Toyama Prefectural Institute for Pharmaceutical Research

Type 2 diabetes mellitus (T2DM) and metabolic dysfunction-associated steatohepatitis (MASH) frequently coexist and are mechanistically linked via insulin resistance, lipotoxicity, and chronic inflammation. Cholic acid (CA), a primary bile acid (BA), modulates metabolic and immune pathways by influencing BA composition and the gut microbiota. In this study, we examined the role of dietary CA in the modulation of T2DM and MASH in Tsumura Suzuki obese diabetes (TSOD) mice, a model of spontaneous obesity and diabetes. Mice were fed a high-fat, high-cholesterol diet with or without CA (iHFC and CA(-) iHFC diets, respectively). CA supplementation significantly improved hyperglycemia and hyperinsulinemia, independent of adipose tissue inflammation. These metabolic benefits were associated with an increased intestinal abundance of Akkermansia muciniphila and elevated ileal expression of fibroblast growth factor 15 (FGF-15), suggesting activation of the FXR-FGF-15 axis. However, CA also exacerbated MASH, accompanied by increased hepatic inflammation, fibrosis, and accumulation of hepatotoxic BAs, including deoxycholic acid and taurodeoxycholic acid. The removal of CA mitigated these hepatic changes while abolishing glycemic improvement. Accordingly, CA can exert opposing effects in TSOD mice-ameliorating T2DM while worsening MASH-highlighting the need for caution when targeting BA pathways in metabolic diseases.

Paeonia lactiflora, the roots of which are used as a crude drug, is one of the most widely used and important medicinal plants. The long cultivation period and low proliferation rate of P. lactiflora makes it difficult to propagate large numbers of plants within a short period. We developed a bio-nursery system using plant tissue culture techniques to contribute to the supply of P. lactiflora seeds and seedlings in Japan. Here, we report on the improved tissue culture and acclimation conditions for a more stable and efficient bio-nursery system. We investigated the effect of culture conditions on shoot proliferation and the effect of calcium concentration during root induction and acclimation of cultured plantlets. The results demonstrated that the number of shoots increased under the 15/5°C diurnal temperature changing treatment [15°C, 12 h light (fluorescent light, 80-130 µmol m-2 s-1)/5°C, 12 h dark] compared to a constant temperature of 15°C. A higher calcium concentration (6 mM Ca2+) during root induction resulted in more vigorous growth after transplantation to the soil. In addition, it was found that planting in a closed greenhouse at a constant temperature of 20°C after cold treatment was suitable for acclimation of cultured plantlets. These findings are expected to contribute to the future seedling supply of P. lactiflora.

Intestinal bacteria play a critical role in the regulation of the host immune system and an imbalance in the intestinal bacterial composition induces various host diseases. Therefore, maintaining a balance in the intestinal bacterial composition is crucial for health. Immunoglobulin A (IgA), produced through T cell-dependent and T cell-independent (TI) pathways, is essential for host defense against pathogen invasion and maintaining the balance of intestinal symbiotic bacteria. Interleukin (IL)-5 is constitutively produced by Group 2 innate lymphoid cells (ILC2s) and plays a critical role in the survival and proliferation of B cells and eosinophils. Here, we show the role of IL-5-producing ILC2s in intestinal TI IgA production at steady state using T cell receptor α deficient mice. In this mouse model, ILC2s increased fecal TI IgA levels in a non-inflammatory state in an IL-5-dependent manner. The administration of recombinant IL-33 (rIL-33) increased the amount of TI IgA production, accompanied by an increase in the number of IL-5-producing ILC2s in the large intestine. In addition, rIL-33 treatment increased IL-5-dependent IgA+ cells in isolated lymphoid follicles, the site of TI IgA production. Furthermore, eosinophils recruited by ILC2s were required for the maximal production of IgA in the TI pathway. Moreover, IL-5 increased the frequency of TI IgA-binding intestinal bacteria and was involved in the maintenance of intestinal bacterial composition. These findings indicate that IL-5-producing ILC2s together with eosinophils contribute to TI IgA production. In addition to their role in TI IgA production, IL-5-producing ILC2s may contribute to the homeostasis of intestinal commensal bacteria.

