analytical-determination-mitochondrial-function-excised-solid-tumor

Analytical Determination of Mitochondrial Function of Excised Solid Tumor Homogenates

Mitochondria are essential to the onset and progression of cancer through energy production, reactive oxygen species regulation, and macromolecule ...

Pennington Biomedical Research Center

The traditional view of mitochondria as isolated, spherical, energy producing organelles, is undergoing a revolutionary change. Emerging data show that mitochondria form a dynamic reticulum that is regulated by cycles of fission and fusion. The discovery of proteins that modulate these activities has led to important advances in understanding human disease. Here, we review the latest evidence that connects the emerging field of mitochondrial dynamics to skeletal muscle insulin resistance and propose some potential mechanisms that may explain the long debated link between mitochondria and the development of type 2 diabetes.

Mitochondrial dynamics in skeletal muscle insulin resistance and type 2 diabetes.

Mitochondria exist as a morphologically plastic network driven by cellular bioenergetic demand. Induction of fusion and fission machinery allows the organelle to regulate quality control and substrate flux. Physiological stressors promote fragmentation of the mitochondrial network, a process implicated in the onset of metabolic disease, including type 2 diabetes and obesity. It is well-known that exercise training improves skeletal muscle mitochondrial volume, number, and density. However, the effect of exercise training on muscle mitochondrial dynamics remains unclear.

Exercise training remodels human skeletal muscle mitochondrial fission and fusion machinery towards a pro-elongation phenotype.

Roux-en-Y gastric bypass (RYGB) is associated with remission of type 2 diabetes. However, the cellular and molecular mechanisms remain unknown. We hypothesized that RYGB would increase peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), sirtuin-1 (SIRT1), AMPK/pAMPK, and citrate synthase (CS) protein expression and decrease insulin resistance and these changes would be mediated by sphingolipids, including ceramides and the sphingolipid metabolite sphingosine-1 phosphate (S1P).

Gastric Bypass Surgery Improves the Skeletal Muscle Ceramide/S1P Ratio and Upregulates the AMPK/ SIRT1/ PGC-1α Pathway in Zucker Diabetic Fatty Rats.

Dysregulation of gut microbiota and intestinal barrier function has emerged as potential mechanisms underlying digestive diseases, yet targeted therapies are lacking The purpose of this investigation was to assess the efficacy of UCC118, a characterized probiotic strain, on exercise-induced GI permeability in healthy humans. In a randomized, double-blind, placebo-controlled crossover study, seven healthy adults received 4 weeks of daily UCC118 or placebo supplementation. GI hyperpermeability was induced by strenuous treadmill running performed before and after each supplementation period. While running, participants ingested 5 g of lactulose, rhamnose, and sucrose. Urine was collected before, immediately after, and every hour for 5 h after exercise to assess GI permeability. Metagenomic sequencing was performed on fecal homogenates collected prior to exercise to identify changes in microbial diversity and taxon abundances. Inflammatory biomarkers were assessed from blood and fecal homogenates collected prior to and immediately following the cessation of exercise. Exercise significantly induced intestinal permeability of lactulose, rhamnose, and sucrose (P < 0.001). UCC118 significantly reduced sucrose (Δ = -0.38 ± 0.13 vs. 1.69 ± 0.79; P < 0.05) recovery, with no substantial change in lactulose (Δ = -0.07 ± 0.23 vs. 0.35 ± 0.15; P = 0.16) or rhamnose (Δ = -0.06 ± 0.22 vs. 0.48 ± 0.28; P = 0.22). Taxonomic sequencing revealed 99 differentially regulated bacteria spanning 6 taxonomic ranks (P < 0.05) after UCC118 supplementation. No differences in plasma IL-6 or fecal zonulin were observed after UCC118 supplementation. The results described herein provide proof of principle that 4 weeks of UCC118 supplementation attenuated exercise-induced intestinal hyperpermeability. Further research is warranted to investigate the as-yet-to-be defined molecular processes of intestinal hyperpermeability and the effects of probiotic supplementation.

