (e.g., reduced body weight, body fat, hyperglycemia, insulin resistance, hepatic steatosis, and lipogenesis). These effects of soy protein have been linked to modulation by the gut microbiota; however, the dynamic interplay among these factors remains unclear. Accordingly, we examined the metabolic phenotype, intestinal BA pool, and the gut microbiome of male C57BL/6 mice that were randomized to receive either a regular high-fat diet (HFD) or HFD that contained soybean protein isolate (SPI) in place of dairy protein. The intake of SPI significantly reduced the HFD-induced weight gain and adipose tissue mass accumulation and attenuated hepatic steatosis. Along with an enhancement in the secretion of intestinal Glucagon-like peptide-1 (GLP-1), an enlarged cecal BA pool with an elevated secondary/primary BA ratio was observed in the mice that consumed SPI, while fecal BA excretion remained unaltered. SPI also elicited dramatic changes in the gut microbiome, characterized by an expansion of taxa that may be involved in the biotransformation of BAs. The observed effects were abolished in germ-free (GF) mice, indicating that they were dependent on the microbiota. These findings collectively indicate that the metabolic benefits of SPI under the HFD regime may arise from a microbiota-driven increase in BA transformation and increase in GLP-1 secretion.

Dietary mung bean protein reduces high-fat diet-induced weight gain by modulating host bile acid metabolism in a gut microbiota-dependent manner.

The 8-globulin-rich mung bean protein (MPI) suppresses hepatic lipogenesis in rodent models and reduces fasting plasma glucose and insulin levels in obese adults. However, its effects on mitigating high fat diet (HFD)-induced obesity and the mechanism underlying these effects remain to be elucidated. Herein, we examined the metabolic phenotype, intestinal bile acid (BA) pool, and gut microbiota of conventionally raised (CONV-R) male C57BL/6 mice and germ-free (GF) mice that were randomized to receive either regular HFD or HFD containing mung bean protein isolate (MPI) instead of the dairy protein present in regular HFD. MPI intake significantly reduced HFD-induced weight gain and adipose tissue accumulation, and attenuated hepatic steatosis. Enhancement in the secretion of intestinal glucagon-like peptide-1 (GLP-1) and an enlarged cecal and fecal BA pool of dramatically elevated secondary/primary BA ratio were observed in mice that had consumed MPI. These effects were abolished in GF mice, indicating that the effects were dependent upon the presence of the microbiota. As revealed by 16S rRNA gene sequence analysis, MPI intake also elicited dramatic changes in the gut microbiome, such as an expansion of taxa belonging to the phylum Bacteroidetes along with a reduced abundance of the Firmicutes.

Hamartoma-like lesions in the mouse retina: an animal model of Pten hamartoma tumour syndrome.

PTEN hamartoma tumour syndrome (PHTS) is a heterogeneous group of rare, autosomal dominant disorders associated with PTEN germline mutations. PHTS patients routinely develop hamartomas, which are benign tissue overgrowths comprised of disorganized 'normal' cells. Efforts to generate PHTS animal models have been largely unsuccessful due to the early lethality of homozygous germline mutations in Pten, together with the lack of hamartoma formation in most conditional mutants generated to date. We report herein a novel PHTS mouse model that reproducibly forms hamartoma-like lesions in the central retina by postnatal day 21. Specifically, we generated a Pten conditional knockout (cKO) using a retinal-specific Pax6::Cre driver that leads to a nearly complete deletion of Pten in the peripheral retina but produces a mosaic of 'wild-type' and Pten cKO cells centrally. Structural defects were only observed in the mosaic central retina, including in Müller glia and in the outer and inner limiting membranes, suggesting that defective mechanical integrity partly underlies the hamartoma-like pathology. Finally, we used this newly developed model to test whether rapamycin, an mTOR inhibitor that is currently the only PHTS therapy, can block hamartoma growth. When administered in the early postnatal period, prior to hamartoma formation, rapamycin reduces hamartoma size, but also induces new morphological abnormalities in the Pten cKO retinal periphery. In contrast, administration of rapamycin after hamartoma initiation fails to reduce lesion size. We have thus generated and used an animal model of retinal PHTS to show that, although current therapies can reduce hamartoma formation, they might also induce new retinal dysmorphologies.This article has an associated First Person interview with the first author of the paper.

Heterochronic Pellet Assay to Test Cell-cell Communication in the Mouse Retina.

