Metabolic Inflexibility in Skeletal Muscle With Obesity

The skeletal muscle of obese individuals has a reduced capacity to oxidize lipids. The hypothesis to be tested in this dissertation is that the ability to regulate lipid oxidation in response to lipid exposure is impaired in skeletal muscle of obese individuals. An inability to appropriately respond to metabolic stimuli has been termed "metabolic inflexibility" and has been linked with obesity and insulin resistance. To test this hypothesis, two models of lipid exposure were utilized: a 5 day high fat diet (HFD) and lipid incubation in primary myotubes cultured from lean and obese donors. Trend analyses indicated that mRNA content of genes linked with fat oxidation were collectively up-regulated with the HFD in skeletal muscle of lean but not obese subjects, suggesting a global response that is indicative of skeletal muscle mitochondrial dysfunction in obesity. Specifically, there was a 2-fold increase (P < 0.05) in fasted PDK4 content following the HFD in leans, while the obese participants tended to have decreased PDK4 content; and UCP3 mRNA content decreased by almost half in the obese, but not lean participants. In the second series of studies, a 24 h lipid incubation increased mitochondrial respiration by up to 50% in the presence of lipid and carbohydrate in myotubes from lean donors in both State 3 and uncoupled respiration (P < 0.05), though there was no change in cells cultured from the obese donors. In addition, mitochondrial DNA content increased by 16% (P < 0.05) with lipid exposure in cells from lean subjects but decreased by 13% (P < 0.05) in myotubes from obese subjects. The presence of these defects in culture suggests a genetic or epigenetic origin with obesity. Together, these data support the hypothesis that the skeletal muscle of obese individuals is metabolically inflexible and provides the novel information that this inflexibility extends to the ability to respond to lipid exposure in human skeletal muscle.  Ph.D

Inhibition of lipid oxidation increases glucose metabolism and enhances 2-deoxy-2-[¹⁸F]fluoro-D-glucose uptake in prostate cancer mouse xenografts

Includes bibliographic references.PURPOSE: Prostate cancer (PCa) is the second most common cause of cancer-related death among men in the United States. Due to the lipid-driven metabolic phenotype of Pca, imaging with 2-deoxy-2-[¹⁸F]fluoro-D-glucose ([¹⁸F]FDG) is suboptimal, since tumors tend to have low avidity for glucose. PROCEDURES: We have used the fat oxidation inhibitor etomoxir (2-[6-(4-chlorophenoxy)-hexyl]oxirane-2-carboxylate) that targets carnitine-palmitoyl-transferase-1 (CPT-1) to increase glucose uptake in PCa cell lines. Small hairpin RNA specific for CPT1A was used to confirm the glycolytic switch induced by etomoxir in vitro. Systemic etomoxir treatment was used to enhance [¹⁸F]FDG-positron emission tomography ([¹⁸F]FDG-PET) imaging in PCa xenograft mouse models in 24 h. RESULTS: PCa cells significantly oxidize more of circulating fatty acids than benign cells via CPT-1 enzyme, and blocking this lipid oxidation resulted in activation of the Warburg effect and enhanced [¹⁸F]FDG signal in PCa mouse models. CONCLUSIONS: Inhibition of lipid oxidation plays a major role in elevating glucose metabolism of PCa cells, with potential for imaging enhancement that could also be extended to other cancers

Fossil Gas and the Electromagnetic Precursor of Supermassive Binary Black Hole Mergers

Using a one-dimensional height integrated model, we calculate the evolution of an unequal mass binary black hole with a coplanar gas disk that contains a gap due to the presence of the secondary black hole. Viscous evolution of the outer circumbinary disk initially hardens the binary, while the inner disk drains onto the primary (central) black hole. As long as the inner disk remains cool and thin at low $\dot{M}_{\rm ext}$ (rather than becoming hot and geometrically thick), the mass of the inner disk reaches an asymptotic mass typically \sim 10^{-3}-10^{-4}\Msun. Once the semimajor axis shrinks below a critical value, angular momentum losses from gravitational waves dominate over viscous transport in hardening the binary. The inner disk then no longer responds viscously to the inspiraling black holes. Instead, tidal interactions with the secondary rapidly drive the inner disk into the primary. Tidal and viscous dissipation in the inner disk lead to a late time brightening in luminosity $L\propto t_{\rm minus}^{5/4}$, where $t_{\rm minus}$ is the time prior to the final merger. This late time brightening peaks $\sim 1$ day prior to the final merger at $\sim 0.1 L_{\rm Edd}$. This behavior is relatively robust because of self regulation in the coupled viscous-gravitational evolution of such binary systems. It constitutes a unique electromagnetic signature of a binary supermassive black hole merger and may allow the host galaxy to be identified if used in conjunction with the Laser Interferometric Space Antenna (LISA) localization.Comment: 11 pages, 6 figures, submitted to MNRA

