Fuel oxidation under the control of mitochondrial shape and dynamics.

How mitochondrial shape and substrate-specific metabolism are related has been a difficult question to address. Here, new work by Ngo et al (2023) reports that mitochondrial shape-long versus fragmented-determines the activity of ß-oxidation of long-chain fatty acids, supporting a novel role for mitochondrial fission products as ß-oxidation hubs.

Enhanced Mitochondria-SR Tethering Triggers Adaptive Cardiac Muscle Remodeling.

BACKGROUND: Cardiac contractile function requires high energy from mitochondria, and Ca2+ from the sarcoplasmic reticulum (SR). Via local Ca2+ transfer at close mitochondria-SR contacts, cardiac excitation feedforward regulates mitochondrial ATP production to match surges in demand (excitation-bioenergetics coupling). However, pathological stresses may cause mitochondrial Ca2+ overload, excessive reactive oxygen species production and permeability transition, risking homeostatic collapse and myocyte loss. Excitation-bioenergetics coupling involves mitochondria-SR tethers but the role of tethering in cardiac physiology/pathology is debated. Endogenous tether proteins are multifunctional; therefore, nonselective targets to scrutinize interorganelle linkage. Here, we assessed the physiological/pathological relevance of selective chronic enhancement of cardiac mitochondria-SR tethering. METHODS: We introduced to mice a cardiac muscle-specific engineered tether (linker) transgene with a fluorescent protein core and deployed 2D/3D electron microscopy, biochemical approaches, fluorescence imaging, in vivo and ex vivo cardiac performance monitoring and stress challenges to characterize the linker phenotype. RESULTS: Expressed in the mature cardiomyocytes, the linker expanded and tightened individual mitochondria-junctional SR contacts; but also evoked a marked remodeling with large dense mitochondrial clusters that excluded dyads. Yet, excitation-bioenergetics coupling remained well-preserved, likely due to more longitudinal mitochondria-dyad contacts and nanotunnelling between mitochondria exposed to junctional SR and those sealed away from junctional SR. Remarkably, the linker decreased female vulnerability to acute massive ß-adrenergic stress. It also reduced myocyte death and mitochondrial calcium-overload-associated myocardial impairment in ex vivo ischemia/reperfusion injury. CONCLUSIONS: We propose that mitochondria-SR/endoplasmic reticulum contacts operate at a structural optimum. Although acute changes in tethering may cause dysfunction, upon chronic enhancement of contacts from early life, adaptive remodeling of the organelles shifts the system to a new, stable structural optimum. This remodeling balances the individually enhanced mitochondrion-junctional SR crosstalk and excitation-bioenergetics coupling, by increasing the connected mitochondrial pool and, presumably, Ca2+/reactive oxygen species capacity, which then improves the resilience to stresses associated with dysregulated hyperactive Ca2+ signaling.

Comparison of colorimetric, fluorometric, and liquid chromatography-mass spectrometry assays for acetyl-coenzyme A.

Acetyl-Coenzyme A is a central metabolite in catabolic and anabolic pathways as well as the acyl donor for acetylation reactions. Multiple quantitative measurement techniques for acetyl-CoA have been reported, including commercially available kits. Comparisons between techniques for acetyl-CoA measurement have not been reported. This lack of comparability between assays makes context-specific assay selection and interpretation of results reporting changes in acetyl-CoA metabolism difficult. We compared commercially available colorimetric ELISA and fluorometric enzymatic-based kits to liquid chromatography-mass spectrometry-based assays using tandem mass spectrometry (LC-MS/MS) and high-resolution mass spectrometry (LC-HRMS). The colorimetric ELISA kit did not produce interpretable results even with commercially available pure standards. The fluorometric enzymatic kit produced comparable results to the LC-MS-based assays depending on matrix and extraction. LC-MS/MS and LC-HRMS assays produced well-aligned results, especially when incorporating stable isotope-labeled internal standards. In addition, we demonstrated the multiplexing capability of the LC-HRMS assay by measuring a suite of short-chain acyl-CoAs in a variety of acute myeloid leukemia cell lines and patient cells.

Adaptation of the heart to Frataxin depletion: Evidence that integrated stress response can predominate over mTORC1 activation.

