Mitochondrial oxidative stress causes insulin resistance without disrupting oxidative phosphorylation.

Mitochondrial oxidative stress, mitochondrial dysfunction, or both have been implicated in insulin resistance. However, disentangling the individual roles of these processes in insulin resistance has been difficult because they often occur in tandem, and tools that selectively increase oxidant production without impairing mitochondrial respiration have been lacking. Using the dimer/monomer status of peroxiredoxin isoforms as an indicator of compartmental hydrogen peroxide burden, we provide evidence that oxidative stress is localized to mitochondria in insulin-resistant 3T3-L1 adipocytes and adipose tissue from mice. To dissociate oxidative stress from impaired oxidative phosphorylation and study whether mitochondrial oxidative stress per se can cause insulin resistance, we used mitochondria-targeted paraquat (MitoPQ) to generate superoxide within mitochondria without directly disrupting the respiratory chain. At ≤10 µm, MitoPQ specifically increased mitochondrial superoxide and hydrogen peroxide without altering mitochondrial respiration in intact cells. Under these conditions, MitoPQ impaired insulin-stimulated glucose uptake and glucose transporter 4 (GLUT4) translocation to the plasma membrane in both adipocytes and myotubes. MitoPQ recapitulated many features of insulin resistance found in other experimental models, including increased oxidants in mitochondria but not cytosol; a more profound effect on glucose transport than on other insulin-regulated processes, such as protein synthesis and lipolysis; an absence of overt defects in insulin signaling; and defective insulin- but not AMP-activated protein kinase (AMPK)-regulated GLUT4 translocation. We conclude that elevated mitochondrial oxidants rapidly impair insulin-regulated GLUT4 translocation and significantly contribute to insulin resistance and that MitoPQ is an ideal tool for studying the link between mitochondrial oxidative stress and regulated GLUT4 trafficking.

High dietary fat and sucrose results in an extensive and time-dependent deterioration in health of multiple physiological systems in mice.

Obesity is associated with metabolic dysfunction, including insulin resistance and hyperinsulinemia, and with disorders such as cardiovascular disease, osteoporosis, and neurodegeneration. Typically, these pathologies are examined in discrete model systems and with limited temporal resolution, and whether these disorders co-occur is therefore unclear. To address this question, here we examined multiple physiological systems in male C57BL/6J mice following prolonged exposure to a high-fat/high-sucrose diet (HFHSD). HFHSD-fed mice rapidly exhibited metabolic alterations, including obesity, hyperleptinemia, physical inactivity, glucose intolerance, peripheral insulin resistance, fasting hyperglycemia, ectopic lipid deposition, and bone deterioration. Prolonged exposure to HFHSD resulted in morbid obesity, ectopic triglyceride deposition in liver and muscle, extensive bone loss, sarcopenia, hyperinsulinemia, and impaired short-term memory. Although many of these defects are typically associated with aging, HFHSD did not alter telomere length in white blood cells, indicating that this diet did not generally promote all aspects of aging. Strikingly, glucose homeostasis was highly dynamic. Glucose intolerance was evident in HFHSD-fed mice after 1 week and was maintained for 24 weeks. Beyond 24 weeks, however, glucose tolerance improved in HFHSD-fed mice, and by 60 weeks, it was indistinguishable from that of chow-fed mice. This improvement coincided with adaptive ß-cell hyperplasia and hyperinsulinemia, without changes in insulin sensitivity in muscle or adipose tissue. Assessment of insulin secretion in isolated islets revealed that leptin, which inhibited insulin secretion in the chow-fed mice, potentiated glucose-stimulated insulin secretion in the HFHSD-fed mice after 60 weeks. Overall, the excessive calorie intake was accompanied by deteriorating function of numerous physiological systems.

Metabolomic analysis of insulin resistance across different mouse strains and diets.

