MCPIP1 Deficiency Increases Insulin Sensitivity But Impairs Hepatic Insulin Signal Transduction in Mice
Materials and MethodsMaterials
Polyclonal antibodies directed against phospho-AKT (ser473, Cat #9271), AKT (Cat #9272), insulin receptor substrate- 1(Cat #2832), PI3-kinase subunit p85 (Cat #4292), monoclonal anti-phospho-tyrosine (Cat #9411), anti-Rabbit IgG (Cat #7074), and anti-mouse IgG were purchased from Cell Signaling Technology (Danvers, MA). Polyclonal anti-insulin receptor β (C-19, sc-711) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Regular human injection insulin (Novolin) was purchased from Med-Depot (Kirksville, MO). Protein G sepharose 4 fast flow was acquired from GE healthcare BioSciences (Cat #17-0618-01, Pittsburgh,PA). AccupPrimeTM SuperMix II was purchased from Invitrogen (Cat #12341-012, Grand Island, NY). MCPIP1 primers were ordered from IDT (Integrated DNA Technologies, Iowa). PI3-kinase activity ELISA: Pico was purchased from Echelon Biosciences Inc. (Cat # K-1000s, Salt Lake, UT). All other reagents were obtained from Sigma-Aldrich (St Louis, MO).
Two pairs of MCPIP1 heterozygous mice in C57/BL6 background were cross-bred in our animal facility. MCPIP1 knockout (MCPIP1-/-) mice were verified by PCR. Animals were housed in a room with a controlled photoperiod of 12 hour light: 12 hour darkness and a temperature of 22° ± 2°C. Animals were given free access to a nutritionally balanced diet containing 11% fat and (5015, LabDiet, PMI Feeds, Inc., St Louis, MO, USA) and tap water. This study was carried out in compliance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The animal protocol was approved by the Kirksville College of Osteopathic Medicine Animal Care and Use Committee. All experiments were conducted using mice between ages of 6-8 weeks old since MCPIP1-/- mice start dying at about 8-weeks old.
Measurement of glucose, insulin and protein concentrations
Fasting plasma glucose levels were measured with One- Touch UltraMini glucose meter and test strips (Wal-Mart, Kirksville, MO) by collecting blood from mouse tail veins. Serum insulin and protein concentrations were measured following the Manufacturers’ instruction. Briefly, after overnight fasting, blood was collected by cardiac puncture from anesthetized animals, allowed to clot at room temperature, and centrifuged at 1500 g at 4°C for 10 minutes. Serum was collected and frozen at -80°C until the assay was performed. Serum concentration of insulin was determined using a Rat/ mouse insulin ELISA kit (Cat # EZRMI-13K, Millipore, St. Charles, MO). Protein concentration in serum and tissue lysates was measured using BCA protein assay reagents (Cat #23228 and 23224, Pierce, Rockford, IL).
Glucose tolerance test
Glucose tolerance tests were conducted on animals after 16- 18 hour fast. Animals were injected with D-glucose (2 g/kg body weight) intraperitoneally. Blood glucose values in plasma samples (obtained by tail puncture) were determined at 0, 15, 30, 60, and 120 minutes after the injection .
Following 16-18 hour fasting, animals were subjected to an intraperitoneal injection of regular human injection insulin (0.75 IU/ kg body weight) . Blood glucose levels were measured at 0, 15, 30, 60, and 120 minutes after the injection.Liver and adipose tissue collectionTo study the effects of MCPIP1-/- on insulin-stimulated insulin signal transduction pathway in liver and adipose tissues, 2 IU/kg of insulin or same amount of phosphate saline buffer was injected intraperitoneally after 16-18 hour food withdrawal by following modified protocols [17-19]. Mice were anesthetized with an intraperitoneal injection of sodium pentobarbital (50 mg/kg). Liver and adipose tissues (including inguinal, gonadal, abdominal and interscapular white fat pads) were harvested 15 minutes after insulin injection and weighed. The tissues were snap frozen in liquid nitrogen and kept at -80°C. Protein extraction was conducted following modified published protocol . Briefly, liver and adipose tissues were homogenized in lysis buffer containing 25 mM Tris-HCI (pH 7.4), 100 mM NaF, 10 mM EDTA, 1% Igepal CA-630, and 2 mM sodium vanadate supplemented with 0.25 mM PMSF, 0.2 μM aprotinin, 5.25 μM leupeptin, 9 μM bestatin, 3.75 μM pepstatin A, and 3.5 μM E-64. Tissue lysate was centrifuged at 5,000 rpm for 15 minutes at 4°C. The supernatant was collected, further centrifuged at 32,000 rpm for 120 minutes at 4°C, and stored at -20°C for immunoprecipitation and direct Western blotting experiments respectively.
