Glucokinase Variant Proteins Are Resistant to Fasting-Induced Uridine Diphosphate Glucose-Dependent Degradation in Maturity-Onset Diabetes of the Young Type 2 Patients.

We previously reported that glucokinase undergoes ubiquitination and subsequent degradation, a process mediated by cereblon, particularly in the presence of uridine diphosphate glucose (UDP-glucose). In this context, we hereby present evidence showcasing the resilience of variant glucokinase proteins of maturity-onset diabetes of the young type 2 (MODY2) against degradation and, concomitantly, their influence on insulin secretion, both in cell lines and in the afflicted MODY2 patient. Hence, glucose-1-phodphate promotes UDP-glucose production by UDP-glucose pyrophosphorylase 2; consequently, UDP-glucose-dependent glucokinase degradation may occur during fasting. Next, we analyzed glucokinase variant proteins from MODY2 or persistent hyperinsulinemic hypoglycemia in infancy (PHHI). Among the eleven MODY2 glucokinase-mutated proteins tested, those with a lower glucose-binding affinity exhibited resistance to UDP-glucose-dependent degradation. Conversely, the glucokinaseA456V-mutated protein from PHHI had a higher glucose affinity and was sensitive to UDP-glucose-dependent degradation. Furthermore, in vitro studies involving UDP-glucose-dependent glucokinase variant proteins and insulin secretion during fasting in Japanese MODY2 patients revealed a strong correlation and a higher coefficient of determination. This suggests that UDP-glucose-dependent glucokinase degradation plays a significant role in the pathogenesis of glucose-homeostasis-related hereditary diseases, such as MODY2 and PHHI.

Cellular senescence induction leads to progressive cell death via the INK4a-RB pathway in naked mole-rats.

Naked mole-rats (NMRs) have exceptional longevity and are resistant to age-related physiological decline and diseases. Given the role of cellular senescence in aging, we postulated that NMRs possess unidentified species-specific mechanisms to prevent senescent cell accumulation. Here, we show that upon induction of cellular senescence, NMR fibroblasts underwent delayed and progressive cell death that required activation of the INK4a-retinoblastoma protein (RB) pathway (termed "INK4a-RB cell death"), a phenomenon not observed in mouse fibroblasts. Naked mole-rat fibroblasts uniquely accumulated serotonin and were inherently vulnerable to hydrogen peroxide (H2 O2 ). After activation of the INK4a-RB pathway, NMR fibroblasts increased monoamine oxidase levels, leading to serotonin oxidization and H2 O2 production, which resulted in increased intracellular oxidative damage and cell death activation. In the NMR lung, induction of cellular senescence caused delayed, progressive cell death mediated by monoamine oxidase activation, thereby preventing senescent cell accumulation, consistent with in vitro results. The present findings indicate that INK4a-RB cell death likely functions as a natural senolytic mechanism in NMRs, providing an evolutionary rationale for senescent cell removal as a strategy to resist aging.

Resistance to chemical carcinogenesis induction via a dampened inflammatory response in naked mole-rats.

Naked mole-rats (NMRs) have a very low spontaneous carcinogenesis rate, which has prompted studies on the responsible mechanisms to provide clues for human cancer prevention. However, it remains unknown whether and how NMR tissues respond to experimental carcinogenesis induction. Here, we show that NMRs exhibit extraordinary resistance against potent chemical carcinogenesis induction through a dampened inflammatory response. Although carcinogenic insults damaged skin cells of both NMRs and mice, NMR skin showed markedly lower immune cell infiltration. NMRs harbour loss-of-function mutations in RIPK3 and MLKL genes, which are essential for necroptosis, a type of necrotic cell death that activates strong inflammation. In mice, disruption of Ripk3 reduced immune cell infiltration and delayed carcinogenesis. Therefore, necroptosis deficiency may serve as a cancer resistance mechanism via attenuating the inflammatory response in NMRs. Our study sheds light on the importance of a dampened inflammatory response as a non-cell-autonomous cancer resistance mechanism in NMRs.

Isolation and characterization of neural stem/progenitor cells in the subventricular zone of the naked mole-rat brain.

