The microbiome as a major function of the gastrointestinal tract and its implication in micronutrient metabolism and chronic diseases.

The composition and function of microbes harbored in the human gastrointestinal lumen have been underestimated for centuries because of the underdevelopment of nucleotide sequencing techniques and the lack of humanized gnotobiotic models. Now, we appreciate that the gut microbiome is an integral part of the human body and exerts considerable roles in host health and diseases. Dietary factors can induce changes in the microbial community composition, metabolism, and function, thereby altering the host immune response, and consequently, may influence disease risks. An imbalance of gut microbiome homeostasis (i.e., dysbiosis) has been linked to several chronic diseases, such as inflammatory bowel diseases, obesity, and diabetes. Remarkable progress has recently been made in better understanding the extent to which the influence of the diet-microbiota interaction on host health outcomes in both animal models and human participants. However, the exact causality of the gut microbiome on the development of diseases is still controversial. In this review, we will briefly describe the general structure and function of the intestine and the process of nutrient absorption in humans. This is followed by a summarization of the recent updates on interactions between gut microbiota and individual micronutrients, including carotenoids, vitamin A, vitamin D, vitamin C, folate, iron, and zinc. In the opinion of the authors, these nutrients were identified as representative of vitamins and minerals with sufficient research on their roles in the microbiome. The host responses to the gut microbiome will also be discussed. Future direction in microbiome research, for example, precision microbiome, will be proposed.

The transcriptional coactivators, PGC-1α and ß, cooperate to maintain cardiac mitochondrial function during the early stages of insulin resistance.

We previously demonstrated a cardiac mitochondrial biogenic response in insulin resistant mice that requires the nuclear receptor transcription factor PPARα. We hypothesized that the PPARα coactivator peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) is necessary for mitochondrial biogenesis in insulin resistant hearts and that this response was adaptive. Mitochondrial phenotype was assessed in insulin resistant mouse models in wild-type (WT) versus PGC-1α deficient (PGC-1α(-/-)) backgrounds. Both high fat-fed (HFD) WT and 6 week-old Ob/Ob animals exhibited a significant increase in myocardial mitochondrial volume density compared to standard chow fed or WT controls. In contrast, HFD PGC-1α(-/-) and Ob/Ob-PGC-1α(-/-) hearts lacked a mitochondrial biogenic response. PGC-1α gene expression was increased in 6 week-old Ob/Ob animals, followed by a decline in 8 week-old Ob/Ob animals with more severe glucose intolerance. Mitochondrial respiratory function was increased in 6 week-old Ob/Ob animals, but not in Ob/Ob-PGC-1α(-/-) mice and not in 8 week-old Ob/Ob animals, suggesting a loss of the early adaptive response, consistent with the loss of PGC-1α upregulation. Animals that were deficient for PGC-1α and heterozygous for the related coactivator PGC-1ß (PGC-1α(-/-)ß(+/-)) were bred to the Ob/Ob mice. Ob/Ob-PGC-1α(-/-)ß(+/-) hearts exhibited dramatically reduced mitochondrial respiratory capacity. Finally, the mitochondrial biogenic response was triggered in H9C2 myotubes by exposure to oleate, an effect that was blunted with shRNA-mediated PGC-1 "knockdown". We conclude that PGC-1 signaling is important for the adaptive cardiac mitochondrial biogenic response that occurs during the early stages of insulin resistance. This response occurs in a cell autonomous manner and likely involves exposure to high levels of free fatty acids.

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.

ß-carotene oxygenase 2 deficiency-triggered mitochondrial oxidative stress promotes low-grade inflammation and metabolic dysfunction.