Immnunoprecipitation was performed by following modified Ozcan’s protocol . Supernatants containing equal amounts of protein from liver tissue (3 mg) were incubated with antibodies (3 μg) directed against insulin receptor β (IRβ) or insulin receptor substrate-1(IRS-1) and protein G sepharose beads (25 μl) overnight at 4°C. The precipitates were washed with cold lysis buffer then boiled in 2 x Laemli sample buffer for 5 minutes.
ImmunoblottingFollowing immuniprecipitation, the precipitates were resolved with SDS-PAGE, transferred to a PVDF membrane (Millipore,Bedford, MA), blocked with 3% of nonfat dry milk, probed with anti-phospho-tyrosine (1:1000 dilution, to check tyrosine phosphorylation of IRβ and IRS-1) or anti-p85 (1:500 dilution, following immunoprecipitation with anti-IRS-1 to check IRS-1 and p85 association), and reprobed with either anti-IRβ (1:1000 dilution) or anti-IRS-1 (1:1000 dilution) to check for equal loading. Phosphorylation of AKT was determined by direct Western blotting using anti-phospho-AKT (1:1000 dilution) followed by re-probing with anti-AKT (1:1000 dilution). After incubation with the primary antibodies, membranes were incubated with HRPconjugated secondary antibodies (anti-mouse IgG, 1:15000 dilution; anti-rabbit IgG 1:5000 dilution), developed using Chemiluminescense Reagent Plus (PerkinElmer Life Science, INC. Boston, MA), and detected by autoradiography using XRay film (Midsci, St. Louis, MO). Images were quantified by densitometric analysis using Image-J software. It should be noted that only insulin-induced phosphorylation of AKT and total AKT were assessed in adipose tissues since MCPIP1-/- mice have very limited amount of adipose tissue. We were unable to measure phosphorylation of IRβ, IRS-1, and neither the association of p85 and IRS-1.
PI3-kinase activityPI3-kinase activity from liver samples was measured by following the manufacturer’s instruction. Briefly, 3 mg of liver lysate was incubated with 3 μg of anti-p85 (a subunit of PI3- kinase) for one hour at 4°C followed by the addition of 30 ul of 50% protein G-sepharose beads and rotation at 4°C overnight. The immunoprecipitate was collected by washing three times with ice-cold lysis buffer containing 2 mM sodium orthovanadate followed by three washes with 0.1 M Tris-HCl buffer (pH 7.4) containing 5 mM LiCl and 2 mM sodium orthovanadate, and three washes with 10 mM Tris- HCl (pH 7.4) containing 150 mM NaCl, 5 mM EDTA, and 0.1 mM sodium orthovanadate. The supernatant was aspirated after the last wash. 30 μl of kinase reaction buffer (provided in the kit) and 30 μl of 10 μM PI (4, 5) P2 (substrate) were added. The reaction proceeded for 3 hours at 37°C and was stopped by adding 90 μl of stop solution. The amount of PIP3 (end product of the kinase reaction) in the supernatant was detected using PI3-kinase activity ELISA kit.
