Altering pyrroloquinoline quinone nutritional status modulates mitochondrial, lipid, and energy metabolism in rats

We have reported that pyrroloquinoline quinone (PQQ) improves reproduction, neonatal development, and mitochondrial function in animals by mechanisms that involve mitochondrial related cell signaling pathways. To extend these observations, the influence of PQQ on energy and lipid relationships and apparent protection against ischemia reperfusion injury are described herein. Sprague-Dawley rats were fed a nutritionally complete diet with PQQ added at either 0 (PQQ-) or 2 mg PQQ/Kg diet (PQQ+). Measurements included: 1) serum glucose and insulin, 2) total energy expenditure per metabolic body size (Wt(3/4)), 3) respiratory quotients (in the fed and fasted states), 4) changes in plasma lipids, 5) the relative mitochondrial amount in liver and heart, and 6) indices related to cardiac ischemia. For the latter, rats (PQQ- or PQQ+) were subjected to left anterior descending occlusions followed by 2 h of reperfusion to determine PQQ's influence on infarct size and myocardial tissue levels of malondialdehyde, an indicator of lipid peroxidation. Although no striking differences in serum glucose, insulin, and free fatty acid levels were observed, energy expenditure was lower in PQQ- vs. PQQ+ rats and energy expenditure (fed state) was correlated with the hepatic mitochondrial content. Elevations in plasma di- and triacylglyceride and β-hydroxybutryic acid concentrations were also observed in PQQ- rats vs. PQQ+ rats. Moreover, PQQ administration (i.p. at 4.5 mg/kg BW for 3 days) resulted in a greater than 2-fold decrease in plasma triglycerides during a 6-hour fast than saline administration in a rat model of type 2 diabetes. Cardiac injury resulting from ischemia/reperfusion was more pronounced in PQQ- rats than in PQQ+ rats. Collectively, these data demonstrate that PQQ deficiency impacts a number of parameters related to normal mitochondrial function.     

Pyrroloquinoline quinone preserves mitochondrial function and prevents oxidative injury in adult rat cardiac myocytes

We investigated the ability of pyrroloquinoline quinone (PQQ) to confer resistance to acute oxidative stress in freshly isolated adult male rat cardiomyocytes. Fluorescence microscopy was used to detect generation of reactive oxygen species (ROS) and mitochondrial membrane potential (Deltapsi(m)) depolarization induced by hydrogen peroxide. H(2)O(2) caused substantial cell death, which was significantly reduced by preincubation with PQQ. H(2)O(2) also caused an increase in cellular ROS levels as detected by the fluorescent indicators CM-H2XRos and dihydroethidium. ROS levels were significantly reduced by a superoxide dismutase mimetic Mn (III) tetrakis (4-benzoic acid) porphyrin chloride (MnTBAP) or by PQQ treatment. Cyclosporine-A, which inhibits mitochondrial permeability transition, prevented H(2)O(2)-induced Deltapsi(m) depolarization, as did PQQ and MnTBAP. Our results provide direct evidence that PQQ reduces oxidative stress, mitochondrial dysfunction, and cell death in isolated adult rat cardiomyocytes. These findings provide new insight into the mechanisms of PQQ action in the heart.     

Depletion of mitochondrial DNA alters glucose metabolism in SK-Hep1 cells

Maternally inherited mitochondrial DNA (mtDNA) has been suggested to be a genetic factor for diabetes. Reports have shown a decrease of mtDNA content in tissues of diabetic patients. We investigated the effects of mtDNA depletion on glucose metabolism by use of rho(0) SK-Hep1 human hepatoma cells, whose mtDNA was depleted by long-term exposure to ethidium bromide. The rho(0) cells failed to hyperpolarize mitochondrial membrane potential in response to glucose stimulation. Intracellular ATP content, glucose-stimulated ATP production, glucose uptake, steady-state mRNA and protein levels of glucose transporters, and cellular activities of glucose-metabolizing enzymes were decreased in rho(0) cells compared with parental rho(+) cells. Our results suggest that the quantitative reduction of mtDNA may suppress the expression of nuclear DNA-encoded glucose transporters and enzymes of glucose metabolism. Thus this may lead to diabetic status, such as decreased ATP production and glucose utilization.     

