Christopher Newell

Metabolomic Modeling To Monitor Host Responsiveness to Gut Microbiota Manipulation in the BTBRT+tf/j Mouse

The microbiota, the entirety of microorganisms residing in the gut, is increasingly recognized as an environmental factor in the maintenance of health and the development of disease. The objective of this analysis was to model in vivo interactions between gut microbiota and both serum and liver metabolites. Different genotypic models (C57BL/6 and BTBR(T+tf/j) mice) were studied in combination with significant dietary manipulations (chow vs ketogenic diets) to perturb the gut microbiota. Diet rather than genotype was the primary driver of microbial changes, with the ketogenic diet diminishing total bacterial levels. Fecal but not cecal microbiota profiles were associated with the serum and liver metabolomes. Modeling metabolome-microbiota interactions showed fecal Clostridium leptum to have the greatest impact on host metabolism, significantly correlating with 10 circulating metabolites, including 5 metabolites that did not correlate with any other microbes. C. leptum correlated negatively with serum ketones and positively with glucose and glutamine. Interestingly, microbial groups most strongly correlated with host metabolism were those modulating gut barrier function, the primary site of microbe-host interactions. These results show very robust relationships and provide a basis for future work wherein the compositional and functional associations of the microbiome can be modeled in the context of the metabolome.

Enhanced stem cell engraftment and modulation of hepatic reactive oxygen species production in diet-induced obesity

The impact of dietary‐induced obesity (DIO) on stem cell engraftment and the respective therapeutic potential of stem cell engraftment in DIO have not been reported. The objectives of this study were to examine the impact of DIO on the survival and efficacy of intravenous bone marrow‐derived mesenchymal stem cell (MSC) administration in the conscious C57BL/6 mouse.

Tissue Specific Impacts of a Ketogenic Diet on Mitochondrial Dynamics in the BTBRT+tf/j Mouse

The ketogenic diet (KD) has been utilized as a dietary therapeutic for nearly a century. One experimental model particularly responsive to the KD is the BTBRT+tf/j (BTBR) mouse, which displays phenotypic characteristics of autism spectrum disorder (ASD) and insulin resistance. Recently, the study of impaired mitochondrial function has become a focal point of research investigating the pathophysiology of ASD. As highly dynamic organelles, mitochondria undergo constant fluctuations in morphology, biogenesis, and quality control in order to maintain cellular homeostasis. An important modifier of mitochondrial dynamics is energy availability. Therefore, the aim of this study was to examine the impact of a KD on mitochondrial dynamics in the liver and brain (prefrontal cortex) of the BTBR mouse model of ASD. Juvenile male C57Bl/6 (B6) and BTBR mice were age-matched to 5 weeks of age before being fed standard chow (CD, 13% kcal fat) or a KD (75% kcal fat) for 10–14 days. Analysis of brain tissue identified differences in mitochondrial gene expression but no correlation with protein levels. Unlike in the brain, KD led to decreased levels of mitochondrial proteins in the liver, despite increased gene expression. Consistent with decreased mitochondrial proteins, we also observed decreased mtDNA for all mice on the KD, demonstrating that the KD reduces the total amount of mitochondria in the liver. In order to explain the discrepancy between protein levels and gene expression, we investigated whether mitochondrial turnover via mitophagy was increased. To this end, we examined expression levels of the mitophagy regulator BNIP3 (BCL2/adenovirus E1B 19 kd-interacting protein 3). BNIP3 gene and protein expression were significantly elevated in liver of KD animals (p < 0.05), indicating the potential activation of mitophagy. Therefore, consumption of a KD exerts highly tissue-specific effects, ultimately increasing mitochondrial turnover in the liver, while gene and protein expression in the brain remaining tightly regulated.

Ketogenic diet leads to O-GlcNAc modification in the BTBRT + tf/j mouse model of autism

BACKGROUND Protein O-linked-β-N-acetyl glucosamine (O-GlcNAc) is a post-translational modification to Ser/Thr residues that integrates energy supply with demand. Abnormal O-GlcNAc patterning is evident in several neurological disease states including epilepsy, Alzheimers disease and autism spectrum disorder (ASD). A potential treatment option for these disorders includes the high-fat, low-carbohydrate, ketogenic diet (KD). The goal of this study was to determine whether the KD induces changes in O-GlcNAc in the BTBRT+tf/j (BTBR) mouse model of ASD. METHODS Juvenile male (5weeks), age-matched C57 or BTBR mice consumed a chow diet (13% kcal fat) or KD (75% kcal fat) for 10-14days. Following these diets, brain (prefrontal cortex) and liver were examined for gene expression levels of key O-GlcNAc mediators, global and protein specific O-GlcNAc as well as indicators of energy status. RESULTS The KD reduced global O-GlcNAc in the livers of all animals (p<0.05). Reductions were likely mediated by lower protein levels of O-GlcNAc transferase (OGT) and increased O-GlcNAcase (OGA) (p<0.05). In contrast, no differences in global O-GlcNAc were noted in the brain (p>0.05), yet OGT and OGA expression (mRNA) were elevated in both C57 and BTBR animals (p<0.05). CONCLUSIONS The KD has tissue specific impacts on O-GlcNAc. Although levels of O-GlcNAc play an important role in neurodevelopment, levels of this modification in the juvenile mouse brain were stable with the KD despite large fluctuations in energy status. This suggests that it is unlikely that the KD exerts it therapeutic benefit in the BTBR model of ASD by O-GlcNAc related pathways.