All seven retinal cell types that make up the mature retina are generated from a common, multipotent pool of retinal progenitor cells (RPCs) (Wallace, 2011). One way that RPCs know when sufficient numbers of particular cell-types have been generated is through negative feedback signals, which are emitted by differentiated cells and must reach threshold levels to block additional differentiation of that cell type. A key assay to assess whether negative feedback signals are emitted by differentiated cells is a heterochronic pellet assay in which early stage RPCs are dissociated and labeled with BrdU, then mixed with a 20-fold excess of dissociated differentiated cells. The combined cells are then re-aggregated and cultured as a pellet on a membrane for 7-10 days in vitro. During this time frame, RPCs will differentiate, and the fate of the BrdU+ RPCs can be assessed using cell type-specific markers. Investigators who developed this pellet assay initially demonstrated that neonatal RPCs give rise to rods on an accelerated schedule compared to embryonic RPCs when the two cell types are mixed together (Watanabe and Raff, 1990; Watanabe et al., 1997). We have used this assay to demonstrate that sonic hedgehog (Shh), which we found acts as a negative regulator of retinal ganglion cell (RGC) differentiation, promotes RPC proliferation (Jensen and Wallace, 1997; Ringuette et al., 2014). More recently we modified the heterochronic pellet assay to assess the role of feedback signals for retinal amacrine cells, identifying transforming growth factor ß2 (Tgfß2) as a negative feedback signal, and Pten as a modulator of the Tgfß2 response (Ma et al., 2007; Tachibana et al., 2016). This assay can be adapted to other lineages and tissues to assess cell-cell interactions between two different cell-types (heterotypic) in either an isochronic or heterochronic manner.

Temporal profiling of photoreceptor lineage gene expression during murine retinal development.

Rod and cone photoreceptors are photosensitive cells in the retina that convert light to electrical signals that are transmitted to visual processing centres in the brain. During development, cones and rods are generated from a common pool of multipotent retinal progenitor cells (RPCs) that also give rise to other retinal cell types. Cones and rods differentiate in two distinct waves, peaking in mid-embryogenesis and the early postnatal period, respectively. As RPCs transition from making cones to generating rods, there are changes in the expression profiles of genes involved in photoreceptor cell fate specification and differentiation. To better understand the temporal transition from cone to rod genesis, we assessed the timing of onset and offset of expression of a panel of 11 transcription factors and 7 non-transcription factors known to function in photoreceptor development, examining their expression between embryonic day (E) 12.5 and postnatal day (P) 60. Transcription factor expression in the photoreceptor layer was observed as early as E12.5, beginning with Crx, Otx2, Rorb, Neurod1 and Prdm1 expression, followed at E15.5 with the expression of Thrb, Neurog1, Sall3 and Rxrg expression, and at P0 by Nrl and Nr2e3 expression. Of the non-transcription factors, peanut agglutinin lectin staining and cone arrestin protein were observed as early as E15.5 in the developing outer nuclear layer, while transcripts for the cone opsins Opn1mw and Opn1sw and Recoverin protein were detected in photoreceptors by P0. In contrast, Opn1mw and Opn1sw protein were not observed in cones until P7, when rod-specific Gnat1 transcripts and rhodopsin protein were also detected. We have thus identified four transitory stages during murine retina photoreceptor differentiation marked by the period of onset of expression of new photoreceptor lineage genes. By characterizing these stages, we have clarified the dynamic nature of gene expression during the period when photoreceptor identities are progressively acquired during development.

Dietary Mung Bean Protein Reduces Hepatic Steatosis, Fibrosis, and Inflammation in Male Mice with Diet-Induced, Nonalcoholic Fatty Liver Disease.

BACKGROUND: As the prevalence of nonalcoholic fatty liver disease (NAFLD), including steatosis and nonalcoholic steatohepatitis, is increasing, novel dietary approaches are required for the prevention and treatment of NAFLD. OBJECTIVE: We evaluated the potential of mung bean protein isolate (MuPI) to prevent NAFLD progression. METHODS: In Expts. 1 and 2, the hepatic triglyceride (TG) concentration was compared between 8-wk-old male mice fed a high-fat diet (61% of energy from fat) containing casein, MuPI, and soy protein isolate and an MuPI-constituent amino acid mixture as a source of amino acids (18% of energy) for 4 wk. In Expt. 3, hepatic fatty acid synthase (Fasn) expression was evaluated in 8-wk-old male Fasn-promoter-reporter mice fed a casein- or MuPI-containing high-fat diet for 20 wk. In Expt. 4, hepatic fibrosis was examined in 8-wk-old male mice fed an atherogenic diet (61% of energy from fat, containing 1.3 g cholesterol/100 g diet) containing casein or MuPI (18% of energy) as a protein source for 20 wk. RESULTS: In the high fat-diet mice, the hepatic TG concentration in the MuPI group decreased by 66% and 47% in Expt. 1 compared with the casein group (P < 0.001) and the soy protein isolate group (P = 0.001), respectively, and decreased by 56% in Expt. 2 compared with the casein group (P = 0.011). However, there was no difference between the MuPI-constituent amino acid mixture and casein groups in Expt. 2. In Expt. 3, Fasn-promoter-reporter activity and hepatic TG concentration were lower in the MuPI group than in those fed casein (P < 0.05). In Expt. 4, in mice fed an atherogenic diet, hepatic fibrosis was not induced in the MuPI group, whereas it developed overtly in the casein group. CONCLUSION: MuPI potently reduced hepatic lipid accumulation in mice and may be a potential foodstuff to prevent NAFLD onset and progression.