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• W2793156734 abstract "ZnT7 (Slc30a7) is a widely expressed zinc transporter involved in sequestration of zinc into the Golgi apparatus and vesicular compartments. znt7-knockout (KO) mice are mildly zinc-deficient and lean. Despite their lean phenotype, adult male znt7-KO mice are prone to insulin resistance. We hypothesized that fat partitioning from adipose to nonadipose tissues causes insulin resistance in znt7-KO mice. Here, we used biological and biochemical methods, including fatty acid and oxylipin profiling, EM, immunohistochemistry, quantitative RT-PCR, and Western blot analysis, to identify the underlying mechanism of insulin resistance in znt7-KO mice. We found that insulin resistance in this model was primarily associated with increased intracellular fatty acid levels in the skeletal muscle, which promoted intracellular lipid accumulation and production of bioactive lipid mediators, such as 12,13-dihydroxyoctadecanoic acid (12,13-DiHOME) and 12-hydroxyeicosatetraenoic acid (12-HETE). The expression of fatty acid–binding protein 3 (Fabp3) was dramatically up-regulated in the znt7-KO muscle cells accompanied by increased expression of Cd36, Slc27a1, and Slc27a4, the three major fatty acid transporters in the skeletal muscle. We also demonstrated that znt7-KO muscle cells had increased fatty acid oxidative capacity, indicated by enlarged mitochondria and increased mRNA or protein expression of key enzymes involved in the fatty acid mitochondrial shuttle and β-oxidation. We conclude that increased fatty acid uptake in the znt7-KO skeletal muscle is a key factor that contributes to the excessive intracellular lipid deposit and elevated production of bioactive lipid mediators. These mediators may play pivotal roles in oxidative stress and inflammation, leading to insulin resistance. ZnT7 (Slc30a7) is a widely expressed zinc transporter involved in sequestration of zinc into the Golgi apparatus and vesicular compartments. znt7-knockout (KO) mice are mildly zinc-deficient and lean. Despite their lean phenotype, adult male znt7-KO mice are prone to insulin resistance. We hypothesized that fat partitioning from adipose to nonadipose tissues causes insulin resistance in znt7-KO mice. Here, we used biological and biochemical methods, including fatty acid and oxylipin profiling, EM, immunohistochemistry, quantitative RT-PCR, and Western blot analysis, to identify the underlying mechanism of insulin resistance in znt7-KO mice. We found that insulin resistance in this model was primarily associated with increased intracellular fatty acid levels in the skeletal muscle, which promoted intracellular lipid accumulation and production of bioactive lipid mediators, such as 12,13-dihydroxyoctadecanoic acid (12,13-DiHOME) and 12-hydroxyeicosatetraenoic acid (12-HETE). The expression of fatty acid–binding protein 3 (Fabp3) was dramatically up-regulated in the znt7-KO muscle cells accompanied by increased expression of Cd36, Slc27a1, and Slc27a4, the three major fatty acid transporters in the skeletal muscle. We also demonstrated that znt7-KO muscle cells had increased fatty acid oxidative capacity, indicated by enlarged mitochondria and increased mRNA or protein expression of key enzymes involved in the fatty acid mitochondrial shuttle and β-oxidation. We conclude that increased fatty acid uptake in the znt7-KO skeletal muscle is a key factor that contributes to the excessive intracellular lipid deposit and elevated production of bioactive lipid mediators. These mediators may play pivotal roles in oxidative stress and inflammation, leading to insulin resistance. Cellular zinc homeostasis is tightly regulated through two families of zinc transporters (Slc30a and Slc39a) 3The abbreviations used are:Slcsolute