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Obesity and metabolic diseases tend to go together, and humans who become obese are also prone to type 2 diabetes and cardiovascular problems. Starting with the observation that offspring of germ-free mice tended to become obese on high-fat diets, Kimura et al. investigated how the presence of the microbiota might be protective in mice (see the Perspective by Ferguson). Short-chain fatty acids (SCFAs) from the microbiota are known to suppress insulin signaling and reduce fat deposition in adipocytes. Further experiments showed that SCFAs in the bloodstream were able to pass from a non–germ-free mother's gut microbiota across the placenta and into the developing embryos. The authors found that in the embryos, the SCFA propionate mediates not only insulin levels through GPR43 signaling but also sympathetic nervous system development through GPR41 signaling. A high-fiber diet promoted propionate production from the maternal microbiota, and maternal antibiotic treatment resulted in obese-prone offspring. Science , this issue p. eaaw8429 ; see also p. 978 The mother’s gut microbiota during pregnancy tunes energy homeostasis and sympathetic nervous system development in offspring. Antibiotics and dietary habits can affect the gut microbial community, thus influencing disease susceptibility. Although the effect of microbiota on the postnatal environment has been well documented, much less is known regarding the impact of gut microbiota at the embryonic stage. Here we show that maternal microbiota shapes the metabolic system of offspring in mice. During pregnancy, short-chain fatty acids produced by the maternal microbiota dictate the differentiation of neural, intestinal, and pancreatic cells through embryonic GPR41 and GPR43. This developmental process helps maintain postnatal energy homeostasis, as evidenced by the fact that offspring from germ-free mothers are highly susceptible to metabolic syndrome, even when reared under conventional conditions. Thus, our findings elaborate on a link between the maternal gut environment and the developmental origin of metabolic syndrome.

Eicosapentaenoic acid (EPA), an omega-3 (ω-3) polyunsaturated fatty acid, is an essential nutrient that exhibits antiinflammatory, neuroprotective, and cardiovascular-protective activities. Although EPA is used as a nutrient-based pharmaceutical agent or dietary supplement, its molecular target(s) is debatable. Here, we showed that EPA and its metabolites strongly and reversibly inhibit vesicular nucleotide transporter (VNUT), a key molecule for vesicular storage and release of adenosine triphosphate (ATP) in purinergic chemical transmission. In vitro analysis showed that EPA inhibits human VNUT-mediated ATP uptake at a half-maximal inhibitory concentration (IC50) of 67 nM, acting as an allosteric modulator through competition with Cl⁻. EPA impaired vesicular ATP release from neurons without affecting the vesicular release of other neurotransmitters. In vivo, VNUT −/− mice showed a delay in the onset of neuropathic pain and resistance to both neuropathic and inflammatory pain. EPA potently attenuated neuropathic and inflammatory pain in wild-type mice but not in VNUT −/− mice without affecting the basal nociception. The analgesic effect of EPA was canceled by the intrathecal injection of purinoceptor agonists and was stronger than that of existing drugs used for neuropathic pain treatment, with few side effects. Neuropathic pain impaired insulin sensitivity in previous studies, which was improved by EPA in the wild-type mice but not in the VNUT −/− mice. Our results showed that VNUT is a molecular target of EPA that attenuates neuropathic and inflammatory pain and insulin resistance. EPA may represent a unique nutrient-based treatment and prevention strategy for neurological, immunological, and metabolic diseases by targeting purinergic chemical transmission.

Activation of purinergic receptors in the spinal cord by extracellular ATP is essential for neuropathic hypersensitivity after peripheral nerve injury (PNI). However, the cell type responsible for releasing ATP within the spinal cord after PNI is unknown. Here we show that PNI increases expression of vesicular nucleotide transporter (VNUT) in the spinal cord. Extracellular ATP content ([ATP] e ) within the spinal cord was increased after PNI, and this increase was suppressed by exocytotic inhibitors. Mice lacking VNUT did not show PNI-induced increase in [ATP] e and had attenuated hypersensitivity. These phenotypes were recapitulated in mice with specific deletion of VNUT in spinal dorsal horn (SDH) neurons, but not in mice lacking VNUT in primary sensory neurons, microglia or astrocytes. Conversely, ectopic VNUT expression in SDH neurons of VNUT-deficient mice restored PNI-induced increase in [ATP] e and pain. Thus, VNUT is necessary for exocytotic ATP release from SDH neurons which contributes to neuropathic pain. Purinergic receptor activation by extracellular ATP in the dorsal horn contributes to neuropathic pain, but which cell types release ATP in this context is not known. The authors show in a mouse model of neuropathic pain that ATP is released by dorsal horn neurons, a process requiring the vesicular nucleotide transporter, VNUT.

Leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5) and its homologs (e.g., Lgr6) mark adult stem cells in multiple tissues. Recently, we and others have shown that Lgr5 marks adult taste stem/progenitor cells in posterior tongue. However, the regenerative potential of Lgr5-expressing (Lgr5 ⁺) cells and the identity of adult taste stem/progenitor cells that regenerate taste tissue in anterior tongue remain elusive. In the present work, we describe a culture system in which single isolated Lgr5 ⁺ or Lgr6 ⁺ cells from taste tissue can generate continuously expanding 3D structures (“organoids”). Many cells within these taste organoids were cycling and positive for proliferative cell markers, cytokeratin K5 and Sox2, and incorporated 5-bromo-2’-deoxyuridine. Importantly, mature taste receptor cells that express gustducin, carbonic anhydrase 4, taste receptor type 1 member 3, nucleoside triphosphate diphosphohydrolase-2, or cytokeratin K8 were present in the taste organoids. Using calcium imaging assays, we found that cells grown out from taste organoids derived from isolated Lgr5 ⁺ cells were functional and responded to tastants in a dose-dependent manner. Genetic lineage tracing showed that Lgr6 ⁺ cells gave rise to taste bud cells in taste papillae in both anterior and posterior tongue. RT-PCR data demonstrated that Lgr5 and Lgr6 may mark the same subset of taste stem/progenitor cells both anteriorly and posteriorly. Together, our data demonstrate that functional taste cells can be generated ex vivo from single Lgr5 ⁺ or Lgr6 ⁺ cells, validating the use of this model for the study of taste cell generation. Significance Taste tissue regenerates continuously throughout the life span in mammals. Here, using lineage tracing and a culture system, we show that leucine-rich repeat-containing G protein-coupled receptor 5-expressing and leucine-rich repeat-containing G protein-coupled receptor 6-expressing taste stem/progenitor cells generate mature taste cells in vivo and ex vivo. Importantly, our ex vivo studies show that single-progenitor cells can generate all mature taste cell types and that differentiated taste cells form in the absence of innervation. This ex vivo model mimics the development of taste bud cells in taste papillae, recapitulates the process of taste renewal from adult taste stem cells to mature taste cells, and provides a means to study the regulation of taste cell generation and to understand the origins and cell lineage relationships within taste buds.

Despite the high incidence of neuropathic and inflammatory pain worldwide, effective drugs with few side effects are currently unavailable for the treatment of chronic pain. Recently, researchers have proposed that inhibitors of purinergic chemical transmission, which plays a key role in the pathological pain response, may allow for targeted treatment of pathological neuropathic and inflammatory pain. However, such therapeutic analgesic agents have yet to be developed. In the present study, we demonstrated that clodronate, a first-generation bisphosphonate with comparatively fewer side effects than traditional treatments, significantly attenuates neuropathic and inflammatory pain unrelated to bone abnormalities via inhibition of vesicular nucleotide transporter (VNUT), a key molecule for the initiation of purinergic chemical transmission. In vitro analyses indicated that clodronate inhibits VNUT at a half-maximal inhibitory concentration of 15.6 nM without affecting other vesicular neurotransmitter transporters, acting as an allosteric modulator through competition with Cl⁻. A low concentration of clodronate impaired vesicular ATP release from neurons, microglia, and immune cells. In vivo analyses revealed that clodronate is more effective than other therapeutic agents in attenuating neuropathic and inflammatory pain, as well as the accompanying inflammation, in wild-type but not VNUT−/− mice, without affecting basal nociception. These findings indicate that clodronate may represent a unique treatment strategy for chronic neuropathic and inflammatory pain via inhibition of vesicular ATP release.

Microglia are highly sensitive to even small changes in the brain environment, such as invasion of non-hazardous toxicants or the presymptomatic state of diseases. However, the physiological or pathophysiological consequences of their responses remain unknown. Here, we report that cultured microglia sense low concentrations of the neurotoxicant methylmercury (MeHg low ) and provide neuroprotection against MeHg, for which astrocytes are also required. When exposed to MeHg low , microglia exocytosed ATP via p38 MAPK- and vesicular nucleotide transporter (VNUT)-dependent mechanisms. Astrocytes responded to the microglia-derived ATP via P2Y 1 receptors and released interleukin-6 (IL-6), thereby protecting neurons against MeHg low . These neuroprotective actions were also observed in organotypic hippocampal slices from wild-type mice, but not in slices prepared from VNUT knockout or P2Y 1 receptor knockout mice. These findings suggest that microglia sense and respond to even non-hazardous toxicants such as MeHg low and change their phenotype into a neuroprotective one, for which astrocytic support is required.