X-linked adrenoleukodystrophy (X-ALD) is a severe inherited metabolic disease with cerebral inflammatory demyelination and abnormal accumulation of very long chain fatty acid (VLCFA) in tissues, especially the brain. At present, bone marrow transplantation (BMT) at an early stage of the disease is the only effective treatment for halting disease progression, but the underlying mechanism of the treatment has remained unclear. Here, we transplanted GFP-expressing wild-type (WT) or Abcd1-deficient (KO) bone marrow cells into recipient KO mice, which enabled tracking of the donor GFP+ cells in the recipient mice. Both the WT and KO donor cells were equally distributed throughout the brain parenchyma, and displayed an Iba1-positive, GFAP- and Olig2-negative phenotype, indicating that most of the donor cells were engrafted as microglia-like cells. They constituted approximately 40% of the Iba1-positive cells. Unexpectedly, no decrease of VLCFA in the cerebrum was observed when WT bone marrow cells were transplanted into KO mice. Taken together, murine study suggests that bone marrow-derived microglia-like cells engrafted in the cerebrum of X-ALD patients suppress disease progression without evidently reducing the amount of VLCFA in the cerebrum.

Nicotinamide adenine dinucleotide (NAD) is an important coenzyme that regulates various metabolic pathways, including glycolysis, β-oxidation, and oxidative phosphorylation. Additionally, NAD serves as a substrate for poly(ADP-ribose) polymerase (PARP), sirtuin, and NAD glycohydrolase, and it regulates DNA repair, gene expression, energy metabolism, and stress responses. Many studies have demonstrated that NAD metabolism is deeply involved in aging and aging-related diseases. Previously, we demonstrated that nicotinamide guanine dinucleotide (NGD) and nicotinamide hypoxanthine dinucleotide (NHD), which are analogs of NAD, are significantly increased in Nmnat3-overexpressing mice. However, there is insufficient knowledge about NGD and NHD in vivo. In the present study, we aimed to investigate the metabolism and biochemical properties of these NAD analogs. We demonstrated that endogenous NGD and NHD were found in various murine tissues, and their synthesis and degradation partially rely on Nmnat3 and CD38. We have also shown that NGD and NHD serve as coenzymes for alcohol dehydrogenase (ADH) in vitro, although their affinity is much lower than that of NAD. On the other hand, NGD and NHD cannot be used as substrates for SIRT1, SIRT3, and PARP1. These results reveal the basic metabolism of NGD and NHD and also highlight their biological function as coenzymes.

Obesity-associated activation of the renin-angiotensin-aldosterone system is implicated in the pathogenesis of insulin resistance; however, influences of mineralocorticoid receptor (MR) inhibition remain unclear. Therefore, we aimed to clarify the anti-inflammatory mechanisms of MR inhibition using eplerenone, a selective MR antagonist, in C57BL/6 mice fed a high-fat diet (HFD) for 12 weeks. Eplerenone prevented excessive body weight gain and fat accumulation, ameliorated glucose intolerance and insulin resistance and enhanced energy metabolism. In the epididymal white adipose tissue (eWAT), eplerenone prevented obesity-induced accumulation of F4/80+CD11c+CD206--M1-adipose tissue macrophage (ATM) and reduction of F4/80+CD11c-CD206+-M2-ATM. Interestingly, M1-macrophage exhibited lower expression levels of MR, compared with M2-macrophage, in the ATM of eWAT and in vitro-polarized bone marrow-derived macrophages (BMDM). Importantly, eplerenone and MR knockdown attenuated the increase in the expression levels of proIl1b, Il6 and Tnfa, in the eWAT and liver of HFD-fed mice and LPS-stimulated BMDM. Moreover, eplerenone suppressed IL1b secretion from eWAT of HFD-fed mice. To reveal the anti-inflammatory mechanism, we investigated the involvement of NLRP3-inflammasome activation, a key process of IL1b overproduction. Eplerenone suppressed the expression of the inflammasome components, Nlrp3 and Caspase1, in the eWAT and liver. Concerning the second triggering factors, ROS production and ATP- and nigericin-induced IL1b secretion were suppressed by eplerenone in the LPS-primed BMDM. These results indicate that eplerenone inhibited both the priming and triggering signals that promote NLRP3-inflammasome activation. Therefore, we consider MR to be a crucial target to prevent metabolic disorders by suppressing inflammasome-mediated chronic inflammation in the adipose tissue and liver under obese conditions.