Friedreich's ataxia (FRDA) is an inherited disorder caused by depletion of frataxin (FXN), a mitochondrial protein required for iron-sulfur cluster (ISC) biogenesis. Cardiac dysfunction is the main cause of death. Yet pathogenesis, and, more generally, how the heart adapts to FXN loss, remain poorly understood, though are expected to be linked to an energy deficit. We modified a transgenic (TG) mouse model of inducible FXN depletion that permits phenotypic evaluation of the heart at different FXN levels, and focused on substrate-specific bioenergetics and stress signaling. When FXN protein in the TG heart was 17% of normal, bioenergetics and signaling were not different from control. When, 8 weeks later, FXN was ~ 97% depleted in the heart, TG heart mass and cardiomyocyte cross-sectional area were less, without evidence of fibrosis or apoptosis. mTORC1 signaling was activated, as was the integrated stress response, evidenced by greater phosphorylation of eIF2α relative to total eIF2α, and decreased protein translation. We interpret these results to suggest that, in TG hearts, an anabolic stimulus was constrained by eIF2α phosphorylation. Cardiac contractility was maintained in the 97%-FXN-depleted hearts, possibly contributed by an unexpected preservation of ß-oxidation, though pyruvate oxidation was lower. Bioenergetics alterations were matched by changes in the mitochondrial proteome, including a non-uniform decrease in abundance of ISC-containing proteins. Altogether, these findings suggest that the FXN depleted heart can suppress a major ATP demanding process such as protein translation, which, together with some preservation of ß-oxidation, could be adaptive, at least in the short term.

Mitochondrial Calcium Uniporter Affects Neutrophil Bactericidal Activity during Staphylococcus aureus Infection.

Neutrophils simultaneously restrict Staphylococcus aureus dissemination and facilitate bactericidal activity during infection through the formation of neutrophil extracellular traps (NETs). Neutrophils that produce higher levels of mitochondrial superoxide undergo enhanced terminal NET formation (suicidal NETosis) in response to S. aureus; however, mechanisms regulating mitochondrial homeostasis upstream of neutrophil antibacterial processes are not fully resolved. Here, we demonstrate that mitochondrial calcium uptake 1 (MICU1)-deficient (MICU1-/-) neutrophils accumulate higher levels of calcium and iron within the mitochondria in a mitochondrial calcium uniporter (MCU)-dependent manner. Corresponding with increased ion flux through the MCU, mitochondrial superoxide production is elevated, thereby increasing the propensity for MICU1-/- neutrophils to undergo suicidal NETosis rather than primary degranulation in response to S. aureus. Increased NET formation augments macrophage killing of bacterial pathogens. Similarly, MICU1-/- neutrophils alone are not more antibacterial toward S. aureus, but rather, enhanced suicidal NETosis by MICU1-/- neutrophils facilitates increased bactericidal activity in the presence of macrophages. Similarly, mice with a deficiency in MICU1 restricted to cells expressing LysM exhibit lower bacterial burdens in the heart with increased survival during systemic S. aureus infection. Coinciding with the decrease in S. aureus burdens, MICU1-/- neutrophils in the heart produce higher levels of mitochondrial superoxide and undergo enhanced suicidal NETosis. These results demonstrate that ion flux by the MCU affects the antibacterial function of neutrophils during S. aureus infection.

Multiple mitochondrial thioesterases have distinct tissue and substrate specificity and CoA regulation, suggesting unique functional roles.

Acyl-CoA thioesterases (Acots) hydrolyze fatty acyl-CoA esters. Acots in the mitochondrial matrix are poised to mitigate ß-oxidation overload and maintain CoA availability. Several Acots associate with mitochondria, but whether they all localize to the matrix, are redundant, or have different roles is unresolved. Here, we compared the suborganellar localization, activity, expression, and regulation among mitochondrial Acots (Acot2, -7, -9, and -13) in mitochondria from multiple mouse tissues and from a model of Acot2 depletion. Acot7, -9, and -13 localized to the matrix, joining Acot2 that was previously shown to localize there. Mitochondria from heart, skeletal muscle, brown adipose tissue, and kidney robustly expressed Acot2, -9, and -13; Acot9 levels were substantially higher in brown adipose tissue and kidney mitochondria, as was activity for C4:0-CoA, a unique Acot9 substrate. In all tissues, Acot2 accounted for about half of the thioesterase activity for C14:0-CoA and C16:0-CoA. In contrast, liver mitochondria from fed and fasted mice expressed little Acot activity, which was confined to long-chain CoAs and due mainly to Acot7 and Acot13 activities. Matrix Acots occupied different functional niches, based on substrate specificity (Acot9 versus Acot2 and -13) and strong CoA inhibition (Acot7, -9, and -13, but not Acot2). Interpreted in the context of ß-oxidation, CoA inhibition would prevent Acot-mediated suppression of ß-oxidation, while providing a release valve when CoA is limiting. In contrast, CoA-insensitive Acot2 could provide a constitutive siphon for long-chain fatty acyl-CoAs. These results reveal how the family of matrix Acots can mitigate ß-oxidation overload and prevent CoA limitation.

