Bax Inhibitor-1 preserves pancreatic ß-cell proteostasis by limiting proinsulin misfolding and programmed cell death.

The prevalence of diabetes steadily increases worldwide mirroring the prevalence of obesity. Endoplasmic reticulum (ER) stress is activated in diabetes and contributes to ß-cell dysfunction and apoptosis through the activation of a terminal unfolded protein response (UPR). Our results uncover a new role for Bax Inhibitor-One (BI-1), a negative regulator of inositol-requiring enzyme 1 (IRE1α) in preserving ß-cell health against terminal UPR-induced apoptosis and pyroptosis in the context of supraphysiological loads of insulin production. BI-1-deficient mice experience a decline in endocrine pancreatic function in physiological and pathophysiological conditions, namely obesity induced by high-fat diet (HFD). We observed early-onset diabetes characterized by hyperglycemia, reduced serum insulin levels, ß-cell loss, increased pancreatic lipases and pro-inflammatory cytokines, and the progression of metabolic dysfunction. Pancreatic section analysis revealed that BI-1 deletion overburdens unfolded proinsulin in the ER of ß-cells, confirmed by ultrastructural signs of ER stress with overwhelmed IRE1α endoribonuclease (RNase) activity in freshly isolated islets. ER stress led to ß-cell dysfunction and islet loss, due to an increase in immature proinsulin granules and defects in insulin crystallization with the presence of Rod-like granules. These results correlated with the induction of autophagy, ER phagy, and crinophagy quality control mechanisms, likely to alleviate the atypical accumulation of misfolded proinsulin in the ER. In fine, BI-1 in ß-cells limited IRE1α RNase activity from triggering programmed ß-cell death through apoptosis and pyroptosis (caspase-1, IL-1ß) via NLRP3 inflammasome activation and metabolic dysfunction. Pharmaceutical IRE1α inhibition with STF-083010 reversed ß-cell failure and normalized the metabolic phenotype. These results uncover a new protective role for BI-1 in pancreatic ß-cell physiology as a stress integrator to modulate the UPR triggered by accumulating unfolded proinsulin in the ER, as well as autophagy and programmed cell death, with consequences on ß-cell function and insulin secretion. In pancreatic ß-cells, BI-1-/- deficiency perturbs proteostasis with proinsulin misfolding, ER stress, terminal UPR with overwhelmed IRE1α/XBP1s/CHOP activation, inflammation, ß-cell programmed cell death, and diabetes.

Cholesterol efflux pathways hinder KRAS-driven lung tumor progenitor cell expansion.

Cholesterol efflux pathways could be exploited in tumor biology to unravel cancer vulnerabilities. A mouse model of lung-tumor-bearing KRASG12D mutation with specific disruption of cholesterol efflux pathways in epithelial progenitor cells promoted tumor growth. Defective cholesterol efflux in epithelial progenitor cells governed their transcriptional landscape to support their expansion and create a pro-tolerogenic tumor microenvironment (TME). Overexpression of the apolipoprotein A-I, to raise HDL levels, protected these mice from tumor development and dire pathologic consequences. Mechanistically, HDL blunted a positive feedback loop between growth factor signaling pathways and cholesterol efflux pathways that cancer cells hijack to expand. Cholesterol removal therapy with cyclodextrin reduced tumor burden in progressing tumor by suppressing the proliferation and expansion of epithelial progenitor cells of tumor origin. Local and systemic perturbations of cholesterol efflux pathways were confirmed in human lung adenocarcinoma (LUAD). Our results position cholesterol removal therapy as a putative metabolic target in lung cancer progenitor cells.

SUN2 regulates mitotic duration in response to extracellular matrix rigidity.

How cells adjust their growth to the spatial and mechanical constraints of their surrounding environment is central to many aspects of biology. Here, we examined how extracellular matrix (ECM) rigidity affects cell division. We found that cells divide more rapidly when cultured on rigid substrates. While we observed no effect of ECM rigidity on rounding or postmitotic spreading duration, we found that changes in matrix stiffness impact mitosis progression. We noticed that ECM elasticity up-regulates the expression of the linker of nucleoskeleton and cytoskeleton (LINC) complex component SUN2, which in turn promotes metaphase-to-anaphase transition by acting on mitotic spindle formation, whereas when cells adhere to soft ECM, low levels of SUN2 expression perturb astral microtubule organization and delay the onset of anaphase.

