Deficiency of ß-carotene oxygenase 2 induces mitochondrial fragmentation and activates the STING-IRF3 pathway in the mouse hypothalamus.

Hypothalamic inflammation has been linked to various aspects of central metabolic dysfunction and diseases in humans, including hyperphagia, altered energy expenditure, and obesity. We previously reported that loss of ß-carotene oxygenase 2 (BCO2), a mitochondrial inner membrane protein, causes the alteration of the hypothalamic metabolome, low-grade inflammation, and an increase in food intake in mice at an early age, e.g., 3-6 weeks. Here, we determined the extent to which the deficiency of BCO2 induces hypothalamic inflammation in BCO2 knockout mice. Mitochondrial proteomics, electron microscopy, and immunoblotting were used to assess the changes in hypothalamic mitochondrial dynamics and mitochondrial DNA sensing and signaling. The results showed that deficiency of BCO2 altered hypothalamic mitochondrial proteome and respiratory supercomplex assembly by enhancing the expression of NADH:ubiquinone oxidoreductase subunit A11 protein and improved cardiolipin synthesis. BCO2 deficiency potentiated mitochondrial fission but suppressed mitophagy and mitochondrial biogenesis. Furthermore, deficiency of BCO2 resulted in inactivation of mitochondrial MnSOD enzyme, excessive production of reactive oxygen species, and elevation of protein levels of stimulator of interferon genes (STING) and interferon regulatory factor 3 (IRF3) in the hypothalamus. The data suggest that BCO2 is essential for hypothalamic mitochondrial dynamics. BCO2 deficiency induces mitochondrial fragmentation and mitochondrial oxidative stress, which may lead to mitochondrial DNA release into the cytosol and subsequently sensing by activation of the STING-IRF3 signaling pathway in the mouse hypothalamus.

Astaxanthin-Shifted Gut Microbiota Is Associated with Inflammation and Metabolic Homeostasis in Mice.

BACKGROUND: Astaxanthin is a red lipophilic carotenoid that is often undetectable in human plasma due to the limited supply in typical Western diets. Despite its presence at lower than detectable concentrations, previous clinical feeding studies have reported that astaxanthin exhibits potent antioxidant properties. OBJECTIVE: We examined astaxanthin accumulation and its effects on gut microbiota, inflammation, and whole-body metabolic homeostasis in wild-type C57BL/6 J (WT) and ß-carotene oxygenase 2 (BCO2) knockout (KO) mice. METHODS: Six-wk-old male and female BCO2 KO and WT mice were provided with either nonpurified AIN93M (e.g., control diet) or the control diet supplemented with 0.04% astaxanthin (wt/wt) ad libitum for 8 wk. Whole-body energy expenditure was measured by indirect calorimetry. Feces were collected from individual mice for short-chain fatty acid assessment. Hepatic astaxanthin concentrations and liver metabolic markers, cecal gut microbiota profiling, inflammation markers in colonic lamina propria, and plasma samples were assessed. Data were analyzed by 3-way ANOVA followed by Tukey's post hoc analysis. RESULTS: BCO2 KO but not WT mice fed astaxanthin had ∼10-fold more of this compound in liver than controls (P < 0.05). In terms of the microbiota composition, deletion of BCO2 was associated with a significantly increased abundance of Mucispirillum schaedleri in mice regardless of gender. In addition to more liver astaxanthin in male KO compared with WT mice fed astaxanthin, the abundance of gut Akkermansia muciniphila was 385% greater, plasma glucagon-like peptide 1 was 27% greater, plasma glucagon and IL-1ß were 53% and 30% lower, respectively, and colon NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome activation was 23% lower (all P < 0.05) in male KO mice than the WT mice. CONCLUSIONS: Astaxanthin affects the gut microbiota composition in both genders, but the association with reductions in local and systemic inflammation, oxidative stress, and improvement of metabolic homeostasis only occurs in male mice.

