Hypophyseal Lipoapoptosis: Diabetes (db/db) Mutation-Associated Cytolipidemia Promotes Pituitary Cellular Disruption and Dysfunction

Expression of the diabetes (db/db) mutation in C57BL/KsJ mice suppresses the female pituitary-gonadal axis via progressive cytolipidemic disruption of hypophyseal gonadotropin release, culminating in premature involution of the reproductive tract and manifest infertility. The current studies define the systemic, endocrine, cytochemical and structural apoptotic changes that result from pituitary hypercytolipidemia induced by db/db mutation expression in this Type II diabetes-obesity syndrome (DOS) model. Adult female C57BL/KsJ control (+/?; genotype) and db/db littermates were monitored for systemic and cellular alterations in LH-, FSH- and gonadal steroid-secretion, and coincident pituitary apoptosis, as indexed by TUNEL labeled 3′ nuclear DNA-fragmentation, associated with cytolipid depositions. Obesity, hyperglycemia and hyperinsulinemia characterized all db/db-mutants relative to +/?; groups. Serum progesterone (P) and estradiol (E2) concentrations were suppressed in db/db mutants coincident with decreased plasma LH and FSH concentrations relative to +/?; values. Cytochemical analysis of anterior (AP) pituitary cell subtypes indicated that db/db mutants demonstrated prominent hypercytolipidemia relative to +/?; pituitary cytoarchitecture. Cytolipidemic vacuoles were localized within protein vesiculateddb/db hypophyseal basophilic and acidophilic cell populations. Hypophyseal cytoadiposity in db/db AP cells was co-localized with prominent cellular apoptotic TUNEL labeling of nuclear 3′-DNA fragments in cells demonstrating vesicular depopulation and cytolytic vacuolization. These data represent the first demonstration of co-localized hypercytolipidemic and cytoapoptotic disruptive events occurring concurrently in a hypopituitary-hypogonadal syndrome model following expression of the Type II (NIDDM) diabetes-obesity syndrome in db/db-mutants. The coincident and progressive vascular-, interstitial- and cyto-lipidemic alterations in hypophyseal cytoarchitecture correlated with the concurrent apoptotic disruption of pituitary endocrine cytoarchitecture and supressed gonadal steroid synthesis, influences which collectively contribute to the premature involution of the pituitary-gonadal axis in C57BL/KsJ-db/db mice.

     

Homing of the bone marrow-derived interstitial cells of Cajal is decreased in diabetic mouse intestine

Background: Interstitial cells of Cajal (ICCs), which express c‐Kit receptor tyrosine kinase (KIT), play an important role in gastrointestinal motility. Loss of ICCs likely contributes to diabetic gastrointestinal motility disorder, however, the mechanism of attrition remains unknown. Here, we test the hypothesis that the bone marrow‐derived progenitors are an important source of intestinal ICCs and that decreased homing of these progenitors in diabetes contributes to ICC diminution. Methods: Wild type mice were X‐ray irradiated, transplanted with bone marrow (BMT) from green fluorescence protein (GFP)‐transgenic (TG)‐mice and subsequently made diabetic by streptozotocin (STZ) injection. Intestinal homing of GFP‐positive bone marrow‐derived cells was examined 2 or 5 months after STZ treatment. Results: In the BMT‐mice, we found many GFP‐positive bone marrow‐derived cells (BMDCs) in most parts of the intestinal area, the number of BMDCs was significantly decreased in diabetic mice compared with nondiabetic controls. As a representative area, we further examined the myenteric plexus of the proximal small intestine, and found that the cell numbers of ICCs marked by c‐Kit‐positive immunoreactivity were decreased by more than 40% in diabetic versus nondiabetic mice. Furthermore, numbers of c‐Kit+/GFP+ and c‐Kit+/GFP‐ cells were similar in nondiabetic mice, and decreased by 45.8% and 42.0%, respectively, in diabetic mice. Conclusion: These results suggest that the decreased homing from the bone marrow is a major cause of ICC loss in the intestine in diabetes mellitus.     

