| BackgroundLoss of protein stores and a decline in lean body mass are associated with morbidity and mortality, making this a major clinical problem. There is evidence that loss of lean body mass is usually caused by activation of the ubiquitin-proteasome proteolytic pathway (UPP) in muscle.Insulin deficiency (e.g. Type 1 diabetes mellitus (DM)) results in accelerated protein degradation by the ubiquitin-proteasome proteolytic pathway. Increased muscle atrophy has been documented in clinical conditions that are characterized by insulin resistance, including uremia, heart failure, sepsis, cancer, trauma and cachexia, but whether type 2 diabetes (insulin resistance) without other confounding conditions (e.g. uremia, sepsis) is associated with muscle atrophy and the musclea trophy's mechanism of type 2 diabetes mellitus(DM)) remain unclearObjective1. To study wethere type 2 diabetes mellitus have muscle wasting. 2. To explor the muscle atrophy's mechanism of type 2 diabetes mellitus.Materials and methods1. Animal:We studied db/db mice (BKS. Cg-m/Lepr db; Jackson Laboratories, Bar Harbor, ME), and wild-type (WT) (C57BLKS/J< m+/m+>) littermates were studied as controls. Four-week-old male db/db and control mice were weighted and then housed in the animal care facility in 12-h light,12-h dark cycles and fed ad libitum. In another experiment, mice were fed the standard or a diet supplemented with rosiglitazone maleate (8 mg/kg d; GlaxoSmithKline, Pittsburgh, PA).2. Nine-week-old male db/db and control mice were weighted, measured body length, then we collected blood samples from control and db/db mice heart, to measure the blood glucose and plasma insulin. At the end of different experimental manipulations, db/db and control mice were anesthetized, and the soleus, the extensor digitorum longus (EDL), and plan-taris muscles were removed to measure protein degradation, the 14-kDa actin fragment, muscle fiber size, proteasome activation.3. Cell cultureMouse 3T3-L1 preadipocytes were studied between passages 3 and 10. Initially, they were grown in standard media containing DMEM containing 10% bovine calf serum, penicillin (200 U/ml), and streptomycin (200μg/ml) in a humidified atmosphere (95%O2; 5%CO2). After 2 days, cells were switched to differentiation media with 1μM dexamethasone,10μg/ml insulin, and 0.5 mM 3-methyl-l-isobutylxanthine for 2 days and then grown in post-differentiation medium containing 10μg/ml insulin for 5 days. The cells were then placed in standard media for another 2 days. Subsequently, we determined the influence of exposing cells to 10μM rosiglitazon for 16 h.4. Real-time PCRFor measuring RT-PCR, we isolated total RNA from mouse muscle and from differentiated 3T3-L1 cells using Trizol reagent. RT was performed using the GeneAmp RNA PCR Core kit according to the manufacturer's instructions.Total RNA was extracted using the Purelink RNA purification kit and 2μg RNA sample was used for RT by oligo 9-mer primers and Superscript II. Real-time PCR was performed with SYBR Green PCR reagents (Bio-Rad, Hercules, CA) and the Option DNA Engine using the following cycle parameters:94℃for 2 min and 40 cycles at 94℃for 15 sec,55℃for 30 sec,and 72℃for 30 sec with a final extension at 72℃for 10 min.5. Statistical analysis:Rusults were anlysized by statistics software SPSS 17.0. Results are presented as mean±se. Comparisons of results between groups were by ANOVA using Systat Software ANOVA, and P<0.05 was considered significant.Results1. At 9 week of age, the body weight, blood sugar, plasma insulin levels of Type 2 diabetic mice were much higher than those in wild type mice2. At 9 week of age, the soleus, EDL and plantaris muscles of Type 2 diabetic mice weighed significantly less than that in wild type mice3. The cross-sectional area of gastrocnemius muscle fibers in Type 2 diabetic mice was significantly smaller than that in wild type mice。4. The density of the 14-kDa actin fragment in gastrocnemius muscles of Type 2 diabetic mice was 2.1-fold higher than that in wild type mice.5. The rate of protein degradation in the soleus muscle of Type 2 diabetic mice was 28.1% higher than the rate measured in soleus muscles of wild type mice. Protein degradation in EDL muscles of Type 2 diabetic mice was 43.5% higher and the rate in plantaris muscles was 34.1% higher than that in muscle of wild type mice.6. The proteasome chymotryptic-like peptidase activity in gastrocnemius musc-les of Type 2 diabetic mice was significantly increased than that in muscle of wild type mice..7. The activity of PI3K in the muscles of type 2 diabetic mice was lower than in those of wild type mice, and the lower PI3K activity was accompanied by an increase in phosphorylation of IRS-1 at serine 307 and a decrease in Akt phosphorylated on serine 473.8. The hyperglycemia and hyperinsulinemia of type 2 diabetic mice were corrected in rosiglitazone-fed type 2 diabetic mice after 35 d, reaching levels found in wild type mice, however, the weight gain was unaffected.9. The production of corticosterone of type 2 diabetic mice was decreases and the circulating levels of adiponectin of type 2 diabetic mice was decreases after rosiglitazone treatmented.10. After rosiglitazone treatmented, the phosphor-ylation of Akt in gastrocnem-ius muscle of type 2 diabetic mice was increased,the phosphorylation of serine307 on IRS-1 was suppressed.11. The protein degradation and proteasome activity in muscle of type 2 diabetic mice was decreased after Rosiglitazone treatmented.12. Rosiglitazone treatment increased the mRNA level of adiponectin but decre-ases the levels of TNF and IL-6 mRNAs in cultured differentiated adipocytes.13. Rosiglitazone treatment increased the activity of PI3K and the phosphory-lation of the forkhead transcription factor FOXO1 and suppressed mRNA expression of the E3 ubiquitin-conjugating enzymes atrogin-1/MAFbx and MuRF1.Conclusion1. Type 2 diabetic mice have serious muscle wasting.2. The 26S and 20S proteasome proteolysis pathway is activated in type 2 diabetics mice, which might be one of the mechanism resulting in mucscle wasting.3. PI3K/Akt cellular signaling is impaired in muscle of db/db mice, which might be the mechanism of mucscle wasting.4. Rosiglitazone and potential processes prevent type 2 diabetics mice's muscle protein loss. |
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