动物造模,2型糖尿病,高脂血症 z-bio 2022-05-18 597

　　Introduction: The increasing prevalence of obesity is emerging as a medical and public health problem as obesity is closely associated with metabolic abnormalities including hyperlipidemia, nonalcoholic fatty liver disease (NAFLD), type 2 diabetes (T2D) and cardiovascular disease . A large number of studies have shown that obesity plays an important role in the occurrence and development of insulin resistance (IR) and T2D. It is generally believed that dyslipidemia caused by obesity is the main factor leading to the occurrence and development of IR and T2D, which is characterized by increased blood lipid content, especially Triacylglycerol (TG) and free fatty acid (FFA) T2D types. However, the exact mechanisms linking dyslipidemia to IR and T2D remain poorly understood. Therefore, it is particularly important and urgent to clarify the key factors of obesity-induced IR and T2D, especially in the early stage of disease progression. Non-human primates (NHPs) such as rhesus monkeys (Macaca mulatta) and cynomolgus monkeys (macacafcicularis) are particularly suitable for biomedical research due to their high genetic and physiological similarity to humans. Although high-calorie diets can easily induce obesity, numerous reports have convincingly demonstrated that spontaneous obesity is common in NHPs and that they display obesity-related physiological changes, including increased abdominal fat, body mass index (BMI), and Dyslipidemia IR and T2D, similar to humans. Therefore, NHPs are frequently used as valuable research models for obesity and obesity-related metabolic diseases. Lipidomics represents a global pattern of lipid species and provides many promising novel lipid biomarkers and valuable information to elucidate the pathogenesis of common complex diseases such as dyslipidemia and obesity. With the advent of novel detection and analysis technologies, comprehensive lipidomic profiling in plasma and tissues has become possible. Previously, we screened a limited number of spontaneously obese and diabetic rhesus monkeys. Three obese female monkeys with a BMI of 40 kg/m2 or higher and aged 12 to 18 years were selected and their metabolic markers were subsequently monitored for 1 year, and they showed dyslipidemia, fatty liver and IR, similar to early T2D in humans. In the present study, we performed plasma liposome analysis in normal and spontaneously obese monkeys to identify potential lipid biomarkers or disease factors for obesity and insulin resistance.

　　Animal and plasma sample collection: We described in a previous study the criteria used to screen monkeys for spontaneous obesity, defined as a body mass index of 40 kg/m2 or higher. Three individually obese (OB) female monkeys and three normal (CK) female monkeys of similar age (12-18 years old) were identified for this study. All monkeys had ad libitum access to water and food, comprising 21.6% of calories (protein), 5.4% (fat) and 56.6% (carbohydrate). The monkeys were euthanized with ketamine at a dose of 20 mg/kg. Under fasting conditions, blood was collected from the femoral vein into tubes containing ethylenediaminetetraacetic acid (EDTA), followed by centrifugation at 3000 rpm for 5 min at room temperature. After centrifugation, plasma was stored at -80°C for further studies.

　　Gas chromatography/mass spectrometry (GC/MS) analysis of plasma fatty acid composition: Plasma fatty acids were methylated as previously reported with minor modifications. Briefly, 20 μL of hexane internal standard containing 1 mg/mL methyl heptanoate, 0.5 mg/mL methyl tricarboxylate, and 28 mg/mL butylated hydroxytoluene (BHT) was added to a Pyrex tube , followed by the addition of 50 μL of plasma and 1 mL of methanol/hexane mixture (4:1, v/v). After cooling the tubes in a homemade liquid nitrogen bath for 10 min, 100 μl of pre-chilled acetyl chloride was added to the mixture, followed by a brief nitrogen flush. The tubes were then capped and kept in the dark for 24 hours at room temperature. The resulting mixture was placed in an ice bath for 10 minutes, then 2.5 mL of 6% K2CO3 solution was gradually added (shaking) to neutralize. The mixture was allowed to stand for 10 minutes and the top layer was transferred to a glass vial. This extraction process was repeated two further times and the combined supernatants were evaporated to dryness. The resulting residue was redissolved in 50 μL of hexane and then subjected to GC-flame ionization detector (FID)/MS analysis. Methylated fatty acids were determined on a Shimadzu GC/MS-QP2010 PLUS spectrometer equipped with an electron impact (EI) ion source and a hydrogen flame ionization detector. An Agilent DB-225 capillary gas chromatography column (10 m, 0.1 mm inner diameter, 0.1 μm film thickness) was used, the injection volume was 1 μL, and the split ratio was 1:60. Helium was used as carrier and supplementary gas. The inlet and detector temperatures were both set to 230°C. The column temperature was set to 55°C for 1 minute and then increased to 205°C at a rate of 30°C/min. The column temperature was then held at 205°C for 3°min and then increased to 230°C (5°C/min). Mass spectra were obtained under the conditions of EI voltage of 70 eV and m/z range of 45-450. Methylated fatty acids were identified by comparison to the chromatograms of a mixture of 37 known standards and further confirmed with their mass spectrometry data. Each fatty acid was quantified using signal integration and FID data of the internal standard. Results are expressed as μmol of fatty acids per liter of plasma. Analysis of plasma free fatty acids using ultra-high performance liquid chromatography/mass spectrometry. Plasma phospholipids and sphingolipids were analyzed using UPLC-MS.