HistologyMice were anesthetized with an intraperitoneal injection of sodium pentobarbital (50 mg/kg) and fixed by perfusion with 3.7% of formaldehyde through the left ventricle. Liver tissues were dissected, further fixed in 3.7% of formaldehyde, processed using a Leica TP1020 semi-enclosed benchtop tissue processor (Leica Microsystems, Nussloch, Germany), and embedded in paraffin using Leico embedding station. Specimens were sectioned using a Leica RM2255 microtome. Cross-sections of 8 μm were stained with hematoxylin- eosin and trichrome. Images were captured with a Leica DFC400 digital microscope camera mounted on a Leica DM4000B microscope using Surveyor Software (objective imaging Ltd, Cambridge, United Kingdom).
Statistical analysisResults are presented as means ± S.E.M. Data were analyzed with one-way ANOVA followed by the Newman-Keuls Multiple Comparison Test and Unpaired Student’s t-test using GraphPad Prism version 5 by GraphPad Software, Inc. (San Diego, CA, USA). The area under the curve (AUC) and inverse area under the curve were calculated using the AUC option in GraphPad Prizm and analyzed by Unpaired Student’s ttest. p<0.05 was considered as statistically significant.
It was previously reported that MCPIP1-/- mice exhibited growth retardation, systemic inflammation, autoimmune response, and premature death [12, 16], however, metabolic changes in these mice are unknown. As shown in the Table, comparing to wild-type littermates, fasting blood glucose and insulin concentrations from MCPIP1-/- mice were significantly lower (30% and 54% of reduction, respectively, p<0.05), suggesting that insulin sensitivity may be increased in MCPIP1-/- mice. In contrast to the basal levels of plasma glucose and insulin, the total serum protein concentration was considerably higher in MCPIP1-/- mice when compared to that in wild-type mice (about 53% of increase, p<0.05).
Figure 2. Insulin tolerance test. 0.75 IU/kg of insulin was administered via intraperitoneal injection after 16-18 hour fasting. Blood glucose levels were measured using one-touch glucose meter by collecting blood from mouse tail vein. Results are expressed as the concentrations of glucose (2a.) and inverse area under the curve (inverse AUCglucose, 2b.). The graph shows the mean ± S.E.M from 6 mice. *p<0.05 when compared to wild-type mice.
Hepatic insulin signal transductionNext, we examined the effect of MCPIP1 deficiency on hepatic insulin signal transduction. As shown in Figure 3, insulin- stimulated IRβ phosphorylation was reduced by 82% (p<0.01) in livers from MCPIP1-/- mice when compared to that from wild-type mice. No difference was observed in total IR expression levels. However, as shown in Figure 4, total IRS-1 expression levels in livers from MCPIP-/- mice were decreased to 39% of those from wild-type mice (Figure 4c). This decrease was consistent with a significant reduction in insulin-induced phosphorylation of IRS-1 (Figure 4b). In addition, insulin-induced serine phosphorylation of AKT in livers from MCPIP1-/- mice was markedly reduced compared to that from wild-type mice (about 59.8% of reduction, p<0.05, Figure 5). No difference was found in total AKT protein expression levels.
Following 16-18 hour fasting, mice received 2 IU/kg of insulin intraperitoneally.Liver tissues were removed 15 minutes after the injection. Tissues were homogenized and proteins were extracted in lysis buffer. Supernatants containing equal amounts of protein (3 mg) were immunoprecipitated with anti-IR-β. Proteins were separated with SDS-PAGE, probed with anti-phosphotyrosine, stripped, and re-probed with anti-IR-β. The upper panel shows a representative blot and the lower panel shows a graph summarizing the results from 4 independent experiments. Results were calculated as the ratio of phosphorylated IRβ to total IRβ and expressed as mean ± S.E.M. **p<0.05 compared to wild-type control, ##p<0.05 compared to wild-type insulin.
