Mitochondrial Respiration in Female Zucker Rats: Effects of Obesity and Short-Term Metformin Treatment

Abstract Objectives As childhood obesity rates climb, so has the incidence of type 2 diabetes among children and adolescents. Metformin, an FDA approved anti-hyperglycemic drug, is thought to target the mitochondria, but its effects on mitochondrial function in obesity have not been well studied. We used an obese Zucker rat model to investigate the effects of obesity and short-term metformin treatment on mitochondrial respiration. Methods Five-week old female Zucker rats (n = 16 lean, n = 16 obese) were fed AIN-93 G diet for 8 weeks before equally randomized to receive metformin (mixed in diet at 1 g/kg of feed); thus forming 4 groups with 8 rats each: lean +/– metformin and obese +/– metformin. Rats were sacrificed 10 weeks post-metformin treatment and spleens, perigonadal visceral adipose tissue (VAT) and skeletal muscle (SM; gracilis) collected. Mitochondrial respiration was measured in splenocytes by extracellular flux analysis and in VAT and SM fibers by high resolution respirometry. Results Effects of obesity on mitochondrial respiration were found in VAT and SM, but not splenocytes. In VAT, obese rats exhibited increased OXPHOS capacity over lean rats when octanoylcarnitine and malate were provided as substrates (obese vs lean: 1.33 vs 0.76 pmol O2/s/mg; SEdiff = 0.18, P = .005), and after subsequent additions of pyruvate (P = .012), glutamate (P = .009), and succinate (P = .045). In SM, OXPHOS capacity was increased when octanoylcarnitine and malate were provided as substrates (obese vs lean: 12.18 vs 5.45 pmol O2/s/mg; SEdiff = 2.31, P = .011) in obese vs lean rats. Metformin effects were observed only in splenocytes: coupling efficiency was decreased (metformin vs no metformin; 56.2% vs 69.8%; SEdiff = 4.1%, P = 0.005) and proton leak was increased (P < .001) in metformin treated rats as compared to rats not treated with metformin. Conclusions We found obesity was associated with increased mitochondrial respiration, particularly fatty acid oxidation, in VAT and SM. Short-term metformin treatment did not alter mitochondrial respiration in VAT or SM, but was found to increase proton leak and reduce coupling efficiency in splenocytes. Funding Sources Arkansas Children's Research Institute, Arkansas Biosciences Institute (R.H). S.R. and E.C. are also supported by the National Institute of General Medical Sciences of the National Institutes of Health.

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The profiles of mitochondrial respiration and glycolysis using extracellular flux analysis in porcine enterocyte IPEC-J2

Animal Nutrition ◽

10.1016/j.aninu.2015.08.004 ◽

2015 ◽

Vol 1 (3) ◽

pp. 239-243 ◽

Cited By ~ 17

Author(s):

Bie Tan ◽

Hao Xiao ◽

Fengna Li ◽

Liming Zeng ◽

Yulong Yin

Keyword(s):

Mitochondrial Respiration ◽

Flux Analysis ◽

Extracellular Flux

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Effects of Short-Term Metformin Treatment on Gut Microbiota Profile Using Female Obese Zucker Rat Model

Current Developments in Nutrition ◽

10.1093/cdn/nzab055_023 ◽

2021 ◽

Vol 5 (Supplement_2) ◽

pp. 1213-1213

Author(s):

Reza Hakkak ◽

Chris Randolph ◽

Sirish Bennuri ◽

Michael Robeson

Keyword(s):