The Inhibitory Properties of Ethanol Extracts of Some Culinary-Medicinal Mushrooms on the Secretion of Interleukin-8 and Vascular Endothelial Growth Factor by PC3 Cancer Cells.

Interleukin (IL)-8, a cytokine produced by immune and non-immune cells, induces angiogenesis via increased vascular endothelial growth factor (VEGF) secretion; both cytokines promote tumor growth. IL-8 and VEGF plasma levels correlate with prostate cancer severity, suggesting that therapeutic options aimed at their downregulation may modulate tumor growth. Available data suggest that Agaricus bisporus (white button mushroom [WBM]) extracts inhibit cancer cell proliferation through aromatase inhibition. However, the extent to which they affect IL-8 and VEGF remains to be elucidated. The aims of this study were to (1) investigate the antiproliferative properties of WBM, brown A. bisporus (portabella), and Lentinus edodes (shiitake mushroom) on PC3 cancer cells; (2) demonstrate that these properties are exerted through the regulation of both IL-8 and VEGF; and (3) determine the role of NFκB activation in the antiproliferative process of mushroom extracts. Cytokine secretion in the supernatant, NFκB activity, and cell proliferation were measured in PC3 cells incubated with 0-100 µg/mL of ethanol extracts of mushrooms. Mushroom extracts decreased IL-8 secretion and cell proliferation (P < .05), and also tended to decrease VEGF (P < .09). Decreased cell proliferation did not appear to result from cell death because trypan blue exclusion tests showed comparable cell viability among cultures. Mushroom extracts also decreased nuclear and total NFκB activity, and the ratio of nuclear to cytoplasmic activity (P < .05) suggesting altered translocation from the cytoplasm to the nucleus. Our data suggest that the three types of studied mushrooms may modulate tumor growth through inhibition of IL-8, VEGF, and NFκB pathways.

Montmorency tart cherry protects against age-related bone loss in female C57BL/6 mice and demonstrates some anabolic effects.

PURPOSE: Age-related bone loss is a consequence of endocrine and immune changes that disrupt bone remodeling. Functional foods (e.g., tart cherries) with antioxidant, anti-inflammatory and prebiotic activity can potentially counter this age-related phenomenon. The aim of this study was to determine if Montmorency tart cherry protects against early age-related bone loss and the culpable alterations in bone metabolism. METHODS: Female, 5-month-old, C57BL/6 mice were assigned to baseline or treatment groups: AIN-93M diet supplemented with 0, 1, 5, or 10% tart cherry for 90 days. Bone mineral density (BMD) and trabecular and cortical bone microarchitecture were assessed. Treatment effects on bone metabolism and regulators of bone formation, resorption and mineralization were determined. RESULTS: Mice consuming the 5% and 10% doses had higher vertebral and tibial BMD (p < 0.05) compared to controls. The age-related decrease in trabecular bone volume (BV/TV) of the distal femur was prevented with these doses. Vertebral trabecular BV/TV and cortical bone thickness of the femur mid-diaphysis were greater (p < 0.05) in the groups receiving the 5% and 10% cherry than the control diet. Notably, these improvements were significantly greater than the baseline controls, consistent with an anabolic response. Although no differences in systemic biomarkers of bone formation or resorption were detected at 90 days, local increases in Phex and decreases in Ppar-γ suggest a bone environment that supports increased mineralization. CONCLUSIONS: These findings demonstrate that cherry supplementation (5% and 10%) improves BMD and some indices of trabecular and cortical bone microarchitecture; these effects are likely attributed to increased bone mineralization.

Ablation of ß,ß-carotene-9',10'-oxygenase 2 remodels the hypothalamic metabolome leading to metabolic disorders in mice.