Mitochondrial Hâ‚‚Oâ‚‚ emission and cellular redox state link excess fat intake to insulin resistance in both rodents and humans

High dietary fat intake leads to insulin resistance in skeletal muscle, and this represents a major risk factor for type 2 diabetes and cardiovascular disease. Mitochondrial dysfunction and oxidative stress have been implicated in the disease process, but the underlying mechanisms are still unknown. Here we show that in skeletal muscle of both rodents and humans, a diet high in fat increases the Hâ‚‚Oâ‚‚-emitting potential of mitochondria, shifts the cellular redox environment to a more oxidized state, and decreases the redox-buffering capacity in the absence of any change in mitochondrial respiratory function. Furthermore, we show that attenuating mitochondrial Hâ‚‚Oâ‚‚ emission, either by treating rats with a mitochondrial-targeted antioxidant or by genetically engineering the overexpression of catalase in mitochondria of muscle in mice, completely preserves insulin sensitivity despite a high-fat diet. These findings place the etiology of insulin resistance in the context of mitochondrial bioenergetics by demonstrating that mitochondrial Hâ‚‚Oâ‚‚ emission serves as both a gauge of energy balance and a regulator of cellular redox environment, linking intracellular metabolic balance to the control of insulin sensitivity. Original version available at http://www.jci.org/articles/view/3704

Characterization of the 1st and 2nd EF-hands of NADPH oxidase 5 by fluorescence, isothermal titration calorimetry, and circular dichroism

<p>Abstract</p> <p>Background</p> <p>Superoxide generated by non-phagocytic NADPH oxidases (NOXs) is of growing importance for physiology and pathobiology. The calcium binding domain (CaBD) of NOX5 contains four EF-hands, each binding one calcium ion. To better understand the metal binding properties of the 1^stand 2^ndEF-hands, we characterized the N-terminal half of CaBD (NCaBD) and its calcium-binding knockout mutants.</p> <p>Results</p> <p>The isothermal titration calorimetry measurement for NCaBD reveals that the calcium binding of two EF-hands are loosely associated with each other and can be treated as independent binding events. However, the Ca²⁺binding studies on NCaBD(E31Q) and NCaBD(E63Q) showed their binding constants to be 6.5 × 10⁵and 5.0 × 10²M^-1with ΔHs of -14 and -4 kJ/mol, respectively, suggesting that intrinsic calcium binding for the 1^stnon-canonical EF-hand is largely enhanced by the binding of Ca²⁺to the 2^ndcanonical EF-hand. The fluorescence quenching and CD spectra support a conformational change upon Ca²⁺binding, which changes Trp residues toward a more non-polar and exposed environment and also increases its α-helix secondary structure content. All measurements exclude Mg²⁺-binding in NCaBD.</p> <p>Conclusions</p> <p>We demonstrated that the 1^stnon-canonical EF-hand of NOX5 has very weak Ca²⁺binding affinity compared with the 2^ndcanonical EF-hand. Both EF-hands interact with each other in a cooperative manner to enhance their Ca²⁺binding affinity. Our characterization reveals that the two EF-hands in the N-terminal NOX5 are Ca²⁺specific.</p> <p>Graphical abstract</p> <p><display-formula><graphic file="1752-153X-6-29-i1.gif"/></display-formula></p

Gestational Diabetes Is Characterized by Reduced Mitochondrial Protein Expression and Altered Calcium Signaling Proteins in Skeletal Muscle

The rising prevalence of gestational diabetes mellitus (GDM) affects up to 18% of pregnant women with immediate and long-term metabolic consequences for both mother and infant. Abnormal glucose uptake and lipid oxidation are hallmark features of GDM prompting us to use an exploratory proteomics approach to investigate the cellular mechanisms underlying differences in skeletal muscle metabolism between obese pregnant women with GDM (OGDM) and obese pregnant women with normal glucose tolerance (ONGT). Functional validation was performed in a second cohort of obese OGDM and ONGT pregnant women. Quantitative proteomic analysis in rectus abdominus skeletal muscle tissue collected at delivery revealed reduced protein content of mitochondrial complex I (C-I) subunits (NDUFS3, NDUFV2) and altered content of proteins involved in calcium homeostasis/signaling (calcineurin A, α1-syntrophin, annexin A4) in OGDM (n = 6) vs. ONGT (n = 6). Follow-up analyses showed reduced enzymatic activity of mitochondrial complexes C-I, C-III, and C-IV (−60–75%) in the OGDM (n = 8) compared with ONGT (n = 10) subjects, though no differences were observed for mitochondrial complex protein content. Upstream regulators of mitochondrial biogenesis and oxidative phosphorylation were not different between groups. However, AMPK phosphorylation was dramatically reduced by 75% in the OGDM women. These data suggest that GDM is associated with reduced skeletal muscle oxidative phosphorylation and disordered calcium homeostasis. These relationships deserve further attention as they may represent novel risk factors for development of GDM and may have implications on the effectiveness of physical activity interventions on both treatment strategies for GDM and for prevention of type 2 diabetes postpartum