Gut Microbiota ◽

Rat Model ◽

Zucker Rats ◽

Metformin Treatment ◽

Zucker Rat ◽

Short Term ◽

Fecal Samples ◽

Obese Zucker Rat ◽

Significant Difference ◽

Obese Control

Abstract Objectives The correlation of short-term metformin treatment and specific alterations to the gut microbiota in obese models is less known. So, the objectives of this experiment was to investigate the effects of short-term metformin treatment on population of gut microbiota profile in obese rat model. Methods Five week old obese (n = 16) female Zucker rats after one week of acclimation, received AIN-93 G diet for 8 weeks and then rats were randomly assigned 8 rats/group): to 1) obese without metformin (ObC), or 2) obese with metformin (ObMet). Metformin were mixed with AIN-93G diet at 1000 mg/kg of diet. Rats were weighed twice per week. All rats were sacrificed 10 weeks post-metformin treatment and fecal samples were collected and kept at − 80c. Total microbial DNA were collected directly from the fecal samples using a PowerSoil® DNA isolation kit. Isolated DNA were used for shotgun-metagenomics data collection using Illumina NextSeq500 and analyzed using MetaPlAn and HUMAnN. DEICODE and Songbird used calculate log-ratios and differential ranks of taxa and functional pathways associated with metformin treatment respectively. The were then visualized using Qurro. Results There was no significant difference between ObC vs. ObMet group body weight (P = 0.20). Overall microbial beta-diversity (DEICODE), showed significant separation between the obese control and metformin samples (P = 0.0007). Differential ranking (Songbird) of Bacteroides dorei and B. massiliensis vs. all other Bacteroides spp., revealed that B. dorei and B. massiliensis were enriched in the obese metformin group, while the remaining Bacteroides spp. where enriched in the obese control group (P = 0.002). The differential ranking of pathway diversity contributed by the Bacteroides were also associated with treatment group (P = 0.008). Conclusions In summary, in the obese zucker rat model, short-term metformin treatment changes the gut microbiota profile. Funding Sources Arkansas Biosciences Institute.

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ALDH2 Activity Reduces Mitochondrial Oxygen Reserve Capacity in Endothelial Cells and Induces Senescence Properties

Oxidative Medicine and Cellular Longevity ◽

10.1155/2018/9765027 ◽

2018 ◽

Vol 2018 ◽

pp. 1-13 ◽

Cited By ~ 9

Author(s):

G. Nannelli ◽

E. Terzuoli ◽

V. Giorgio ◽

S. Donnini ◽

P. Lupetti ◽

...

Keyword(s):

Endothelial Cells ◽

Mitochondrial Respiration ◽

Reserve Capacity ◽

Flux Analysis ◽

P53 Expression ◽

Senescent Phenotype ◽

Extracellular Flux ◽

Age Related ◽

And Migration ◽

Endothelial Dysfunctions

Endothelial cells (ECs) are dynamic cells that turn from growth into senescence, the latter being associated with cellular dysfunction, altered metabolism, and age-related cardiovascular diseases. Aldehyde dehydrogenase 2 (ALDH2) is a mitochondrial enzyme metabolizing acetaldehyde and other toxic aldehydes, such as 4-hydroxynonenal (4-HNE). In conditions in which lipid peroxidation products and reactive oxygen species (ROS) are accumulated, ECs become dysfunctional and significantly contribute to the progression of vascular-dependent diseases. The aim of the present study has been to investigate whether inhibition of ALDH2 alters endothelial functions together with the impairment of bioenergetic functions, accelerating the acquisition of a senescent phenotype. HUVECs transfected with siRNA targeting ALDH2 or treated with daidzin, an ALDH2 inhibitor, were used in this study. We observed an alteration in cell morphology associated with endothelial dysfunctions. Loss of ALDH2 reduced cell proliferation and migration and increased paracellular permeability. To assess bioenergetic function in intact ECs, extracellular flux analysis was carried out to establish oxygen consumption rates (OCR). We observed a decrease in mitochondrial respiration and reserve capacity that coincided with SA-β-Gal accumulation and an increase in p21 and p53 expression in siALDH2 or daidzin-treated HUVECs. Treatment with N-acetyl-L-cysteine (NAC) reduced endothelial dysfunctions mediated by siALDH2, indicating that oxidative stress downstream to siALDH2 plays an instrumental role. Our results highlight that ALDH2 impairment accelerates the acquisition of a premature senescent phenotype, a change likely to be associated with the observed reduction of mitochondrial respiration and reserve capacity.