ß,ß-Carotene-9',10'-oxygenase 2 (BCO2) is a protein localized to the inner membrane of mitochondria. It was initially discovered as an enzyme that catalyzes the asymmetric cleavage of carotenoids. Systemic depletion of BCO2 causes increased food intake and impaired hepatic lipid metabolism in mice. The aim of this current study was to determine the extent to which BCO2 exerts its role in hypothalamic nutrient metabolism and feeding behavior through remodeling the hypothalamic metabolome in mice. Male BCO2 knockout (KO) and the isogenic wild-type 129S6 (WT) mice at 6 weeks of age were used for metabolic and cytokine and hypothalamic metabolomics and biochemical analysis. Compared to the WT, BCO2 KO mice exhibited widespread disruptions in metabolism and metabolite homeostasis, an increase in fasting blood glucose, a decrease in circulating glucagon and leptin, an elevation of plasma interleukin 1 beta and tumor necrosis factor alpha, and impaired AMP-activated protein kinase signaling. The global hypothalamic metabolomic results revealed that depletion of BCO2 resulted in striking metabolic changes, including suppression of long-chain fatty acids transport into mitochondria, inhibition of the metabolism of dipeptides and sulfur-containing amino acids, and stimulation of local oxidative stress and inflammation in the hypothalamus of BCO2 KO mice. These findings suggest that BCO2 regulates hypothalamic mitochondrial function, nutrient metabolism, and local oxidative stress and inflammation. Complex interplay between the hormone signaling and impaired lipid and glucose metabolism could account for initiation of oxidative stress, inflammation and eventual metabolic disorders in BCO2 KO mice.

Zinc Supplementation in a Randomized Controlled Trial Decreased ZIP4 and ZIP8 mRNA Abundance in Peripheral Blood Mononuclear Cells of Adult Women.

Zinc plays an integral role in numerous cellular processes including regulation of gene expression. This randomized placebo-controlled trial in adult women evaluated the effects of 20 mg Zn for 23 days. The mRNA abundance of zinc transporters (ZnT1/ZIP3/ZIP4/ZIP8) and metallothionein (MT1) from peripheral blood mononuclear cells was determined by real-time quantitative polymerase chain reaction. In paired samples (n = 6-9), the ZIP4 (P = 0.036) and ZIP8 (P = 0.038) mRNA abundance decreased following zinc supplementation. ZnT1, ZIP3, and MT1 mRNA abundance did not change significantly. The mean ± standard deviation plasma zinc concentration (by inductively coupled plasma mass spectrometry) at baseline was 680 ± 110 µg/L for the zinc group (n = 24) and 741 ± 92 µg/L for the placebo group (n = 23). At endpoint, plasma zinc in the zinc group increased to 735 ± 80 µg/L (P < 0.01) while in the placebo group (717 ± 100 µg/L) it did not change significantly from baseline. The change in mRNA abundance highlights the importance of further investigating ZIP4 and ZIP8 mRNA abundance as potential zinc status biomarkers.

Zinc supplementation reduced DNA breaks in Ethiopian women.

Assessment of zinc status remains a challenge largely because serum/plasma zinc may not accurately reflect an individual's zinc status. The comet assay, a sensitive method capable of detecting intracellular DNA strand breaks, may serve as a functional biomarker of zinc status. We hypothesized that effects of zinc supplementation on intracellular DNA damage could be assessed from samples collected in field studies in Ethiopia using the comet assay. Forty women, from villages where reported consumption of meat was less than once per month and phytate levels were high, received 20 mg zinc as zinc sulfate or placebo daily for 17 days in a randomized placebo-controlled trial. Plasma zinc concentrations were determined by inductively coupled plasma mass spectrometry. Cells from whole blood at the baseline and end point of the study were embedded in agarose, electrophoresed, and stained before being scored by an investigator blinded to the treatments. Although zinc supplementation did not significantly affect plasma zinc, mean (± SEM) comet tail moment measurement of supplemented women decreased from 39.7 ± 2.7 to 30.0 ± 1.8 (P< .005), indicating a decrease in DNA strand breaks in zinc-supplemented individuals. These findings demonstrated that the comet assay could be used as a functional assay to assess the effects of zinc supplementation on DNA integrity in samples collected in a field setting where food sources of bioavailable zinc are limited. Furthermore, the comet assay was sufficiently sensitive to detect changes in zinc status as a result of supplementation despite no significant changes in plasma zinc.