Metabolic reprogramming of murine cardiomyocytes during autophagy requires the extracellular nutrient sensor decorin.

The extracellular matrix is a master regulator of tissue homeostasis in health and disease. Here we examined how the small, leucine-rich, extracellular matrix proteoglycan decorin regulates cardiomyocyte metabolism during fasting in vivo First, we validated in Dcn-/- mice that decorin plays an essential role in autophagy induced by fasting. High-throughput metabolomics analyses of cardiac tissue in Dcn-/- mice subjected to fasting revealed striking differences in the hexosamine biosynthetic pathway resulting in aberrant cardiac O-ß-N-acetylglycosylation as compared with WT mice. Functionally, Dcn-/- mice maintained cardiac function at a level comparable with nonfasted animals whereas fasted WT mice showed reduced ejection fraction. Collectively, our results suggest that reduced sensing of nutrient deprivation in the absence of decorin preempts functional adjustments of cardiac output associated with metabolic reprogramming.

Arsenic-induced metabolic shift triggered by the loss of miR-199a-5p through Sp1-dependent DNA methylation.

Inorganic arsenic is an environmental carcinogen that poses a major global public health risk. A high percentage of drinking water from wells in the U.S. contains higher-than-normal levels of arsenic, suggesting an increased risk of arsenic-induced deleterious effects. In addition to primary preventive measures, therapeutic strategies need to effectively address and integrate multiple molecular mechanisms underlying arsenic-induced carcinogenesis. We previously showed that the loss of miR-199a-5p in arsenic-transformed cells is pivotal to promote arsenic-induced angiogenesis and tumor growth in lung epithelial cells. In this study, we further showed that subacute or chronic exposure to arsenic diminished miR-199a-5p levels largely due to DNA methylation, which was achieved by increased DNA methyltransferase-1 (DNMT1) activity, mediated by the formation of specific protein 1 (Sp1)/DNMT1 complex. In addition to the DNA hypermethylation, arsenic exposure also repressed miR-199a transcription through a transcriptional repressor Sp1. We further identified an association between miR-199a-5p repression and the arsenic-mediated energy metabolic shift, as reflected by mitochondria defects and a switch to glycolysis, in which a glycolytic enzyme pyruvate kinase 2 (PKM2) was a functional target of miR-199a-5p. Taken together, the repression of miR-199a-5p through both Sp1-dependent DNA methylation and Sp1 transcriptional repression promotes an arsenic-mediated metabolic shift from mitochondria respiration to aerobic glycolysis via PKM2.

Mitochondrial functions modulate neuroendocrine, metabolic, inflammatory, and transcriptional responses to acute psychological stress.

The experience of psychological stress triggers neuroendocrine, inflammatory, metabolic, and transcriptional perturbations that ultimately predispose to disease. However, the subcellular determinants of this integrated, multisystemic stress response have not been defined. Central to stress adaptation is cellular energetics, involving mitochondrial energy production and oxidative stress. We therefore hypothesized that abnormal mitochondrial functions would differentially modulate the organism's multisystemic response to psychological stress. By mutating or deleting mitochondrial genes encoded in the mtDNA [NADH dehydrogenase 6 (ND6) and cytochrome c oxidase subunit I (COI)] or nuclear DNA [adenine nucleotide translocator 1 (ANT1) and nicotinamide nucleotide transhydrogenase (NNT)], we selectively impaired mitochondrial respiratory chain function, energy exchange, and mitochondrial redox balance in mice. The resulting impact on physiological reactivity and recovery from restraint stress were then characterized. We show that mitochondrial dysfunctions altered the hypothalamic-pituitary-adrenal axis, sympathetic adrenal-medullary activation and catecholamine levels, the inflammatory cytokine IL-6, circulating metabolites, and hippocampal gene expression responses to stress. Each mitochondrial defect generated a distinct whole-body stress-response signature. These results demonstrate the role of mitochondrial energetics and redox balance as modulators of key pathophysiological perturbations previously linked to disease. This work establishes mitochondria as stress-response modulators, with implications for understanding the mechanisms of stress pathophysiology and mitochondrial diseases.