Insulin resistance is a major risk factor for many diseases. However, its underlying mechanism remains unclear in part because it is triggered by a complex relationship between multiple factors, including genes and the environment. Here, we used metabolomics combined with computational methods to identify factors that classified insulin resistance across individual mice derived from three different mouse strains fed two different diets. Three inbred ILSXISS strains were fed high-fat or chow diets and subjected to metabolic phenotyping and metabolomics analysis of skeletal muscle. There was significant metabolic heterogeneity between strains, diets, and individual animals. Distinct metabolites were changed with insulin resistance, diet, and between strains. Computational analysis revealed 113 metabolites that were correlated with metabolic phenotypes. Using these 113 metabolites, combined with machine learning to segregate mice based on insulin sensitivity, we identified C22:1-CoA, C2-carnitine, and C16-ceramide as the best classifiers. Strikingly, when these three metabolites were combined into one signature, they classified mice based on insulin sensitivity more accurately than each metabolite on its own or other published metabolic signatures. Furthermore, C22:1-CoA was 2.3-fold higher in insulin-resistant mice and correlated significantly with insulin resistance. We have identified a metabolomic signature composed of three functionally unrelated metabolites that accurately predicts whole-body insulin sensitivity across three mouse strains. These data indicate the power of simultaneous analysis of individual, genetic, and environmental variance in mice for identifying novel factors that accurately predict metabolic phenotypes like whole-body insulin sensitivity.

Proteomic Analysis of GLUT4 Storage Vesicles Reveals Tumor Suppressor Candidate 5 (TUSC5) as a Novel Regulator of Insulin Action in Adipocytes.

Insulin signaling augments glucose transport by regulating glucose transporter 4 (GLUT4) trafficking from specialized intracellular compartments, termed GLUT4 storage vesicles (GSVs), to the plasma membrane. Proteomic analysis of GSVs by mass spectrometry revealed enrichment of 59 proteins in these vesicles. We measured reduced abundance of 23 of these proteins following insulin stimulation and assigned these as high confidence GSV proteins. These included established GSV proteins such as GLUT4 and insulin-responsive aminopeptidase, as well as six proteins not previously reported to be localized to GSVs. Tumor suppressor candidate 5 (TUSC5) was shown to be a novel GSV protein that underwent a 3.7-fold increase in abundance at the plasma membrane in response to insulin. siRNA-mediated knockdown of TUSC5 decreased insulin-stimulated glucose uptake, although overexpression of TUSC5 had the opposite effect, implicating TUSC5 as a positive regulator of insulin-stimulated glucose transport in adipocytes. Incubation of adipocytes with TNFα caused insulin resistance and a concomitant reduction in TUSC5. Consistent with previous studies, peroxisome proliferator-activated receptor (PPAR) γ agonism reversed TNFα-induced insulin resistance. TUSC5 expression was necessary but insufficient for PPARγ-mediated reversal of insulin resistance. These findings functionally link TUSC5 to GLUT4 trafficking, insulin action, insulin resistance, and PPARγ action in the adipocyte. Further studies are required to establish the exact role of TUSC5 in adipocytes.

The incidence and implications of anti-heparin-platelet factor 4 antibody formation in a pediatric cardiac surgical population.

BACKGROUND: The incidence and implications of anti-heparin-platelet factor 4 (PF4) antibody seroconversion in the pediatric cardiac surgical population remain largely unexplored. We sought to prospectively characterize the incidence of seroconversion in two populations: neonates undergoing primary cardiac surgery and children undergoing reoperative cardiac surgery with a history of unfractionated heparin (UFH) exposure. METHODS: One hundred and thirty-five consecutive patients were studied: Neonatal = 60 neonates, first time cardiac surgery. Reoperative (ReOp) = 75 children, reoperative cardiac surgery. Preoperative and postoperative day (POD) 5 and 10 blood samples were used to determine the presence of PF4 immunoglobulin (Ig)G, IgA, and IgM antibodies with enzyme-linked immunosorbent assay. RESULTS: No anti-heparin/PF4 antibodies were detected preoperatively in either group. On POD 5, antibodies were present in 1 of 60 (1.7%) Neonatal; and in 12 of 75 (16%) ReOp; P = 0.006. On POD 10, antibodies were present in 1 of 60 (1.7%) Neonatal; and in 39 of 75 (52%) ReOp; P < 0.001. Seroconversion in ReOp patients on POD 10 was significantly associated (P = 0.03) with previous UFH exposures. Heparin-induced thrombocytopenia (HIT) was not diagnosed in any Neonatal patients. One ReOp patient (1.3%) seroconverted and developed HIT without thrombosis or skin lesions. CONCLUSIONS: HIT is a rare occurrence in pediatric cardiac surgical patients. The incidence of anti-heparin-PF4 antibody seroconversion in children undergoing reoperation is approximately 50% at 10 days postoperatively, a finding similar to that reported in adult cardiac surgical patients. Both age and previous UFH exposure correlate with this rate of seroconversion. In contrast, the rate of seroconversion in neonates undergoing first time surgery is substantially lower.