Nucleosome occupancy reveals regulatory elements of the CFTR promoter

Access to regulatory elements of the genome can be inhibited by nucleosome core particles arranged along the DNA strand. Hence, sites that are accessible by transcription factors may be located by using nuclease digestion to identify the relative nucleosome occupancy of a genomic region. In order to define novel cis regulatory elements in the ∼2.7-kb promoter region of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, we define its nucleosome occupancy. This profile reveals the precise positions of nucleosome-free regions (NFRs), both cell-type specific and others apparently unrelated to CFTR-expression level and offer the first high-resolution map of the chromatin structure of the entire CFTR promoter in relevant cell types. Several of these NFRs are strongly bound by nuclear factors in a sequence-specific manner, and directly influence CFTR promoter activity. Sequences within the NFR1 and NFR4 elements are highly conserved in many human gene promoters. Moreover, NFR1 contributes to promoter activity of another gene, angiopoietin-like 3 (ANGPTL3), while NFR4 is constitutively nucleosome-free in promoters genome wide. Conserved motifs within NFRs of the CFTR promoter also show a high level of protection from DNase I digestion genome-wide, and likely have important roles in the positioning of nucleosome core particles more generally

Precision stratification of prognostic risk factors associated with outcomes in gestational diabetes mellitus:a systematic review

BackgroundThe objective of this systematic review is to identify prognostic factors among women and their offspring affected by gestational diabetes mellitus (GDM), focusing on endpoints of cardiovascular disease (CVD) and type 2 diabetes (T2D) for women, and cardiometabolic profile for offspring.MethodsThis review included studies published in English language from January 1st, 1990, through September 30th, 2021, that focused on the above outcomes of interest with respect to sociodemographic factors, lifestyle and behavioral characteristics, traditional clinical traits, and 'omics biomarkers in the mothers and offspring during the perinatal/postpartum periods and across the lifecourse. Studies that did not report associations of prognostic factors with outcomes of interest among GDM-exposed women or children were excluded.ResultsHere, we identified 109 publications comprising 98 observational studies and 11 randomized-controlled trials. Findings indicate that GDM severity, maternal obesity, race/ethnicity, and unhealthy diet and physical activity levels predict T2D and CVD in women, and greater cardiometabolic risk in offspring. However, using the Diabetes Canada 2018 Clinical Practice Guidelines for studies, the level of evidence was low due to potential for confounding, reverse causation, and selection biases.ConclusionsGDM pregnancies with greater severity, as well as those accompanied by maternal obesity, unhealthy diet, and low physical activity, as well as cases that occur among women who identify as racial/ethnic minorities are associated with worse cardiometabolic prognosis in mothers and offspring. However, given the low quality of evidence, prospective studies with detailed covariate data collection and high fidelity of follow-up are warranted.Gestational diabetes mellitus (GDM) occurs when levels of sugar in the blood are high during pregnancy. We sought to identify factors associated with short- and long-term cardiometabolic disease risk, health conditions that involve heart-related issues and complications in bodily function, among women with GDM and their offspring. We reviewed publications on factors related to type 2 diabetes (T2D) and cardiovascular disease (CVD) risk among women with GDM, and additionally assessed body composition in offspring of women with GDM. We found that GDM severity, maternal obesity, self-identified race/ethnicity, poor diet, and low physical activity levels predict postpartum T2D and CVD in the women, and unfavorable long-term cardiometabolic disease risk in offspring. The quality of evidence was poor, emphasizing a need for high-quality research capturing detailed short- and long-term outcome data to facilitate preventative interventions to improve health of women and children.Semnani-Azad et al. review the evidence on prognostic factors that predict cardiovascular disease and type 2 diabetes for women, and cardiometabolic profile in offspring subsequent to gestational diabetes. The evidence was of low quality, but some maternal characteristics were predictive of unfavourable outcomes in women and their offspring