A comparative study of the bone metabolic response to dried plum supplementation and PTH treatment in adult, osteopenic ovariectomized rat.

Dried plum has been reported to have potent effects on bone in osteopenic animal models, but the mechanisms through which bone metabolism is altered in vivo remain unclear. To address this issue, a study comparing the metabolic response of dried plum to the anabolic agent, parathyroid hormone (PTH), was undertaken. Six month-old female Sprague Dawley rats (n=84) were sham-operated (SHAM) or ovariectomized (OVX) and maintained on a control diet for 6wks until osteopenia was confirmed. Treatments were initiated consisting of a control diet (AIN-93M) supplemented with dried plum (0, 5, 15 or 25%; w/w) or a positive control group receiving PTH. At the end of 6wks of treatment, whole body and femoral bone mineral density (BMD) were restored by the two higher doses of dried plum to the level of the SHAM group. Trabecular bone volume and cortical thickness were also improved with these two doses of dried plum. Dried plum suppressed the OVX-induced increase in bone turnover as indicated by systemic biomarkers of bone metabolism, N-terminal procollagen type 1 (P1NP) and deoxypyridinoline (DPD). Dynamic bone histomorphometric analysis of the tibial metaphysis revealed that dried plum restored the OVX-induced increase in cancellous bone formation rate (BFR) and mineralizing surface (MS/BS) to the SHAM group, but some doses of dried plum increased endocortical mineral apposition rate (MAR). As expected, PTH significantly increased endocortical MAR and BFR, periosteal BFR, and trabecular MAR and BFR beyond that of the OVX and maintained the accelerated rate of bone resorption associated with OVX. Dried plum up-regulated bone morphogenetic protein 4 (Bmp4) and insulin-like growth factor 1 (Igf1) while down-regulating nuclear factor T cell activator 1 (Nfatc1). These findings demonstrate that in the adult osteopenic OVX animal, the effects of dried plum differ from that of PTH in that dried plum primarily suppressed bone turnover with the exception of the indices of bone formation at the endocortical surface.

White button, portabella, and shiitake mushroom supplementation up-regulates interleukin-23 secretion in acute dextran sodium sulfate colitis C57BL/6 mice and murine macrophage J.744.1 cell line.

Interleukin-23 (IL-23), a cytokine produced primarily by dendritic cells, is involved in host defense against gut pathogens and promotes innate immunity and inflammatory responses through the IL-23/interleukin-17 axis. We previously reported that extracts from edible mushrooms enhanced antimicrobial α-defensin production n HL60 cells. Because IL-23 is involved in defensin production, we hypothesized that edible mushrooms may modulate its secretion and gut inflammation. Eight-week-old C57BL/6 mice were fed the AIN76 diet or the same diet supplemented with 5% white button (WBM), portabella, or shiitake mushrooms. To assess in vivo and in vitro cytokine secretion, 7 to 8 mice per group received 3% dextran sodium sulfate (DSS) in drinking water during the last 5 days of the 6-week feeding period. To delineate the mechanisms by which mushrooms alter IL-23 secretion, J.744.1 cells were incubated with (100 µg/mL) WBM, portabella, and shiitake extracts without and with 100 µg/mL curdlan (a dectin-1 agonist) or 1 mg/mL laminarin (a dectin-1 antagonist). The dectin-1 receptor is a pattern-recognition receptor found in phagocytes, and its activation promotes antimicrobial innate immunity and inflammatory responses. In DSS-untreated mice, mushrooms significantly increased IL-23 plasma levels but decreased those of interleukin-6 (IL-6) (P < .05). In DSS-treated mice, mushroom-supplemented diets increased IL-6 and IL-23 levels (P < .05). Mushroom extracts potentiated curdlan-induced IL-23 secretion, and mushroom-induced IL-23 secretion was not blocked by laminarin in vitro, suggesting the involvement of both dectin-1-dependent and dectin-1-independent pathways. Although all mushrooms tended to increase IL-6 in the colon, only WBM and shiitake tended to increase IL-23 levels. These data suggest that edible mushrooms may enhance gut immunity through IL-23.

