Disruption of klotho gene causes an abnormal energy homeostasis in mice

klotho mice, which genetically lack klotho gene expression, are characterized with various systemic phenotypes resembling human aging, and also with growth retardation. Here we show that klotho mice have a barely detectable amount of the white adipose tissue but their brown adipose tissue (BAT) is comparably preserved. Glucose tolerance and insulin sensitivity in klotho mice are increased compared to those in wild-type mice as revealed by intraperitoneal glucose and insulin tolerance tests. Uncoupling protein-1 gene expression of BAT and body temperature in klotho mice are lower than those in wild-type mice, suggesting that klotho mice have less energy expenditure than wild-type mice. Histological examination suggests that klotho mice possess less energy storage than wild-type mice with respect to glycogen in the liver and lipid in BAT. All these changes of parameters for energy homeostasis in klotho mice are very similar to those reported under food-restricted conditions. However, the amount of food intake is not different between klotho and wild-type mice when normalized for body weight. The present study elucidates the importance of klotho gene expression for the maintenance of normal energy homeostasis.     

Disruption of growth hormone receptor gene causes diminished pancreatic islet size and increased insulin sensitivity in mice

Growth hormone, acting through its receptor (GHR), plays an important role in carbohydrate metabolism and in promoting postnatal growth. GHR gene-deficient (GHR(-/-)) mice exhibit severe growth retardation and proportionate dwarfism. To assess the physiological relevance of growth hormone actions, GHR(-/-) mice were used to investigate their phenotype in glucose metabolism and pancreatic islet function. Adult GHR(-/-) mice exhibited significant reductions in the levels of blood glucose and insulin, as well as insulin mRNA accumulation. Immunohistochemical analysis of pancreatic sections revealed normal distribution of the islets despite a significantly smaller size. The average size of the islets found in GHR(-/-) mice was only one-third of that in wild-type littermates. Total beta-cell mass was reduced 4.5-fold in GHR(-/-) mice, significantly more than their body size reduction. This reduction in pancreatic islet mass appears to be related to decreases in proliferation and cell growth. GHR(-/-) mice were different from the human Laron syndrome in serum insulin level, insulin responsiveness, and obesity. We conclude that growth hormone signaling is essential for maintaining pancreatic islet size, stimulating islet hormone production, and maintaining normal insulin sensitivity and glucose homeostasis.     

Heart extracellular matrix gene expression profile in the vitamin D receptor knockout mice

1,25-Dihydroxyvitamin D₃ [1,25D] deficiency and vitamin D receptor [VDR] genotypes are risk factors for several diseases and disorders including heart diseases. Extracellular matrix (ECM) remodeling mediated by matrix metalloproteinases [MMPs] contributes to progressive left ventricular remodeling, dilation, and heart failure. In the present study, we used high-density oligonucleotide microarray to examine gene expression profile in wild type [WT] and vitamin D receptor knockout mice (VDR KO) which was further validated by RT-PCR. Microarray analysis revealed tissue inhibitors of metalloproteinases [TIMP-1 and TIMP-3] were significantly under expressed in VDR KO mice as compared to WT mice which was further validated by RT-PCR. Zymography and RT-PCR showed that MMP-2 and MMP-9 were up regulated in VDR KO mice. In addition, cross-sectional diameter and longitudinal width of the VDR KO heart myofibrils showed highly significant cellular hypertrophy. Trichrome staining showed marked increase in fibrotic lesions in the VDR KO mice. Heart weight to body weight ratio showed 41% increase in VDR KO mice when compared to WT mice. This data supports a role for 1,25D in heart ECM metabolism and suggests that MMPs and TIMPs expression may be modulated by vitamin D.     