carrierznt7zinc transporter 7Cd36cluster of differentiation 36Slc27a1 and Slc27a4solute carrier family 27 members 1 and 4Fabp3fatty acid–binding protein 3Acsl1long-chain fatty acid coenzyme A ligase 1Ephxepoxide hydrolaseAloxlipoxygenaseAcadlacyl-CoA dehydrogenaseHadhbhydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase/enoyl-CoA hydratase β subunitCpt1bcarnitine palmitoyltransferase 1bAcacbacetyl-CoA carboxylase β12-HETE12S-hydroxyeicosatetraenoic acid12,13-DiHOME12,13-dihydroxyoctadecanoic acidIPGTTintraperitoneal glucose tolerance testIPITTintraperitoneal insulin tolerance testROSreactive oxygen speciesSFAsaturated fatty acidMUFAmonounsaturated fatty acidPUFApolyunsaturated fatty acidPparαproliferator-activated receptor-αMEMminimum essential mediumKRHKrebs-Ringer solution, Hepes-buffered. (1Huang L. Tepaamorndech S. The SLC30 family of zinc transporters—a review of current understanding of their biological and pathophysiological roles.Mol. Aspect Med. 2013; 34 (23506888): 548-56010.1016/j.mam.2012.05.008Crossref PubMed Scopus (237) Google Scholar, 2Jeong J. Eide D.J. The SLC39 family of zinc transporters.Mol. Aspects Med. 2013; 34 (23506894): 612-61910.1016/j.mam.2012.05.011Crossref PubMed Scopus (279) Google Scholar) and other small proteins, such as metallothioneins (3Thirumoorthy N. Shyam Sunder A. Manisenthil Kumar K. Senthil Kumar M. Ganesh G. Chatterjee M. A review of metallothionein isoforms and their role in pathophysiology.World J. Surg. Oncol. 2011; 9 (21599891): 5410.1186/1477-7819-9-54Crossref PubMed Scopus (174) Google Scholar). In general, Slc30a family members (Slc30a1–10) are involved in sequestration of zinc into intracellular compartments or export of zinc out of the cell when cellular zinc is in excess. In contrast, Slc39a family members (Slc39a1–14) play roles in either zinc uptake from the extracellular space or zinc release from the intracellular stores into the cytoplasm when cellular zinc concentration is low (1Huang L. Tepaamorndech S. The SLC30 family of zinc transporters—a review of current understanding of their biological and pathophysiological roles.Mol. Aspect Med. 2013; 34 (23506888): 548-56010.1016/j.mam.2012.05.008Crossref PubMed Scopus (237) Google Scholar, 2Jeong J. Eide D.J. The SLC39 family of zinc transporters.Mol. Aspects Med. 2013; 34 (23506894): 612-61910.1016/j.mam.2012.05.011Crossref PubMed Scopus (279) Google Scholar). The expression of zinc transporters can be ubiquitous or tissue- or cell-specific. For example, Slc30a1 is expressed ubiquitously, whereas Slc30a8 is abundantly and almost exclusively expressed in the pancreatic islet. Between the two extremes, expression of many other zinc transporters can be widespread at various levels. For example, ZnT7 is ubiquitously expressed among tissues/cell types; however, the expression level differs greatly. As a result, the impact of a null mutation in an Slc30a or Slc39a gene on zinc metabolism varies due to differences in expression levels in the body. solute carrier zinc transporter 7 cluster of differentiation 36 solute carrier family 27 members 1 and 4 fatty acid–binding protein 3 long-chain fatty acid coenzyme A ligase 1 epoxide hydrolase lipoxygenase acyl-CoA dehydrogenase hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase/enoyl-CoA hydratase β subunit carnitine palmitoyltransferase 1b acetyl-CoA carboxylase β 12S-hydroxyeicosatetraenoic acid 12,13-dihydroxyoctadecanoic acid intraperitoneal glucose tolerance test intraperitoneal insulin tolerance test reactive oxygen species saturated fatty acid monounsaturated fatty acid polyunsaturated fatty acid proliferator-activated receptor-α minimum essential medium Krebs-Ringer solution, Hepes-buffered. We reported previously that ZnT7 was expressed in mouse skeletal muscle (4Huang