Dried plum's unique capacity to reverse bone loss and alter bone metabolism in postmenopausal osteoporosis model.

Interest in dried plum has increased over the past decade due to its promise in restoring bone and preventing bone loss in animal models of osteoporosis. This study compared the effects of dried plum on bone to other dried fruits and further explored the potential mechanisms of action through which dried plum may exert its osteoprotective effects. Adult osteopenic ovariectomized (OVX) C57BL/6 mice were fed either a control diet or a diet supplemented with 25% (w/w) dried plum, apple, apricot, grape or mango for 8 weeks. Whole body and spine bone mineral density improved in mice consuming the dried plum, apricot and grape diets compared to the OVX control mice, but dried plum was the only fruit to have an anabolic effect on trabecular bone in the vertebra and prevent bone loss in the tibia. Restoration of biomechanical properties occurred in conjunction with the changes in trabecular bone in the spine. Compared to other dried fruits in this study, dried plum was unique in its ability to down-regulate osteoclast differentiation coincident with up-regulating osteoblast and glutathione (GPx) activity. These alterations in bone metabolism and antioxidant status compared to other dried fruits provide insight into dried plum's unique effects on bone.

Dietary supplementation with dried plum prevents ovariectomy-induced bone loss while modulating the immune response in C57BL/6J mice.

This study was designed to investigate the effects of dried plum on the changes in bone metabolism and the immune response associated with ovarian hormone deficiency. Adult female C57BL/6J mice were either sham-operated (Sham) and fed AIN-93 diet (control) or ovariectomized (OVX) and fed a control diet with 0%, 5%, 15% or 25% dried plum (w/w), corresponding to control, low- (LDP), medium- (MDP) and high (HDP)-dose dried plum. Four weeks of HDP supplementation prevented the decrease in spine bone mineral density and content induced by OVX. The OVX compromise in trabecular bone of the vertebra and proximal tibia was prevented by the higher doses of dried plum, and in the vertebra these effects resulted in greater (P<.05) bone strength and stiffness. In the bone marrow, OVX suppressed granulocyte and committed monocyte populations and increased the lymphoblast population, but the MDP and HDP restored these myeloid and lymphoid populations to the level of the Sham. Dried plum also suppressed lymphocyte tumor necrosis factor (TNF)-α production ex vivo by splenocytes, in response to concanavalin (Con) A stimulation. These data indicate that dried plum's positive effects on bone structural and biomechanical properties coincide with the restoration of certain bone marrow myeloid and lymphoid populations, and suppressed splenocyte activation occurring with ovarian hormone deficiency.

Differential effects of shiitake- and white button mushroom-supplemented diets on hepatic steatosis in C57BL/6 mice.

Shiitake mushrooms (SMs) have been used in Asia for treatment and/or prevention of chronic diseases and hypercholesterolemia. Previously, we observed a diet supplemented with 5% SM resulted in a twofold increase in plasma IL-6 levels in DBA arthritic mice. An elevation in plasma IL-6 has also been implicated in the pathogenesis fatty liver disease. Thus, the aim of this study was to investigate the effect of SM supplemented-diet on hepatic steatosis. In study 1, eight-week old female C57BL/6 mice were randomly assigned to the following groups for 6 weeks: the AIN-93 diet; 5% SM, and 5% white button mushroom (WBM) supplemented diets (12/group). In study 2, mice were fed either the AIN-93 diet or SM (20/group). After 6 weeks, 13 mice fed SM diet were given the AIN93 diet for 8 or 15 days. Unlike other groups, all mice fed the SM diet developed fatty liver (mean histopathology score 4.5 vs <1 in the other groups; p<0.001) without fibrosis and inflammation. Fifteen days post withdrawal of SM completely normalized liver histology. To the best of our knowledge, this is the first report that chronic consumption of SM is associated with the development of fatty liver. The mechanism by which SM causes hepatic steatosis warrants further investigation.