Vestibular dysfunction in vitamin D receptor mutant mice

The vitamin D endocrine system is essential for calcium and bone homeostasis. Vitamin D deficits are associated with muscle weakness and osteoporosis, whereas vitamin D supplementation may improve muscle function, body sway and frequency of falls, growth and mineral homeostasis of bones. The loss of muscle strength and mass, as well as deficits in bone formation, lead to poor balance. Poor balance is one of the main causes of falls, and may lead to dangerous injuries. Here we examine balance functions in vitamin D receptor deficient (VDR−/−) mice, an animal model of vitamin D-dependent rickets type II, and in 1α-hydroxylase deficient (1α-OHase−/−) mice, an animal model of pseudovitamin D-deficiency rickets. Recently developed methods (tilting box, rotating tube test), swim test, and modified accelerating rotarod protocol were used to examine whether the absence of functional VDR, or the lack of a key vitamin D-activating enzyme, could lead to mouse vestibular dysfunctions. Overall, VDR−/− mice, but not 1α-OHase−/− mice, showed shorter latency to fall from the rotarod, smaller fall angle in the tilting box test, and aberrant poor swimming. These data suggest that VDR deficiency in mice is associated with decreased balance function, and may be relevant to poorer balance/posture control in humans with low levels of vitamin D.     

Premature aging in vitamin D receptor mutant mice

Hypervitaminosis vitamin D₃ has been recently implicated in premature aging through the regulation of 1alpha hydroxylase expression by klotho and fibroblast growth factor-23 (Fgf-23). Here we examined whether the lack of hormonal function of vitamin D₃ in mice is linked to aging phenomena. For this, we used vitamin D₃ receptor (VDR) “Tokyo” knockout (KO) mice (fed with a special rescue diet) and analyzed their growth, skin and cerebellar morphology, as well as overall motor performance. We also studied the expression of aging-related genes, such as Fgf-23, nuclear factor kappaB (NF-kappaB), p53, insulin like growth factor 1 (IGF1) and IGF1 receptor (IGF1R), in liver, as well as klotho in liver, kidney and prostate tissues. Overall, VDR KO mice showed several aging related phenotypes, including poorer survival, early alopecia, thickened skin, enlarged sebaceous glands and development of epidermal cysts. There was no difference either in the structure of cerebellum or in the number of Purkinje cells. Unlike the wildtype controls, VDR KO mice lose their ability to swim after 6 months of age. Expression of all the genes was lower in old VDR KO mice, but only NF-kappaB, Fgf-23, p53 and IGF1R were significantly lower. Since the phenotype of aged VDR knockout mice is similar to mouse models with hypervitaminosis D₃, our study suggests that VDR genetic ablation promotes premature aging in mice, and that vitamin D₃ homeostasis regulates physiological aging.     

Disruption of the phosphatidylserine decarboxylase gene in mice causes embryonic lethality and mitochondrial defects

Most of the phosphatidylethanolamine (PE) in mammalian cells is synthesized by two pathways, the CDP-ethanolamine pathway and the phosphatidylserine (PS) decarboxylation pathway, the final steps of which operate at spatially distinct sites, the endoplasmic reticulum and mitochondria, respectively. We investigated the importance of the mitochondrial pathway for PE synthesis in mice by generating mice lacking PS decarboxylase activity. Disruption of Pisd in mice resulted in lethality between days 8 and 10 of embryonic development. Electron microscopy of Pisd-/- embryos revealed large numbers of aberrantly shaped mitochondria. In addition, fluorescence confocal microscopy of Pisd-/- embryonic fibroblasts showed fragmented mitochondria. PS decarboxylase activity and mRNA levels in Pisd+/- tissues were approximately one-half of those in wild-type mice. However, heterozygous mice appeared normal, exhibited normal vitality, and the phospholipid composition of livers, testes, brains, and of mitochondria isolated from livers, was the same as in wild-type littermates. The amount and activity of a key enzyme of the CDP-ethanolamine pathway for PE synthesis, CTP:phosphoethanolamine cytidylyltransferase, were increased by 35-40 and 100%, respectively, in tissues of Pisd+/- mice, as judged by immunoblotting; PE synthesis from [3H]ethanolamine was correspondingly increased in hepatocytes. We conclude that the CDP-ethanolamine pathway in mice cannot substitute for a lack of PS decarboxylase during development. Moreover, elimination of PE production in mitochondria causes fragmented, misshapen mitochondria, an abnormality that likely contributes to the embryonic lethality.     