Natural and Induced Mitochondrial Phosphate Carrier Loss: DIFFERENTIAL DEPENDENCE OF MITOCHONDRIAL METABOLISM AND DYNAMICS AND CELL SURVIVAL ON THE EXTENT OF DEPLETION.

The relevance of mitochondrial phosphate carrier (PiC), encoded by SLC25A3, in bioenergetics is well accepted. However, little is known about the mechanisms mediating the cellular impairments induced by pathological SLC25A3 variants. To this end, we investigated the pathogenicity of a novel compound heterozygous mutation in SLC25A3 First, each variant was modeled in yeast, revealing that substituting GSSAS for QIP within the fifth matrix loop is incompatible with survival on non-fermentable substrate, whereas the L200W variant is functionally neutral. Next, using skin fibroblasts from an individual expressing these variants and HeLa cells with varying degrees of PiC depletion, PiC loss of ∼60% was still compatible with uncompromised maximal oxidative phosphorylation (oxphos), whereas lower maximal oxphos was evident at ∼85% PiC depletion. Furthermore, intact mutant fibroblasts displayed suppressed mitochondrial bioenergetics consistent with a lower substrate availability rather than phosphate limitation. This was accompanied by slowed proliferation in glucose-replete medium; however, proliferation ceased when only mitochondrial substrate was provided. Both mutant fibroblasts and HeLa cells with 60% PiC loss showed a less interconnected mitochondrial network and a mitochondrial fusion defect that is not explained by altered abundance of OPA1 or MFN1/2 or relative amount of different OPA1 forms. Altogether these results indicate that PiC depletion may need to be profound (>85%) to substantially affect maximal oxphos and that pathogenesis associated with PiC depletion or loss of function may be independent of phosphate limitation when ATP requirements are not high.

TP53-inducible Glycolysis and Apoptosis Regulator (TIGAR) Metabolically Reprograms Carcinoma and Stromal Cells in Breast Cancer.

A subgroup of breast cancers has several metabolic compartments. The mechanisms by which metabolic compartmentalization develop in tumors are poorly characterized. TP53 inducible glycolysis and apoptosis regulator (TIGAR) is a bisphosphatase that reduces glycolysis and is highly expressed in carcinoma cells in the majority of human breast cancers. Hence we set out to determine the effects of TIGAR expression on breast carcinoma and fibroblast glycolytic phenotype and tumor growth. The overexpression of this bisphosphatase in carcinoma cells induces expression of enzymes and transporters involved in the catabolism of lactate and glutamine. Carcinoma cells overexpressing TIGAR have higher oxygen consumption rates and ATP levels when exposed to glutamine, lactate, or the combination of glutamine and lactate. Coculture of TIGAR overexpressing carcinoma cells and fibroblasts compared with control cocultures induce more pronounced glycolytic differences between carcinoma and fibroblast cells. Carcinoma cells overexpressing TIGAR have reduced glucose uptake and lactate production. Conversely, fibroblasts in coculture with TIGAR overexpressing carcinoma cells induce HIF (hypoxia-inducible factor) activation with increased glucose uptake, increased 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 (PFKFB3), and lactate dehydrogenase-A expression. We also studied the effect of this enzyme on tumor growth. TIGAR overexpression in carcinoma cells increases tumor growth in vivo with increased proliferation rates. However, a catalytically inactive variant of TIGAR did not induce tumor growth. Therefore, TIGAR expression in breast carcinoma cells promotes metabolic compartmentalization and tumor growth with a mitochondrial metabolic phenotype with lactate and glutamine catabolism. Targeting TIGAR warrants consideration as a potential therapy for breast cancer.

The retinal pigment epithelium utilizes fatty acids for ketogenesis.