UCC118 supplementation reduces exercise-induced gastrointestinal permeability and remodels the gut microbiome in healthy humans.

We aimed to determine the immediacy of exercise intervention on liver-specific metabolic processes in nonalcoholic fatty liver disease.

Exercise Training Rapidly Increases Hepatic Insulin Extraction in NAFLD.

Individuals with idiopathic pulmonary arterial hypertension (PAH) display reduced oral glucose tolerance. This may involve defects in pancreatic function or insulin sensitivity but this hypothesis has not been tested; moreover, fasting nutrient metabolism remains poorly described in PAH. Thus, we aimed to characterise fasting nutrient metabolism and investigated the metabolic response to hyperglycaemia in PAH.12 participants (six PAH, six controls) were administered a hyperglycaemic clamp, while 52 (21 PAH, 31 controls) underwent plasma metabolomic analysis. Glucose, insulin, C-peptide, free fatty acids and acylcarnitines were assessed from the clamp. Plasma metabolomics was conducted on fasting plasma samples.The clamp verified a reduced insulin response to hyperglycaemia in PAH (-53%  control), but with similar pancreatic insulin secretion. Skeletal muscle insulin sensitivity was unexpectedly greater in PAH. Hepatic insulin extraction was elevated in PAH (+11%  control). Plasma metabolomics identified 862 metabolites: 213 elevated, 145 reduced in PAH (p<0.05). In both clamp and metabolomic cohorts, lipid oxidation and ketones were elevated in PAH. Insulin sensitivity, fatty acids, acylcarnitines and ketones correlated with PAH severity, while hepatic extraction and fatty acid:ketone ratio correlated with longer six-min walk distance.Poor glucose control in PAH could not be explained by pancreatic β-cell function or skeletal muscle insulin sensitivity. Instead, elevated hepatic insulin extraction emerged as an underlying factor. In agreement, nutrient metabolism in PAH favours lipid and ketone metabolism at the expense of glucose control. Future research should investigate the therapeutic potential of reinforcing lipid and ketone metabolism on clinical outcomes in PAH.

Lipids and ketones dominate metabolism at the expense of glucose control in pulmonary arterial hypertension: a hyperglycaemic clamp and metabolomics study.

Nonalcoholic fatty liver disease (NAFLD) is characterized by hepatic fat accumulation and impaired insulin sensitivity. Reduced hepatic ketogenesis may promote these pathologies, but data are inconclusive in humans and the link between NAFLD and reduced insulin sensitivity remains obscure. We investigated individuals with obesity-related NAFLD and hypothesized that β-hydroxybutyrate (βOHB; the predominant ketone species) would be reduced and related to hepatic fat accumulation and insulin sensitivity. Furthermore, we hypothesized that ketones would impact skeletal muscle mitochondrial respiration in vitro. Hepatic fat was assessed by H-MRS in 22 participants in a parallel design, case control study [Control:  = 7, age 50 ± 6 yr, body mass index (BMI) 30 ± 1 kg/m; NAFLD:  = 15, age 57 ± 3 yr, BMI 35 ± 1 kg/m]. Plasma assessments were conducted in the fasted state. Whole body insulin sensitivity was determined by the gold-standard hyperinsulinemic-euglycemic clamp. The effect of ketone dose (0.5-5.0 mM) on mitochondrial respiration was conducted in human skeletal muscle cell culture. Fasting βOHB, a surrogate measure of hepatic ketogenesis, was reduced in NAFLD (-15.6%,  < 0.01) and correlated negatively with liver fat ( = 0.21,  = 0.03) and positively with insulin sensitivity ( = 0.30,  = 0.01). Skeletal muscle mitochondrial oxygen consumption increased with low-dose ketones, attributable to increases in basal respiration (135%,  < 0.05) and ATP-linked oxygen consumption (136%,  < 0.05). NAFLD pathophysiology includes impaired hepatic ketogenesis, which is associated with hepatic fat accumulation and impaired insulin sensitivity. This reduced capacity to produce ketones may be a potential link between NAFLD and NAFLD-associated reductions in whole body insulin sensitivity, whereby ketone concentrations impact skeletal muscle mitochondrial respiration.