Prevention of decline in renal function in the diabetic db/db mouse

Summary We recently reported that when diabetic db/db mice, which develop glomerular pathology resembling that in human diabetes mellitus, are treated with monoclonal antibodies (A717) that neutralize the effects of excess glycated albumin, there is an amelioration of mesangial expansion, renal overexpression of mRNAs encoding for the extracellular matrix proteins collagen IV and fibronectin, and proteinuria. These findings suggested that A717 might also retard the development of compromised renal function in this animal model. To examine this possibility, serum creatinine and blood urea nitrogen (BUN) were measured in diabetic db/db mice and their non-diabetic db/m littermates before and after an 8-week course of treatment with A717 or irrelevant murine immunoglobulin (MIg). Early in the course of diabetes, BUN and serum creatinine concentrations did not significantly differ from those in the db/m littermates, but were significantly increased after 10 weeks of sustained hyperglycaemia. Treatment of db/db mice with A717 prevented the rise in creatinine and attenuated the elevation in BUN. A717 also prevented the decrease in creatinine clearance observed in diabetic compared with non-diabetic animals (2.2±0.8 vs 4.1±0.3 vs 5.0±1.1 ml/h in db/db vs db/db-A717 vs db/m, respectively). MIg did not alter the change in renal function with time in db/db mice. Taken together with our previous results, the present findings indicate that the diabetic db/db mouse develops changes in renal function and structure that parallel the course of human diabetic nephropathy in nature and chronology and demonstrate, for the first time, that therapy directed against increased glycated albumin can prevent the decline in renal function in this rodent model of genetic diabetes.Abbreviations MIg Murine immunoglobulin - BUN blood urea nitrogen - ACE angiotensin converting enzyme - AGE advanced glycation end product     

Deficiency of KIT-positive cells in the colon of patients with diabetes mellitus

BACKGROUND: Diabetes mellitus is a well-known cause of gastrointestinal dysmotility. The pathogenesis of diabetic gastroenteropathy is mainly considered to be a neuropathy, but the cause of dysmotility remains unknown. Interstitial cells of Cajal (ICC), which express c-kit receptor tyrosine kinase (KIT), are considered to be pacemaker cells for the gastrointestinal movement. Therefore, we investigated a possible involvement of ICC in the pathogenesis of diabetic gastroenteropathy in humans. METHODS: The KIT-positive cells in the proper muscle layer of the colon were detected by immunohistochemistry in patients with diabetes mellitus and normal control subjects. Mast cells, which are also known to express KIT, were detected by staining with Alcian blue. The numbers of KIT-positive cells and Alcian blue-positive cells in the proper muscle layer were counted under the microscope and the number of KIT-positive cells apart from Alcian blue-positive cells was calculated. RESULTS: In the normal control subjects, KIT-positive cells were located at the myenteric plexus region and in the circular muscle layer of the colon. Their distribution pattern was similar to that of ICC. The average number of KIT-positive cells, apart from mast cells (which reflects the number of ICC), in patients with diabetes mellitus was approximately 40% of that found in normal subjects. CONCLUSIONS: Deficiency of ICC might be related to the pathogenesis of diabetic gastroenteropathy in humans.     

Combination treatment of db/db mice with exendin‐4 and gastrin preserves β‐cell mass by stimulating β‐cell growth and differentiation

Aim/Introduction: Preservation of β‐cell mass is crucial for maintaining long‐term glucose homeostasis. Therapies based on incretin and its mimetics are expected to achieve this goal through various biological functions, particularly the restoration of β‐cell mass. Here we tested the effects of gastrin and exendin‐4 in type 2 diabetic animals. Materials and Methods: The effects of exendin‐4 and gastrin on β‐cell function and mass were examined in 8‐week‐old db/db mice. INS‐1 beta cells and AR42J cells were used to determine the molecular mechanism underlying the effects of the two agents. Immunohistochemistry, western blotting and RT‐PCR assays were used to assess the biological effects of the two agents. Results: Two weeks of combination administration of exendin‐4 plus gastrin resulted in a significant improvement of glucose tolerance associated with a marked preservation of β‐cell mass in db/db mice. Immunohistochemical analysis showed that such treatment resulted in the appearance of numerous irregularly‐shaped small islets and single insulin‐positive cells. While gastrin had little biological effect on INS‐1 β‐cells consistent with low expression of its intrinsic receptor on these cells, it caused differentiation of AR42J cells into insulin‐producing cells. Co‐stimulation with exendin‐4 significantly enhanced gastrin‐induced endocrine differentiation of AR42J precursor cells. These findings were further supported by enhanced expression of key genes involved in β‐cell differentiation and maturation, such as neurogenin3 (Ngn3) and MafA. Conclusions: These results suggest that combination treatment of db/db mice with exendin‐4 and gastrin preserves β‐cell mass by stimulating β‐cell growth and differentiation. (J Diabetes Invest, doi: 10.1111/j.2040‐1124.00044.x, 2010)     