Low-grade inflammation is a critical pathological factor contributing to the development of metabolic disorders. ß-carotene oxygenase 2 (BCO2) was initially identified as an enzyme catalyzing carotenoids in the inner mitochondrial membrane. Mutations in BCO2 are associated with inflammation and metabolic disorders in humans, yet the underlying mechanisms remain unknown. Here, we used loss-of-function approaches in mice and cell culture models to investigate the role of BCO2 in inflammation and metabolic dysfunction. We demonstrated decreases in BCO2 mRNA and protein levels and suppression of mitochondrial respiratory complex I proteins and mitochondrial superoxide dismutase levels in the liver of type 2 diabetic human subjects. Deficiency of BCO2 caused disruption of assembly of the mitochondrial respiratory supercomplexes, such as supercomplex III2+IV in mice, and overproduction of superoxide radicals in primary mouse embryonic fibroblasts. Further, deficiency of BCO2 increased protein carbonylation and populations of natural killer cells and M1 macrophages, and decreased populations of T cells, including CD4+ and/or CD8+ in the bone marrow and white adipose tissues. Elevation of plasma inflammatory cytokines and adipose tissue hypertrophy and inflammation were also characterized in BCO2 deficient mice. Moreover, BCO2 deficient mice were more susceptible to high-fat diet-induced obesity and hyperglycemia. Double knockout of BCO2 and leptin receptor genes caused a significantly greater elevation of the fasting blood glucose level in mice at 4 weeks of age, compared to the age- and sex-matched leptin receptor knockout. Finally, administration of Mito-TEMPO, a mitochondrial specific antioxidant attenuated systemic low-grade inflammation induced by BCO2 deficiency. Collectively, these findings suggest that BCO2 is essential for mitochondrial respiration and metabolic homeostasis in mammals. Loss or decreased expression of BCO2 leads to mitochondrial oxidative stress, low-grade inflammation, and the subsequent development of metabolic disorders.

Assessing mitochondria biogenesis.

Mitochondria have their own DNA (mtDNA) and hence biogenesis of mitochondria requires a coordination of nuclear and mtDNA, both of which encode for mitochondria proteins. Our understanding of the molecular control of mitochondria biogenesis has increased in recent years, providing key signatures of the process. To determine whether or not a tissue or an organ of human or animal origin is undergoing mitochondria biogenesis, multiple parameters should be analyzed. First and foremost is visualization and measurement of mitochondria mass/volume in histological sections using fluorescent mitochondria dyes and light microscopy or transmission electron microscopy to yield quantitative results. To confirm or extend these types of analysis, biochemical markers of mitochondria biogenesis should also be included, including assessment of mtDNA copy number, steady-state levels of biogenesis-related transcription factors (e.g. mitochondria transcription factor A, mitochondrial transcription specificity factors, nuclear respiratory factors 1 and 2, and peroxisome proliferator activated receptor gamma coactivator-1-alpha), mtDNA-encoded transcripts and proteins, and rates of mitochondria translation. These techniques are described in isolation and in the context of transgenic and dietary animal models that have been used as tools to study the regulation of mitochondria biogenesis and its role in disease pathology.

Insulin-resistant heart exhibits a mitochondrial biogenic response driven by the peroxisome proliferator-activated receptor-alpha/PGC-1alpha gene regulatory pathway.

