| As of 2019,there were 463 million people with diabetes worldwide and this number is expected to rise to 700 million by 2045.Type 2 diabetes mellitus(T2DM)is a chronic,metabolic disease owing to relatively insufficient insulin secretion and/or insulin resistance(IR)and is characterized by continuous hyperglycemia.The occurrence of T2DM easily causes the metabolism disorder including glucolipid,protein,mineral,and vitamin or easily induces inflammation causing skeletal muscle atrophy.It is well-known that the most common way to increase insulin sensitivity and convert hyperglycemia is with oral hypoglycemic drugs.Nevertheless,most of these medicines have much side effects.Therefore,it is urgent to develop of alternative and new natural compounds for T2DM treatment with high efficiency and low toxicity.Huajiao(Zanthoxylum bungeanum Maxim)is a plant fruit belonging to the Zanthoxylum genus(Rutaceae).Z.bungeanum,a cooking sauce,is one of the eight traditional condiments in China.The primary factor for tongue numbness in Z.bungeanum is Zanthoxylum alkylamides.Z.alkylamides have a series of bioactivities including antioxidant activities,anti-tumor,anti-inflammatory,analgesia,hypoglycemic activity,decreading cholesterol,and anaesthesia.And in recent years,researchers have turned their attention to the effect of Z.alkylamides on protein metabolism.The previous study showed that Z.alkylamides improved the protein metabolism in both healthy Sprague Dawley(SD)rats and T1DM rats induced by streptozocin(STZ).However,it is not clear that whether Z.alkylamides could have the same effect on T2DM rats.Therefore,based on the previous research studying on the effect of Z.alkylamides on protein metabolism in T1DM rats,the present study was performed with T2DM rats model induced with high fat and sugar fodder combined with low-dose of STZ(40 mg/kg·bw).According to the body weight and fasting blood glucose,the T2DM rats were then separated into 5 groups(n=6 per group)as follows:(1)Diabetes model group(DM),(2)Metformin group(ME,135 mg/kg·bw metformin daily),(3)Low-dose group(LDG,2mg/kg·bw Z.alkylamides daily),(4)Medium-dose group(MDG,4 mg/kg·bw Z.alkylamides daily),(5)High-dose group(HDG,8 mg/kg·bw Z.alkylamides daily).The rats fed with standard fodder were regarded as normal group(CN).The CN and DM rats were treated with soybean oil at a dose of 0.2 m L/100 g daily,which was the same dose of soybean oil without Z.alkylamides.All the rats were treated for 28 d and the body weight and the fasting blood glucose were measured weekly.On the last day of the experiment,the plasma,jejunum,liver,and skeletal muscle of experimental rats were collected and measured to explore the effect and mechanism of Z.alkylamides on protein metabolism of T2DM rats,providing the scientific evidence for finding the natural drug for T2DM treatment.The main results of the present study were as follows:(1)The effect of Z.alkylamides on the basic physiological and biochemical indexes of T2DM ratsThe result showed that compared with the rats in DM group,Z.alkylamides could significantly resist(p<0.01)the body weight reduction induced by T2DM and remarkably decreased(p<0.05)both the liver and kidney indexes,improving hepatomegaly and kidney enlargement induced by T2DM.Z.alkylamides also significantly decreased(p<0.05)the hyperglycemia and significantly promoted insulin sensitivity of T2DM rats.Z.alkylamides could not only decrease(p<0.05)the levels of triglyceride,total cholesterol,and low density lipoprotein cholesterin but also increase the high density lipoprotein cholesterin level in plasma of T2DM rats,with best effect discovered in high-dose of Z.alkylamides and metformin.Z.alkylamides could also significantly increase(p<0.05)the levels of total protein(TP)and albumin and decrease(p<0.05)the level of global proteins(GPs)of T2DM rats,but have no effect on the values of albumin/GPs(p>0.05)with comparison