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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

The cardiac copper chaperone proteins Sco1 and CCS are up-regulated, but Cox 1 and Cox4 are down-regulated, by copper deficiency.

Copper is ferried in a cell complexed to chaperone proteins, and in the heart much copper is required for cytochrome c oxidase (Cox). It is not completely understood how copper status affects the levels of these proteins. Here we determined if dietary copper deficiency could up- or down-regulate select copper chaperone proteins and Cox subunits 1 and 4 in cardiac tissue of rats. Sixteen weanling male Long-Evans rats were randomized into treatment groups, one group receiving a copper-deficient diet (<1 mg Cu/kg diet) and one group receiving a diet containing adequate copper (6 mg Cu/kg diet) for 5 weeks. Hearts were removed, weighed, and non-myofibrillar proteins separated to analyze for levels of CCS, Sco1, Ctr1, Cox17, Cox1, and Cox4 by SDS-PAGE and Western blotting. No changes were observed in the concentrations of CTR1 and Cox17 between copper-adequate and copper-deficient rats. CCS and Sco1 were up-regulated and Cox1 and Cox4 were both down-regulated as a result of copper deficiency. These data suggest that select chaperone proteins and may be up-regulated, and Cox1 and 4 down-regulated, by a dietary copper deficiency, whereas others appear not to be affected by copper status.

Mitochondrial and sarcoplasmic protein changes in hearts from copper-deficient rats: up-regulation of PGC-1alpha transcript and protein as a cause for mitochondrial biogenesis in copper deficiency.

Changes in mitochondrial and sarcoplasmic proteins using proteinomics and Western blotting in hearts from copper-deficient rats were explored in this study. Also, key enzymes that are involved in cardiac energy metabolism via glycolysis and fatty acid oxidation and related transcription factors were determined. Rats were fed one of two diets: a copper-adequate diet containing 6 mg Cu/kg diet or a diet with less than 1 mg Cu/kg diet for 5 weeks. Copper deficiency was confirmed by low liver copper levels, decreased hematocrit levels and cardiac hypertrophy. Proteinomic data revealed that of the more than 50 proteins identified from the mitochondrial fraction of heart tissue, six were significantly down-regulated and nine were up-regulated. The proteins that were decreased were beta enolase 3, carbonic anhydrase 2, aldose reductase 1, glutathione peroxidase, muscle creatine kinase and mitochondrial aconitase 2. The proteins that were up-regulated were isocitrate dehydrogenase, dihydrolipoamide dehydrogenase, transferrin, subunit d of ATP synthase, transthyretin, preproapolipoprotein A-1, GRP 75, alpha-B crystalline and heat shock protein alpha. Follow-up Western blots on rate-limiting enzymes in glycolysis (phosphofructose kinase), fatty acid oxidation (medium chain acyl dehydrogenase, peroxisome proliferator-actvator receptor-alpha or PPARalpha) and gluconeogenesis (phosphoenolpyruvate carboxykinase) did not reveal changes in metabolic enzymes. However, a significant increase in peroxisome proliferator-activated receptor-gamma coactivator (PGC)-1alpha protein, as well as the transcript, which increased 2.5-fold, was observed. It would appear that increased mitochondrial biogenesis known to occur in copper deficiency hearts is caused by an increased expression in the master regulator of mitochondrial biogenesis, PGC-1alpha.

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.

Transcriptional coactivators PGC-1alpha and PGC-lbeta control overlapping programs required for perinatal maturation of the heart.

Oxidative tissues such as heart undergo a dramatic perinatal mitochondrial biogenesis to meet the high-energy demands after birth. PPARgamma coactivator-1 (PGC-1) alpha and beta have been implicated in the transcriptional control of cellular energy metabolism. Mice with combined deficiency of PGC-1alpha and PGC-1beta (PGC-1alphabeta(-/-) mice) were generated to investigate the convergence of their functions in vivo. The phenotype of PGC-1beta(-/-) mice was minimal under nonstressed conditions, including normal heart function, similar to that of PGC-1alpha(-/-) mice generated previously. In striking contrast to the singly deficient PGC-1 lines, PGC-1alphabeta(-/-) mice died shortly after birth with small hearts, bradycardia, intermittent heart block, and a markedly reduced cardiac output. Cardiac-specific ablation of the PGC-1beta gene on a PGC-1alpha-deficient background phenocopied the generalized PGC-1alphabeta(-/-) mice. The hearts of the PGC-1alphabeta(-/-) mice exhibited signatures of a maturational defect including reduced growth, a late fetal arrest in mitochondrial biogenesis, and persistence of a fetal pattern of gene expression. Brown adipose tissue (BAT) of PGC-1alphabeta(-/-) mice also exhibited a severe abnormality in function and mitochondrial density. We conclude that PGC-1alpha and PGC-1beta share roles that collectively are necessary for the postnatal metabolic and functional maturation of heart and BAT.

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.