Figure 4. IRS-1 expression and insulin-induced IRS-1 phosphorylation in liver tissue. Following 16-18 hour fasting, mice received 2 IU/kg of insulin intraperitoneally. Livers were removed and homogenized and proteins were extracted in lysis buffer. Supernatants containing equal amounts of protein (3 mg) were immunoprecipitated with anti-IRS-1. Proteins were separated with SDS-PAGE, probed with anti-phosphotyrosine, stripped, and reprobed with anti-IRS-1. The image (Figure 4a) shows a representative blot of IRS-1 protein expression and insulin-induced IRS-1 phosphorylation. The graphs show the results of insulin-induced IRS-1 phosphorylation (4b, n=4) and total IRS-1 protein expression (4c, n=4). Results were calculated as arbitrary density of the bands and expressed as mean ± S.E.M. *p<0.05 compared to wild-type control, #p<0.05 compared to wild-type insulin.
Basal and insulin-stimulated hepatic PI3 kinase activityAs shown in Figure 6a, intraperitoneal injection of insulin in wild-type mice increased production of PIP3, an end product of PI3 kinase which serves as an indication of PI3 kinase activation. However, the same concentration of insulin failed to activate PI3 kinase in MCPIP1-/- mice. It should also be noted that the baseline activity of PI3 kinase in livers from MCPIP1-/- mice was also significantly lower than those from wild-type mice. No differences were observed in PI3 kinase protein expression between wild-type and MCPIP1-/- mice (Figure 6b).
Insulin-stimulated association of IRS-1 with p85 kinase in the liverAs shown in Figure 7, the basal level of association of IRS- 1 with p85 in the liver tissue was significantly lower in MCPIP1-/- mice when compared to wild-type mice. Administration of insulin failed to affect the association of IRS-1 with p85 in wild-type mice and MCPIP1-/- mice.
Following 16-18 hour fasting, mice received 2 IU/kg of insulin intraperitoneally. 15 minutes after insulin injection, livers were removed and homogenized and proteins were extracted in lysis buffer. 3 mg of liver lysate was immunoprecipitaed with 3 μg of anti- IRS-1. The supernatant was collected and proteins were separated by SDS-PAGE and probed with anti-p85. The upper panel shows a representative blot and the lower panel shows a graph summarizing the results from 4 independent experiments. Results were calculated as arbitrary units of the band density and expressed as mean ± S.E.M. *p<0.05 compared to wild-type control.
As shown in figure 8, liver sections from MCPIP1-/- mice showed reduced hepatocytes and multiple foci containing collagen and bile ducts in the interlobular area in HE staining. Masson-Trichrome staining revealed matrix deposition mainly in the centrilobular area and portal tract.
Insulin-induced AKT phosphorylation in adipose tissue
No differences in insulin-induced AKT phosphorylation were observed in adipose tissue as it occurred in liver tissue between wild type and MCPIP1-/- mice (Figure 9).
Following 16-18 hour fasting, mice received 2 IU/kg of insulin intraperitoneally. Adipose tissues were removed 15 minutes after the injection. Tissues were homogenized and proteins were extracted in lysis buffer. Supernatants containing equal amounts of protein (100 μg) were used to detect AKT phosphorylation levels. Proteins were separated with SDS-PAGE, probed with anti-phospho-AKT (ser473), stripped, and re-probed with anti-AKT. The upper panel shows a representative blot and the lower panel shows a graph summarizing the results from 4 independent experiments. Results were calculated as the ratio of phosphorylated AKT to total AKT and expressed as mean ± S.E.M. *p<0.05 compared to wild-type control, #p<0.05 compared to wild-type insulin.
This work was supported by Warner/Fermaturo grant awarded by A.T. Still University Board of Trustees and the grant awarded by A.T. Still University Biomedical Graduate Program.
The authors do not have anything to disclose.
Participated in research design: Yingzi Chang
Performed data analysis: Yingzi Chang and Brett Holdaway Wrote or contributed to the writing of the manuscript: Yingzi Chang, Brett Holdaway, Joshua Moody, Mingui Fu, and Lauren Brooks.Acknowledgement
Authors are grateful to the faculty and staff in Department of Pharmacology at A.T. Still University for their scientific and technical support. We would also like to acknowledge Dr. Maria Evans and Dr. Peter Kondrashov for lending their expertise to explaining the pathological results. We thank Michael Cramberg and Kelly Rogers for providing their valuable technical assistance.
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