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Plate-Based Respirometry to Assess Thermal Sensitivity of Zebrafish Embryo Bioenergetics in situ

Frontiers in Physiology ◽

10.3389/fphys.2021.746367 ◽

2021 ◽

Vol 12 ◽

Author(s):

Erik Rollwitz ◽

Martin Jastroch

Keyword(s):

Oxygen Consumption ◽

Thermal Sensitivity ◽

Coupling Efficiency ◽

Temperature Acclimation ◽

Effect Of Temperature ◽

Proton Leak ◽

Zebrafish Embryos ◽

Atp Production ◽

Extracellular Flux

Oxygen consumption allows measuring the metabolic activity of organisms. Here, we adopted the multi-well plate-based respirometry of the extracellular flux analyzer (Seahorse XF96) to investigate the effect of temperature on the bioenergetics of zebrafish embryos (Danio rerio) in situ. We show that the removal of the embryonic chorion is beneficial for oxygen consumption rates (OCR) and penetration of various mitochondrial inhibitors, and confirm that sedation reduces the variability of OCR. At 48h post-fertilization, embryos (maintained at a routine temperature of 28°C) were exposed to different medium temperatures ranging from 18°C to 37°C for 20h prior OCR measurement. Measurement temperatures from 18°C to 45°C in the XF96 were achieved by lowering the room temperature and active in-built heating. At 18°C assay temperature, basal OCR was low due to decreased ATP-linked respiration, which was not limited by mitochondrial power, as seen in substantial spare respiratory capacity. Basal OCR of the embryos increased with assay temperature and were stable up to 37°C assay temperature, with pre-exposure of 37°C resulting in more thermo-resistant basal OCR measured at 41°C. Adverse effects of the mitochondrial inhibitor oligomycin were seen at 37°C and chemical uncouplers disrupted substrate oxidation gradually with increasing assay temperature. Proton leak respiration increased at assay temperatures above 28°C and compromised the efficiency of ATP production, calculated as coupling efficiency. Thus, temperature impacts mitochondrial respiration by reduced cellular ATP turnover at lower temperatures and by increased proton leak at higher temperatures. This conclusion is coherent with the assessment of heart rate, an independent indicator of systemic metabolic rate, which increased with exposure temperature, peaking at 28°C, and decreased at higher temperatures. Collectively, plate-based respirometry allows assessing distinct parts of mitochondrial energy transduction in zebrafish embryos and investigating the effect of temperature and temperature acclimation on mitochondrial bioenergetics in situ.

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Thyroid Hormone at Near Physiologic Concentrations Acutely Increases Oxygen Consumption and Extracellular Acidification in LH86 Hepatoma Cells

Endocrinology ◽

10.1210/en.2015-1221 ◽

2015 ◽

Vol 156 (11) ◽

pp. 4325-4335 ◽

Cited By ~ 3

Author(s):

Elizabeth A. McAninch ◽

Bradford T. Miller ◽

Cintia B. Ueta ◽

Sungro Jo ◽

Brian W. Kim

Keyword(s):

Thyroid Hormone ◽

Experimental System ◽

Hepatoma Cells ◽

Nucleotide Metabolism ◽

Acid Oxidation ◽

Flux Analysis ◽

Potential Candidate ◽

O2 Consumption ◽

Human Hepatoma Cells ◽

Extracellular Flux

Thyroid hormone (T3) has been known to regulate the basal metabolic rate for more than a century, but mechanistic understanding is lacking both at the level of the intact organism and in terms of how T3 alters energy expenditure in individual tissues. The current studies investigate the question of which metabolically relevant genes respond acutely as T3 concentrations increase through the physiologic range in liver cells. Because this has been technically unfeasible historically, we developed a modified protocol for extracellular flux analysis using a 96-well Extracellular Flux Analyzer (Seahorse Bioscience). Using a modified extracellular flux protocol and LH86 human hepatoma cells, we established an experimental system where small but significant changes in O2 consumption could be reproducibly quantified as hypothyroid cells were exposed to near-physiologic final concentrations of T3 approximately 2 orders of magnitude lower than most studies (0.04nM free T3), in only 6–7 hours. Taking advantage of the nondestructive nature of 96-well Extracellular Flux Analyzer measurements, the acute, direct, transcriptional changes that occur were measured in the exact same cells demonstrating increased O2 consumption. An unbiased, genome-wide microarray analysis identified potential candidate genes related to fatty acid oxidation, angiogenesis, nucleotide metabolism, immune signaling, mitochondrial respiration, and cell proliferation. The identified transcriptome is likely enriched in the genes most important for mediating the energetic effects of T3 in hepatoma cells.

