| As diet and lifestyle changes, the incidence of obesity and diabetes increase year byyear. And with the number of aged and obesity women growing, the incidence ofgestational diabetes millus (GDM) is also arising quickly with an serious results of moreType2diabetes mellitus (T2DM) women and obesity children after pregnancy; GDM isclosely related to the deficiency expansion of pancreatic β cells, so many researchers paytheir attentions on the expansion of β cells in pregnancy, and they think it’s important tolook for a drug that can prevent and promote β cells from damage, restore β cells function,and inhibit their apoptosis function, which may be an important direction to deal withGDM and other diabetes.Histone deacetylase inhibitors (HDACIs) show promising properties, which canpromote β cells function, enhance insulin production and secretion, prevent β cells frominflammatory damage, improve peripheral insulin resistance, keep immune system balancein pathologic process of T1DM and T2DM. Sodium butyrate (CH3CH2CH2COONa) isone member of HDACIs, it’s also a fatty acid derivative found in foods, such asresistant starch and cheese, and it can produced physiologically in large amounts fromfermentation of dietary fiber in the large intestine, it can be used as substrates energyproduction for intestinal and ruminal epithelial cells, which can meet5-10%basic energyneed of human. So in this study, we explored whether sodium butyrate has an influence onmetabolism, pancreatic β-cell function and microenvironment in islet in mice model ofobesity pregnancy.1. Established obesity pregnancy animal model and explored whetherhigh fat diet with butyrate can improve metabolic impairmentsSPF C57BL/6J60female mouse (4weeks old) were randomly divided into threegroups with three different diets after1W quarantine, diets were the high fat food (HF)(40%fat,20%protein,40%carbohydrate); High-fat diet with sodium butyrate food (HSF)was fed the above high-fat diet with sodium butyrate at5%wt/wt; control food (CF)(14%fat,26%protein,60%carbohydrate) was fed a control food. Body weight was measured once a week; Blood glucose was determined by glucometer at indicated time points fromcutting tails after overnight fasted12h every2weeks; Insulin of blood samples werecollected by retro orbital sinus puncture after overnight fasted12h after14weeks dietaryintervention for insulin analysis. And after14weeks dietary intervention, conventionalintraperitoneal glucose tolerance test (IPGTT) and intraperitoneal Insulin tolerance test(IPITT)were performed after fasting for12h and4h respectively; Results showed the bodyweight had great difference in groups, HF showed a higher blood glucose and with asignificant insulin resistance state: insulin level of HF was1.580±0.23ng/ml (P<0.001), aswhile HSF was at a0.690±0.120ng/ml (P<0.05), CF was at0.343±0.06ng/ml. Then after1week rest, mouse were mating with male mouse, combined with the obesity and GD14.5status, the establishment of obesity mouse model is the most close to the pathogenesis ofhuman GDM. To explore whether high fat diet with butyrate can improve metabolicimpairments, dams of pregnant mouse of groups were sacrificed at GD14.5, maternalserum was analyzed for triglyceride, total cholesterol, insulin, TNF-α, and IL-1β levelsrespectively. In our results: at GD14.5, the body weight of HF gained24g, HSF gained14.8g, CF gained11g (in groups P<0.001). Blood glucose levels showed a progressiveincrease in10thW of HF feeding(P<0.001), but not showed in CF or HSF groups, glucoselevels in HSF were elevated compared to CF, but were still lower compared to HF(p>0.05).IPGTT demonstrated that glucose clearance is significantly retarded in HF mice, and IPITTof HF also exhibited much worse glucose clearance response to insulin stimulus at all timepoints compared to HSF and CF. HSF had improved insulin resistance someway. AtGD14.5, Groups of fast insulin were all a little increase compared before pregnancy, butthe statistical differences between groups were not changed. Our results showed sodiumbutyrate can clear away triglyceride and total cholesterol（P<0.001）: HF triglyceride(235.21±29.08mg/dl); CF(140.45±18.02mg/dl); total cholesterol: HF351.08±59.07mg/dl, CF236.8±41.8mg/dl (P<0.001), triglyceride and total cholesterol in HSF:176.6±13.92mg/dl and244.42±25.17; and compared to HF, HSF has a lower IL-1β(P<0.001）and TNF-α level (P<0.05).2. Butyrate influenced β cells insulin expression and anti-apoptosisfunction in obesity pregnancy miceIn part I, butyrate showed an important regulation on metabolic impairments ofobesity, we wonder if butyrate can influence β cells, so based on this obesity pregnantmouse model, at the GD14.5, in which time β cell has the peak mass with much excited proliferation