Pyrroloquinoline quinone attenuates iNOS gene expression in the injured spinal cord

Pyrroloquinoline quinone (PQQ) is a naturally occurring redox cofactor that acts as an essential nutrient, antioxidant, and redox modulator. PQQ has been demonstrated to oxidize the redox modulatory site of N-methyl-d-aspartic acid (NMDA) receptors. Such agents are known to be neuroprotective in experimental stroke models. Therefore, we examined the possible ameliorating effect of PQQ on spinal cord injury (SCI) in adult rats. Intraperitoneal administration of PQQ effectively promoted the functional recovery of SCI rats after hemi-transection, which was preceded by the attenuation of the expression of inducible nitric oxide (NO) synthase (iNOS) mRNA in the injury site. NO is involved in the secondary detrimental mechanisms and has been implicated in NMDA receptor-mediated neurotoxicity. In fact, administration of PQQ induced significantly decreased lesion size and increased axon density adjoining the lesion area. These observations suggest that PQQ protects against the secondary damage by reducing iNOS expression following primary physical injury to the spinal cord.     

Dependence with nutritional status of the ethanol effects on fatty acid metabolism in rat hepatocytes

1. The effects of ethanol on fatty acid synthesis, esterification and oxidation were studied in hepatocytes isolated from fed and 24 hr fasted rats. 2. [3H]H2O was preferentially incorporated into the glycerol backbone of triglycerides and phospholipids. Addition of ethanol markedly increased the incorporation of this label in both classes of glycerolipids; the increase was higher in fasted rat hepatocytes, both in the glycerol backbone and acyl groups of glycerolipids. 3. Ethanol increased [U-14C]palmitate incorporation into triglycerides only in hepatocytes from fasted rats. 4. [14C]CO2 and total acid soluble product formation from [1-14C]palmitate resulted inhibited by ethanol both in the fed and the fasted state.     

Tolbutamide alters glucose transport and metabolism in the embryonic mouse heart

BACKGROUND: Tolbutamide is a sulfonylurea oral hypoglycemic agent widely used for the treatment of non insulin-dependent diabetes mellitus. Tolbutamide produces dysmorphogenesis in rodent embryos and becomes concentrated in the embryonic heart after maternal oral dosing. Tolbutamide increases glucose metabolism in extra-pancreatic adult tissues, but this has not previously been examined in embryonic heart. METHODS: CD-1 mouse embryos were exposed on GD 9.5 to tolbutamide (0, 100, 250, or 500 microg/ml) for 6, 12, or 24 hr in whole-embryo culture. Isolated hearts were evaluated for (3)H-2DG uptake and conversion of (14)C-glucose to (14)C-lactate. Glut-1, HKI, and GRP78 protein levels were determined by Western analysis, and Glut-1 mRNA was measured by RT-PCR. RESULTS: Cardiac (3)H-2DG uptake increased after exposure to 500 microg/ml tolbutamide for 6 hr, and 100, 250, or 500 microg/ml tolbutamide for 24 hr, compared to controls. Glycolysis increased after exposure to 500 microg/ml tolbutamide for 6 or 24 hr compared to controls. Glut-1 protein levels increased in hearts exposed to 500 microg/ml tolbutamide for 12 or 24 hr, and Glut-1 mRNA increased in hearts exposed to 500 microg/ml tolbutamide for 24 hr compared to controls. HKI protein levels increased in hearts exposed to 500 microg/ml tolbutamide for 6 hr, but not 12 or 24 hr. There was no effect on GRP78 protein levels in hearts exposed to tolbutamide for 6, 12, or 24 hr. CONCLUSIONS: Tolbutamide stimulates glucose uptake and metabolism in the embryonic heart, as occurs in adult extra-pancreatic tissues. Glut-1 and HKI, but not GRP78, are likely involved in tolbutamide-induced cardiac dysmorphogenesis.     

Revisiting the mouse mitochondrial DNA sequence

The existence of reliable mtDNA reference sequences for each species is of great relevance in a variety of fields, from phylogenetic and population genetics studies to pathogenetic determination of mtDNA variants in humans or in animal models of mtDNA-linked diseases. We present compelling evidence for the existence of sequencing errors on the current mouse mtDNA reference sequence. This includes the deletion of a full codon in two genes, the substitution of one amino acid on five occasions and also the involvement of tRNA and rRNA genes. The conclusions are supported by: (i) the re-sequencing of the original cell line used by Bibb and Clayton, the LA9 cell line, (ii) the sequencing of a second L-derivative clone (L929), and (iii) the comparison with 12 other mtDNA sequences from live mice, 10 of them maternally related with the mouse from which the L cells were generated. Two of the latest sequences are reported for the first time in this study (Balb/cJ and C57BL/6J). In addition, we found that both the LA9 and L929 mtDNAs also contain private clone polymorphic variants that, at least in the case of L929, promote functional impairment of the oxidative phosphorylation system. Conse quently, the mtDNA of the strain used for the mouse genome project (C57BL/6J) is proposed as the new standard for the mouse mtDNA sequence.     