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Effects of Obesity and Short-Term Metformin Treatment on Liver Steatosis in Female Zucker Rats

Current Developments in Nutrition ◽

10.1093/cdn/nzaa063_038 ◽

2020 ◽

Vol 4 (Supplement_2) ◽

pp. 1640-1640

Author(s):

Reza Hakkak ◽

David Irby ◽

Shannon Rose ◽

Sirish Bennuri ◽

Beverly Spray ◽

...

Keyword(s):

Liver Disease ◽

Rat Model ◽

Liver Steatosis ◽

Serum Markers ◽

Zucker Rats ◽

Metformin Treatment ◽

Zucker Rat ◽

Short Term ◽

Obese Zucker Rat ◽

Obese Rats

Abstract Objectives Non-alcoholic fatty liver disease (NAFLD) is the leading cause of liver disease in adolescents in the US and World, and the risk has increased with the rise in obesity. We reported that obesity increases Fatty liver (steatosis) using an obese Zucker rat model. Metformin is an oral anti-hyperglycemic agent approved by the FDA to treat type 2 diabetes (T2D) in adults and children older than 10 years of age. There is insufficient evidence regarding the effects of metformin in pediatric liver steatosis. The objective of this study was to investigate the effects of short-term metformin treatment on liver steatosis and related serum markers for liver damage. Methods Five week old lean (n = 16) and obese (n = 16) female Zucker rats after one week of acclimation, received AIN-93 G diet for 8 weeks to induce NAFLD. After 8 weeks, lean and obese rats were randomly assigned to the following four groups (8 rats/group): 1) lean without metformin (LC), 2) lean with metformin (LMet), 3) obese without metformin (ObC), and 4) obese with metformin (ObMet). Metformin were mixed with AIN-93 G diet at 1000 mg/kg of diet. Rats were weighed twice per week. All rats were sacrificed 10 weeks post-metformin treatment and serum and livers were collected. Steatosis was semiquantitated as a score of 1 to 4 based upon the relative degree of steatosis within hepatocytes: 1) < 25%, 2) 25–50%, 3) 50–75%, and 4) >75%. Serums were collected to measure the levels of Aspartate Aminotransferase (AST), Alanine Aminotransferase (ALT) on a clinical analyzer. Results Obese rats gained significantly more weight (P < .001) than lean rats for both control and Metformin treatment groups and there was no significant difference between ObC vs. ObMet group (P = 0.20). The mean + SD liver steatosis scores for the LC, LMet, ObC and ObMet groups were 0.13 + o.3, 0.13 + o.3, 3.67 + 0.52 and 3.00 + 0.82. The ObMet treated rats had lower (P < 0.04) liver steatosis than ObC rats. There were no significant differences for the serum ALT and AST levels between groups. However, obesity increased significantly (P < 0.01) serum AST levels compared to LC but not in the metformin group. Conclusions In summary, in the obese zucker rat model, short-term metformin treatment decreased liver steatosis but did not impact serum markers of liver steatosis. Funding Sources Arkansas Children Research Institute/Arkansas Bioscience Institute.

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Peroxisomal-mitochondrial oxidation in a rodent model of obesity-associated insulin resistance

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00399.2006 ◽

2007 ◽

Vol 293 (4) ◽

pp. E986-E1001 ◽

Cited By ~ 46

Author(s):

Robert C. Noland ◽

Tracey L. Woodlief ◽

Brian R. Whitfield ◽

Steven M. Manning ◽

Jasper R. Evans ◽

...