to the DM group.Meanwhile,Z.alkylamides could remarkably decrease(p<0.05)the levels of blood urea nitrogen(BUN)and creatinine(Cr)in plasma but increase(p<0.01)the levels of C-peptide and insulin growth factor 1(IGF-1)of T2DM rats.Z.alkylamides significantly decreased(p<0.05)the levels of free triiodothyronine(FT3)and free thyroxine(FT4)but increased(p<0.05)the asprosin(ASP)level in plasma of T2DM rats.Additionally,Z.alkylamides significantly decreased(p<0.05)the plasma levels of proinflammation factor including IL-6 and C-reactive protein,and increased(p<0.05)the values of anti-inflammation factor including IL-10 and adiponectin(ADP)of T2DM rats.Z.alkylamides could also decrease(p<0.05)the level of aspartate aminotransferase(AST)and alanine aminotransferase(ALT)in liver and could increase(p<0.05)the content of 6 kinds of short chain fatty acid in the faeces of T2DM rats.These results suggested that Z.alkylamides,as well as metformin,could ameliorate the weight loss and hyperglycemia of T2DM rats to enhance insulin sensitivity and improve insulin resistance.The above basic physiological and biochemical indexes in both plasma and liver indicated that the levels of both diagnostic markers of protein metabolism(TP,BUN,Cr,insulin,IGF-1,et al.)and the diagnostic markers of energy metabolism(FT3,FT4,ASP,short chain fatty acid,et al.)were disordered.However,Z.alkylamides could effectively improve the above symptom with a dose dependent manner,suggesting that Z.alkylamides could be considered as the potential substance to improve protein metabolism of T2DM rats.(2)The effect of Z.alkylamides on amino acid levels and amino acid transporter m RNA in plasma and different tissues of T2DM rats.The analysis of amino acid levels in plasma,jejunum,liver and skeletal muscle of T2DM rats was carried out using HPLC and the expression of amino acid transporter related genes was investigated at m RNA level using RT-q PCR.The results showed that Glu and Ala were the major components affecting the amino acid levels in plasma,jejunum,and liver of T2DM rats,while His,Pro,Thr,Vla,and Cystine were the main components affecting the amino acid level in skeletal muscle of T2DM rats.Comparing to the rats in DM group,metformin and Z.alkylamides could significantly decrease(p<0.05)the levels of Glu and BCAA in plasma.Z.alkylamides could also decrease(p<0.05)the levels of BCAA,Met,and Cys but increase(p<0.05)the values of Gln,Ala,and Gly in jejunum of T2DM rats.Moreover,Z.alkylamides could remarkably decrease(p<0.05)the levels of BCAA,Val,Met,and Glu but increase(p<0.05)those of Ala and Gln in liver of T2DM rats.Z.alkylamides could aslo significantly decrease(p<0.05)the levels of BCAA,Leu,Cystine,and Thr but increase(p<0.05)Gln,Cys,and Gly levels in skeletal muscle of T2DM rats.In addition,comparing to the DM group,Z.alkylamides,as well as metformin,could significantly up-regulate(p<0.05)the m RNA relative expression of cationic amino acid transporters(b0,+LAT,CAT1,and y+LAT1)in jejunum and that of CAT2 in liver.And in accordance with the result,the corresponding amino acid levels were improved in different degrees.Z.alkylamides could also significantly down-regulate(p<0.05)m RNA relative expression of neutral amino acid transporter LAT1 in jejunum,but could up-regulate(p<0.05)the value of SNAT2 in jejunum and skeletal muscle of T2DM rats.Meanwhile,the netural amino acid levels in plasma and each tissue were ameliorated.Z.alkylamides could also significantly up-regulate(p<0.05)the m RNA relative expression of PAT1 in jejunum and skeletal muscle of T2DM rats,regulating the disorder level of Pro,Ala,Gly,and Ser.The above results suggested that the disorder of amino acid metabolism in plasma and different tissues(jejunum,liver,and skeletal muscle)was mediated by different amino acids.Like metformin,Z.alkylamides could regulate the m RNA expressions of amino acid transporters in jejunum,liver,and skeletal muscle of T2DM rats to promote the transportation of amino acid,further indicating that Z.alkylamides may be the potential substance for protein metabolism improvement of T2DM rats.