L. Kirschke C.P. Lay Y.A. Levy L.B. Lamirande D.E. Zhang P.H. Znt7-null mice are more susceptible to diet-induced glucose intolerance and insulin resistance.J. Biol. Chem. 2012; 287 (22854958): 33883-3389610.1074/jbc.M111.309666Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar), adipose tissue (5Tepaamorndech S. Kirschke C.P. Pedersen T.L. Keyes W.R. Newman J.W. Huang L. Zinc transporter 7 deficiency affects lipid synthesis in adipocytes by inhibiting insulin-dependent Akt activation and glucose uptake.FEBS J. 2016; 283 (26524605): 378-39410.1111/febs.13582Crossref PubMed Scopus (24) Google Scholar), β-cells in the islet of the pancreas (6Huang L. Yan M. Kirschke C.P. Over-expression of ZnT7 increases insulin synthesis and secretion in pancreatic β-cells by promoting insulin gene transcription.Exp. Cell Res. 2010; 316 (20599947): 2630-264310.1016/j.yexcr.2010.06.017Crossref PubMed Scopus (45) Google Scholar), epithelium of the small intestine (7Yu Y.Y. Kirschke C.P. Huang L. Immunohistochemical analysis of ZnT1, 4, 5, 6, and 7 in the mouse gastrointestinal tract.J. Histochem. Cytochem. 2007; 55 (17101726): 223-23410.1369/jhc.6A7032.2006Crossref PubMed Scopus (67) Google Scholar), and prostate (8Kirschke C.P. Huang L. Expression of the ZNT (SLC30) family members in the epithelium of the mouse prostate during sexual maturation.J. Mol. Histol. 2008; 39 (18548323): 359-37010.1007/s10735-008-9174-1Crossref PubMed Scopus (20) Google Scholar). Consequently, the znt7-knockout (KO) mouse displays phenotypes that are closely associated with its protein expression level in a given tissue or cell type (9Tepaamorndech S. Huang L. Kirschke C.P. A null-mutation in the Znt7 gene accelerates prostate tumor formation in a transgenic adenocarcinoma mouse prostate model.Cancer Lett. 2011; 308 (21621325): 33-4210.1016/j.canlet.2011.04.011Crossref PubMed Scopus (25) Google Scholar). A reduced capability of maintaining cellular zinc level at a normal range contributes to zinc deficiency in znt7-KO mice, which results from decreased dietary zinc absorption in the gut and consequently reduced zinc accumulation in organs, such as the bone, liver, and kidney (10Huang L. Yu Y.Y. Kirschke C.P. Gertz E.R. Lloyd K.K. Znt7 (Slc30a7)-deficient mice display reduced body zinc status and body fat accumulation.J. Biol. Chem. 2007; 282 (17954933): 37053-3706310.1074/jbc.M706631200Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). We have also demonstrated that male adult znt7-KO mice are susceptible to diet-induced insulin resistance. We showed that the abnormality in glucose metabolism was attributed to the disruption of insulin metabolism in pancreatic β-cells in znt7-KO mice (4Huang L. Kirschke C.P. Lay Y.A. Levy L.B. Lamirande D.E. Zhang P.H. Znt7-null mice are more susceptible to diet-induced glucose intolerance and insulin resistance.J. Biol. Chem. 2012; 287 (22854958): 33883-3389610.1074/jbc.M111.309666Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 6Huang L. Yan M. Kirschke C.P. Over-expression of ZnT7 increases insulin synthesis and secretion in pancreatic β-cells by promoting insulin gene transcription.Exp. Cell Res. 2010; 316 (20599947): 2630-264310.1016/j.yexcr.2010.06.017Crossref PubMed Scopus (45) Google Scholar) and reduced insulin sensitivity in skeletal muscle, a tissue responsible for 70–80% of glucose disposal following a carbohydrate load (4Huang L. Kirschke C.P. Lay Y.A. Levy L.B. Lamirande D.E. Zhang P.H. Znt7-null mice are more susceptible to diet-induced glucose intolerance and insulin resistance.J. Biol. Chem. 2012; 287 (22854958): 33883-3389610.1074/jbc.M111.309666Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). The underlying molecular mechanism by which the znt7-null mutation leads to peripheral insulin resistance in znt7-knockout