Mitochondrial substrate specificity in beetle flight muscle: assessing respiratory oxygen flux in small samples from Dermestes maculatus and Tenebrio molitor

In the present study, the permeabilized fibre approach is adapted to investigate substrate utilization patterns in the flight muscle mitochondria of Dermestes maculatus (Coleoptera: Dermestidae; a carrion scavenger beetle) and Tenebrio molitor (Coleoptera: Tenebrionidae; a phytophagous scavenger beetle). Respiration in saponin‐permeabilized fibres is measured during titration of palmitoyl‐carnitine (Palm‐C), pyruvate (Pyr) or l‐glycerol 3‐phosphate (G3‐P). Michaelis–Menten‐type enzyme kinetics for oxygen consumption are observed as a function of substrate concentration in Pyr and G3‐P, from which substrate‐specific apparent Km (sensitivity) and Vmax (capacity) are determined. Compared with D. maculatus, the apparent Km in T. molitor is lower (P < 0.001) for Pyr, and Vmax is greater for G3‐P (P < 0.001). In D. maculatus, the apparent Km for G3‐P is greater (P < 0.001), and respiratory Vmax is lower (P < 0.001), than kinetics for Pyr. Robust respiration with l‐proline (Pro) is also observed in both beetle species tested; however, it is over 2.5‐fold greater in D. maculatus than T. molitor (P < 0.05). These results demonstrate that respiration in beetle flight muscle mitochondria can be assessed in small samples (i.e. at the individual beetle level) using the approach adapted for the present study. The results of the present study also highlight the substrate oxidative capacity patterns in both D. maculatus and T. molitor, which rank Palm‐C < G3‐P < Pyr < Pro.

Plasma-derived cell-free mitochondrial DNA: A novel non-invasive methodology to identify mitochondrial DNA haplogroups in humans

BACKGROUND Mitochondrial diseases are a clinically heterogeneous group of diseases caused by mutations in either nuclear or mitochondrial DNA (mtDNA). The diagnosis is challenging and has frequently required a tissue biopsy to obtain a sufficient quantity of mtDNA. Less-invasive sources mtDNA, such as peripheral blood leukocytes, urine sediment, or buccal swab, contain a lower quantity of mtDNA compared to tissue sources which may reduce sensitivity. Cellular apoptosis of tissues and hematopoetic cells releases fragments of DNA and mtDNA into the circulation and these molecules can be extracted from plasma as cell-free DNA (cfDNA). However, entire mtDNA has not been successfully identified from the cell free fraction previously. We hypothesized that the circular nature of mtDNA would prevent its degradation and a higher sensitivity method, such as next generation sequencing, could identify intact cf-mtDNA from human plasma. METHODS Plasma was obtained from patients with mitochondrial disease diagnosed from skeletal muscle biopsy (n = 7) and healthy controls (n = 7) using a specially cfDNA collection tube (Streck Inc.; La Vista, NE). To demonstrate the presence of mtDNA within these samples, we amplified the isolated DNA using custom PCR primers specific to overlapping fragments of mtDNA. cfDNA samples were then sequenced using the Illumina MiSeq sequencing platform. RESULTS We confirmed the presence of mtDNA, demonstrating that the full mitochondrial genome is in fact present within the cell-free plasma fraction of human blood. Sequencing identified the mitochondrial haplogroup matching with the tissue specimen for all patients. CONCLUSION We report the existence of full length mtDNA in cell-free human plasma that was successfully used to perform haplogroup matching. Clinical applications for this work include patient monitoring for heteroplasmy status after mitochondrially-targeted therapies or haplogroup monitoring as a measure of stem cell transplantation.

Mesenchymal stem cells shift mitochondrial dynamics and enhance oxidative phosphorylation in recipient cells

Mesenchymal stem cells (MSCs) are the most commonly used cells in tissue engineering and regenerative medicine. MSCs can promote host tissue repair through several different mechanisms including donor cell engraftment, release of cell signaling factors, and the transfer of healthy organelles to the host. In the present study, we examine the specific impacts of MSCs on mitochondrial morphology and function in host tissues. Employing in vitro cell culture of inherited mitochondrial disease and an in vivo animal experimental model of low-grade inflammation (high fat feeding), we show human-derived MSCs to alter mitochondrial function. MSC co-culture with skin fibroblasts from mitochondrial disease patients rescued aberrant mitochondrial morphology from a fission state to a more fused appearance indicating an effect of MSC co-culture on host cell mitochondrial network formation. In vivo experiments confirmed mitochondrial abundance and mitochondrial oxygen consumption rates were elevated in host tissues following MSC treatment. Furthermore, microarray profiling identified 226 genes with differential expression in the liver of animals treated with MSC, with cellular signaling, and actin cytoskeleton regulation as key upregulated processes. Collectively, our data indicate that MSC therapy rescues impaired mitochondrial morphology, enhances host metabolic capacity, and induces widespread host gene shifting. These results highlight the potential of MSCs to modulate mitochondria in both inherited and pathological disease states.