Malat1 deficiency prevents hypoxia-induced lung dysfunction by protecting the access to alveoli.

Hypoxia is common in lung diseases and a potent stimulator of the long non-coding RNA Metastasis-Associated Lung Adenocarcinoma Transcript 1 (MALAT1). Herein, we investigated the impact of Malat1 on hypoxia-induced lung dysfunction in mice. Malat1-deficient mice and their wild-type littermates were tested after 8 days of normoxia or hypoxia (10% oxygen). Hypoxia decreased elastance of the lung by increasing lung volume and caused in vivo hyperresponsiveness to methacholine without altering the contraction of airway smooth muscle. Malat1 deficiency also modestly decreased lung elastance but only when tested at low lung volumes and without altering lung volume and airway smooth muscle contraction. The in vivo responsiveness to methacholine was also attenuated by Malat1 deficiency, at least when elastance, a readout sensitive to small airway closure, was used to assess the response. More impressively, in vivo hyperresponsiveness to methacholine caused by hypoxia was virtually absent in Malat1-deficient mice, especially when hysteresivity, a readout sensitive to small airway narrowing heterogeneity, was used to assess the response. Malat1 deficiency also increased the coefficient of oxygen extraction and decreased ventilation in conscious mice, suggesting improvements in gas exchange and in clinical signs of respiratory distress during natural breathing. Combined with a lower elastance at low lung volumes at baseline, as well as a decreased propensity for small airway closure and narrowing heterogeneity during a methacholine challenge, these findings represent compelling evidence suggesting that the lack of Malat1 protects the access to alveoli for air entering the lung.

T cells accumulate in non-diabetic islets during ageing.

BACKGROUND: The resident immune population of pancreatic islets has roles in islet development, beta cell physiology, and the pathology of diabetes. These roles have largely been attributed to islet macrophages, comprising 90% of islet immune cells (in the absence of islet autoimmunity), and, in the case of type 1 diabetes, to infiltrating autoreactive T cells. In adipose, tissue-resident and recruited T and B cells have been implicated in the development of insulin resistance during diet-induced obesity and ageing, but whether this is paralleled in the pancreatic islets is not known. Here, we investigated the non-macrophage component of resident islet immune cells in islets isolated from C57BL/6 J male mice during ageing (3 to 24 months of age) and following similar weight gain achieved by 12 weeks of 60% high fat diet. Immune cells were also examined by flow cytometry in cadaveric non-diabetic human islets. RESULTS: Immune cells comprised 2.7 ± 1.3% of total islet cells in non-diabetic mouse islets, and 2.3 ± 1.7% of total islet cells in non-diabetic human islets. In 3-month old mice on standard diet, B and T cells each comprised approximately 2-4% of the total islet immune cell compartment, and approximately 0.1% of total islet cells. A similar amount of T cells were present in non-diabetic human islets. The majority of islet T cells expressed the αß T cell receptor, and were comprised of CD8-positive, CD4-positive, and regulatory T cells, with a minor population of γδ T cells. Interestingly, the number of islet T cells increased linearly (R2 = 0.9902) with age from 0.10 ± 0.05% (3 months) to 0.38 ± 0.11% (24 months) of islet cells. This increase was uncoupled from body weight, and was not phenocopied by a degree similar weight gain induced by high fat diet in mice. CONCLUSIONS: This study reveals that T cells are a part of the normal islet immune population in mouse and human islets, and accumulate in islets during ageing in a body weight-independent manner. Though comprising only a small subset of the immune cells within islets, islet T cells may play a role in the physiology of islet ageing.

Loss of Malat1 does not modify age- or diet-induced adipose tissue accretion and insulin resistance in mice.

Several studies have suggested that signals emerging from white adipose tissue can contribute to the control of longevity. In turn, aging is associated with perturbed regulation and partitioning of fat depots and insulin resistance. However, the exact mechanisms involved in these relationships remain undetermined. Using RAP-PCR on adipose tissue of young and old male mice coupled with qPCR validation, we have uncovered the long non-coding RNA Malat1 as a gene robustly downregulated in visceral white adipose tissue (vWAT) during normal aging in male mice and men. Reductions in Malat1 expression in subcutaneous WAT (scWAT) were also observed in genetic (ob and db) as well as diet-induced models of obesity. Based on these findings, Malat1+/+ and Malat1-/- mouse littermates were thus probed to detect whether loss of Malat1 would impact age or diet-induced gain in fat mass and development of glucose intolerance. Contrary to this hypothesis, male and female Malat1-deficient mice gained as much weight, and developed insulin resistance to a similar extent as their Malat1+/+ littermates when studied up to eight months old on regular chow or a high-fat, high-sucrose diet. Moreover, we observed no marked difference in oxygen consumption, food intake, or lipid profiles between Malat1+/+ and Malat1-/- mice. Therefore, we conclude that the overall metabolic impact of the absence of Malat1 on adipose tissue accretion and glucose intolerance is either physiologically not relevant upon aging and obesity, or that it is masked by as yet unknown compensatory mechanisms.