(3)The effect of Z.alkylamides on protein anabolism of T2DM rats.The results indicated that compared with the rats in DM group,Z.alkylamides could significantly up-regulate(p<0.05 or p<0.01)the m RNA relative expressions of IGF-1,IGF-1R,PI3K,Akt and m TOR in both liver and skeletal muscle of T2DM rats.Z.alkylamides could also significantly up-regulate(p<0.05 or p<0.01)the relative protein expression levels of PI3K p85,PI3K p110,p-Akt,p-Akt/Akt,p-m TOR and p-m TOR/m TOR in skeletal muscle of T2DM rats.Additionally,Z.alkylamides could remarkably up-regulate(p<0.05 or p<0.01)the m RNA relative expressions of AMPK,PPAR?,and GLUT4 and the relative protein expressions of p-AMPK,p-AMPK/AMPK,PPAR?,and GLUT4 in skeletal muscle of T2DM rats.The results suggested that Z.alkylamides could up-regulate the expression of IGF-1 and IGF-IR,which then promoted the downstream signal factor PI3K expression to phosphorylate and activate the expression of Akt to further up-regulate the expression of m TOR,promoting protein synthesis to thus ameliorate T2DM.Meanwhile,Z.alkylamides could phosphorylate and activate the expression of AMPK to regulate the transportation of GLUT4 to promote glucose transportation and energy metabolism to eventually ameliorate protein anabolism.Moreover,Z.alkylamides could up-regulate the expression of PPAR?to improve energy metabolism,but the specific mechanism of it should be further investigated.(4)The effect of Z.alkylamides on protein catabolism of T2DM rats.The results showed that Z.alkylamides could significantly down-regulate(p<0.05or p<0.01)the m RNA relative expressions of Fox O1,Fox O3a,Mu RF1,MAFbx,TNF-?,and NF?B and down-regulate(p<0.05 or p<0.01)the relative protein expression levels of Fox O1,Fox O3a,Mu RF1,MAFbx,TNF-?,p-NF?B and p-NF?B/NF?B in skeletal muscle of T2DM rats.The results indicated that Z.alkylamides could inhibit the activation of Fox Os by activating PI3K and Akt.This then down-regulated the expression of MAFbx and Mu RF1 to inhibit protein degradation through ubiquitin-proteasome system(UPS)to ultimately improve protein catabolism and T2DM.Meanwhile,Z.alkylamides could down-regulate the expression of TNF-?to inhibit the expression of MSTN,up-regulating the expression of Myo D to promote protein synthesis.Z.alkylamides could also inhibit the activation of NF?B by down-regulating the expression of TNF-?,inhibiting the secretion of IL-6 and C-reactive protein to improve inflammation.The inhibition of TNF-?could inhibit the transfer of NF?B into the nuclear and thus down-regulates the expression of Mu RF1 and MAFbx to inhibit protein degradation through UPS pathway.According to the above study,the results of this study were as follows:(1)Z.alkylamides could resist the body weight loss and have hypoglycemic activity.Z.alkylamides could improve the level of Ins and increase the content of IGF-1,activating the transduction of PI3K/Akt/m TOR signaling pathway mediated by Ins/IGF-1 to promote protein synthesis in skeletal muscle of T2DM rats;Z.alkylamides could ameliorate energy metabolism to provide energy for protein synthesis through regulating AMPK and PPAR?signaling pathways.(2)Z.alkylamides could decrease the level of inflammation factors in plasma and inhibit the inflammation reaction through restraining the transduction of TNF-?/NF-?B signaling pathway;Z.alkylamides could promote protein synthesis through inhibiting the transduction of TNF-?/MSTN signaling pathway;Z.alkylamides could also inhibit protein degradation in skeletal muscle to ameliorate T2DM through down-regulating the ubiquitin-proteasome system mediated by TNF-?/NF-?B and PI3K/Akt/Fox Os signaling pathways. |
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