Unravelling the sex-specific diversity and functions of adrenal gland macrophages.

Despite the ubiquitous function of macrophages across the body, the diversity, origin, and function of adrenal gland macrophages remain largely unknown. We define the heterogeneity of adrenal gland immune cells using single-cell RNA sequencing and use genetic models to explore the developmental mechanisms yielding macrophage diversity. We define populations of monocyte-derived and embryonically seeded adrenal gland macrophages and identify a female-specific subset with low major histocompatibility complex (MHC) class II expression. In adulthood, monocyte recruitment dominates adrenal gland macrophage maintenance in female mice. Adrenal gland macrophage sub-tissular distribution follows a sex-dimorphic pattern, with MHC class IIlow macrophages located at the cortico-medullary junction. Macrophage sex dimorphism depends on the presence of the cortical X-zone. Adrenal gland macrophage depletion results in altered tissue homeostasis, modulated lipid metabolism, and decreased local aldosterone production during stress exposure. Overall, these data reveal the heterogeneity of adrenal gland macrophages and point toward sex-restricted distribution and functions of these cells.

Non-canonical glutamine transamination sustains efferocytosis by coupling redox buffering to oxidative phosphorylation.

Macrophages rely on tightly integrated metabolic rewiring to clear dying neighboring cells by efferocytosis during homeostasis and disease. Here we reveal that glutaminase-1-mediated glutaminolysis is critical to promote apoptotic cell clearance by macrophages during homeostasis in mice. In addition, impaired macrophage glutaminolysis exacerbates atherosclerosis, a condition during which, efficient apoptotic cell debris clearance is critical to limit disease progression. Glutaminase-1 expression strongly correlates with atherosclerotic plaque necrosis in patients with cardiovascular diseases. High-throughput transcriptional and metabolic profiling reveals that macrophage efferocytic capacity relies on a non-canonical transaminase pathway, independent from the traditional requirement of glutamate dehydrogenase to fuel ɑ-ketoglutarate-dependent immunometabolism. This pathway is necessary to meet the unique requirements of efferocytosis for cellular detoxification and high-energy cytoskeletal rearrangements. Thus, we uncover a role for non-canonical glutamine metabolism for efficient clearance of dying cells and maintenance of tissue homeostasis during health and disease in mouse and humans.

Brown adipose tissue monocytes support tissue expansion.

Monocytes are part of the mononuclear phagocytic system. Monocytes play a central role during inflammatory conditions and a better understanding of their dynamics might open therapeutic opportunities. In the present study, we focused on the characterization and impact of monocytes on brown adipose tissue (BAT) functions during tissue remodeling. Single-cell RNA sequencing analysis of BAT immune cells uncovered a large diversity in monocyte and macrophage populations. Fate-mapping experiments demonstrated that the BAT macrophage pool requires constant replenishment from monocytes. Using a genetic model of BAT expansion, we found that brown fat monocyte numbers were selectively increased in this scenario. This observation was confirmed using a CCR2-binding radiotracer and positron emission tomography. Importantly, in line with their tissue recruitment, blood monocyte counts were decreased while bone marrow hematopoiesis was not affected. Monocyte depletion prevented brown adipose tissue expansion and altered its architecture. Podoplanin engagement is strictly required for BAT expansion. Together, these data redefine the diversity of immune cells in the BAT and emphasize the role of monocyte recruitment for tissue remodeling.

REDD1 deficiency protects against nonalcoholic hepatic steatosis induced by high-fat diet.

