Skeletal muscle‐specific overexpression of heat shock protein 72 improves skeletal muscle insulin‐stimulated glucose uptake but does not alter whole body metabolism

Marshall, Jessica P.; Estévez, Emma; Kammoun, Hélène L.; King, E.; Bruce, Clinton R.; Drew, Brian G.; Qian, Hongwei; Iliades, Peter; Gregorevic, Paul; Febbraio, Mark A.; Henstridge, Darren C.

doi:10.1111/dom.13319

Cited by 17 publications

(9 citation statements)

References 31 publications

(104 reference statements)

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“…It has been shown previously that saturated fatty acid palmitate reduces insulin sensitivity and induces insulin resistance in skeletal muscle [18]. In this experiment, the inhibitory effect of palmitate on the insulin signaling pathway in C2C12 myotubes was tested.…”

Section: Resultsmentioning

confidence: 99%

“…The experimental procedures for all animal work has been previously described and the mice used in these experiments represent a sub group of a previously published cohort of animals [18]. All experiments involving the use of animals for research were approved by the Alfred Medical Research Education Precinct Animal Ethics Committee and were conducted in accordance with the National Health and Medical Research Council of Australia guidelines.…”

Section: Methodsmentioning

confidence: 99%

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The Zinc Transporter Zip7 Is Downregulated in Skeletal Muscle of Insulin-Resistant Cells and in Mice Fed a High-Fat Diet

Norouzi

Adulcikas

Henstridge

et al. 2019

Cells

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Background: The zinc transporter Zip7 modulates zinc flux and controls cell signaling molecules associated with glucose metabolism in skeletal muscle. The present study evaluated the role of Zip7 in cell signaling pathways involved in insulin-resistant skeletal muscle and mice fed a high-fat diet. Methods: Insulin-resistant skeletal muscle cells were prepared by treatment with an inhibitor of the insulin receptor, HNMPA-(AM)3 or palmitate, and Zip7 was analyzed along with pAkt, pTyrosine and Glut4. Similarly, mice fed normal chow (NC) or a high-fat diet (HFD) were also analyzed for protein expression of Glut4 and Zip7. An overexpression system for Zip7 was utilized to determine the action of this zinc transporter on several genes implicated in insulin signaling and glucose control. Results: We identified that Zip7 is upregulated by glucose in normal skeletal muscle cells and downregulated in insulin-resistant skeletal muscle. We also observed (as expected) a decrease in pAkt and Glut4 in the insulin-resistant skeletal muscle cells. The overexpression of Zip7 in skeletal muscle cells led to the modulation of key genes involved in the insulin signaling axis and glucose metabolism including Akt3, Dok2, Fos, Hras, Kras, Nos2, Pck2, and Pparg. In an in vivo mouse model, we identified a reduction in Glut4 and Zip7 in the skeletal muscle of mice fed a HFD compared to NC controls. Conclusions: These data suggest that Zip7 plays a role in skeletal muscle insulin signaling and is downregulated in an insulin-resistant, and HFD state. Understanding the molecular mechanisms of Zip7 action will provide novel opportunities to target this transporter therapeutically for the treatment of insulin resistance and type 2 diabetes.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

The Zinc Transporter Zip7 Is Downregulated in Skeletal Muscle of Insulin-Resistant Cells and in Mice Fed a High-Fat Diet

Norouzi

Adulcikas

Henstridge

et al. 2019

Cells

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“…Similar to the liver, the virus requires high purity and a high viral titer for injection of small volumes into the muscle. We examined the tissue-specific expression of AAV2-6-shGlrx-mVenus since this capsid has a better tissue tropism for skeletal muscle 59–62 . Also, using a different capsid further supports the general applicability of our protocol.…”

Section: Resultsmentioning

confidence: 99%

Production of adeno-associated virus vectors for in vitro and in vivo applications