mice is still unknown. We reported previously that the znt7-null mutation decreased lipid accumulation in mouse adipose tissue (5Tepaamorndech S. Kirschke C.P. Pedersen T.L. Keyes W.R. Newman J.W. Huang L. Zinc transporter 7 deficiency affects lipid synthesis in adipocytes by inhibiting insulin-dependent Akt activation and glucose uptake.FEBS J. 2016; 283 (26524605): 378-39410.1111/febs.13582Crossref PubMed Scopus (24) Google Scholar). Hence, we hypothesized that insulin resistance in the muscle of znt7-KO mice might be the result of fat partitioning from adipose to nonadipose tissues. According to the ZnT7 protein expression pattern in the skeletal muscle (abundant) and liver (undetectable in hepatocytes), we further hypothesized that the lipid partitioning in the znt7-KO mouse mainly occurred in the skeletal muscle but not the liver. In this study, we used fatty acid and oxylipin profiling, EM, immunohistochemistry, quantitative RT-PCR, and Western blot analysis to probe the underlying mechanism of insulin resistance in the skeletal muscle of znt7-KO mice. Here, we provide evidence that insulin resistance in znt7-KO mice is associated with increased uptake and binding of free fatty acids in the muscle cell. Bioactive lipid mediators were elevated in the skeletal muscle of znt7-KO mice and showed the potential to drive insulin resistance. In addition, we demonstrated that the fatty acid oxidative capacity of znt7-KO muscle cells was increased, evident by the enlarged mitochondria, increased mRNA and protein expression of key enzymes involved in fatty acid mitochondrial shuttle and β-oxidation, and reactive oxygen species (ROS) production. We previously demonstrated that adult male znt7-KO mice were more sensitive to high fat diet–induced abnormalities in glucose and insulin metabolism than the WT control mice (4Huang L. Kirschke C.P. Lay Y.A. Levy L.B. Lamirande D.E. Zhang P.H. Znt7-null mice are more susceptible to diet-induced glucose intolerance and insulin resistance.J. Biol. Chem. 2012; 287 (22854958): 33883-3389610.1074/jbc.M111.309666Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). We have also shown that znt7-KO mice gain less body weight than the controls mainly due to reduced body fat accumulation (10Huang L. Yu Y.Y. Kirschke C.P. Gertz E.R. Lloyd K.K. Znt7 (Slc30a7)-deficient mice display reduced body zinc status and body fat accumulation.J. Biol. Chem. 2007; 282 (17954933): 37053-3706310.1074/jbc.M706631200Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 11Tepaamorndech S. Kirschke C.P. Huang L. Linking cellular zinc status to body weight and fat mass: mapping quantitative trait loci in Znt7 knockout mice.Mamm. Genome. 2014; 25 (24770585): 335-35310.1007/s00335-014-9512-4Crossref PubMed Scopus (7) Google Scholar). Hence, we hypothesized that znt7-KO mice had dysregulation of lipid metabolism and that this genetically predisposed factor might render the znt7-KO mouse sensitive to high fat diet–induced glucose intolerance. We therefore studied a cohort of 18.5-week-old male znt7-KO and age-matched male WT littermates to determine the underlying mechanism of insulin resistance in metabolically relevant tissues, such as the skeletal muscle and liver. znt7-KO mice used in this study were congenic mice with a C57BL/6J (B6) genetic background in which the znt7 allele (129P2) was backcrossed to the B6 strain for at least 12 generations. The KO and WT mice were littermates; therefore, both the znt7-KO and WT mice were genetically identical except for the znt7 allele and its flanking regions on chromosome 3. The study design is laid out in Fig. 1A. Both genotypes of mice were fed a semipurified diet containing 30 mg of zinc/kg of diet starting at 5 weeks of age. Difference in body weight was seen around 9–10 weeks of age and