Methionine restriction improves renal insulin signalling in aged kidneys.

Dietary methionine restriction (MR) leads to loss of adiposity, improved insulin sensitivity and lifespan extension. The possibility that dietary MR can protect the kidney from age-associated deterioration has not been addressed. Aged (10-month old) male and female mice were placed on a MR (0.172% methionine) or control diet (0.86% methionine) for 8-weeks and blood glucose, renal insulin signalling, and gene expression were assessed. Methionine restriction lead to decreased blood glucose levels compared to control-fed mice, and enhanced insulin-stimulated phosphorylation of PKB/Akt and S6 in kidneys, indicative of improved glucose homeostasis. Increased expression of lipogenic genes and downregulation of PEPCK were observed, suggesting that kidneys from MR-fed animals are more insulin sensitive. Interestingly, renal gene expression of the mitochondrial uncoupling protein UCP1 was upregulated in MR-fed animals, as were the anti-ageing and renoprotective genes Sirt1, FGF21, klotho, and β-klotho. This was associated with alterations in renal histology trending towards reduced frequency of proximal tubule intersections containing vacuoles in mice that had been on dietary MR for 190days compared to control-fed mice, which exhibited a pre-diabetic status. Our results indicate that dietary MR may offer therapeutic potential in ameliorating the renal functional decline related to ageing and other disorders associated with metabolic dysfunction by enhancing renal insulin sensitivity and renoprotective gene expression

A Cellular Pathway Involved in Clara Cell to Alveolar Type II Cell Differentiation after Severe Lung Injury

Regeneration of alveolar epithelia following severe pulmonary damage is critical for lung function. We and others have previously shown that Scgb1a1-expressing cells, most likely Clara cells, can give rise to newly generated alveolar type 2 cells (AT2s) in response to severe lung damage induced by either influenza virus infection or bleomycin treatment. In this study, we have investigated cellular pathway underlying the Clara cell to AT2 differentiation. We show that the initial intermediates are bronchiolar epithelial cells that exhibit Clara cell morphology and express Clara cell marker, Scgb1a1, as well as the AT2 cell marker, pro-surfactant protein C (pro-SPC). These cells, referred to as pro-SPC[superscript +] bronchiolar epithelial cells (or SBECs), gradually lose Scgb1a1 expression and give rise to pro-SPC[superscript +] cells in the ring structures in the damaged parenchyma, which appear to differentiate into AT2s via a process sharing some features with that observed during alveolar epithelial development in the embryonic lung. These findings suggest that SBECs are intermediates of Clara cell to AT2 differentiation during the repair of alveolar epithelia following severe pulmonary injury.Singapore-MIT Alliance for Research and Technology Center. Infectious Disease Research Grou

Extensions of MADM (Mosaic Analysis with Double Markers) in Mice

Mosaic Analysis with Double Markers (MADM) is a method for generating genetically mosaic mice, in which sibling mutant and wild-type cells are labeled with different fluorescent markers. It is a powerful tool that enables analysis of gene function at the single cell level in vivo. It requires transgenic cassettes to be located between the centromere and the mutation in the gene of interest on the same chromosome. Here we compare procedures for introduction of MADM cassettes into new loci in the mouse genome, and describe new approaches for expanding the utility of MADM. We show that: 1) Targeted homologous recombination outperforms random transgenesis in generation of reliably expressed MADM cassettes, 2) MADM cassettes in new genomic loci need to be validated for biallelic and ubiquitous expression, 3) Recombination between MADM cassettes on different chromosomes can be used to study reciprocal chromosomal deletions/duplications, and 4) MADM can be modified to permit transgene expression by combining it with a binary expression system. The advances described in this study expand current, and enable new and more versatile applications of MADM

In vivo imaging of systemic transport and elimination of xenobiotics and endogenous molecules in mice

We describe a two-photon microscopy-based method to evaluate the in vivo systemic transport of compounds. This method comprises imaging of the intact liver, kidney and intestine, the main organs responsible for uptake and elimination of xenobiotics and endogenous molecules. The image quality of the acquired movies was sufficient to distinguish subcellular structures like organelles and vesicles. Quantification of the movement of fluorescent dextran and fluorescent cholic acid derivatives in different organs and their sub-compartments over time revealed significant dynamic differences. Calculated half-lives were similar in the capillaries of all investigated organs but differed in the specific sub-compartments, such as parenchymal cells and bile canaliculi of the liver, glomeruli, proximal and distal tubules of the kidney and lymph vessels (lacteals) of the small intestine. Moreover, tools to image immune cells, which can influence transport processes in inflamed tissues, are described. This powerful approach provides new possibilities for the analysis of compound transport in multiple organs and can support physiologically based pharmacokinetic modeling, in order to obtain more precise predictions at the whole body scale