BACKGROUND: The naked mole-rat (NMR) is the longest-lived rodent with a maximum lifespan of more than 37 years and shows a negligible senescence phenotype, suggesting that tissue stem cells of NMRs are highly capable of maintaining homeostasis. However, the properties of NMR tissue stem cells, including neural stem cells (NSCs), are largely unclear. METHODS: Neural stem/progenitor cells (NS/PCs) were isolated from the subventricular zone of the neonate NMR brain (NMR-NS/PCs) and cultured in neurosphere and adherent culture conditions. Expression of NSC markers and markers of neurons, astrocytes, and oligodendrocytes was analyzed by immunocytochemistry. In adherent culture conditions, the proliferation rate and cell cycle of NMR-NS/PCs were assessed and compared with those of NS/PCs from mice (mouse-NS/PCs). The DNA damage response to γ-irradiation was analyzed by immunocytochemistry and reverse transcription-quantitative PCR. RESULTS: NMR-NS/PCs expressed several NSC markers and differentiated into neurons, astrocytes, and oligodendrocytes. NMR-NS/PCs proliferated markedly slower than mouse-NS/PCs, and a higher percentage of NMR-NS/PCs than mouse-NS/PCs was in G0/G1 phase. Notably, upon γ-irradiation, NMR-NS/PCs exhibited a faster initiation of the DNA damage response and were less prone to dying than mouse-NS/PCs. CONCLUSIONS: NMR-NS/PCs were successfully isolated and cultured. The slow proliferation of NMR-NS/PCs and their resistance to DNA damage may help to prevent stem cell exhaustion in the brain during the long lifespan of NMRs. Our findings provide novel insights into the mechanism underlying delayed aging of NMRs. Further analysis of NMR tissue stem cells may lead to the development of new strategies that can prevent aging in humans.

Induced Pluripotent Stem Cells from Cancer-Resistant Naked Mole-Rats.

Stem cells play essential roles in the development and tissue homeostasis of animals and are closely associated with carcinogenesis and aging. Also, the somatic cell reprogramming process to induced pluripotent stem (iPS) cells shares several characteristics with carcinogenesis. In this chapter, we focus on iPS cells and the reprogramming process of somatic cells in the naked mole-rat (NMR), the longest-living rodent with remarkable cancer resistance capabilities. NMR somatic cells show resistance to reprogramming induction, and generated NMR-iPS cells have a unique tumor-resistant phenotype. This phenotype is regulated by expressional activation of the tumor suppressor ARF gene and loss-of-function mutation in oncogene ERAS. Notably, it was also found that NMR somatic cells undergo senescence when ARF is suppressed during reprogramming, which would contribute to the resistance to both reprogramming and cancer in NMR somatic cells. Further studies on reprogramming resistance in NMR somatic cells and their concomitant tumor resistance in NMR-iPS cells would contribute to a better understanding of both cancer resistance and delayed aging in NMRs. In addition, NMR-iPS cells can be used as a new and important cell source for advancing research concerning several extraordinary physiological characteristics of NMR. Furthermore, study of NMR-iPS cells could lead to the development of safer regenerative therapies in the future.

Binding activity of Valeriana fauriei root extract on GABAA receptor flunitrazepam sites and distribution of its active ingredients in the brain of mice - A comparison with that of V. officinalis root.