Studies in the diabetic mutant mouse: V. Glucose tolerance in mice homozygous and heterozygous for the diabetes (db) gene

Summary Mice homozygous and heterozygous for the diabetes (db) gene were studied to determine: 1. whether latent carbohydrate intolerance is present in young normoglycemic diabetic mutants (db/db); 2. whether normoglycemic food restricted diabetic mutants are carbohydrate intolerant; and 3. whether mice heterozygous for thedb gene (db/+) manifest abnormalities in glucose tolerance, serum IRI levels or body weights. Blood glucose levels were determined 0, 1/2, 1, 2 and 3 h following intraperitoneal administration of 2 mg glucose/g body weight. Normals (+/+) and diabetics (db/db) showed similar glucose tolerance curves during the first two weeks of life; however, both were markedly glucose intolerant compared to normal adult mice. At 3 weeks a small number of mutants had higher 3 h levels than any achieved in normal mice. By 4 weeks the average value for diabetics prior to glucose loading (0 time) was significantly (P < 0.02) elevated (db/db — 144 mg glucose/100 ml, +/+ = 124 mg glucose/100 ml). Although food restriction reduced blood glucose concentration at 0 time, persistence of carbohydrate intolerance was readily demonstrable following glucose loading. — Abnormalities in heterozygotes (db/+), 3 to 16 months of age, were primarily restricted to male mice, which showed moderate, but statistically significant elevations in blood glucose both at 0 time and following glucose administration. Forty percent of male heterozygotes had higher serum IRI levels than any observed in normal control males. Body weights of male heterozygotes were significantly greater (P<0.01) than those in agematched normals.USPHS Research Career Development Awardee, Grant K4-AM-7394.     

干细胞因子对糖尿病小鼠小肠Cajal间质细胞的影响

目的探讨外源性干细胞因子（stem cell factor,SCF）对糖尿病（diabetes mellitus,DM）小鼠小肠Cajal间质细胞的影响。方法将雄性Balb/c小鼠随机分为正常对照组、糖尿病组（DM组）、糖尿病＋外源性SCF组（DM＋SCF组）,DM组和DM＋SCF组一次性腹腔注射（ip）链脲佐菌素（STZ,150 mg/kg）造模,选取成功模型;DM＋SCF组再ip SCF 0.2μg/（kg.d）;正常组和DM组每天ip等量的磷酸盐缓冲液（pH=7.4）。所有小鼠干预6周结束后,给予印度墨水灌胃测定小肠传输速率,免疫组化观察小肠c-kit的表达,Western blot观察c-kit蛋白的表达。结果饲养6周后,DM＋SCF组的体质量（g）、推进率、c-kit阳性细胞数（个/hpf）、c-kit蛋白的表达均比DM组明显增加（P均〈0.05）,仍低于正常组（P均〈0.05）;DM＋SCF组的血糖（mmol/L）比DM组明显降低（P〈0.01）,仍高于正常组（P〈0.05）。结论外源性SCF可以逆转Cajal间质细胞的异常改变,并对糖尿病小鼠的小肠动力障碍有一定的改善作用。     

Metformin opposes impaired AMPK and SIRT1 function and deleterious changes in core clock protein expression in white adipose tissue of genetically-obese db/db mice