and anti-apoptosis functions during pregnancy, and wonder how β cell to meetthese double (obesity and pregnancy) insulin resistance state, the influence on β cellswhich butyrate played at this time can be easily found. Dams of pregnant mouse weresacrificed at gestational14.5d, entire pancreata were removed and weighed quickly, onepart of tissues snapped frozen in OCT, and stored in-70°C after frozen section. Insulinexpression as a marker of the β cell and β cell mass was examined byimmunohistochemistry, PCNA and Bcl-2were chosen to be marker protein of proliferationand anti-apoptosis respectively to incubation with insulin to assess the influence of sodiumbutyrate on β cells. Then morphometrical analysis was explored by Image-Pro Plussoftware, the insulin expressions were measured by determining the average pixel value ofstaining per islet, and the size of β cell, the area of β cells in pancreases, the rate of PCNAand Bcl-2in β cells were all measured and counted. The results showed that the averagepixel value of insulin of CF was0.17±0.03, and HF was0.037±0.007, HSF0.09±0.01,(P<0.001). The area of β each cell was biggest in HF112.1±7.18um2, as HSF94.5±4.21um2and CF:90.6±6.73um2, but there was no statistically difference between last twogroups. The β cell mass in HF was2.67±0.17mg, HSF:2.18±0.19mg，CF:1.94±0.11, βcell mass of HF was significantly different from HSF and CF (P<0.001), and there only alitter difference between HSF and CF (P<0.05). The percentage of PCNA positive β cells,with no difference between CF and HSF groups, PCNA levels were higher in HF（1.70±0.27）%compared to HSF（1.45±0.15）%,(P<0.05), CF（1.4±0.11）%, there wasno significant difference between HSF and CF. On the other hand, the number of Bcl-2positive β cells was obviously lower in the HF group compared to HSF and CF (HF:1.26±0.49vs. HSF:4.32±0.81and CF:4.46±0.49, P<0.001), and there was still nosignificant difference between HSF and CF.3. The influence of sodium butyrate on pancreatal islet inflammatoryresponse and mother and infant mouse development.And more and more studies have confirmed that the microenvironment in islet is veryimportant, higher release of inflammatory cytokines in islets can affect the function andsurvival of β cells. GDM is also closely related with islet inflammation. The pathologicalprocess can be relieved or treated by inhibiting the inflammatory reaction. Besides abovefunctions of regulate metabolic disorder and enhance β cells functions, sodium butyratehad found that can suppress autoimmune diseases in numerous studies.To futher exploer the influence on metabolism, based on the model, we also chose part pancreatic tissue to explore the expression of NF-κB, pNF-κB to evaluate isletinflammatory of groups, since the activation of NF-κB is an important and classical markerof inflammatory response. Dams of pregnant mouse of groups were sacrificed at GD14.5,the other part pancreata left from the part II experiment was fixed in4%paraformaldehyde for24h, and then laid flat for paraffin section, then detected theexpression of NF-κB, pNF-κB by immunohistochemistry method. The assessment wasperformed by determining the number of positive cells per islet section by Image-Pro Plussoftware. Results in our study showed: NF-κB positive cells in islet of HF were greaterthan that of HSF and CF (HF:6.48±2.39VS. HSF2.92±0.73and CF0.56±0.15,p<0.001), and inflammatory response in HSF islet was greater than that in CF (P<0.05), asmeasuring pNF-ΚB positive cells in islet, HF still showed increased inflammatoryresponse compared to HSF and CF (HF:2.07±0.78vs. HSF:0.78±0.15, P<0.005; HF vs.CF:0.286±0.08, P<0.001); the ratio of pNF-ΚB to NF-ΚB positive cells in HF was higherthan that of other groups (HF:0.35±0.052VS. HSF:0.27±0.03, P<0.001; HFVS.CF:0.2666±0.04, P<0.001), but there was no statistically significant differencebetween CF and HSF.3pregnant mouse of each group were let them born without humanintervention, as litters were born from mothers of HSF, it did not showany visible deformity to the naked eye were not found and any change of pregnancyduration.Therefore, in our research we established obesity pregnant mouse model, when theobesity mouse has significant difference on body weight, glucose glucose toleranceimpaired and insulin resistance, with above symptoms GDM models were thought to besuccessfully established. And based on this model, we found sodium butyrate has an effecton metabolism, and β cells biological behaviors such as anti-apoptosis, and inhibited isletinflammatory response without any toxicity found in pregnancy and new born mouse, sobutyrate was presented itself as a new therapeutic target for GDM, T1DM, T2DM andother metabolic diseases. |
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