Nonalcoholic fatty liver disease is a chronic liver disease which is associated with obesity and insulin resistance. We investigated the implication of REDD1 (Regulated in development and DNA damage response-1), a stress-induced protein in the development of hepatic steatosis. REDD1 expression was increased in the liver of obese mice and morbidly obese patients, and its expression correlated with hepatic steatosis and insulin resistance in obese patients. REDD1 deficiency protected mice from the development of hepatic steatosis induced by high-fat diet (HFD) without affecting body weight gain and glucose intolerance. This protection was associated with a decrease in the expression of lipogenic genes, SREBP1c, FASN, and SCD-1 in liver of HFD-fed REDD1-KO mice. Healthy mitochondria are crucial for the adequate control of lipid metabolism and failure to remove damaged mitochondria is correlated with liver steatosis. Expression of markers of autophagy and mitophagy, Beclin, LC3-II, Parkin, BNIP3L, was enhanced in liver of HFD-fed REDD1-KO mice. The number of mitochondria showing colocalization between LAMP2 and AIF was increased in liver of HFD-fed REDD1-KO mice. Moreover, mitochondria in liver of REDD1-KO mice were smaller than in WT. These results are correlated with an increase in PGC-1α and CPT-1 expression, involved in fatty acid oxidation. In conclusion, loss of REDD1 protects mice from the development of hepatic steatosis.

Mesothelial cell CSF1 sustains peritoneal macrophage proliferation.

Macrophages play a central role during infection, inflammation and tissue homeostasis maintenance. Macrophages have been identified in all organs and their core transcriptomic signature and functions differ from one tissue to another. Interestingly, macrophages have also been identified in the peritoneal cavity and these cells have been extensively used as a model for phagocytosis, efferocytosis and polarization. Peritoneal macrophages are involved in B-cell IgA production, control of inflammation and wound healing following thermal-induced liver surface injury. These cells presumably require and interact with the omentum, where milky spot stromal cells have been proposed to secrete CSF1 (colony stimulating factor 1). Peritoneal macrophages depend on CSF1 for their generation and survival, but the identity of CSF1 producing cells inside the large peritoneal cavity remains unknown. Here we investigated peritoneal macrophage localization and their interaction with mesothelial cells, the major cell type predicted to secrete CSF1. Our data revealed that mesothelial cells produce membrane bound and secreted CSF1 that both sustain peritoneal macrophage growth.

Leukocyte RhoA exchange factor Arhgef1 mediates vascular inflammation and atherosclerosis.

Abnormal activity of the renin-angiotensin-aldosterone system plays a causal role in the development of hypertension, atherosclerosis, and associated cardiovascular events such as myocardial infarction, stroke, and heart failure. As both a vasoconstrictor and a proinflammatory mediator, angiotensin II (Ang II) is considered a potential link between hypertension and atherosclerosis. However, a role for Ang II-induced inflammation in atherosclerosis has not been clearly established, and the molecular mechanisms and intracellular signaling pathways involved are not known. Here, we demonstrated that the RhoA GEF Arhgef1 is essential for Ang II-induced inflammation. Specifically, we showed that deletion of Arhgef1 in a murine model prevents Ang II-induced integrin activation in leukocytes, thereby preventing Ang II-induced recruitment of leukocytes to the endothelium. Mice lacking both LDL receptor (LDLR) and Arhgef1 were protected from high-fat diet-induced atherosclerosis. Moreover, reconstitution of Ldlr-/- mice with Arhgef1-deficient BM prevented high-fat diet-induced atherosclerosis, while reconstitution of Ldlr-/- Arhgef1-/- with WT BM exacerbated atherosclerotic lesion formation, supporting Arhgef1 activation in leukocytes as causal in the development of atherosclerosis. Thus, our data highlight the importance of Arhgef1 in cardiovascular disease and suggest targeting Arhgef1 as a potential therapeutic strategy against atherosclerosis.

Cardiac Protective Effects of Moringa oleifera Seeds in Spontaneous Hypertensive Rats.