DNA content of microcells prepared from rat kangaroo and mouse cells

Enucleation of animal cells in which nuclear fragmentation (micronucleation) has been induced by treatment with mitotic inhibitors results in the formation of subdiploid microcells consisting of one or several micronuclei, some cytoplasm and surrounded by a plasma membrane. Microcells were prepared from rat kangaroo cells (12 chromosomes) and a polyoma virus transformed mouse cell line (61 chromosomes) and analysed for DNA content. Microspectrophotometric DNA measurements and the appearance of micronuclei at mitosis show that small micronuclei contain genetic information equivalent to single chromosomes. A large proportion of the micronuclei and the microcells, however, contains DNA corresponding to several chromosomes. Heterogeneous mixtures of microcells can be fractionated by a unit gravity sedimentation procedure so as to isolate the small microcells. These can afterwards be fused with intact normal or mutant cells.     

Nuclear and mitochondrial DNA synthesis and energy metabolism in primary rat glial cell cultures

DNA synthesis in nuclei and mitochondria purified from serum-supplemented rat glial cell cultures at different days after plating was studied. Furthermore in mitochondria, some enzymatic activities related to energy transduction (citrate synthase, malate dehydrogenase, total NADH-cytochromec reductase, cytochrome oxidase and glutamate dehydrogenase) were measured. For DNA labeling [methyl-³H]thymidine was added to the culture medium at different days after plating. During the culture times studied the specific activity of total, nuclear, and mitochondrial DNA decreased from 8 days in vitro (DIV) to 21 DIV and increased at 30 DIV. The specific activity of nuclear DNA was always higher than that of mitochondrial DNA. The specific activity of the above mentioned mitochondrial enzymes increased from 8 DIV up to 21 DIV and decreased at 30 DIV, suggesting a relationship between the energy metabolism and the differentiation of glial cells in culture.The AA. would like to dedicate this paper to the memory of Dr. Ida Serra, Associate Professor of Biochemistry at the Medical Faculty, University of Catania, who prematurely died, after this paper was submitted for publication.     

Embryo quality,and not chromosome nondiploidy,affects mitochondrial DNA content in mouse blastocysts

It has been shown recently that there is premature mitochondria biosynthesis in blastocysts from older women whose egg or embryo quality is poor and that aneuploid blastocysts also have a high number of mitochondrial DNA (mtDNA) copies. Whether nondiploidy/aneuploidy or reduced egg or embryo quality causes premature mitochondrial biosynthesis is not known. This study constructed haploid, diploid, triploid, and tetraploid blastocysts by parthenogenetic activation, intracytoplasmic sperm injection with one or two sperm heads, blastomere electrofusion, respectively, and generated reduced cytoplasm quality embryos from diabetic mouse and in vitro fertilization of aged oocytes, and examined whether nondiploidy or reduced cytoplasm quality causes premature mitochondrial biosynthesis. MtDNA numbers of each blastocyst from different models were tested by absolute quantitative real-time polymerase chain reaction. It was found that mtDNA content in preimplantation embryos was not associated with their chromosome ploidy, while mtDNA copy numbers in embryos with suboptimal quality were increased. Therefore, it might be the reduced cytoplasmic quality, and not chromosome nondiploidy, that causes premature mitochondria biosynthesis in blastocysts.     

Downhill exercise alters immunoproteasome content in mouse skeletal muscle

Content of the immunoproteasome, the inducible form of the standard proteasome, increases in atrophic muscle suggesting it may be associated with skeletal muscle remodeling. However, it remains unknown if the immunoproteasome responds to stressful situations that do not promote large perturbations in skeletal muscle proteolysis. The purpose of this study was to determine how an acute bout of muscular stress influences immunoproteasome content. To accomplish this, wild-type (WT) and immunoproteasome knockout lmp7 ^?/? /mecl1 ^?/? (L7M1) mice were run downhill on a motorized treadmill. Soleus muscles were excised 1 and 3 days post-exercise and compared to unexercised muscle (control). Ex vivo physiology, histology and biochemical analyses were used to assess the effects of immunoproteasome knockout and unaccustomed exercise. Besides L7M1 muscle being LMP7/MECL1 deficient, no other major biochemical, histological or functional differences were observed between the control muscles. In both strains, the downhill run shifted the force-frequency curve to the right and reduced twitch force; however, it did not alter tetanic force or inflammatory markers. In the days post-exercise, several of the proteasome’s catalytic subunits were upregulated. Specifically, WT muscle increased LMP7 while L7M1 muscle instead increased β5. These findings indicate that running mice downhill results in subtle contractile characteristics that correspond to skeletal muscle injury, yet it does not appear to induce a significant inflammatory response. Interestingly, this minor stress activated the production of specific immunoproteasome subunits that if knocked out were replaced by components of the standard proteasome. These data suggest that the immunoproteasome may be involved in maintaining cellular homeostasis.     