BACKGROUND: Obesity and diabetes mellitus are complex metabolic problems of pandemic proportion, contributing to significant cardiovascular mortality. Recent studies have shown altered mitochondrial function in the hearts of diabetic animals. We hypothesized that regulatory events involved in the control of mitochondrial function are activated in the prediabetic, insulin-resistant stage. METHODS AND RESULTS: Morphometric analyses demonstrated that cardiac myocyte mitochondrial volume density was increased in insulin-resistant uncoupling protein-diphtheria toxin A (UCP-DTA) transgenic mice, a murine model of metabolic syndrome, compared with littermate controls. Mitochondrial DNA content and expression of genes involved in multiple mitochondrial pathways were also increased in insulin-resistant UCP-DTA hearts. The nuclear receptor, peroxisome proliferator-activated receptor-alpha (PPARalpha), is known to activate metabolic genes in the diabetic heart. Therefore, we evaluated the role of PPARalpha in the observed mitochondrial biogenesis response in the insulin-resistant heart. Insulin-resistant UCP-DTA mice crossed into a PPARalpha-null background did not exhibit evidence of mitochondrial biogenesis or induction of mitochondrial gene expression. Conversely, transgenic mice with cardiac-specific overexpression of PPARalpha exhibited signatures of cardiac mitochondrial biogenesis. A screen for candidate mediators of the PPARalpha-driven mitochondrial biogenic response revealed that expression of PPARgamma coactivator-1alpha (PGC-1alpha), a known regulator of mitochondrial biogenesis, was activated in wild-type UCP-DTA mice but not in PPARalpha-deficient UCP-DTA mice. CONCLUSIONS: These results demonstrate that mitochondrial biogenesis occurs early in the development of diabetic cardiac dysfunction through a transcriptional regulatory circuit that involves activation of PGC-1alpha gene expression by the fatty acid-activated nuclear receptor PPARalpha.

PGC-1alpha deficiency causes multi-system energy metabolic derangements: muscle dysfunction, abnormal weight control and hepatic steatosis.

The gene encoding the transcriptional coactivator peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha) was targeted in mice. PGC-1alpha null (PGC-1alpha(-/-)) mice were viable. However, extensive phenotyping revealed multi-system abnormalities indicative of an abnormal energy metabolic phenotype. The postnatal growth of heart and slow-twitch skeletal muscle, organs with high mitochondrial energy demands, is blunted in PGC-1alpha(-/-) mice. With age, the PGC-1alpha(-/-) mice develop abnormally increased body fat, a phenotype that is more severe in females. Mitochondrial number and respiratory capacity is diminished in slow-twitch skeletal muscle of PGC-1alpha(-/-) mice, leading to reduced muscle performance and exercise capacity. PGC-1alpha(-/-) mice exhibit a modest diminution in cardiac function related largely to abnormal control of heart rate. The PGC-1alpha(-/-) mice were unable to maintain core body temperature following exposure to cold, consistent with an altered thermogenic response. Following short-term starvation, PGC-1alpha(-/-) mice develop hepatic steatosis due to a combination of reduced mitochondrial respiratory capacity and an increased expression of lipogenic genes. Surprisingly, PGC-1alpha(-/-) mice were less susceptible to diet-induced insulin resistance than wild-type controls. Lastly, vacuolar lesions were detected in the central nervous system of PGC-1alpha(-/-) mice. These results demonstrate that PGC-1alpha is necessary for appropriate adaptation to the metabolic and physiologic stressors of postnatal life.

Differential expression of genes involved with apoptosis, cell cycle, connective tissue proteins, fuel substrate utilization, inflammation and mitochondrial biogenesis in copper-deficient rat hearts: implication of a role for Nfkappab1.

We hypothesized that the increase in mitochondrial proliferation in hearts from copper-deficient rats is due to an increase in expression of the transcriptional factor peroxisomal-like proliferating related coactivator 1alpha (Ppargc1a), which regulates transcriptional activity for many of the genes that encode for mitochondrial proteins. In addition to several transcriptional factors implicated in mitochondrial biogenesis, we also looked at a number of genes involved in cell cycle regulation and fuel substrate utilization. Long-Evans rats were placed on either a copper-adequate (n=4) or copper-deficient (n=4) diet 3 days post weaning and remained on the diet for 5 weeks; their copper deficiency status was confirmed using previously established assays. Custom oligo arrays spotted with genes pertinent to mitochondrial biogenesis were hybridized with cRNA probes synthesized from the collected heart tissue. Chemiluminescent array images from both groups were analyzed for gene spot intensities and differential gene expression. Our results did not demonstrate any significant increase in Ppargc1a or its implicated targets, as we had predicted. However, consistent with previous data, an up-regulation of genes that encode for collagen type 3, fibronectin and elastin were found. Interestingly, there was also a significant increase in the expression of the transcriptional factor nuclear factor kappaB1 (Nfkappab1) in the copper-deficient treatment animals, compared to the control group, and this was confirmed by real time quantitative polymerase chain reaction. The results of this study merit the further investigation of the role of reactive oxidative species with regard to Nfkappab1 in the copper deficient rat heart.