BACKGROUND: Hypertension is characterized by a maintained high blood pressure leading to cardiac complications such as left ventricular hypertrophy and fibrosis and an increased risk of heart failure and myocardial infarction. This study investigated the cardiac effects of oral administration of Moringa oleifera (MOI) seed powder in spontaneous hypertensive rats (SHR). METHODS: SHR received food containing MOI seed powder (750mg/d, 8 weeks) or normal food. In vivo measurement of hemodynamic parameters by telemetry and cardiac structure and function analysis by echocardiography were performed. Histological studies were performed to determine fibrosis and protein expression. RESULTS: MOI treatment did not modify blood pressure in SHR but reduced nocturnal heart rate and improved cardiac diastolic function (reduction of isovolumetric relaxation time and deceleration time of the E wave, increase of ejection volume and cardiac output compared to nontreated SHR). Left ventricular anterior wall thickness, interseptal thickness on diastole, and relative wall thickness were reduced after MOI treatment. Furthermore, we found a significant reduction of fibrosis in the left ventricle of MOI-treated SHR. This antihypertrophic and antifibrotic effect of MOI was associated with increased expression of peroxisome proliferator-activated receptor (PPAR)-α and δ, reduced cardiac triglyceride level, and enhanced plasmatic prostacyclins. CONCLUSIONS: Our data show a beneficial effect of MOI on the cardiac structure and function in SHR associated with an upregulation of PPAR-α and δ signaling. This study thus provides scientific rational support for the empirical use of MOI in the traditional Malagasy medicine against cardiac diseases associated with blood pressure overload.

Taxonomy, Physiology, and Natural Products of Actinobacteria.

Actinobacteria are Gram-positive bacteria with high G+C DNA content that constitute one of the largest bacterial phyla, and they are ubiquitously distributed in both aquatic and terrestrial ecosystems. Many Actinobacteria have a mycelial lifestyle and undergo complex morphological differentiation. They also have an extensive secondary metabolism and produce about two-thirds of all naturally derived antibiotics in current clinical use, as well as many anticancer, anthelmintic, and antifungal compounds. Consequently, these bacteria are of major importance for biotechnology, medicine, and agriculture. Actinobacteria play diverse roles in their associations with various higher organisms, since their members have adopted different lifestyles, and the phylum includes pathogens (notably, species of Corynebacterium, Mycobacterium, Nocardia, Propionibacterium, and Tropheryma), soil inhabitants (e.g., Micromonospora and Streptomyces species), plant commensals (e.g., Frankia spp.), and gastrointestinal commensals (Bifidobacterium spp.). Actinobacteria also play an important role as symbionts and as pathogens in plant-associated microbial communities. This review presents an update on the biology of this important bacterial phylum.

PCSK9-deficiency does not alter blood pressure and sodium balance in mouse models of hypertension.

OBJECTIVE: Proprotein convertase subtilisin/kexin type 9 (PCSK9) is highly expressed in the kidney, where its function remains unclear. In vitro data suggested that PCSK9 could impair the trafficking of the epithelial Na channel (ENaC). Here, we aimed at determining the consequences of PCSK9-deficiency on blood pressure, sodium balance and ENaC function in vivo in mice. METHODS: Blood pressure was measured using non-invasive tail-cuff system or radiotelemetry under basal conditions in male and female PCSK9(+/+) and PCSK9(-/-) mice, as well as in models of hypertension: l-NAME (2 mg/kg/day), angiotensin II (1 mg/kg/day) and deoxycorticosterone acetate (DOCA)-salt in male mice only. Plasma and urine electrolytes (Na(+), K(+), Cl(-)) were collected under basal conditions, after DOCA-salt and amiloride treatment. Renal expression of ENaC subunits was assessed by western blotting. RESULTS: PCSK9-deficiency did not alter both basal blood pressure and its increase in salt-insensitive (l-NAME) and salt-sensitive (Ang-II and DOCA-salt) hypertension models. Plasma PCSK9 concentrations were increased by 2.8 fold in DOCA-salt-induced hypertension. The relative expression of the cleaved, active, 30-kDa αENaC subunit was significantly increased by 32% in kidneys of PCSK9(-/-) mice under basal, but not under high-Na(+) diet or DOCA-salt conditions. Amiloride increased urinary Na(+) excretion to similar level in both genotypes, indicating that ENaC activity was not affected by PCSK9-deficiency. CONCLUSIONS: Despite an increase of cleaved αENaC under basal condition, PCSK9(-/-) mice display normal sodium balance and blood pressure regulation. Altogether, these data are reassuring regarding the development of PCSK9 inhibitors in hypercholesterolemia.