　　Results: Elevated FFA Levels in Obese Monkeys: Our previous report identified a group of spontaneously obese rhesus monkeys with significantly increased body weight, BMI, serum and liver TG content, and mild insulin resistance. To identify potential lipid biomarkers or disease factors associated with obesity and insulin resistance, blood samples from three obese (OB) and normal (CK) monkeys previously used for liver proteome analysis were also collected simultaneously for lipid Quantitative analysis. The types of fatty acids were determined by gas chromatography-mass spectrometry (GC/MS). Although there were no differences in plasma total fatty acid levels, palmitoleic acid (C16:1) and arachidonic acid (C20:4) levels were significantly higher in obese monkeys than in normal monkeys. Many studies have reported that elevated levels of FFAs are associated with the pathogenesis of insulin resistance. We therefore measured plasma free fatty acid levels. Compared with normal monkeys, FFA levels were significantly elevated in obese monkeys. Consistent with the human results, among the eight free fatty acids in monkey plasma, palmitic acid (C16:0) was the major constituent fatty acid. It is worth noting that, except for the low content of C20:4 and C20:5, the content of five fatty acids C16:0, C16:1, C18:1, C18:2 and C20:6 in obese monkeys is higher than that in normal monkeys . Taken together, these results indicate that FFA levels are also increased in obese monkeys, which is consistent with human and rodent models of obesity.

　　Distribution of plasma phospholipids and sphingolipids: Plasma phospholipids (PL) and sphingolipids (SM) in normal and obese monkeys were detected by UPLC/MS. From phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylglycerol (PG), lysophosphatidylcholine (LPC), lysophosphatidic acid A total of 129 lipids and SMs were identified, quantified and classified in (LPA). Among the eight lipid classes, PC was the most abundant lipid class, followed by SM, LPC and PI. In conclusion, compared with normal monkeys, the plasma PC, SM and LPA contents of obese monkeys were increased, while the plasma PE contents were slightly decreased.

　　Increased phosphatidylcholine (PC) content in obese monkeys: A total of 42 PCs were detected, of which 17 had an average concentration of more than 50 nmol/mL, and 25 had an average concentration of less than 50 mmol/mL. In addition, the concentrations of 8 PCs containing C14:0 fatty acids were all lower than 10 nmol/mL. In general, obese monkeys had slightly increased levels of most PC species compared with normal monkeys. As mentioned above, fatty acid C16:1 and C20:4 levels were significantly elevated in obese monkeys. PC levels of all four C20:4 and half of C16:1 were significantly increased in obese monkeys compared to normal monkeys. These results suggest that elevated levels of fatty acid C20:4 may originate from PC rather than FFA. Phosphatidylethanolamine (PE) levels were decreased in obese monkeys. Increased sphingomyelin (SM) content in obese monkeys.

　　Conclusions: In conclusion, many of our findings in rhesus monkeys are consistent with rodent and human reports. At the same time, we also discovered a new lipid species, 16:0-LPA, with elevated levels in obese monkeys. Alterations in these lipid species, especially FFA C16:0 and 16:0-LPA, may be the subject of diagnosis and research in obesity-related diseases.