The Proteolipid Protein Promoter Drives Expression outside of the Oligodendrocyte Lineage during Embryonic and Early Postnatal Development

The proteolipid protein (Plp) gene promoter is responsible for driving expression of one of the major components of myelin – PLP and its splice variant DM-20. Both products are classically thought to express predominantly in oligodendrocytes. However, accumulating evidence suggests Plp expression is more widespread than previously thought. In an attempt to create a mouse model for inducing oligodendrocyte-specific gene deletions, we have generated transgenic mice expressing a Cre recombinase cDNA under control of the mouse Plp promoter. We demonstrate Plp promoter driven Cre expression is restricted predominantly to mature oligodendrocytes of the central nervous system (CNS) at postnatal day 28. However, crosses into the Rosa26LacZ and mT/mG reporter mouse lines reveal robust and widespread Cre activity in neuronal tissues at E15.5 and E10.5 that is not strictly oligodendrocyte lineage specific. By P28, all CNS tissues examined displayed high levels of reporter gene expression well outside of defined white matter zones. Importantly, our study reinforces the emerging idea that Plp promoter activity is not restricted to the myelinating cell lineage, but rather, has widespread activity both during embryonic and early postnatal development in the CNS. Specificity of the promoter to the oligodendrocyte cell lineage, as shown through the use of a tamoxifen inducible Plp-CreERt line, occurs only at later postnatal stages. Understanding the temporal shift in Plp driven expression is of consequence when designing experimental models to study oligodendrocyte biology

Glucocorticoid receptor in astrocytes regulates midbrain dopamine neurodegeneration through connexin hemichannel activity

The precise contribution of astrocytes in neuroinflammatory process occurring in Parkinson's disease (PD) is not well characterized. In this study, using GR(Cx30CreERT2) mice that are conditionally inactivated for glucocorticoid receptor (GR) in astrocytes, we have examined the actions of astrocytic GR during dopamine neuron (DN) degeneration triggered by the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The results show significantly augmented DN loss in GR(Cx30CreERT2) mutant mice in substantia nigra (SN) compared to controls. Hypertrophy of microglia but not of astrocytes was greatly enhanced in SN of these astrocytic GR mutants intoxicated with MPTP, indicating heightened microglial reactivity compared to similarly-treated control mice. In the SN of GR astrocyte mutants, specific inflammation-associated transcripts ICAM-1, TNF-alpha and Il-1 beta as well as TNF-alpha protein levels were significantly elevated after MPTP neurotoxicity compared to controls. Interestingly, this paralleled increased connexin hemichannel activity and elevated intracellular calcium levels in astrocytes examined in acute midbrain slices from control and mutant mice treated with MPP+. The increased connexin-43 hemichannel activity was found in vivo in MPTP-intoxicated mice. Importantly, treatment of MPTP-injected GR(Cx30CreERT2) mutant mice with TAT-Gap19 peptide, a specific connexin-43 hemichannel blocker, reverted both DN loss and microglial activation; in wild-type mice there was partial but significant survival effect. In the SN of postmortem PD patients, a significant decrease in the number of astrocytes expressing nuclear GR was observed, suggesting the participation of astrocytic GR deregulation of inflammatory process in PD. Overall, these data provide mechanistic insights into GR-modulated processes in vivo, specifically in astrocytes, that contribute to a pro-inflammatory state and dopamine neurodegeneration in PD pathology

Humanin, a Cytoprotective Peptide, Is Expressed in Carotid Artherosclerotic Plaques in Humans