Targeting PI3Kγ activity decreases vascular trauma-induced intimal hyperplasia through modulation of the Th1 response.

Interventional strategies to treat atherosclerosis, such as transluminal angioplasty and stent implantation, often cause vascular injury. This leads to intimal hyperplasia (IH) formation that induces inflammatory and fibroproliferative processes and ultimately restenosis. We show that phosphoinositide 3-kinase γ (PI3Kγ) is a key player in IH formation and is a valid therapeutic target in its prevention/treatment. PI3Kγ-deficient mice and mice expressing catalytically inactive PI3Kγ (PI3Kγ KD) showed reduced arterial occlusion and accumulation of monocytes and T cells around sites of vascular lesion. The transfer of PI3Kγ KD CD4(+) T cells into Rag2-deficient mice greatly reduced vascular occlusion compared with WT cells, clearly demonstrating the involvement of PI3Kγ in CD4(+) T cells during IH formation. In addition we found that IH is associated with increased levels of Th1 and Th17 cytokines. A specific decrease in the Th1 response was observed in the absence of PI3Kγ activity, leading to decreased CXCL10 and RANTES production by smooth muscle cells. Finally, we show that treatment with the PI3Kγ inhibitor AS-605240 is sufficient to decrease IH in both mouse and rat models, reinforcing the therapeutic potential of PI3Kγ inhibition. Altogether, these findings demonstrate a new role for PI3Kγ activity in Th1-controlled IH development.

Photosynthetic activity of Lolium perenne as a function of endophyte status and zinc nutrition.

Grass infection by endophyte fungi can confer host resistance to different types of stress, but limited evidence is available on the related effects on the photosynthetic mechanism of the grasses. Zinc has direct and indirect effects on this mechanism and is one of the more important environmental pollutants. To measure whether photosynthesis of the host plant is affected by fungal infection when endophyte-free and endophyte-infected grasses contain similar excess zinc concentrations in their leaves, two batches of Lolium perenne L. cv. Apollo plants were established, one batch was infected with Neotyphodium lolii, the other was not. Both batches were then treated for 8 d with a nutrient solution containing 0, 1, 5, 10 or 20 mm ZnSO4. The increase in zinc concentration induced some reduction in photosystem II (PSII) activity but not enough to account for the total drop in the net photosynthetic rate. Endophyte fungus favoured maintenance of the PSII activity, but did not significantly modify the net photosynthesis and similar zinc concentration levels were observed in leaves of both types of plant. Interactive effects of zinc and light induced less photodamage to the PSII of the host, which is able to react to an increase in photon flux density (PFD). In endophyte-free plants, the reaction centre contributed more than antenna complexes to energy dissipation. In endophyte-infected plants, the quenching of the reaction centre and antenna complexes rose simultaneously and at a constant rate, as zinc concentrations increased.

Use of commercial plant species in a hydroponic system to treat domestic wastewaters.

The objectives in this work were to investigate a conceptual layout for an inexpensive and simple system that would treat primary municipal wastewater to discharge standards. A commercial hydroponic system was adapted for this study and the wastewater was used to irrigate wooly digitalis (Digitalis lanata Ehrh.) and foxglove (Digitalis purpurea L.). These plants are medicinal and produce cardenolide compounds. Influent and effluent samples were collected once a month for six months and analyzed to determine the various parameters relating to water quality. The legal discharge levels for total suspended solids (SS), biochemical oxygen demand (BOD5), and chemical oxygen demand (COD) were reached for the two tested plants after 48 h of wastewater treatment; the removal was 82, 93, and 79%, respectively, for wooly digitalis and 92, 92, and 84%, respectively, for foxglove. Similar results were obtained during a 6-mo period although the sewage composition varied widely. The system tended to be unable to remove N and P to concentrations below regulated levels. Compared with the nutrient solution composition, the wastewater was more concentrated in Na+ and Cl- and less in N, K+, and Ca2+. These variations can lead to the decline of wooly digitalis plants. Foxglove developed a significant root system to increase mineral absorption wastewater being used as the unique nutritive source. After 10 wk all the wooly digitalis seedlings were dead. Despite this fact, however, the root system remained in place for a significant time (< 4 mo), thus continuing to filter wastewater and to be used as a bacterial support thus making it possible to have a security period to replace the dead plants.