ETHNOPHARMACOLOGICAL RELEVANCE: Valeriana fauriei root (VF) is a crude drug registered in the Japanese Pharmacopeia 17th Edition and a known substitute for V. officinalis (VO). Although VO has been pharmacologically evaluated for its sedative effects and mechanism of action, data regarding VF remain scarce. AIM OF THE STUDY: We compared the binding affinity of VF and VO extracts, as well as examined the active ingredients in the VF extract, on flunitrazepam sites of γ-aminobutyric acid receptor type A (GABAA receptor). Furthermore, we confirmed whether these active ingredients were distributed in the brain of mice orally administered the VF extract. MATERIALS AND METHODS: We prepared the assay system to evaluate the binding activity of flunitrazepam sites of GABAA receptor using a 96-well plate and assessed the activities of VF and VO extracts. We then analyzed their constituents using HPLC with principal component analysis (PCA) and evaluated active ingredients correlated with their activities. The distribution of active ingredients in the plasma and brain of mice orally administered the VF extract prepared with different emulsifiers were analyzed by LC-MS/MS. RESULTS: The ethanol extract of VF exhibited significantly higher activity on flunitrazepam sites of GABAA receptor than VO. For the VF extract, kessyl glycol diacetate (KGD) was markedly associated with the binding activities; however, active ingredients included KGD, kessyl glycol 8-acetate (KG8), α-kessyl acetate (α-KA), and coniferyl isovalerate (CI). For VO, valerenic acid and five other compounds were associated with the binding affinity on flunitrazepam sites of GABAA receptor. On emulsifying the VF extract with a fat-soluble glycerin fatty acid ester, the plasma and brain distributions of KGD tended to be higher, those of KG8 were significantly more than 10-times higher, and those of α-KA was lower than those of the VF extract emulsified with water-soluble gum arabic, after oral administration in mice. CONCLUSIONS: Based on the binding activity on flunitrazepam sites of GABAA receptor and brain distribution, KGD, KG8, and α-KA can be considered active ingredients of VF. The addition of a fat-soluble emulsifier promoted the absorption of KGD, the main active ingredient, and KGD was metabolized to KG8 in the body. The present results suggest a possible mechanism underlying the sedative effect for VF, and these three compounds can be used as marker compounds to evaluate the quality of VF products.

Characterization of brown adipose tissue thermogenesis in the naked mole-rat (Heterocephalus glaber), a heterothermic mammal.

The naked mole-rat (NMR) is a heterothermic mammal that forms eusocial colonies consisting of one reproductive female (queen), several reproductive males, and subordinates. Despite their heterothermy, NMRs possess brown adipose tissue (BAT), which generally induces thermogenesis in cold and some non-cold environments. Previous studies suggest that NMR-BAT induces thermogenesis by cold exposure. However, detailed NMR-BAT characteristics and whether NMR-BAT thermogenesis occurs in non-cold environments are unknown. Here, we show beta-3 adrenergic receptor (ADRB3)-dependent thermogenic potential of NMR-BAT, which contributes to thermogenesis in the isolated queen in non-cold environments (30 °C). NMR-BAT expressed several brown adipocyte marker genes and showed noradrenaline-dependent thermogenic activity in vitro and in vivo. Although our ADRB3 inhibition experiments revealed that NMR-BAT thermogenesis slightly delays the decrease in body temperature in a cold environment (20 °C), it was insufficient to prevent the decrease in the body temperatures. Even at 30 °C, NMRs are known to prevent the decrease of and maintain their body temperature by heat-sharing behaviors within the colony. However, isolated NMRs maintained their body temperature at the same level as when they are in the colony. Interestingly, we found that queens, but not subordinates, induce BAT thermogenesis in this condition. Our research provides novel insights into NMR thermoregulation.

Tumour resistance in induced pluripotent stem cells derived from naked mole-rats.

The naked mole-rat (NMR, Heterocephalus glaber), which is the longest-lived rodent species, exhibits extraordinary resistance to cancer. Here we report that NMR somatic cells exhibit a unique tumour-suppressor response to reprogramming induction. In this study, we generate NMR-induced pluripotent stem cells (NMR-iPSCs) and find that NMR-iPSCs do not exhibit teratoma-forming tumorigenicity due to the species-specific activation of tumour-suppressor alternative reading frame (ARF) and a disruption mutation of the oncogene ES cell-expressed Ras (ERAS). The forced expression of Arf in mouse iPSCs markedly reduces tumorigenicity. Furthermore, we identify an NMR-specific tumour-suppression phenotype-ARF suppression-induced senescence (ASIS)-that may protect iPSCs and somatic cells from ARF suppression and, as a consequence, tumorigenicity. Thus, NMR-specific ARF regulation and the disruption of ERAS regulate tumour resistance in NMR-iPSCs. Our findings obtained from studies of NMR-iPSCs provide new insight into the mechanisms of tumorigenicity in iPSCs and cancer resistance in the NMR.