Every day, shortly after light onset, photoreceptor cells shed approximately a tenth of their outer segment. The adjacent retinal pigment epithelial (RPE) cells phagocytize and digest shed photoreceptor outer segment, which provides a rich source of fatty acids that could be utilized as an energy substrate. From a microarray analysis, we found that RPE cells express particularly high levels of the mitochondrial HMG-CoA synthase 2 (Hmgcs2) compared with all other tissues (except the liver and colon), leading to the hypothesis that RPE cells, like hepatocytes, can produce ß-hydroxybutyrate (ß-HB) from fatty acids. Using primary human fetal RPE (hfRPE) cells cultured on Transwell filters with separate apical and basal chambers, we demonstrate that hfRPE cells can metabolize palmitate, a saturated fatty acid that constitutes .15% of all lipids in the photoreceptor outer segment, to produce ß-HB. Importantly, we found that hfRPE cells preferentially release ß-HB into the apical chamber and that this process is mediated primarily by monocarboxylate transporter isoform 1 (MCT1). Using a GC-MS analysis of (13)C-labeled metabolites, we showed that retinal cells can take up and metabolize (13)C-labeled ß-HB into various TCA cycle intermediates and amino acids. Collectively, our data support a novel mechanism of RPE-retina metabolic coupling in which RPE cells metabolize fatty acids to produce ß-HB, which is transported to the retina for use as a metabolic substrate.

The mitochondrial phosphate carrier: Role in oxidative metabolism, calcium handling and mitochondrial disease.

The mitochondrial phosphate carrier (PiC) is a mitochondrial solute carrier protein, which is encoded by SLC25A3 in humans. PiC delivers phosphate, a key substrate of oxidative phosphorylation, across the inner mitochondrial membrane. This transport activity is also relevant for allowing effective mitochondrial calcium handling. Furthermore, PiC has also been described to affect cell survival mechanisms via interactions with cyclophilin D and the viral mitochondrial-localized inhibitor of apoptosis (vMIA). The significance of PiC has been supported by the recent discovery of a fatal human condition associated with PiC mutations. Here, we present first the early studies that lead to the discovery and molecular characterization of the PiC, then discuss the very recently developed mouse models for PiC and pathological mutations in the human SLC25A3 gene.

IL-15Rα is a determinant of muscle fuel utilization, and its loss protects against obesity.

IL-15Rα is the widely expressed primary binding partner for IL-15. Because of the wide distribution in nonlymphoid tissues like skeletal muscle, adipose, or liver, IL-15/IL-15Rα take part in physiological and metabolic processes not directly related to immunity. In fast muscle, lack of IL-15Rα promotes an oxidative switch, with increased mitochondrial biogenesis and fatigue resistance. These effects are predicted to reproduce some of the benefits of exercise and, therefore, improve energy homeostasis. However, the direct effects of IL-15Rα on metabolism and obesity are currently unknown. We report that mice lacking IL-15Rα (IL-15Rα(-/-)) are resistant to diet-induced obesity (DIO). High-fat diet-fed IL-15Rα(-/-) mice have less body and liver fat accumulation than controls. The leaner phenotype is associated with increased energy expenditure and enhanced fatty acid oxidation by muscle mitochondria. Despite being protected against DIO, IL-15Rα(-/-) are hyperglycemic and insulin-resistant. These findings identify novel roles for IL-15Rα in metabolism and obesity.

The SIRT1 deacetylase protects mice against the symptoms of metabolic syndrome.

Type 2 diabetes, hepatic steatosis, and gut dysbiosis are pathophysiological consequences of obesity. Sirtuin (SIRT)-1 is a protein deacetylase implicated in the regulation of metabolic activity. We set out to determine whether the catalytic activity of SIRT1 plays a role in the development of metabolic syndrome, hepatic steatosis, and the distribution of gut microbiota. We challenged with a high-fat diet (HFD) a strain of mice homozygous for a Sirt1 allele carrying a point mutation that ablates the deacetylase activity of SIRT1. When compared to wild-type animals, mice lacking SIRT1 catalytic activity rapidly accumulated excessive hepatic lipid while fed the HFD, an effect evident within 2 wk of HFD feeding. Both white and brown adipose depots became hypertrophic, and the animals developed insulin resistance. The ratio of the major phyla of gut microbiota (Firmicutes and Bacteroidetes) increased rapidly in the SIRT1-deficient mice after HFD challenge. We conclude that the deacetylase activity of SIRT1 plays an important role in regulating glucose and hepatic lipid homeostasis. In addition, the composition of gut microbiota is influenced by both the animals' Sirt1 genotype and diet composition.