Kimura

Ferrán

Tsukahara

et al. 2019

Sci Rep

105

101

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Delivering and expressing a gene of interest in cells or living animals has become a pivotal technique in biomedical research and gene therapy. Among viral delivery systems, adeno-associated viruses (AAVs) are relatively safe and demonstrate high gene transfer efficiency, low immunogenicity, stable long-term expression, and selective tissue tropism. Combined with modern gene technologies, such as cell-specific promoters, the Cre/lox system, and genome editing, AAVs represent a practical, rapid, and economical alternative to conditional knockout and transgenic mouse models. However, major obstacles remain for widespread AAV utilization, such as impractical purification strategies and low viral quantities. Here, we report an improved protocol to produce serotype-independent purified AAVs economically. Using a helper-free AAV system, we purified AAVs from HEK293T cell lysates and medium by polyethylene glycol precipitation with subsequent aqueous two-phase partitioning. Furthermore, we then implemented an iodixanol gradient purification, which resulted in preparations with purities adequate for in vivo use. Of note, we achieved titers of 1010–1011 viral genome copies per µl with a typical production volume of up to 1 ml while requiring five times less than the usual number of HEK293T cells used in standard protocols. For proof of concept, we verified in vivo transduction via Western blot, qPCR, luminescence, and immunohistochemistry. AAVs coding for glutaredoxin-1 (Glrx) shRNA successfully inhibited Glrx expression by ~66% in the liver and skeletal muscle. Our study provides an improved protocol for a more economical and efficient purified AAV preparation.

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“…Oral glucose tolerance tests (oGTT) were performed at three different time points, at a dose of 2 mg/kg of lean mass as determined by EchoMRI (see Supplementary Figure S1). All oGTTs were performed as previously described by Marshall et al (2018).…”

Section: Methodsmentioning

confidence: 99%

The Antioxidant Moiety of MitoQ Imparts Minimal Metabolic Effects in Adipose Tissue of High Fat Fed Mice

et al. 2019

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Mitochondrial dysfunction is associated with a diverse array of diseases ranging from dystrophy and heart failure to obesity and hepatosteatosis. One of the major biochemical consequences of impaired mitochondrial function is an accumulation of mitochondrial superoxide, or reactive oxygen species (ROS). Excessive ROS can be detrimental to cellular health and is proposed to underpin many mitochondrial diseases. Accordingly, much research has been committed to understanding ways to therapeutically prevent and reduce ROS accumulation. In white adipose tissue (WAT), ROS is associated with obesity and its subsequent complications, and thus reducing mitochondrial ROS may represent a novel strategy for treating obesity related disorders. One therapeutic approach employed to reduce ROS abundance is the mitochondrial-targeted coenzyme Q (MitoQ), which enables mitochondrial specific delivery of a CoQ10 antioxidant via its triphenylphosphonium bromide (TPP+) cation. Indeed, MitoQ has been successfully shown to accumulate at the outer mitochondrial membrane and prevent ROS accumulation in several tissues in vivo ; however, the specific effects of MitoQ on adipose tissue metabolism in vivo have not been studied. Here we demonstrate that mice fed high-fat diet with concomitant administration of MitoQ, exhibit minimal metabolic benefit in adipose tissue. We also demonstrate that both MitoQ and its control agent dTPP+ had significant and equivalent effects on whole-body metabolism, suggesting that the dTPP+ cation rather than the antioxidant moiety, was responsible for these changes. These findings have important implications for future studies using MitoQ and other TPP+ compounds.

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Skeletal muscle‐specific overexpression of heat shock protein 72 improves skeletal muscle insulin‐stimulated glucose uptake but does not alter whole body metabolism

Cited by 17 publications

References 31 publications

The Zinc Transporter Zip7 Is Downregulated in Skeletal Muscle of Insulin-Resistant Cells and in Mice Fed a High-Fat Diet

The Zinc Transporter Zip7 Is Downregulated in Skeletal Muscle of Insulin-Resistant Cells and in Mice Fed a High-Fat Diet

Production of adeno-associated virus vectors for in vitro and in vivo applications

The Antioxidant Moiety of MitoQ Imparts Minimal Metabolic Effects in Adipose Tissue of High Fat Fed Mice

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