Pyrroloquinoline quinone improves growth and reproductive performance in mice fed chemically defined diets

Growth, reproductive performance, and indices of collagen maturation and expression were investigated in Balb/c mice fed chemically defined, amino acid-based diets with or without the addition 6 micro Mpyrroloquinoline quinone (PQQ)/kg diet. The diets were fed to virgin mice for 8 weeks before breeding. At weaning, the pups from successful pregnancies were fed the same diet as their respective dams. Reproductive performance was compromised in mice fed diets devoid of PQQ, and their offspring grew at slower rates than offspring from mice fed diets supplemented with PQQ. Successful mating (confirmed vaginal plugs) was not affected by the presence or absence of PQQ; however, pup viability (number of pups at parturition/number of pups at Day 4 of lactation) was decreased in PQQ-deprived mice. Conception (percentage of females giving live births) and fertility (percentage of births) were also decreased in PQQ-deprived mice. The slower rates of growth in offspring from PQQ-deprived mice were associated with decreased steady-state mRNA levels for Type I procollagen alpha(1)-chains in skin and lungs from neonatal mice. Values for lysyl oxidase accumulation as protein in PQQ-deficient mice also tended to be lower than corresponding values from PQQ-supplemented or -replete mice. Skin collagen solubility was increased in PQQ-deprived mice. These results indicate that PQQ supplementation can improve reproductive performance, growth, and may modulate indices of neonatal extracellular matrix production and maturation in mice fed chemically defined, but otherwise nutritionally complete diets.     

Pyrroloquinoline quinone (coenzyme PQQ) and the oxidation of SH residues in proteins.

Pyrroloquinoline quinone (PQQ) catalyzes the oxidation of cysteamine at neutral pH with a second order rate constant K2 = 0.45 M-1 s-1. The reduction of PQQ was monitored by absorption and fluorescence spectroscopy, whereas the oxidation of cysteamine to cystamine was followed by titration with 5,5'-dithiobis(2-nitrobenzoic acid). PQQ also catalyzes the oxidation of thiol groups critically connected with the function of two proteins, i.e. thioredoxin and phosphoribulose kinase. The reaction of PQQ with reduced thioredoxin brings about the oxidation of two thiol groups of the oxireductase, whereas the enzyme phosphoribulose kinase is inactivated at 25 degrees C. The oxidized disulfide bond of phosphoribulose kinase is reduced by dithiothreitol and the enzyme recovers catalytic activity. The ability of PQQ to catalyze the oxidation of vicinal cysteinyl residues to generate disulfide bonds under mild experimental conditions can be exploited to define the precise role of modified thiol residues in either catalysis or stabilization of protein structure.     

Dietary pyrroloquinoline quinone (PQQ) alters indicators of inflammation and mitochondrial-related metabolism in human subjects

Pyrroloquinoline quinone (PQQ) influences energy-related metabolism and neurologic functions in animals. The mechanism of action involves interactions with cell signaling pathways and mitochondrial function. However, little is known about the response to PQQ in humans. Using a crossover study design, 10 subjects (5 females, 5 males) ingested PQQ added to a fruit-flavored drink in two separate studies. In study 1, PQQ was given in a single dose (0.2 mg PQQ/kg). Multiple measurements of plasma and urine PQQ levels and changes in antioxidant potential [based on total peroxyl radical-trapping potential and thiobarbituric acid reactive product (TBAR) assays] were made throughout the period of 48 h. In study 2, PQQ was administered as a daily dose (0.3 mg PQQ/kg). After 76 h, measurements included indices of inflammation [plasma C-reactive protein, interleukin (IL)-6 levels], standard clinical indices (e.g., cholesterol, glucose, high-density lipoprotein, low-density lipoprotein, triglycerides, etc.) and ¹H-nuclear magnetic resonance estimates of urinary metabolites related in part to oxidative metabolism. The standard clinical indices were normal and not altered by PQQ supplementation. However, dietary PQQ exposure (Study 1) resulted in apparent changes in antioxidant potential based on malonaldehyde-related TBAR assessments. In Study 2, PQQ supplementation resulted in significant decreases in the levels of plasma C-reactive protein, IL-6 and urinary methylated amines such as trimethylamine N-oxide, and changes in urinary metabolites consistent with enhanced mitochondria-related functions. The data are among the first to link systemic effects of PQQ in animals to corresponding effects in humans.     

Gut colonization by Proteobacteria alters host metabolism and modulates cocaine neurobehavioral responses

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