Taste cells undergo constant turnover throughout life; however, the molecular mechanisms governing taste cell generation are not well understood. Using RNA-Seq, we systematically surveyed the transcriptome landscape of taste organoids at different stages of growth. Our data show the staged expression of a variety of genes and identify multiple signaling pathways underlying taste cell differentiation and taste stem/progenitor cell proliferation. For example, transcripts of taste receptors appear only or predominantly in late-stage organoids. Prior to that, transcription factors and other signaling elements are upregulated. RNA-Seq identified a number of well-characterized signaling pathways in taste organoid cultures, such as those involving Wnt, bone morphogenetic proteins (BMPs), Notch, and Hedgehog (Hh). By pharmacological manipulation, we demonstrate that Wnt, BMPs, Notch, and Hh signaling pathways are necessary for taste cell proliferation, differentiation and cell fate determination. The temporal expression profiles displayed by taste organoids may also lead to the identification of currently unknown transducer elements underlying sour, salt, and other taste qualities, given the staged expression of taste receptor genes and taste transduction elements in cultured organoids.

In the intestine, the innate immune system excludes harmful substances and invading microorganisms. Tuft cells are taste-like chemosensory cells found in the intestinal epithelium involved in the activation of group 2 innate lymphoid cells (ILC2). Although tuft cells in other tissues secrete the neurotransmitter acetylcholine (ACh), their function in the gut remains poorly understood. In this study, we investigated changes in the expression of genes and cell differentiation of the intestinal epithelium by stimulation with interleukin-4 (IL-4) or IL-13 in macaque intestinal organoids. Transcriptome analysis showed that tuft cell marker genes were highly expressed in the IL-4- and IL-13-treated groups compared with the control, and the gene expression of choline acetyltransferase (ChAT), a synthesis enzyme of ACh, was upregulated in IL-4- and IL-13-treated groups. ACh accumulation was observed in IL-4-induced organoids using high-performance liquid chromatography-mass spectrometry (HPLC/MS), and ACh strongly released granules from Paneth cells. This study is the first to demonstrate ACh upregulation by IL-4 induction in primates, suggesting that IL-4 plays a role in Paneth cell granule secretion via paracrine stimulation.

Adequate nutrition is required to maintain healthy skin integrity, and malnourished patients with poor protein diet often experience delayed wound healing. Understanding the cellular mechanisms of protein malnutrition will justify the importance of optimal protein diets in health and disease defense. Therefore in the present study, we examined the effects of changes in wound fluid composition and its function caused by protein malnutrition on wound healing. Rats were fed a control (CO; 20% protein) diet or a protein-free (PF) diet for 2 weeks; we then created full-thickness wounds on the dorsolateral skin. On day 5 after wounding, frozen sections of the wounds were created to investigate the state of granulation tissues, and wound fluid obtained from the rats was collected to examine variations in cytokine levels and its function. Wound closure was significantly delayed from day 4 until total wound closure in rats fed a PF diet. Thickness of granulation tissue, which is composed of mainly dermal fibroblasts, and Ki67 immunohistochemical staining were significantly decreased in rats fed PF diets. PF diets decreased insulin-like growth factor (IGF)-I, which promotes wound healing, and increased IGF-binding protein-1, which inhibits IGF-I bioavailability, in wound fluid. Wound fluid obtained from rats fed a PF diet suppressed dermal fibroblast proliferation. Furthermore, the wound fluid remarkably decreased the phosphorylation level of IGF-I receptor β (IGF-IR) and extracellular signal-regulated kinase (ERK)(1/2) in dermal fibroblasts. These results show that wound fluid of rats fed PF diets delays wound healing by inhibiting granulation tissue formation through the suppression of the IGF-1/ERK(1/2) signaling pathway.

Protein malnutrition is largely associated with a delay or failure of the healing process. However, the effect of dietary protein quality on wound healing is largely unknown. This study aimed to reveal the effect of dietary protein quality on wound healing and elucidate the regulatory mechanisms in a rat model of full-thickness cutaneous wounds. Rats were fed a normal diet for a week, and then they were divided into three groups that were fed the following diet for the experimental period: casein diet, gluten diet and gluten + lysine diet. The gluten diet significantly decreased body weight and wound healing compared with the casein diet, but this effect was reversed by supplementation with lysine. The numbers of leukocytes were significantly higher in the skin of the gluten group than those in the casein group. The wounded skin tissues of the gluten group showed lower amounts of collagen deposition compared with that in the casein group. Our results also showed that both matrix metalloproteinase (MMP) 2 activity and MMP14 mRNA levels were significantly increased in the skin of the gluten group, compared with the casein group. In summary, this study suggests low-quality protein diets have negative effects on wound healing via modulation of MMP2 activity in rats.