Functional mitochondrial analysis in acute brain sections from adult rats reveals mitochondrial dysfunction in a rat model of migraine.

Mitochondrial dysfunction has been implicated in many neurological disorders that only develop or are much more severe in adults, yet no methodology exists that allows for medium-throughput functional mitochondrial analysis of brain sections from adult animals. We developed a technique for quantifying mitochondrial respiration in acutely isolated adult rat brain sections with the Seahorse XF Analyzer. Evaluating a range of conditions made quantifying mitochondrial function from acutely derived adult brain sections from the cortex, cerebellum, and trigeminal nucleus caudalis possible. Optimization of this technique demonstrated that the ideal section size was 1 mm wide. We found that sectioning brains at physiological temperatures was necessary for consistent metabolic analysis of trigeminal nucleus caudalis sections. Oxygen consumption in these sections was highly coupled to ATP synthesis, had robust spare respiratory capacities, and had limited nonmitochondrial respiration, all indicative of healthy tissue. We demonstrate the effectiveness of this technique by identifying a decreased spare respiratory capacity in the trigeminal nucleus caudalis of a rat model of chronic migraine, a neurological disorder that has been associated with mitochondrial dysfunction. This technique allows for 24 acutely isolated sections from multiple brain regions of a single adult rat to be analyzed simultaneously with four sequential drug treatments, greatly advancing the ability to study mitochondrial physiology in adult neurological disorders.

Acyl-CoA thioesterase-2 facilitates mitochondrial fatty acid oxidation in the liver.

Acyl-CoA thioesterase (Acot)2 localizes to the mitochondrial matrix and hydrolyses long-chain fatty acyl-CoA into free FA and CoASH. Acot2 is expressed in highly oxi-dative tissues and is poised to modulate mitochondrial FA oxidation (FAO), yet its biological role is unknown. Using a model of adenoviral Acot2 overexpression in mouse liver (Ad-Acot2), we show that Acot2 increases the utilization of FA substrate during the daytime in ad libitum-fed mice, but the nighttime switch to carbohydrate oxidation is similar to control mice. In further support of elevated FAO in Acot2 liver, daytime serum ketones were higher in Ad-Acot2 mice, and overnight fasting led to minimal hepatic steatosis as compared with control mice. In liver mitochondria from Ad-Acot2 mice, phosphorylating O2 consumption was higher with lipid substrate, but not with nonlipid substrate. This increase depended on whether FA could be activated on the outer mitochondrial membrane, suggesting that the FA released by Acot2 could be effluxed from mitochondria then taken back up again for oxidation. This circuit would prevent the build-up of inhibitory long-chain fatty acyl-CoA esters. Altogether, our findings indicate that Acot2 can enhance FAO, possibly by mitigating the accumulation of FAO intermediates within the mitochondrial matrix.

Muscle uncoupling protein 3 overexpression mimics endurance training and reduces circulating biomarkers of incomplete ß-oxidation.

Exercise substantially improves metabolic health, making the elicited mechanisms important targets for novel therapeutic strategies. Uncoupling protein 3 (UCP3) is a mitochondrial inner membrane protein highly selectively expressed in skeletal muscle. Here we report that moderate UCP3 overexpression (roughly 3-fold) in muscles of UCP3 transgenic (UCP3 Tg) mice acts as an exercise mimetic in many ways. UCP3 overexpression increased spontaneous activity (∼40%) and energy expenditure (∼5-10%) and decreased oxidative stress (∼15-20%), similar to exercise training in wild-type (WT) mice. The increase in complete fatty acid oxidation (FAO; ∼30% for WT and ∼70% for UCP3 Tg) and energy expenditure (∼8% for WT and 15% for UCP3 Tg) in response to endurance training was higher in UCP3 Tg than in WT mice, showing an additive effect of UCP3 and endurance training on these two parameters. Moreover, increases in circulating short-chain acylcarnitines in response to acute exercise in untrained WT mice were absent with training or in UCP3 Tg mice. UCP3 overexpression had the same effect as training in decreasing long-chain acylcarnitines. Outcomes coincided with a reduction in muscle carnitine acetyltransferase activity that catalyzes the formation of acylcarnitines. Overall, results are consistent with the conclusions that circulating acylcarnitines could be used as a marker of incomplete muscle FAO and that UCP3 is a potential target for the treatment of prevalent metabolic diseases in which muscle FAO is affected.