became significant at 13 weeks between the two genotype groups (Fig. 1B). We observed that mice in both znt7-KO and WT groups gained limited weight 1 week after they were singly housed at 11 weeks of age. But the body weight gain resumed from 13 weeks until euthanization at 18.5 weeks (Fig. 1B). Table 1 lists the measurements of body weights, fat pad weights, and organ weights at necrosis. As we expected, when compared with the WT controls, znt7-KO mice accumulated less body fat as measured by individual or total fat pad weights (epididymal, subcutaneous, and retroperitoneal fat pads). When adjusting the body fat (total measured fat pad weights) to the body weight, znt7-KO mice appeared leaner than the WT controls at 18.5 weeks (Table 1), consistent with our previous findings in znt7-KO mice with a mixed B6 and 129P2 genetic background (10Huang L. Yu Y.Y. Kirschke C.P. Gertz E.R. Lloyd K.K. Znt7 (Slc30a7)-deficient mice display reduced body zinc status and body fat accumulation.J. Biol. Chem. 2007; 282 (17954933): 37053-3706310.1074/jbc.M706631200Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 11Tepaamorndech S. Kirschke C.P. Huang L. Linking cellular zinc status to body weight and fat mass: mapping quantitative trait loci in Znt7 knockout mice.Mamm. Genome. 2014; 25 (24770585): 335-35310.1007/s00335-014-9512-4Crossref PubMed Scopus (7) Google Scholar). The weights of other organs, including the liver, heart, and brain, were not apparently different between the two genotypes (Table 1). Food intake was decreased in the KO group (Fig. 1C). However, when the food intake was adjusted for the body weight gain, the food intake per gram of body weight gain was comparable between the two genotype groups (Fig. 1D), suggesting a similar growth efficiency.Table 1Body and tissue weights in znt7-KO and WT control miceznt7-KO (n = 6)WT (n = 6)p valueaStudent's t test was used for calculation of p values;Body weight (BW)24.75 ± 0.5426.10 ± 0.540.070Fat mass (mg)Epididymal fat201.03 ± 13.22292.25 ± 34.530.016*statistically significant.Femoral subcutaneous fat161.93 ± 10.95236.25 ± 19.010.003*statistically significant.Retroperitoneal fat32.38 ± 5.2563.22 ± 12.270.022*statistically significant.Percentage of total fat pads to BW1.80 ± 0.092.51 ± 0.240.011*statistically significant.Liver (mg)871.55 ± 19.74938.13 ± 44.470.101Percentage of liver to BW3.98 ± 0.043.99 ± 0.190.484Heart (mg)116.55 ± 5.87116.00 ± 5.890.474Brain (mg)378.90 ± 12.30407.85 ± 10.940.055a Student's t test was used for calculation of p values;* statistically significant. Open table in a new tab Overnight fasting plasma triglycerides and cholesterol levels were slightly low in znt7-KO mice compared with the WT controls, whereas overnight fasting glucose and total fatty acid levels were similar between the two genotypes (Table 2). In contrast, an elevated blood glucose level (17%, p < 0.01) was observed in znt7-KO mice compared with the controls after short-term fasting (6 h), suggesting abnormal glucose metabolism in znt7-KO mice. Nevertheless, the increased 6-h fasting glucose level in znt7-KO mice was not accompanied by an elevated blood insulin level (Table 2). The effect of the znt7-KO on the whole-body glucose metabolism was further confirmed by glucose and insulin tolerance tests. As shown in Fig. 2A, znt7-KO mice had a higher blood glucose level than the controls at 120 min (p < 0.05) after a glucose load (Fig. 2A). Plasma insulin levels in znt7-KO mice during glucose tolerance tests were not significantly changed compared with the controls (Fig. 2B). Furthermore, blood glucose levels in znt7-KO mice during insulin tolerance tests were higher than those of the controls, suggesting a resistance to the blood glucose-lowering effect of exogenously administered insulin in znt7-KO mice (Fig. 2C). This resistance was also reflected by the area under the curve glucose measurements of the insulin tolerance test (Fig. 2D). Taken together, our data suggest that znt7-KO mice had impaired glucose tolerance, likely due to insulin resistance.Table 2Blood chemistry parameters in znt7-KO and WT control miceznt7-KO (n = 6)WT (n = 6)p valueaStudent's t test was used for calculation of p values;Blood glucose, 16 h fasting (mg/dl)92.67 ± 3.8487.30 ± 14.690.366Blood glucose, 6 h fasting (mg/dl)217.17 ± 5.68185.50 ± 8.84<0.01*statistically significant.Plasma insulin, 6 h fasting (ng/ml)0.35 ± 0.120.31 ± 0.030.269Plasma triglycerides, 16 h fasting (mg/dl)47.06 ± 1.3151.69 ± 2.17<0.05*statistically significant.Plasma cholesterol, 16 h fasting (mg/dl)71.88 ± 1.6781.88 ± 3.210.05Plasma total fatty acids, 16 h fasting (mm)8.50 ± 2.897.95 ± 2.990.103a Student's t test was used for calculation of p values;* statistically significant. Open table in a new tab The homeostatic control of blood glucose levels is largely accomplished by the skeletal muscle, liver, and adipose tissue in which the muscle takes up and utilizes about 70–80% of daily absorbed glucose (12DeFronzo R.A. Tripathy D. Skeletal muscle insulin resistance is the primary defect in type 2 diabetes.Diabetes Care. 2009; 32 (19875544): S157-S16310.2337/dc09-S302Crossref PubMed Google Scholar). We have shown that the activity of the insulin signaling pathway and glucose uptake are compromised by znt7 knockdown in adipocytes, leading to a reduction in fat accumulation (5Tepaamorndech S. Kirschke C.P. Pedersen T.L. Keyes W.R. Newman J.W. Huang L. Zinc transporter 7 deficiency affects lipid synthesis in adipocytes by inhibiting insulin-dependent Akt activation and glucose uptake.FEBS J. 2016; 283 (26524605): 378-39410.1111/febs.13582Crossref PubMed Scopus (24) Google Scholar). Thus, we hypothesized that fat partitioning from adipose to nonadipose tissues might be the underlying mechanism of insulin resistance observed in znt7-KO mice. Hence, we performed metabolomic profiling of fatty acids in the skeletal muscle and liver from znt7-KO and the control mice. As shown in Fig. 3A, the total fatty acid content in the skeletal muscle of znt7-KO mice was 35% higher than that of the control mice (p < 0.05). The sum of concentrations of saturated (SFA), monounsaturated (MUFA), and polyunsaturated fatty acids (PUFA) was elevated in the skeletal muscle of znt7-KO mice compared with that of the controls. The ratio of ω-3 PUFA to ω-6 PUFA was decreased (KO, 0.56 versus WT, 0.66), suggesting an increased risk for insulin resistance in the skeletal muscle of znt7-KO mice. In addition, fatty acid composition analysis indicated that elevated fatty acids were mostly concentrated in the long-chain fatty acid group (Fig. 3C). The relative abundance of C18:1(n-9) to C18:0 was increased in the muscle of znt7-KO mice (p = 0.038), consistent with elevated stearoyl-CoA desaturase 1 activity, which has been reported to contribute to abnormal lipid partitioning and insulin resistance (13Hulver M.W. Berggren J.R. Carper M.J. Miyazaki M. Ntambi J.M. Hoffman E.P. Thyfault J.P. Stevens R. Dohm G.L. Houmard J.A. Muoio D.M. Elevated stearoyl-CoA desaturase-1 expression in skeletal muscle contributes to abnormal fatty acid partitioning in obese humans.Cell Metab. 2005; 2 (16213227): 251-26110.1016/j.cmet.2005.09.002Abstract Full Text Full Text PDF PubMed Scopus (301) Google Scholar). Arachidonic acid (C20:4(n-6)), a key inflammatory intermediate in skeletal muscle (14Korotkova M. Lundberg I.E. The skeletal muscle arachidonic acid cascade in health and inflammatory disease.Nat. Rev. Rheumatol. 