Loss of OcaB Prevents Age-Induced Fat Accretion and Insulin Resistance by Altering B-Lymphocyte Transition and Promoting Energy Expenditure.

The current demographic shift toward an aging population has led to a robust increase in the prevalence of age-associated metabolic disorders. Recent studies have demonstrated that the etiology of obesity-related insulin resistance that develops with aging differs from that induced by high-calorie diets. Whereas the role of adaptive immunity in changes in energy metabolism driven by nutritional challenges has recently gained attention, its impact on aging remains mostly unknown. Here we found that the number of follicular B2 lymphocytes and expression of the B-cell-specific transcriptional coactivator OcaB increase with age in spleen and in intra-abdominal epididymal white adipose tissue (eWAT), concomitantly with higher circulating levels of IgG and impaired glucose homeostasis. Reduction of B-cell maturation and Ig production-especially that of IgG2c-by ablation of OcaB prevented age-induced glucose intolerance and insulin resistance and promoted energy expenditure by stimulating fatty acid utilization in eWAT and brown adipose tissue. Transfer of wild-type bone marrow in OcaB-/- mice replenished the eWAT B2-cell population and IgG levels, which diminished glucose tolerance, insulin sensitivity, and energy expenditure while increasing body weight gain in aged mice. Thus these findings demonstrate that upon aging, modifications in B-cell-driven adaptive immunity contribute to glucose intolerance and fat accretion.

Hypoxia upregulates Malat1 expression through a CaMKK/AMPK/HIF-1α axis.

Increased expression levels of the long non-coding RNA metastasis-associated lung adenocarcinoma transcript 1 (Malat1) have been associated with enhanced proliferation and metastasis of several cancer cell types. Hypoxia, a hallmark characteristic of solid tumors, has been linked to an increase in the activity of the ATP-generating AMPK protein. Since Malat1 was recently shown to be upregulated during hypoxia, the objective of this study was to determine the contribution of AMPK in the mechanistic pathways regulating Malat1 expression in low oxygen conditions. Compared to those cultured in 21% O2 conditions, HeLa cells incubated in 1.5% O2 expressed more Malat1 transcripts. This observation was mimicked in HEK293T cells using a synthetic reporter construct containing 5.6 kb of the human Malat1 promoter, suggesting that hypoxia directly impacted Malat1 gene transcription. Interestingly, pharmacological stimulation of AMPK increased Malat1 promoter transactivation in 21% O2 conditions, whereas inhibition of either AMPK or its upstream activator CaMKK completely abolished the augmentation of Malat1 under hypoxia. Pharmacological modulation of LKB1, another major regulator of AMPK, had no impact on Malat1 promoter transactivation, suggesting that calcium inputs are important in the control of Malat1 expression by AMPK. Overexpression of hypoxia-inducible factor-1α (HIF-1α) increased Malat1 expression in 21% O2 conditions, whereas pharmacological inhibition of HIF-1α blocked the impact of hypoxia on the Malat1 promoter. Taken together, these findings strongly suggest that Malat1 expression is regulated in hypoxic conditions by a CaMKK/AMPK/HIF-1α axis. More research is needed in physiological settings to test the clinical relevance of this pathway.

Morphological behaviour and metabolic capacity of cryopreserved human primary hepatocytes cultivated in a perfused multiwell device.

1. The quantitative prediction of the pharmacokinetic parameters of a drug from data obtained using human in vitro systems remains a significant challenge i.e. prediction of metabolic clearance in humans and estimation of the relative contribution of enzymes involved in the clearance. This has become particularly problematic for low turnover compounds. 2. Having human hepatocytes with stable cellular function over several days that adequately mimic the complexity of the physiological environment would be a major advance. Thus, we evaluated human hepatocytes, maintained in culture during 7 days in the microfluidic LiverChip™ system, in terms of morphological appearance, relative mRNA expression of phase I and II enzymes and transporters as a function of time, and metabolic capacity using probe substrates. 3. The results showed that mRNA levels of the major genes for enzymes involved in drug metabolism were well-maintained over a 7-day period of culture. Furthermore, after 4 days of culture, in the Liverchip™ device, human hepatocytes exhibited higher or similar CYPs activities compared to 1 day of culture in 2D-static conditions. 4. The functional data were supported by light/electron microscopies and immunohistochemistry showing viable tissue structure and well-differentiated human hepatocytes: presence of cell junctions, glycogen storage, and bile canaliculi.