Perspectives on the Role and Relevance of Copper in Cardiac Disease.

Cardiac hypertrophy as a result of dietary copper deficiency has been studied for 40 plus years and is the subject of this review. While connective tissue anomalies occur, a hallmark pathology is cardiac hypertrophy, increased mitochondrial biogenesis, with disruptive cristae, vacuolization of mitochondria, and deposition of lipid droplets. Electrocardiogram abnormalities have been demonstrated along with biochemical changes especially as it relates to the copper-containing enzyme cytochrome c oxidase. The master controller of mitochondrial biogenesis, PGC1-α expression and protein, along with other proteins and transcriptional factors that play a role are upregulated. Nitric oxide, vascular endothelial growth factor, and cytochrome c oxidase all may enhance the upregulation of mitochondrial biogenesis. Marginal copper intakes reveal similar pathologies in the absence of cardiac hypertrophy. Reversibility of the copper-deficient rat heart with a copper-replete diet has resulted in mixed results, depending on both the animal model used and temporal relationships. New information has revealed that copper supplementation may rescue cardiac hypertrophy induced by pressure overload.

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.

Targeted Metabolomics Reveals Abnormal Hepatic Energy Metabolism by Depletion of ß-Carotene Oxygenase 2 in Mice.

ß-carotene oxygenase 2 (BCO2) is a carotenoid cleavage enzyme located in the inner mitochondrial membrane. Ablation of BCO2 impairs mitochondrial function leading to oxidative stress. Herein, we performed a targeted metabolomics study using ultrahigh performance liquid chromatography-tandem mass spectroscopy and gas chromatography-mass spectroscopy to discriminate global metabolites profiles in liver samples from six-week-old male BCO2 systemic knockout (KO), heterozygous (Het), and wild type (WT) mice fed a chow diet. Principal components analysis revealed distinct differences in metabolites in the livers of KO mice, compared to WT and Het mice. However, no marked difference was found in the metabolites of the Het mouse liver compared to the WT. We then conducted random forest analysis to classify the potential biomarkers to further elucidate the different metabolomics profiles. We found that systemic ablation of BCO2 led to perturbations in mitochondrial function and metabolism in the TCA cycle, amino acids, carnitine, lipids, and bile acids. In conclusion, BCO2 is essential to macronutrient and mitochondrial metabolism in the livers of mice. The ablation of BCO2 causes dysfunctional mitochondria and altered energy metabolism, which further leads to systemic oxidative stress and inflammation. A single functional copy of BCO2 largely rescues the hepatic metabolic homeostasis in mice.

Lack of ß, ß-carotene-9', 10'-oxygenase 2 leads to hepatic mitochondrial dysfunction and cellular oxidative stress in mice.

SCOPE: ß,ß-Carotene-9',10'-dioxygenase 2 (BCO2) is a carotenoid cleavage enzyme localized to the inner mitochondrial membrane in mammals. This study was aimed to assess the impact of genetic ablation of BCO2 on hepatic oxidative stress through mitochondrial function in mice. METHODS AND RESULTS: Liver samples from 6-wk-old male BCO2-/- knockout (KO) and isogenic wild-type (WT) mice were subjected to proteomics and functional activity assays. Compared to the WT, KO mice consumed more food (by 18%) yet displayed significantly lower body weight (by 12%). Mitochondrial proteomic results demonstrated that loss of BCO2 was associated with quantitative changes of the mitochondrial proteome mainly shown by suppressed expression of enzymes and/or proteins involved in fatty acid ß-oxidation, the tricarboxylic acid cycle, and the electron transport chain. The mitochondrial basal respiratory rate, proton leak, and electron transport chain complex II capacity were significantly elevated in the livers of KO compared to WT mice. Moreover, elevated reactive oxygen species and increased mitochondrial protein carbonylation were also demonstrated in liver of KO mice. CONCLUSIONS: Loss of BCO2 induces mitochondrial hyperactivation, mitochondrial stress, and changes of the mitochondrial proteome, leading to mitochondrial energy insufficiency. BCO2 appears to be critical for proper hepatic mitochondrial function.