β-Hydroxybutyrate is reduced in humans with obesity-related NAFLD and displays a dose-dependent effect on skeletal muscle mitochondrial respiration in vitro.

Exercise seems to enhance the beneficial effect of bariatric (Roux-en-Y gastric bypass [RYGB]) surgery on insulin resistance. We hypothesized that skeletal muscle extracellular matrix (ECM) remodeling may underlie these benefits. Women were randomized to either a combined aerobic and resistance exercise training program following RYGB (RYGB + ET) or standard of care (RYGB). Insulin sensitivity was assessed by oral glucose tolerance test. Muscle biopsy specimens were obtained at baseline and 3 and 9 months after surgery and subjected to comprehensive phenotyping, transcriptome profiling, molecular pathway identification, and validation in vitro. Exercise training improved insulin sensitivity beyond surgery alone (e.g., Matsuda index: RYGB 123% vs. RYGB + ET 325%;  ≤ 0.0001). ECM remodeling was reduced by surgery alone, with an additive benefit of surgery and exercise training (e.g., collagen I: RYGB -41% vs. RYGB + ET -76%;  ≤ 0.0001). Exercise and RYGB had an additive effect on enhancing insulin sensitivity, but surgery alone did not resolve insulin resistance and ECM remodeling. We identified candidates modulated by exercise training that may become therapeutic targets for treating insulin resistance, in particular, the transforming growth factor-β1/SMAD 2/3 pathway and its antagonist follistatin. Exercise-induced increases in insulin sensitivity after bariatric surgery are at least partially mediated by muscle ECM remodeling.

Exercise-Induced Increases in Insulin Sensitivity After Bariatric Surgery Are Mediated By Muscle Extracellular Matrix Remodeling.

Obesity is a leading cause of preventable death worldwide. Despite this, current strategies for the treatment of obesity remain ineffective at achieving long-term weight control. This is due, in part, to difficulties in identifying tolerable and efficacious small molecules or biologics capable of regulating systemic nutrient homeostasis. Here, we demonstrate that BAM15, a mitochondrially targeted small molecule protonophore, stimulates energy expenditure and glucose and lipid metabolism to protect against diet-induced obesity. Exposure to BAM15 in vitro enhanced mitochondrial respiratory kinetics, improved insulin action, and stimulated nutrient uptake by sustained activation of AMPK. C57BL/6J mice treated with BAM15 were resistant to weight gain. Furthermore, BAM15-treated mice exhibited improved body composition and glycemic control independent of weight loss, effects attributable to drug targeting of lipid-rich tissues. We provide the first phenotypic characterization and demonstration of pre-clinical efficacy for BAM15 as a pharmacological approach for the treatment of obesity and related diseases.

BAM15-mediated mitochondrial uncoupling protects against obesity and improves glycemic control.

Reductions in β-cell number and function contribute to the onset type 2 diabetes (T2D). Roux-en-Y gastric bypass (RYGB) surgery can resolve T2D within days of operation, indicating a weight-independent mechanism of glycemic control. We hypothesized that RYGB normalizes glucose homeostasis by restoring β-cell structure and function.

Roux-en-Y Gastric Bypass Restores Islet Function and Morphology Independent of Body Weight in ZDF Rats.

Patients with type 2 diabetes experience resolution of hyperglycemia within days after Roux-en-Y gastric bypass (RYGB) surgery. This is attributed, in part, to enhanced secretion of hindgut factors following exclusion of the gastric remnant and proximal intestine during surgery. However, evidence of the mechanisms of remission remain limited due to the challenges of metabolic evaluation during the early post-operative period. The purpose of this investigation was to determine the role of foregut exclusion in the resolution of type 2 diabetes after RYGB.

Foregut Exclusion Enhances Incretin and Insulin Secretion After Roux-en-Y Gastric Bypass in Adults with Type 2 Diabetes.

A diminution in skeletal muscle mitochondrial function due to ectopic lipid accumulation and excess nutrient intake is thought to contribute to insulin resistance and the development of type 2 diabetes. However, the functional integrity of mitochondria in insulin-resistant skeletal muscle remains highly controversial.