Mango modulates body fat and plasma glucose and lipids in mice fed a high-fat diet.

Consumption of fruits and vegetables has been investigated for their role in the prevention of many chronic conditions. Among the fruits, mango provides numerous bioactive compounds such as carotenoids, vitamin C and phenolic compounds, which have been shown to have antioxidant and anti-inflammatory properties. The present study examined the effects of dietary supplementation of freeze-dried mango pulp, in comparison with the hypolipidaemic drug, fenofibrate, and the hypoglycaemic drug, rosiglitazone, in reducing adiposity and alterations in glucose metabolism and lipid profile in mice fed a high-fat (HF) diet. Male C57BL/6J mice were randomly divided into six treatment groups (eight to nine/group): control (10 % energy from fat); HF (60 % energy from fat); HF+1 or 10 % freeze-dried mango (w/w); HF+fenofibrate (500 mg/kg diet); HF+rosiglitazone (50 mg/kg diet). After 8 weeks of treatment, mice receiving the HF diet had a higher percentage body fat (P = 0·0205) and epididymal fat mass (P = 0·0037) compared with the other treatment groups. Both doses of freeze-dried mango, similar to fenofibrate and rosiglitazone, prevented the increase in epididymal fat mass and the percentage of body fat. Freeze-dried mango supplementation at the 1 % dose improved glucose tolerance as shown by approximately 35 % lower blood glucose area under the curve compared with the HF group. Moreover, freeze-dried mango lowered insulin resistance, as indicated by the homeostasis model assessment of insulin resistance, to a similar extent as rosiglitazone and modulated NEFA. The present findings demonstrate that incorporation of freeze-dried mango in the diet of mice improved glucose tolerance and lipid profile and reduced adiposity associated with a HF diet.

White button and shiitake mushrooms reduce the incidence and severity of collagen-induced arthritis in dilute brown non-agouti mice.

Exotic mushrooms have been used in ancient Chinese medicine due to their immunomodulatory properties for the treatment and/or prevention of chronic diseases. However, only limited data exist on the health benefits of white button mushrooms (WBM), the most common in the American diet. In the current study, we investigated the effects of WBM and shiitake mushrooms (SM) on collagen-induced arthritis (CIA) using a 2 x 3 factorial design in 8-wk-old female dilute brown non-agouti mice that were fed a control diet (n = 37) or the same diet supplemented with 5% lyophilized WBM or SM (n = 27) for 6 wk. CIA was induced by immunizing mice with 100 µg bovine collagen followed by 50 µg LPS on d 20 post-collagen injection. CIA was assessed by mononuclear cell infiltration, bone erosion, plasma IL-6, TNFα, and intercellular adhesion molecule-1 (sICAM-1) concentrations. Compared with the control diet, WBM and SM tended to reduce the CIA index from 5.11 ± 0.82 to 3.15 ± 0.95 (P = 0.06) (median, 6-9 to 1-2) 31 d post-collagen injection. Whereas 58% of control mice had a CIA index ≥ 7, only 23% of WBM and 29% of SM mice did (P = 0.1). Although both types of mushrooms reduced plasma TNFα (34%, WBM; 64%, SM), only SM increased plasma IL-6 by 1.3-fold (P < 0.05). The CIA index was positively correlated with sICAM1 (r = 0.55; P < 0.05) but negatively correlated with TNFα (r = 0.34; P < 0.05). Whether mushrooms are beneficial for arthritis management remains to be investigated. To our knowledge, this is the first report demonstrating a possible health benefit of WBM in arthritis treatment.