Genetic analysis of lung function in inbred mice suggests vitamin D receptor as a candidate gene

Vitamin D receptor (VDR) polymorphisms are associated with an increased asthma incidence in human populations; however, observations in Vdr knockout mice are unclear. The aim of our study was to determine the influence of the genetic variation in Vdr among inbred strains on lung resistance (i.e., dynamic and airway resistance). In an intercross between the strains C57BL/6J (B6) and KK/HlJ (KK), we identified that a significant QTL for dynamic resistance on Chr X was interacting with a QTL on Chr 15. The Chr 15 QTL peak was located in close proximity to the Vdr locus. We further examined if phenotypes of several inbred strains with varying Vdr genotypes differed. Strains with a B6-like genotype on the Vdr locus had significantly lower airway resistance than strains with a KK-like genotype. Vdr knockout mice were examined for dynamic resistance and showed significantly higher resistance than mice with one (i.e., heterozygous) or both copies (i.e., wild-type) of the Vdr. In comparison to B6, the strain A/J is more resistant but carries the same genotype at the Vdr locus. Dietary vitamin D manipulation in the strain A/J did not rescue the high airway resistance phenotype. Finally, we observed that serum vitamin D does not correlate significantly with lung resistance parameters in a survey of 18 strains. Conclusively, Vdr contributes to the phenotypic variation of lung resistance in inbred mice but other molecules in the Vdr pathway and extended network [i.e., Chr X gene(s)] may contribute as well.     

Osteoblasts lacking the vitamin D receptor display enhanced osteogenic potential in vitro

1,25-dihydroxyvitamin D plays an important role in the regulation of osteoblast gene expression, regulating the expression of bone matrix proteins as well as that of Runx2, a key regulator of osteoblast differentiation. Studies in mice lacking the vitamin D receptor (VDR) have revealed that the actions of the VDR on the skeleton are not required in the setting of normal mineral ion homeostasis. Since paracrine and endocrine factors can compensate for gene defects in vivo, studies were performed to determine whether ablation of the VDR alters the program of osteoblast differentiation in vitro. Studies in primary calvarial cultures revealed that ablation of the VDR enhanced osteoblast differentiation. The cells from the VDR null mice exhibited an earlier onset and increased magnitude of alkaline phosphatase activity, as well as an earlier and sustained increase in mineralized matrix formation, demonstrating that this enhancement persists throughout the program of osteoblast differentiation. The expression of bone sialoprotein, which enhances mineralization, was also increased in the VDR null cultures. To determine whether the increase in osteoblast differentiation was associated with an increase in the number of osteogenic progenitors, the number of osteoblastic colony forming units (CFU-OB) was evaluated. There was a twofold increase in the number of CFU-OB in the cultures isolated from the VDR null mice. Furthermore, the VDR null CFU-OB demonstrated an earlier onset and higher magnitude of expression of alkaline phosphatase activity when compared to the CFU-OB from their wild-type control littermates. These studies demonstrate that the VDR attenuates osteoblast differentiation in vitro and suggest that other endocrine and paracrine factors modulate the effect of the VDR on osteoblast differentiation in vivo.     