Keyword(s):

Insulin Resistance ◽

Clinical Importance ◽

Product Distribution ◽

Zucker Rats ◽

Acid Oxidation ◽

Insulin Resistant ◽

Malonyl Coa ◽

Skeletal Muscle Lipid ◽

Mitochondrial Oxidation ◽

Cpt I

Peroxisomal oxidation yields metabolites that are more efficiently utilized by mitochondria. This is of potential clinical importance because reduced fatty acid oxidation is suspected to promote excess lipid accumulation in obesity-associated insulin resistance. Our purpose was to assess peroxisomal contributions to mitochondrial oxidation in mixed gastrocnemius (MG), liver, and left ventricle (LV) homogenates from lean and fatty ( fa/fa) Zucker rats. Results indicate that complete mitochondrial oxidation (CO2production) using various lipid substrates was increased approximately twofold in MG, unaltered in LV, and diminished ∼50% in liver of fa/fa rats. In isolated mitochondria, malonyl-CoA inhibited CO2production from palmitate 78%, whereas adding isolated peroxisomes reduced inhibition to 21%. These data demonstrate that peroxisomal products may enter mitochondria independently of CPT I, thus providing a route to maintain lipid disposal under conditions where malonyl-CoA levels are elevated, such as in insulin-resistant tissues. Peroxisomal metabolism of lignoceric acid in fa/fa rats was elevated in both liver and MG (LV unaltered), but peroxisomal product distribution varied. A threefold elevation in incomplete oxidation was solely responsible for increased hepatic peroxisomal oxidation (CO2unaltered). Alternatively, only CO2was detected in MG, indicating that peroxisomal products were exclusively partitioned to mitochondria for complete lipid disposal. These data suggest tissue-specific destinations for peroxisome-derived products and emphasize a potential role for peroxisomes in skeletal muscle lipid metabolism in the obese, insulin-resistant state.

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Bioenergetic and Proteomic Profiling of Immune Cells in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome Patients: An Exploratory Study

Biomolecules ◽

10.3390/biom11070961 ◽

2021 ◽

Vol 11 (7) ◽

pp. 961

Author(s):

Paula Fernandez-Guerra ◽

Ana C. Gonzalez-Ebsen ◽

Susanne E. Boonen ◽

Julie Courraud ◽

Niels Gregersen ◽

...

Keyword(s):

Chronic Fatigue Syndrome ◽

Complex Disease ◽

Mononuclear Cells ◽

Coupling Efficiency ◽

Well Being ◽

Chronic Fatigue ◽

Myalgic Encephalomyelitis ◽

Flux Analysis ◽

Proteomic Profiling ◽

Fatigue Syndrome

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a heterogeneous, debilitating, and complex disease. Along with disabling fatigue, ME/CFS presents an array of other core symptoms, including autonomic nervous system (ANS) dysfunction, sustained inflammation, altered energy metabolism, and mitochondrial dysfunction. Here, we evaluated patients' symptomatology and the mitochondrial metabolic parameters in peripheral blood mononuclear cells (PBMCs) and plasma from a clinically well-characterised cohort of six ME/CFS patients compared to age- and gender-matched controls. We performed a comprehensive cellular assessment using bioenergetics (extracellular flux analysis) and protein profiles (quantitative mass spectrometry-based proteomics) together with self-reported symptom measures of fatigue, ANS dysfunction, and overall physical and mental well-being. This ME/CFS cohort presented with severe fatigue, which correlated with the severity of ANS dysfunction and overall physical well-being. PBMCs from ME/CFS patients showed significantly lower mitochondrial coupling efficiency. They exhibited proteome alterations, including altered mitochondrial metabolism, centred on pyruvate dehydrogenase and coenzyme A metabolism, leading to a decreased capacity to provide adequate intracellular ATP levels. Overall, these results indicate that PBMCs from ME/CFS patients have a decreased ability to fulfill their cellular energy demands.

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Cell-Permeable Succinate Rescues Mitochondrial Respiration in Cellular Models of Statin Toxicity

International Journal of Molecular Sciences ◽

10.3390/ijms22010424 ◽

2021 ◽

Vol 22 (1) ◽

pp. 424

Author(s):

Vlad F. Avram ◽

Imen Chamkha ◽

Eleonor Åsander-Frostner ◽

Johannes K. Ehinger ◽

Romulus Z. Timar ◽

...