Aim: AMPK activates SIRT1 in liver and skeletal muscle. Impaired circadian function is associated with development of obesity. SIRT1 regulates circadian function and is suppressed in white adipose tissue (WAT) of obese patients. We examined the potential role of AMPK and SIRT1 in regulation of circadian components in WAT of obese db/db mice and in mice fed a high‐fat diet (HFD), and investigated whether metformin‐mediated activation of AMPK opposed any deleterious changes in the WAT clock mechanism. Methods: db/+ and db/db mice were administered metformin (250 mg/kg/day; 7 days). Separately, mice were fed HFD for 16‐weeks. 3T3‐L1 adipocytes were incubated with metformin, EX527 or FK866, inhibitors of SIRT1 and NAMPT, respectively. Gene and protein expression were measured by qRT‐PCR and immunoblotting. Results: AMPK activity, NAMPT expression and SIRT1 expression were decreased in WAT of db/db and HFD mice, in association with suppressed expression of the core circadian components CLOCK and BMAL1. Expression of Pparγ and the adipogenic repressors Irf3 and Irf4 were also suppressed. Metformin increased AMPK activity in WAT of db/db mice and in metformin‐treated adipocytes, with increased NAMPT, SIRT1 and circadian component expression. Metformin‐mediated induction of Clock mRNA in adipocytes was blocked by inhibition of NAMPT and SIRT1. Conclusions: Decreased AMPK‐SIRT1 signalling in db/db and HFD mice impacts WAT circadian function causing dysregulated lipid regulation, favouring an obese phenotype. Metformin mediates a phenotypic shift away from lipid accretion through AMPK‐NAMPT‐SIRT1 mediated changes in clock components, supporting chronotherapeutic treatment approaches for obesity.     

Intranasal delivery of mouse [D-Leu-4]-OB3, a synthetic peptide amide with leptin-like activity, improves energy balance, glycaemic control, insulin sensitivity and bone formation in leptin-resistant C57BLK/6-m db/db mice

Background: We have recently shown that intranasal administration of mouse [D‐Leu‐4]‐OB3 reconstituted in Intravail^® to male Swiss Webster mice resulted in significantly higher uptake and bioavailability when compared with commonly used injection methods of delivery. Aim and Methods: In this study, we examined the effects of intranasal delivery of mouse [D‐Leu‐4]‐OB3 in Intravail^® on energy balance, glucose regulation, insulin secretion and serum levels of osteocalcin, a specific and sensitive marker of bone formation. Genetically obese C57BLK/6‐m db/db mice were allowed food and water ad libitum and given either Intravail^® alone or mouse [D‐Leu‐4]‐OB3 in Intravail^® for 14 days by intranasal instillation. Results: Mouse [D‐Leu‐4]‐OB3 reduced body weight gain, daily food intake, daily water intake and serum glucose by 11.5, 2.2, 4.0 and 61.9%, respectively. Serum insulin levels in db/db mice given mouse [D‐Leu‐4]‐OB3 were approximately threefold lower than those in mice receiving Intravail^® alone. Mouse [D‐Leu‐4]‐OB3 elevated serum osteocalcin in db/db mice by 28.7% over Intravail^® treated control mice. Conclusions: The results of our study indicate that intranasal delivery of biologically active mouse [D‐Leu‐4]‐OB3 in Intravail^® is feasible and has significant effects on regulating body weight gain, food and water intake, serum glucose, insulin sensitivity and bone formation in leptin‐resistant C57BLK/6‐m db/db mice.     

Glomerular hyperfiltration during the onset of diabetes mellitus in two strains of diabetic mice (C57BL/6Jdb/db and C57BL/KsJdb/db)

Summary GFR estimated by the total clearence of⁵¹Cr-EDTA increases from 41 to 60 ml/min·m² at the onset and during the development of marked hyperglycaemia and obesity in female diabetic mice (db/db) of the C57BL/6J and the C57BL/KsJ strains (blood sugar rises from 160 to 400 mg/100 ml). GFR decreases slowly in older diabetic mice (>120 days old) approaching the control values (42±7 ml/ min·m²) and then decreases still further. The total clearance of¹⁴C-hippuric acid is unaltered indb/db mice and controls between 45 and 150 days of age (96±20 ml/min·m²). This suggests no alteration of RPF during the development of the diabetic syndrome. The glomerular hyperfiltration of diabetic mice lasts until they are 120 days old and shows no correlation with the different blood glucose levels typical for diabetic mice (db/db) of the two strains.     