雌激素改善维生素D受体基因敲除雌性小鼠的骨、钙代谢

以维生素 D 受体基因敲除雌性小鼠为模型,研究雌激素对骨、钙代谢的调节作用。外源性给予雌二醇一个月后,观察小鼠血钙水平的变化,同时测定小鼠骨密度,并利用胫骨非脱钙 von Kossa 染色观察钙化的骨小梁和未钙化的类骨质面积的变化。结果显示,外源性给予雌二醇一个月后,维生素 D 受体基因敲除小鼠的血钙水平,从(2.10±0.37) mmol/L 上升到(2.80±0.41) mmol/L (P<0.05); 骨密度从(0.037±0.006) g/cm2增高到(0.048±0.007) g/cm2,显著改善(P<0.05) ;钙化骨小梁面积显著增加,未钙化的类骨质面积显著缩小。结果提示,外源性雌二醇对骨、钙代谢具有非依赖于维生素 D 的正向调节作用。     

Disruption of FADS2 gene in mice impairs male reproduction and causes dermal and intestinal ulceration

Delta-6 desaturase (D6D) catalyzes the first step in the synthesis of highly unsaturated fatty acids (HUFA) such as arachidonic (AA), docosapentaenoic (DPAn-6), and docosahexaenoic (DHA) acids, as well as the last desaturation of DPAn-6 and DHA. We created D6D-null mice (−/−), which enabled us to study HUFA deficiency without depleting their precursors. In −/−, no in vivo AA synthesis was detected after administration of [U-¹³C]linoleic acid (LA), indicating absence of D6D isozyme. Unexpectedly, all of the −/− developed ulcerative dermatitis when fed a purified diet lacking D6D products but containing ample LA. The −/− also exhibited splenomegaly and ulceration in duodenum and ileocecal junction. Male −/− lacked normal spermatozoa with a severe impairment of spermiogenesis. Tissue HUFAs in −/− declined differentially: liver AA and DHA by 95%, and a smaller decrease in brain and testes. Dietary AA completely prevented dermatitis and intestinal ulcers in −/−. DPAn-6 was absent in −/− brain under AA supplementation, indicating absence of D6D isozyme for DPAn-6 synthesis from AA. This study demonstrated a distinct advantage of the D6D-null mice (−/−) to elucidate (1) AA function without complication of LA deprivation and (2) DHA function in the nervous system without AA depletion or DPAn-6 replacement seen in traditional models.—Stroud, C. K., T. Y. Nara, M. Roqueta-Rivera, E. C. Radlowski, P. Lawrence, Y. Zhang, B. H. Cho, M. Segre, R. A. Hess, J. T. Brenna, W. M. Haschek, and M. T. Nakamura. Disruption of FADS2 gene in mice impairs male reproduction and causes dermal and intestinal ulceration.     

Affinity labeling of the nuclear vitamin D receptor with nonsteroidal alkylating agents

Synthesis of an affinity alkylating non steroidal mimic of 1alpha,25-dihydroxyvitamin D(3) and its radiolabeled counterpart is presented. We also report the affinity labeling of the VDR-ligand binding domain (VDR-LBD) with this analogue.     

Aberrant nest building and prolactin secretion in vitamin D receptor mutant mice

1,25(OH)₂D₃, the hormonal form of vitamin D, is a neuroactive seco-steroid hormone with multiple functions in the brain. Most of these effects are mediated through the nuclear vitamin D receptor (VDR), widely distributed in the central nervous system. Our earlier studies showed that mutant mice lacking functional VDR have specific behavioural abnormalities, including anxiety and aberrant maternal behaviour, which may be hormonally regulated. Here we describe impaired nest building behaviour in VDR mutant mice. Since prolactin plays a key role in the regulation of nest building in both sexes, we also examine whether VDR mutant mice have altered prolactin levels. Overall, serum prolactin levels were increased in VDR mutant mice, accompanied by marked impairments in their nest building activity. In contrast, there were no differences in prolactin mRNA expression levels between wildtype control mice and VDR mutant mice. Collectively, these data suggest that partial genetic ablation of VDR affects prolactin system in mice, and that altered serum prolactin levels in VDR mutants may underlie some of their behavioural abnormalities, such as impaired nest building.     

Vitamin D receptor gene variants and serum vitamin D in childhood autism spectrum disorder

Molecular Biology Reports - This study aimed to examine the correlation between polymorphisms in vitamin D receptor (VDR) gene and serum vitamin D, and to determine their role in predicting...