Keyword(s):

Oxygen Consumption ◽

Mitochondrial Dysfunction ◽

Mitochondrial Respiration ◽

Complex I ◽

Ex Vivo ◽

Coupling Efficiency ◽

Human Platelets ◽

Lipid Lowering ◽

Lipid Lowering Therapy ◽

Cellular Models

Statins are the cornerstone of lipid-lowering therapy. Although generally well tolerated, statin-associated muscle symptoms (SAMS) represent the main reason for treatment discontinuation. Mitochondrial dysfunction of complex I has been implicated in the pathophysiology of SAMS. The present study proposed to assess the concentration-dependent ex vivo effects of three statins on mitochondrial respiration in viable human platelets and to investigate whether a cell-permeable prodrug of succinate (complex II substrate) can compensate for statin-induced mitochondrial dysfunction. Mitochondrial respiration was assessed by high-resolution respirometry in human platelets, acutely exposed to statins in the presence/absence of the prodrug NV118. Statins concentration-dependently inhibited mitochondrial respiration in both intact and permeabilized cells. Further, statins caused an increase in non-ATP generating oxygen consumption (uncoupling), severely limiting the OXPHOS coupling efficiency, a measure of the ATP generating capacity. Cerivastatin (commercially withdrawn due to muscle toxicity) displayed a similar inhibitory capacity compared with the widely prescribed and tolerable atorvastatin, but did not elicit direct complex I inhibition. NV118 increased succinate-supported mitochondrial oxygen consumption in atorvastatin/cerivastatin-exposed platelets leading to normalization of coupled (ATP generating) respiration. The results acquired in isolated human platelets were validated in a limited set of experiments using atorvastatin in HepG2 cells, reinforcing the generalizability of the findings.

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Prominent Indomethacin-Induced Enteropathy in Fcgriib Defi-cient lupus Mice: An Impact of Macrophage Responses and Immune Deposition in Gut

International Journal of Molecular Sciences ◽

10.3390/ijms22031377 ◽

2021 ◽

Vol 22 (3) ◽

pp. 1377

Author(s):

Thansita Bhunyakarnjanarat ◽

Kanyarat Udompornpitak ◽

Wilasinee Saisorn ◽

Bhumdhanin Chantraprapawat ◽

Peerapat Visitchanakun ◽

...

Keyword(s):

Cytokine Production ◽

High Dose ◽

Flux Analysis ◽

Wild Type ◽

Fc Gamma Receptor ◽

Serum Cytokines ◽

Extracellular Flux ◽

Gamma Receptor ◽

Gut Leakage ◽

Gastrointestinal Permeability

A high dose of NSAIDs, a common analgesic, might induce lupus activity through several NSAIDs adverse effects including gastrointestinal permeability defect (gut leakage) and endotoxemia. Indomethacin (25 mg/day) was orally administered for 7 days in 24-wk-old Fc gamma receptor IIb deficient (FcgRIIb-/-) mice, an asymptomatic lupus model (increased anti-dsDNA without lupus nephritis), and age-matched wild-type (WT) mice. Severity of indomethacin-induced enteropathy in FcgRIIb-/- mice was higher than WT mice as demonstrated by survival analysis, intestinal injury (histology, immune-deposition, and intestinal cytokines), gut leakage (FITC-dextran assay and endotoxemia), serum cytokines, and lupus characteristics (anti-dsDNA, renal injury, and proteinuria). Prominent responses of FcgRIIb-/- macrophages toward lipopolysaccharide (LPS) compared to WT cells due to the expression of only activating-FcgRs without inhibitory-FcgRIIb were demonstrated. Extracellular flux analysis indicated the greater mitochondria activity (increased respiratory capacity and respiratory reserve) in FcgRIIb-/- macrophages with a concordant decrease in glycolysis activity when compared to WT cells. In conclusion, gut leakage-induced endotoxemia is more severe in indomethacin-administered FcgRIIb-/- mice than WT, possibly due to the enhanced indomethacin toxicity from lupus-induced intestinal immune-deposition. Due to a lack of inhibitory-FcgRIIb expression, mitochondrial function, and cytokine production of FcgRIIb-/- macrophages were more prominent than WT cells. Hence, lupus disease-activation from NSAIDs-enteropathy-induced gut leakage is possible.

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