Mitochondrial CoQ deficiency is a common driver of mitochondrial oxidants and insulin resistance.

Insulin resistance in muscle, adipocytes and liver is a gateway to a number of metabolic diseases. Here, we show a selective deficiency in mitochondrial coenzyme Q (CoQ) in insulin-resistant adipose and muscle tissue. This defect was observed in a range of in vitro insulin resistance models and adipose tissue from insulin-resistant humans and was concomitant with lower expression of mevalonate/CoQ biosynthesis pathway proteins in most models. Pharmacologic or genetic manipulations that decreased mitochondrial CoQ triggered mitochondrial oxidants and insulin resistance while CoQ supplementation in either insulin-resistant cell models or mice restored normal insulin sensitivity. Specifically, lowering of mitochondrial CoQ caused insulin resistance in adipocytes as a result of increased superoxide/hydrogen peroxide production via complex II. These data suggest that mitochondrial CoQ is a proximal driver of mitochondrial oxidants and insulin resistance, and that mechanisms that restore mitochondrial CoQ may be effective therapeutic targets for treating insulin resistance.

Evidence for ACTN3 as a genetic modifier of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is characterized by muscle degeneration and progressive weakness. There is considerable inter-patient variability in disease onset and progression, which can confound the results of clinical trials. Here we show that a common null polymorphism (R577X) in ACTN3 results in significantly reduced muscle strength and a longer 10 m walk test time in young, ambulant patients with DMD; both of which are primary outcome measures in clinical trials. We have developed a double knockout mouse model, which also shows reduced muscle strength, but is protected from stretch-induced eccentric damage with age. This suggests that α-actinin-3 deficiency reduces muscle performance at baseline, but ameliorates the progression of dystrophic pathology. Mechanistically, we show that α-actinin-3 deficiency triggers an increase in oxidative muscle metabolism through activation of calcineurin, which likely confers the protective effect. Our studies suggest that ACTN3 R577X genotype is a modifier of clinical phenotype in DMD patients.

Adipocyte lipolysis links obesity to breast cancer growth: adipocyte-derived fatty acids drive breast cancer cell proliferation and migration.

BACKGROUND: Obesity is associated with increased recurrence and reduced survival of breast cancer. Adipocytes constitute a significant component of breast tissue, yet their role in provisioning metabolic substrates to support breast cancer progression is poorly understood. RESULTS: Here, we show that co-culture of breast cancer cells with adipocytes revealed cancer cell-stimulated depletion of adipocyte triacylglycerol. Adipocyte-derived free fatty acids were transferred to breast cancer cells, driving fatty acid metabolism via increased CPT1A and electron transport chain complex protein levels, resulting in increased proliferation and migration. Notably, fatty acid transfer to breast cancer cells was enhanced from "obese" adipocytes, concomitant with increased stimulation of cancer cell proliferation and migration. This adipocyte-stimulated breast cancer cell proliferation was dependent on lipolytic processes since HSL/ATGL knockdown attenuated cancer cell responses. CONCLUSIONS: These findings highlight a novel and potentially important role for adipocyte lipolysis in the provision of metabolic substrates to breast cancer cells, thereby supporting cancer progression.

mTORC2 and AMPK differentially regulate muscle triglyceride content via Perilipin 3.

OBJECTIVE: We have recently shown that acute inhibition of both mTOR complexes (mTORC1 and mTORC2) increases whole-body lipid utilization, while mTORC1 inhibition had no effect. Therefore, we tested the hypothesis that mTORC2 regulates lipid metabolism in skeletal muscle. METHODS: Body composition, substrate utilization and muscle lipid storage were measured in mice lacking mTORC2 activity in skeletal muscle (specific knockout of RICTOR (Ric mKO)). We further examined the RICTOR/mTORC2-controlled muscle metabolome and proteome; and performed follow-up studies in other genetic mouse models and in cell culture. RESULTS: Ric mKO mice exhibited a greater reliance on fat as an energy substrate, a re-partitioning of lean to fat mass and an increase in intramyocellular triglyceride (IMTG) content, along with increases in several lipid metabolites in muscle. Unbiased proteomics revealed an increase in the expression of the lipid droplet binding protein Perilipin 3 (PLIN3) in muscle from Ric mKO mice. This was associated with increased AMPK activity in Ric mKO muscle. Reducing AMPK kinase activity decreased muscle PLIN3 expression and IMTG content. AMPK agonism, in turn, increased PLIN3 expression in a FoxO1 dependent manner. PLIN3 overexpression was sufficient to increase triglyceride content in muscle cells. CONCLUSIONS: We identified a novel link between mTORC2 and PLIN3, which regulates lipid storage in muscle. While mTORC2 is a negative regulator, we further identified AMPK as a positive regulator of PLIN3, which impacts whole-body substrate utilization and nutrient partitioning.