SirT1 catalytic activity is required for male fertility and metabolic homeostasis in mice.

The protein encoded by the sirt1 gene is an enzyme, SirT1, that couples the hydrolysis of NAD(+) to the deacetylation of acetyl-lysine residues in substrate proteins. Mutations of the sirt1 gene that fail to encode protein have been introduced into the mouse germ line, and the animals homozygous for these null mutations have various physiological abnormalities. To determine which of the characteristics of these sirt1(-/-) mice are a consequence of the absence of the catalytic activity of the SirT1 protein, we created a mouse strain carrying a point mutation (H355Y) that ablates the catalytic activity but does not affect the amount of the SirT1 protein. Mice carrying point mutations in both sirt1 genes, sirt1(Y/Y), have a phenotype that is overlapping but not identical to that of the sirt1-null animals. The sirt1(Y/Y) phenotype is significantly milder than that seen in the sirt1(-/-) animals. For example, female sirt1(Y/Y) animals are fertile, while sirt1(-/-) females are sterile. On the other hand, both sirt1(-/-) and sirt1(Y/Y) male mice are sterile and hypermetabolic. We report that sirt1(Y/Y) mice respond aberrantly to caloric restriction, although the effects are more subtle than seen in sirt1(-/-) mice. Thus, the SirT1 protein has functions that can be attributed to the catalytic activity of the protein, as well as other functions that are conferred by the protein itself.

Intraperitoneal instillation of bupivacaine for reduction of postoperative pain after laparoscopic hysterectomy: a double-blind randomized controlled trial.

STUDY OBJECTIVE: To evaluate the effect on postoperative pain of intraperitoneal instillation of dilute bupivacaine at the conclusion of laparoscopic hysterectomy. DESIGN: Prospective, randomized, double-blind, placebo-controlled trial (Canadian Task Force classification I). SETTING: Tertiary care, urban, academic teaching hospital. PATIENTS: Women aged 18 to 65 years undergoing total or supracervical laparoscopic hysterectomy with or without salpingo-oophorectomy. INTERVENTION: Randomization to intraperitoneal instillation of bupivacaine vs normal saline solution at the conclusion of laparoscopic hysterectomy performed because of benign indications. MEASUREMENTS AND MAIN RESULTS: A total of 160 patients consented to participate in the study and were randomized to receive either intraperitoneal instillation of 100 mg bupivacaine in 100 mL normal saline solution or 100 mL normal saline solution alone, at the conclusion of laparoscopic hysterectomy. Sixty seven of 77 patients (87%) in the treatment group and 73 of 80 patients (91%) in the placebo group completed the study. There were no significant differences in demographic profile, indication for hysterectomy, or number of previous surgeries between the two groups. All patients were prescribed a standardized routine postoperative analgesic regimen. Pain was measured by patient self-report using a 10-cm visual analog scale (VAS) at 1, 2, 4, 6, 12, and 24 hours postoperatively. Mean VAS scores at all time points were between 2.0 and 4.3 and were highest in the first postoperative hour. VAS scores were not significantly different between the two groups at any time point. None of the measured secondary outcomes were significantly different between the bupivacaine and placebo groups, including total postoperative opioid analgesic use in morphine equivalents (23.2 mg vs 27.5 mg; p = .09), length of hospital stay in hours (23.3 vs 23.0; p = .49), patient satisfaction on a 10-cm VAS (9.0 vs 8.2; p = .12), and complication rates (9% vs 15%; p = .35). CONCLUSION: Intraperitoneal instillation of bupivacaine at the conclusion of laparoscopic hysterectomy does not reduce postoperative pain. Opioid analgesic use, length of hospital stay, overall patient satisfaction, and complication rates are also unchanged. Self-reported postoperative pain was low in both groups after this major gynecologic surgery performed laparoscopically.