2014; 10 (24468934): 295-30310.1038/nrrheum.2014.2Crossref PubMed Scopus (61) Google Scholar), was also increased by ∼25% in the znt7-KO muscle compared with the controls (p = 0.029) (Fig. 3C). Meanwhile, the anti-inflammatory docosahexaenoic acid (C22:6(n-3)) (15Calder P.C. ω-3 fatty acids and inflammatory processes.Nutrients. 2010; 2 (22254027): 355-37410.3390/nu2030355Crossref PubMed Scopus (545) Google Scholar) was increased by ∼30% in the znt7-KO muscle compared with the controls (p = 0.043). Regardless, the balance of the pro- and anti-inflammatory long-chain fatty acids appeared to be unchanged in the znt7-KO muscle (p = 0.5). In contrast to the changes observed in the znt7-KO muscle, little to no change in the total fatty acid content or composition was observed in the liver of znt7-KO compared with the control mice (Fig. 3, B and D). This discrepancy may be associated with the difference in ZnT7 protein expression levels in the two tissues (Fig. 3, C and D, insets). We showed that ZnT7 was readily detectable in the sarcoplasm of the myofibril, whereas ZnT7 was absent from the hepatocyte. Only the sinusoid endothelial cell in the liver stained positive for ZnT7 (Fig. 3, C and D, insets). To examine whether increased fatty acid content leads to an increased lipid accumulation in the skeletal muscle of znt7-KO mice, we stained the femoral skeletal muscle prepared from znt7-KO and WT mice with Oil Red O, a dye that detects neutral lipids. As shown in Fig. 4A, lipid accumulation was noticeably increased in the myofibrils of znt7-KO mice compared with the controls. This observation was further confirmed by triglyceride quantification in the skeletal muscle of znt7-KO and WT mice. We showed that znt7-KO skeletal muscle contained ∼50% higher triglycerides than the control (Fig. 4B). Together, we conclude that znt7-KO impairs lipid metabolism in the skeletal muscle. Next, we examined whether increased fatty acid concentrations in the skeletal muscle of znt7-KO mice would result in an increased capacity for fatty acid utilization. First, we performed EM of the femoral skeletal muscle isolated from znt7-KO and WT mice. As shown in Fig. 5A, in the WT, the mitochondria (dark and ovoid structures) were localized at the level of the I band and centered around the z-line between the myofibrils (16Ogata T. Yamasaki Y. Ultra-high-resolution scanning electron microscopy of mitochondria and sarcoplasmic reticulum arrangement in human red, white, and intermediate muscle fibers.Anat. Rec. 1997; 248 (9185987): 214-223Crossref PubMed Scopus (172) Google Scholar). In contrast, in the znt7-KO muscle, the sarcoplasmic space of the myofibril was inflated with nonuniformly distributed mitochondria. In addition, the size of the mitochondria in the znt7-KO muscle was expanded, covering two or more z-lines along the myofibril (Fig. 5A). High magnification images revealed a high level of lipid accumulation between the mitochondria in the znt7-KO muscle (Fig. 5B). Nevertheless, we did not observe a noticeable internal structure change in the mitochondria of the znt7-KO muscle (Fig. 5B). In addition, mitochondrial DNA contents in the muscle cells between znt7-KO and the WT controls were not different (data not shown). Dysregulation of several factors, such as fatty acid transport, intracellular fatty acid binding, and/or fatty acid oxidation, could increase fatty acid concentrations in the znt7-KO muscle. Thus, we decided to investigate the mRNA expression of genes involved in the fatty acid uptake and oxidation in the skeletal muscle, including Cd36 (fatty acid translocase), Slc27a1 (long-chain fatty acid transport protein 1), Slc27a4, Fabp3 (fatty acid–binding protein 3), Acsl1 (long-chain fatty acid CoA ligase 1), Acacb (acetyl-CoA carboxylase 2), Cpt1b (carnitine palmitoyltransferase 1b), Acadl (long-chain acyl-CoA dehydrogenase), and Hadhb (3-ketoacyl-CoA thiolase, β subunit), in differentiated