Evidence based selection of commonly used RT-qPCR reference genes for the analysis of mouse skeletal muscle.

The ability to obtain accurate and reproducible data using quantitative real-time Polymerase Chain Reaction (RT-qPCR) is limited by the process of data normalization. The use of 'housekeeping' or 'reference' genes is the most common technique used to normalize RT-qPCR data. However, commonly used reference genes are often poorly validated and may change as a result of genetic background, environment and experimental intervention. Here we present an analysis of 10 reference genes in mouse skeletal muscle (Actb, Aldoa, Gapdh, Hprt1, Ppia, Rer1, Rn18s, Rpl27, Rpl41 and Rpl7L1), which were identified as stable either by microarray or in the literature. Using the MIQE guidelines we compared wild-type (WT) mice across three genetic backgrounds (R129, C57BL/6j and C57BL/10) as well as analyzing the α-actinin-3 knockout (Actn3 KO) mouse, which is a model of the common null polymorphism (R577X) in human ACTN3. Comparing WT mice across three genetic backgrounds, we found that different genes were more tightly regulated in each strain. We have developed a ranked profile of the top performing reference genes in skeletal muscle across these common mouse strains. Interestingly the commonly used reference genes; Gapdh, Rn18s, Hprt1 and Actb were not the most stable. Analysis of our experimental variant (Actn3 KO) also resulted in an altered ranking of reference gene suitability. Furthermore we demonstrate that a poor reference gene results in increased variability in the normalized expression of a gene of interest, and can result in loss of significance. Our data demonstrate that reference genes need to be validated prior to use. For the most accurate normalization, it is important to test several genes and use the geometric mean of at least three of the most stably expressed genes. In the analysis of mouse skeletal muscle, strain and intervention played an important role in selecting the most stable reference genes.

The role of the Niemann-Pick disease, type C1 protein in adipocyte insulin action.

The Niemann-Pick disease, type C1 (NPC1) gene encodes a transmembrane protein involved in cholesterol efflux from the lysosome. SNPs within NPC1 have been associated with obesity and type 2 diabetes, and mice heterozygous or null for NPC1 are insulin resistant. However, the molecular mechanism underpinning this association is currently undefined. This study aimed to investigate the effects of inhibiting NPC1 function on insulin action in adipocytes. Both pharmacological and genetic inhibition of NPC1 impaired insulin action. This impairment was evident at the level of insulin signalling and insulin-mediated glucose transport in the short term and decreased GLUT4 expression due to reduced liver X receptor (LXR) transcriptional activity in the long-term. These data show that cholesterol homeostasis through NPC1 plays a crucial role in maintaining insulin action at multiple levels in adipocytes.

Acute mTOR inhibition induces insulin resistance and alters substrate utilization in vivo.

The effect of acute inhibition of both mTORC1 and mTORC2 on metabolism is unknown. A single injection of the mTOR kinase inhibitor, AZD8055, induced a transient, yet marked increase in fat oxidation and insulin resistance in mice, whereas the mTORC1 inhibitor rapamycin had no effect. AZD8055, but not rapamycin reduced insulin-stimulated glucose uptake into incubated muscles, despite normal GLUT4 translocation in muscle cells. AZD8055 inhibited glycolysis in MEF cells. Abrogation of mTORC2 activity by SIN1 deletion impaired glycolysis and AZD8055 had no effect in SIN1 KO MEFs. Re-expression of wildtype SIN1 rescued glycolysis. Glucose intolerance following AZD8055 administration was absent in mice lacking the mTORC2 subunit Rictor in muscle, and in vivo glucose uptake into Rictor-deficient muscle was reduced despite normal Akt activity. Taken together, acute mTOR inhibition is detrimental to glucose homeostasis in part by blocking muscle mTORC2, indicating its importance in muscle metabolism in vivo.