Cardiac-specific induction of the transcriptional coactivator peroxisome proliferator-activated receptor gamma coactivator-1alpha promotes mitochondrial biogenesis and reversible cardiomyopathy in a developmental stage-dependent manner.

Recent evidence has identified the peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1alpha) as a regulator of cardiac energy metabolism and mitochondrial biogenesis. We describe the development of a transgenic system that permits inducible, cardiac-specific overexpression of PGC-1alpha. Expression of the PGC-1alpha transgene in this system (tet-on PGC-1alpha) is cardiac-specific in the presence of doxycycline (dox) and is not leaky in the absence of dox. Overexpression of PGC-1alpha in tet-on PGC-1alpha mice during the neonatal stages leads to a dramatic increase in cardiac mitochondrial number and size coincident with upregulation of gene markers associated with mitochondrial biogenesis. In contrast, overexpression of PGC-1alpha in the hearts of adult mice leads to a modest increase in mitochondrial number, derangements of mitochondrial ultrastructure, and development of cardiomyopathy. The cardiomyopathy in adult tet-on PGC-1alpha mice is characterized by an increase in ventricular mass and chamber dilatation. Surprisingly, removal of dox and cessation of PGC-1alpha overexpression in adult mice results in complete reversal of cardiac dysfunction within 4 weeks. These results indicate that PGC-1alpha drives mitochondrial biogenesis in a developmental stage-dependent manner permissive during the neonatal period. This unique murine model should prove useful for the study of the molecular regulatory programs governing mitochondrial biogenesis and characterization of the relationship between mitochondrial dysfunction and cardiomyopathy and as a general model of inducible, reversible cardiomyopathy.

Copper, iron, and selenium dietary deficiencies negatively impact skeletal integrity: A review.

Nutrients have been known to have a significant role in maintaining the health of the skeleton, both bone and cartilage. The nutrients that have received the majority of the attention are Vitamin D and calcium. However, limited attention has been directed toward three trace elements that may have mechanistic impact upon the skeletal tissues and could compromise skeletal health resulting from inadequate intakes of copper, iron, and selenium. The role of copper and selenium has been known, but the role of iron has only received recent attention. Copper deficiency is thought to impact bone health by a decrease in lysyl oxidase, a copper-containing enzyme, which facilitates collagen fibril crosslinking. Iron deficiency impact upon bone has only recently been discovered but the exact mechanism on how the deficient states enhance bone pathology is speculative. Selenium deficiency has an impact on cartilage thereby having an indirect impact on bone. However, several studies suggest that a mycotoxin when consumed by humans is the culprit in some cartilage disorders and the presence of selenium could attenuate the pathology. This review summarizes the current knowledge base with respect to skeletal integrity when each of these three trace elements are inadequate in diets of both animals and humans.

The P2Y2 receptor mediates uptake of matrix-retained and aggregated low density lipoprotein in primary vascular smooth muscle cells.