The mechanism of atherosclerotic plaque progression leading to instability, rupture, and ischemic manifestation involves oxidative stress and apoptosis. Humanin (HN) is a newly emerging endogenously expressed cytoprotective peptide. Our goal was to determine the presence and localization of HN in carotid atherosclerotic plaques.Plaque specimens from 34 patients undergoing carotid endarterectomy were classified according to symptomatic history. Immunostaining combined with digital microscopy revealed greater expression of HN in the unstable plaques of symptomatic compared to asymptomatic patients (29.42±2.05 vs. 14.14±2.13% of plaque area, p<0.0001). These data were further confirmed by immunoblot (density of HN/β-actin standard symptomatic vs. asymptomatic 1.32±0.14 vs. 0.79±0.11, p<0.01). TUNEL staining revealed a higher proportion of apoptotic nuclei in the plaques of symptomatic patients compared to asymptomatic (68.25±3.61 vs. 33.46±4.46% of nuclei, p<0.01). Double immunofluorescence labeling revealed co-localization of HN with macrophages (both M1 and M2 polarization), smooth muscle cells, fibroblasts, and dendritic cells as well as with inflammatory markers MMP2 and MMP9.The study demonstrates a higher expression of HN in unstable carotid plaques that is localized to multiple cell types within the plaque. These data support the involvement of HN in atherosclerosis, possibly as an endogenous response to the inflammatory and apoptotic processes within the atheromatous plaque

Attenuation of Age-Related Metabolic Dysfunction in Mice With a Targeted Disruption of the Cβ Subunit of Protein Kinase A

The cyclic adenosine monophosphate–dependent protein kinase A (PKA) pathway helps regulate both cell growth and division, and triglyceride storage and metabolism in response to nutrient status. Studies in yeast show that disruption of this pathway promotes longevity in a manner similar to caloric restriction. Because PKA is highly conserved, it can be studied in mammalian systems. This report describes the metabolic phenotype of mice lacking the PKA catalytic subunit Cβ. We confirmed that Cβ has high levels of expression in the brain but also showed moderate levels in liver. Cβ-null animals had reduced basal PKA activity while appearing overtly normal when fed standard rodent chow. However, the absence of Cβ protected mice from diet-induced obesity, steatosis, dyslipoproteinemia, and insulin resistance, without any differences in caloric intake or locomotor activity. These findings have relevant pharmacological implications because aging in mammals is characterized by metabolic decline associated with obesity, altered body fat distribution, and insulin resistance

Notch Lineages and Activity in Intestinal Stem Cells Determined by a New Set of Knock-In Mice

The conserved role of Notch signaling in controlling intestinal cell fate specification and homeostasis has been extensively studied. Nevertheless, the precise identity of the cells in which Notch signaling is active and the role of different Notch receptor paralogues in the intestine remain ambiguous, due to the lack of reliable tools to investigate Notch expression and function in vivo. We generated a new series of transgenic mice that allowed us, by lineage analysis, to formally prove that Notch1 and Notch2 are specifically expressed in crypt stem cells. In addition, a novel Notch reporter mouse, Hes1-EmGFPSAT, demonstrated exclusive Notch activity in crypt stem cells and absorptive progenitors. This roster of knock-in and reporter mice represents a valuable resource to functionally explore the Notch pathway in vivo in virtually all tissues

Accelerated turnover of taste bud cells in mice deficient for the cyclin-dependent kinase inhibitor p27Kip1

Background: Mammalian taste buds contain several specialized cell types that coordinately respond to tastants and communicate with sensory nerves. While it has long been appreciated that these cells undergo continual turnover, little is known concerning how adequate numbers of cells are generated and maintained. The cyclin-dependent kinase inhibitor p27Kip1 has been shown to influence cell number in several developing tissues, by coordinating cell cycle exit during cell differentiation. Here, we investigated its involvement in the control of taste cell replacement by examining adult mice with targeted ablation of the p27Kip1 gene.Results: Histological and morphometric analyses of fungiform and circumvallate taste buds reveal no structural differences between wild-type and p27Kip1-null mice. However, when examined in functional assays, mutants show substantial proliferative changes. In BrdU incorporation experiments, more S-phase-labeled precursors appear within circumvallate taste buds at 1 day post-injection, the earliest time point examined. After 1 week, twice as many labeled intragemmal cells are present, but numbers return to wild-type levels by 2 weeks. Mutant taste buds also contain more TUNEL-labeled cells and 50% more apoptotic bodies than wild-type controls. In normal mice, p27 Kip1 is evident in a subset of receptor and presynaptic taste cells beginning about 3 days post-injection, correlating with the onset of taste cell maturation. Loss of gene function, however, does not alter the proportions of distinct immunohistochemically-identified cell types.Conclusions: p27Kip1 participates in taste cell replacement by regulating the number of precursor cells available for entry into taste buds. This is consistent with a role for the protein in timing cell cycle withdrawal in progenitor cells. The equivalence of mutant and wild-type taste buds with regard to cell number, cell types and general structure contrasts with the hyperplasia and tissue disruption seen in certain developing p27Kip1-null sensory organs, and may reflect a compensatory capability inherent in the regenerative taste system