This is a perspective based on the paper “Cloning of complementary DNA encoding T cell-replacing factor and identity with B cell growth factor II,” by Kinashi et al. (1). We have been interested in understanding the molecular basis of T–B cell cooperation for antibody formation. Although many investigators had described a number of different soluble factors that appeared to have biological relevance to T–B cell interactions, molecular basis of such active substances remained unknown for a long period of time. In this perspective, I will briefly summarize the history of the initial discovery of T cell-replacing factor/B cell growth factor II that appeared to be involved in B cell growth and differentiation, and outline the discovery and characterization of interleukin-5. Studies of interleukin-5 have provided strong evidence that a single cytokine exerts a variety of activities on diverse target cells.

NAD biosynthesis is of substantial interest because of its important roles in regulating various biological processes. Nicotinamide mononucleotide adenylyltransferase 3 (Nmnat3) is considered a mitochondria-localized NAD synthesis enzyme involved in de novo and salvage pathways. Although the biochemical properties of Nmnat3 are well documented, its physiological function in vivo remains unclear. In this study, we demonstrated that Nmnat3 was localized in the cytoplasm of mature erythrocytes and critically regulated their NAD pool. Deficiency of Nmnat3 in mice caused splenomegaly and hemolytic anemia, which was associated with the findings that Nmnat3-deficient erythrocytes had markedly lower ATP levels and shortened lifespans. However, the NAD level in other tissues were not apparently affected by the deficiency of Nmnat3. LC-MS/MS-based metabolomics revealed that the glycolysis pathway in Nmnat3-deficient erythrocytes was blocked at a glyceraldehyde 3-phosphate dehydrogenase (GAPDH) step because of the shortage of the coenzyme NAD. Stable isotope tracer analysis further demonstrated that deficiency of Nmnat3 resulted in glycolysis stall and a shift to the pentose phosphate pathway. Our findings indicate the critical roles of Nmnat3 in maintenance of the NAD pool in mature erythrocytes and the physiological impacts at its absence in mice.

The radioprotective 105 (RP105)/MD-1 complex is a member of the Toll-like receptor (TLR) family of proteins. We have previously reported that this complex cooperates with the essential lipopolysaccharide (LPS) receptor TLR4/MD-2 complex and plays a crucial role in LPS responses by B cells. Recent evidences suggest that TLRs can also recognize endogenous ligands and promote non-infectious chronic inflammation. For instance, TLR4/MD-2 can be ligated by adipose tissue-derived saturated free fatty acids (FAs) and induce adipose tissue inflammation and insulin resistance. Recently, we reported that RP105 knockout (KO) or MD-1 KO mice have less high-fat diet (HFD)-induced obesity, adipose tissue inflammation and insulin resistance than wild-type (WT) or TLR4 KO mice. As RP105/MD-1 is not involved in recognition of palmitic and stearic acids, which are endogenous ligands for TLR4/MD-2, we conclude that RP105/MD-1 is itself a key regulator of diet-induced chronic inflammation in adipose tissue, obesity and insulin resistance that appears to be independent of the TLR4-dependent pathway. In this mini-review, we will highlight the significance of the RP105/MD-1 complex in adipose tissue inflammation and discuss implications for human diseases.