BACKGROUND AND AIMS: The internalization of aggregated low-density lipoproteins (agLDL) mediated by low-density lipoprotein receptor related protein (LRP1) may involve the actin cytoskeleton in ways that differ from the endocytosis of soluble LDL by the LDL receptor (LDLR). This study aims to define novel mechanisms of agLDL uptake through modulation of the actin cytoskeleton, to identify molecular targets involved in foam cell formation in vascular smooth muscle cells (VSMCs). The critical observation that formed the basis for these studies is that under pathophysiological conditions, nucleotide release from blood-derived and vascular cells activates SMC P2Y2 receptors (P2Y2Rs) leading to rearrangement of the actin cytoskeleton and cell motility. Therefore, we tested the hypothesis that P2Y2R activation mediates agLDL uptake by VSMCs. METHODS: Primary VSMCs were isolated from aortas of wild type (WT) C57BL/6 and.P2Y2R-/- mice to investigate whether P2Y2R activation modulates LRP1 expression. Cells were transiently transfected with cDNA encoding a hemagglutinin-tagged (HA-tagged) WT P2Y2R, or a mutant P2Y2R that unlike the WT P2Y2R does not bind the cytoskeletal actin-binding protein filamin-A (FLN-A). RESULTS: P2Y2R activation significantly increased agLDL uptake, and LRP1 mRNA expression decreased in P2Y2R-/- VSMCs versus WT. SMCs, expressing P2Y2R defective in FLN-A binding, exhibit 3-fold lower LDLR expression levels than SMCs expressing WT P2Y2R, while cells transfected with WT P2Y2R show greater agLDL uptake in both WT and P2Y2R-/- VSMCs versus cells transfected with the mutant P2Y2R. CONCLUSIONS: Together, these results show that both LRP1 and LDLR expression and agLDL uptake are regulated by P2Y2R in VSMCs, and that agLDL uptake due to P2Y2R activation is dependent upon cytoskeletal reorganization mediated by P2Y2R binding to FLN-A.

Molecular aspects of ß, ß-carotene-9', 10'-oxygenase 2 in carotenoid metabolism and diseases.

Carotenoids, the carotenes and xanthophylls, are essential components in human nutrition. ß, ß-carotene-9', 10'-oxygenase 2 (BCO2), also named as ß, ß-carotene-9', 10'-dioxygenase 2 (BCDO2) catalyzes the asymmetrical cleavage of carotenoids, whereas ß, ß-carotene-15, 15'-monooxygenase (BCMO1) conducts the symmetrical cleavage of pro-vitamin A carotenoids into retinoid. Unlike BCMO1, BCO2 has a broader substrate specificity and has been considered an alternative way to produce vitamin A. In contrast to BCMO1, a cytoplasmic protein, BCO2 is located in the inner mitochondrial membrane. The difference in cellular compartmentalization may reflect the different substrate specificity and physiological functions with respect to BCMO1 and BCO2. The BCO2 gene mutations are proven to be associated with yellow color of skin and fat tissue and milk in livestock. Mutation in intron 2 of BCO2 gene is also supposed to be related to the expression of IL-18, a pro-inflammatory cytokine associated with obesity, cardiovascular diseases, and type 2 diabetes. Further, BCO2 is associated with the development of mitochondrial oxidative stress, macular degeneration, anemia, and hepatic steatosis. This review of the literature will mostly address recent updates regarding the role of BCO2 in carotenoid metabolism, and discuss the potential impacts of BCO2 protein and the mutations in mammalian diseases.

Mitochondrial membrane potential is reduced in copper-deficient C2C12 cells in the absence of apoptosis.

Mitochondrial membrane potential is reduced in copper-deficient rat hearts, but it is uncertain if this will lead to the onset of apoptosis. To determine if copper deficiency per se leads to apoptosis, C2C12 cells were made copper deficient by treatment with the copper chelator tetraethylenepentamine (TEPA). In TEPA-treated cells, the activity of Cu, Zn-superoxide dismutase and cytochrome-c oxidase decreased dramatically. The protein levels of nuclear-encoded subunits of the cytochromie-c oxidase decreased, but the mitochondrial-encoded subunits remained unchanged. Decreased mitochondrial membrane potential was indicated in TEPA-treated cells, but further investigation of the potential induction of apoptosis by measuring caspase-3 activity, protein concentrations of Bcl-2 and Bax, and DNA fragmentation suggested that apoptosis is not induced in TEPA-treated C2C12 cells. Cells with decreased mitochondrial membrane potential were not destined to apoptosis as a result of copper deficiency.