Treatment of domestic wastewater by an hydroponic NFT system.

The objectives in this work were to investigate a conceptual layout for an inexpensive and simple system that would treat primary municipal wastewater to discharge standards. A commercial hydroponic system was adapted for this study and the wastewater was used to irrigate Datura innoxia plants. Influent and effluent samples were collected once a month for six months and analysed to determine the various parameters relating to the water quality. The legal discharge levels for total suspended, biochemical oxygen demand and chemical oxygen demand were reached with the plant system after 24 h of wastewater treatment. Total nitrogen and total phosphorus reduction were also obtained. NH4(+)-N was reduced by 93% with nitrification proving to be the predominant removal process. Significant nitrification occurred when the BOD5 level dropped 45 mg/l. Similar results were obtained for six months although the sewage composition varied widely. D. innoxia develops and uses the wastewater as the unique nutritive source.

Treatment of domestic wastewater using the nutrient film technique (NFT) to produce horticultural roses.

Removal of organic matter, nitrogen and phosphorus was investigated in a pilot based on the nutrient film technique system used for horticultural production. Rosebushes producing commercial roses were set on an inclined impermeable surface over which a thin film of domestic wastewater flowed directly through the root matrix. The roses produced with wastewater as nutrient solution were qualitatively and quantitatively similar to those produced traditionally and can be marketable. In the presence of the rosebushes, the lowering of chemical oxygen demand (COD), biological oxygen demand (BOD5) and suspended solids (SS) increased with their influent strengths in a linear fashion correlation. Whatever the pollutant load of the influent, within the range studied, the level of organic pollution required to allow the discharge of the effluent into water courses was reached after 24 h of treatment. At this time, the COD reached 39 +/- 13 mg L(-1), the BOD5 7 +/- 4 mg L(-1) and the SS 8 +/- 6 mg L(-1). The removal percentages were 89, 95 and 94, respectively, whereas without plants they were 55, 33 and 53, respectively. The rosebushes had a beneficial effect on the removal of phosphorus from 20% to 23% greater in their presence than without. In presence of plants, the nitrate was the principal form of nitrogen from 48 h of treatment whereas without rosebushes, nitrite represent more than 20% of initial nitrogen whatever the treatment duration. The root matrix served not only as a filter for the organic matter, but also provided a microhabitat suitable for nitrifying bacteria and heterotrophic micro-organisms, which responded rapidly to the pollutant load.

Urban wastewater treatment by a nutrient film technique system with a valuable commercial plant species (Chrysanthemum cinerariaefolium Trev.).

Urban wastewater causes rapid eutrophication of natural waters and requires treatment before discharge. This is expensive and produces huge quantities of sludge. In the European Community, it will no longer be lawful to dispose of this sludge as landfill after 2005 (European Directive 91/271/CEE of May 21, 1991). Wastewater treatment by the Chrysanthemum cinerariaefolium plants in horizontal flow was investigated using the nutrient film technique (NFT), a widely used hydroponic system in the commercial greenhouse industry. After a 48 h plant treatment, the purification efficiency was 95%, 91%, and 99% with respect to suspended solids (SS), biochemical oxygen demand (BOD5), and chemical oxygen demand (COD), and the elimination of nutrients (total nitrogen and total phosphorus) varied between 40% and 80%. SS and thus indirectly BOD5 and COD were removed by filtration and adsorption; the solids trapped in the root systems were then decomposed and mineralized. The system with 25 plants purified 30 L of wastewater in 48 h. One-hundred people communities wastewater could be treated with a 6 m2 area of production. Pyrethrin contents and chlorophyll a fluorescence of plants grown on raw urban waters were not significantly different from those grown on a standard nutrient solution.