Lipids activate skeletal muscle mitochondrial fission and quality control networks to induce insulin resistance in humans.

Mutations in Valosin Containing Protein (VCP) are associated with several degenerative diseases, including multisystem proteinopathy (MSP-1) and amyotrophic lateral sclerosis. However, patients with VCP mutations vary widely in their pathology and clinical penetrance, making it difficult to devise effective treatment strategies. A deeper understanding of how each mutation affects VCP function could enhance the prediction of clinical outcomes and design of personalized treatment options. The power of a genetically tractable model organism coupled with well-established in vivo assays and a relatively short life cycle make Drosophila an attractive system to study VCP disease pathogenesis. Using CRISPR/Cas9, we have generated individual Drosophila knock-in mutants that include nine hereditary VCP disease mutations. Our models display many hallmarks of VCP-mediated degeneration, including progressive decline in mobility, protein aggregate accumulation and defects in lysosomal and mitochondrial function. We also made some novel and unexpected findings, including nuclear morphology defects and sex-specific phenotypic differences in several mutants. Taken together, the Drosophila VCP disease models generated in this study will be useful for studying the etiology of individual VCP patient mutations and testing potential genetic and/or pharmacological therapies.

Article	Year
Mitochondrial dynamics in skeletal muscle insulin resistance and type 2 diabetes. Translational research : the journal of laboratory and clinical medicine\| PubMed ID: 30153426	2018
Exercise training remodels human skeletal muscle mitochondrial fission and fusion machinery towards a pro-elongation phenotype. Acta physiologica (Oxford, England)\| PubMed ID: 30408342	2019
Gastric Bypass Surgery Improves the Skeletal Muscle Ceramide/S1P Ratio and Upregulates the AMPK/ SIRT1/ PGC-1α Pathway in Zucker Diabetic Fatty Rats. Obesity surgery\| PubMed ID: 30809769	2019
UCC118 supplementation reduces exercise-induced gastrointestinal permeability and remodels the gut microbiome in healthy humans. Physiological reports\| PubMed ID: 31758610	2019
Exercise Training Rapidly Increases Hepatic Insulin Extraction in NAFLD. Medicine and science in sports and exercise\| PubMed ID: 32028458	2020
Lipids and ketones dominate metabolism at the expense of glucose control in pulmonary arterial hypertension: a hyperglycaemic clamp and metabolomics study. The European respiratory journal\| PubMed ID: 32108049	2020
β-Hydroxybutyrate is reduced in humans with obesity-related NAFLD and displays a dose-dependent effect on skeletal muscle mitochondrial respiration in vitro. American journal of physiology. Endocrinology and metabolism\| PubMed ID: 32396388	2020
Exercise-Induced Increases in Insulin Sensitivity After Bariatric Surgery Are Mediated By Muscle Extracellular Matrix Remodeling. Diabetes\| PubMed ID: 32409493	2020
BAM15-mediated mitochondrial uncoupling protects against obesity and improves glycemic control. EMBO molecular medicine\| PubMed ID: 32519812	2020
Roux-en-Y Gastric Bypass Restores Islet Function and Morphology Independent of Body Weight in ZDF Rats. American journal of physiology. Endocrinology and metabolism\| PubMed ID: 33427046	2021
Foregut Exclusion Enhances Incretin and Insulin Secretion After Roux-en-Y Gastric Bypass in Adults with Type 2 Diabetes. The Journal of clinical endocrinology and metabolism\| PubMed ID: 33870426	2021
Lipids activate skeletal muscle mitochondrial fission and quality control networks to induce insulin resistance in humans. Metabolism: clinical and experimental\| PubMed ID: 34090870	2021
CRISPR/Cas9-engineered Drosophila knock-in models to study VCP diseases. Disease models & mechanisms\| PubMed ID: 34160014	2021
CRISPR/Cas9-engineered Drosophila knock-in models to study VCP diseases. Disease models & mechanisms\| PubMed ID: 34296742	2021

Christopher L. Axelrod

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