Wolfberries potentiate mitophagy and enhance mitochondrial biogenesis leading to prevention of hepatic steatosis in obese mice: the role of AMP-activated protein kinase α2 subunit.

SCOPE: The aim of this study is to investigate whether AMP-activated protein kinase α2 (AMPKα2) is essential for wolfberry's protective effects on mitochondrial dysfunction and subsequent hepatic steatosis in mice. METHODS AND RESULTS: Six-week-old male AMPKα2 knockout mice and genetic background C57BL/6J (B6) mice were fed a control, high-fat diet (HD, 45% (kilocalorie) fat), and/or HD with 5% (kilocalarie) wolfberry diets for 18 wk. At termination, blood and liver tissues were sampled for analysis by ELISA, HPLC, microscopy, real-time PCR, and Western blot. HD lowered hepatic lutein and zeaxanthin contents, inhibited protein expression of ß,ß-carotene 9',10'-oxygenase 2 (BCO2) and heat shock protein 60 in mitochondria, increased reactive oxygen species level, and suppressed mitophagy and mitochondrial biogenesis as determined by accumulation of p62, inhibited phosphorylation of Unc-51-like kinase 1 on Ser555, and declined expression of peroxisome proliferator-activated receptor γ coactivator 1 α, resulting in hepatic steatosis in B6 and knockout mice. Dietary wolfberry elevated the xanthophyll concentrations and enhanced expression of BCO2 and heat shock protein 60, attenuated mitochondrial oxidative stress, activated AMPKα2, potentiated mitophagy and mitochondrial biogenesis, and enhanced lipid oxidation and secretion in the liver of B6 mice. CONCLUSION: Dietary wolfberry selectively activated AMPKα2, which resulted in enhanced mitochondrial biogenesis and potentiated mitophagy, leading to the prevention of hepatic steatosis in obese mice.

Dietary wolfberry upregulates carotenoid metabolic genes and enhances mitochondrial biogenesis in the retina of db/db diabetic mice.

SCOPE: Our aim was to investigate whether dietary wolfberry altered carotenoid metabolic gene expression and enhanced mitochondrial biogenesis in the retina of diabetic mice. METHODS AND RESULTS: Six-week-old male db/db and wild-type mice were fed the control or wolfberry diets for 8 weeks. At study termination, liver and retinal tissues were collected for analysis by transmission electron microscopy, real-time PCR, immunoprecipitation, Western blot, and HPLC. Wolfberry elevated zeaxanthin and lutein levels in the liver and retinal tissues and stimulated expression of retinal scavenger receptor class B type I, glutathione S-transferase Pi 1, and ß,ß-carotene 9',10'-oxygenase 2, and induced activation and nuclear enrichment of retinal AMP-activated protein kinase α2 (AMPK-α2). Furthermore, wolfberry attenuated hypoxia and mitochondrial stress as demonstrated by declined expression of hypoxia-inducible factor-1-α, vascular endothelial growth factor, and heat shock protein 60. Wolfberry enhanced retinal mitochondrial biogenesis in diabetic retinas as demonstrated by reversed mitochondrial dispersion in the retinal pigment epithelium, increased mitochondrial copy number, elevated citrate synthase activity, and upregulated expression of peroxisome proliferator-activated receptor γ co-activator 1α, nuclear respiratory factor 1, and mitochondrial transcription factor A. CONCLUSION: Consumption of dietary wolfberry could be beneficial to retinoprotection through reversal of mitochondrial function in diabetic mice.