primary myoblasts isolated from skeletal muscles of znt7-KO and WT mice (4Huang L. Kirschke C.P. Lay Y.A. Levy L.B. Lamirande D.E. Zhang P.H. Znt7-null mice are more susceptible to diet-induced glucose intolerance and insulin resistance.J. Biol. Chem. 2012; 287 (22854958): 33883-3389610.1074/jbc.M111.309666Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Although primary isolated skeletal muscle cells might not reflect skeletal muscle in vivo, we used these cells to achieve improved results for quantitative PCR, Western blot assay, fatty acid uptake, and ROS measurements. As shown in Fig. 6A, among the genes examined, the mRNA expression of Fabp3, a gene associated with intracellular fatty acid binding, was significantly higher (25-fold) in the znt7-KO primary myotubes than in the WT controls. Up-regulation of Fabp3 in znt7-KO myotubes was also confirmed at the protein level (Fig. 6B). Four genes that are implicated in fatty acid uptake, Cd36, Slc27a1, Slc27a4, and Acsl1, were moderately up-regulated (2.5–5-fold higher in the znt7-KO muscle than in the controls). We further confirmed that the up-regulation of expression of these fatty acid uptake genes in znt7-KO myotubes led to an increase in fatty acid uptake by ∼27% compared with the controls (Fig. 6C). Interestingly, the mRNA expression of Acacb, an enzyme that converts acetyl-CoA to malonyl-CoA, was also up-regulated in the znt7-KO primary myotubes. Malonyl-CoA is a potent inhibitor of mitochondrial fatty acid uptake through allosteric inhibition of Cpt1 enzymatic activity, a rate-limiting step in fatty acid uptake and oxidation by the mitochondria. Increased mRNA expression of Acacb suggests a negative impact of the znt7-KO on mitochondrial fatty acid oxidation in myotubes even though Acadl and Hadhb were up-regulated in the znt7-KO primary myotubes compared with the controls (Fig. 7, A and B). Taken together, our results suggest that the znt7-null mutation may increase fatty acid uptake and expand the Fabp3-bound fatty acid pool in the skeletal muscle. znt7-KO may also impair fatty acid oxidation in the skeletal muscle. As a result, lipids accumulate in the skeletal muscle of znt7-KO mice.Figure 7.Expression of key enzymes involved in the mitochondrial β-oxidation, bioactive lipid mediator production, and ROS accumulation in primary skeletal myotubes. A, expression of Acadl, Hadhb, Cpt1b, and Acacb mRNAs. B, expression of Hadhb protein. C, expression of Alox12, Ephx1, Ephx2, and Ephx4 mRNAs. Primary myoblasts of znt7-KO and the WT control were allowed to differentiate for 4–6 days before the experiments. The amount of the target mRNA was measured by SYBR-based quantitative RT-PCR. Three micrograms of total proteins were loaded for the Western blotting assay. Actb was used as the internal reference for mRNA measurements or as the loading control for Western blotting assays. Three independent experiments, each with duplicate or triplicate, were performed. A representative image of the Western blot assays is shown. D, ROS levels in living myotubes. Results are means, and error bars represent S.E. (three independent experiments with three to six replicates, n = 12)" @default.
• W2793156734 created "2018-03-29" @default.
• W2793156734 creator A5032577341 @default.
• W2793156734 creator A5035235254 @default.
• W2793156734 creator A5046459372 @default.
• W2793156734 creator A5050683506 @default.
• W2793156734 creator A5059985697 @default.
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• W2793156734 date "2018-05-01" @default.
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• W2793156734 title "Aberrant fatty acid metabolism in skeletal muscle contributes to insulin resistance in zinc transporter 7 (znt7)-knockout mice" @default.
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