Oxidative damage in cerebral vessels of diabetic db/db mice

BACKGROUND: Oxidative stress in diabetes mellitus has recently received increasing attention as it has been proven to be associated with the development of diabetic vascular complications. Our aim was to examine whether microvascular changes, including oxidative damage, were induced in the brains of diabetic animals. METHODS: The expression of 8-hydroxy-2'-deoxyguanosine (8-OHdG), a marker of oxidative DNA damage, the binding of cationized ferritin, a marker for evaluating endothelial glycocalyx, to the endothelial cells of capillaries and vascular permeability of intravenously injected horseradish peroxidase were examined in the cortices of 12- and 20-week-old db/db and db/+m mice. RESULTS: Immunostaining for 8-OHdG was clearly seen in the vessels of the cortex of 20-week-old db/db mice, but was hardly seen in those of mice in the other groups. The immunopositive area of 8-OHdG was significantly increased in the cortex of 20-week-old db/db mice compared with that of 20-week-old db/+m mice. No extravasated leakage of horseradish peroxidase was seen in any groups of mice, while the numbers of cationized ferritin particles binding to the endothelial cells was significantly decreased in 12- and 20-week-old db/db mice compared with that of db/+m mice at the same age, respectively. CONCLUSION: These findings suggest that changes in endothelial glycocalyx are induced in db/db mice and, in addition, the long-term diabetic condition of these mice induces oxidative DNA damage to the cerebral vessels.     

糖尿病大鼠小肠Cajal间质细胞及干细胞因子的变化及半夏泻心汤的干预作用

[目的]观察糖尿病（diabetes mellitus,DM）大鼠小肠Cajal间质细胞（ICC）及干细胞因子（stem cell faetor,SCF）表达的变化及半夏泻心汤的干预作用。[方法]除10只大鼠作为正常组外,其他大鼠采用腹腔注射STZ复制DM模型后随机分为DM组、半夏泻心汤组及西药组。半固体营养糊灌胃测定各组大鼠小肠推进率,免疫组化检测c-Kit及SCF在小肠组织中的表达;Western blot检测小肠组织c-Kit蛋白、SCF蛋白表达。[结果]半夏泻心汤组与西药组大鼠的体质量、小肠推进率、c-Kit及SCF阳性细胞数、c-Kit蛋白及SCF蛋白的表达均较DM组明显增加（均P〈o．05）;半夏泻心汤组与西药组大鼠的血糖值较DM组明显降低（P〈0．05）。半夏泻心汤组与西药组之间差异无统计学意义（均P〉0．05）。[结论]半夏泻心汤可以促进DM大鼠小肠肌间神经丛c-Kit、SCF的表达,提示对受损的DM大鼠小肠ICC、SCF有部分恢复作用,从而对DM大鼠的小肠动力障碍有一定的改善作用。     

PEGylated glucagon-like peptide-1 displays preserved effects on insulin release in isolated pancreatic islets and improved biological activity in db/db mice

Aims/hypothesis The rapid degradation and clearance of glucagon-like peptide-1 (GLP-1) by the enzymes dipeptidyl peptidase-IV and neutral endopeptidase 24.11 are the main impediments to the development of GLP-1 as a potential glucose-lowering agent. In this study, new enzyme-resistant polyethylene glycol (PEG)-conjugated GLP-1 analogues were designed and examined for metabolic stability and biological potency.Materials and methods Two mono-PEGylated GLP-1 analogues, N-terminally modified N-PEG/GLP-1 and Lys-modified Lys-PEG/GLP-1, were prepared. Stability was tested in plasma and tissue extracts. In vitro insulin release studies were performed using isolated rat pancreatic islets, while in vivo glycaemic responses were measured in db/db mice.Results The half-life of Lys-PEG/GLP-1 was 40-, 10- and 28-fold longer than that of GLP-1 in plasma, liver and kidney homogenates, respectively. Lys-PEG/GLP-1 stimulated insulin secretion in the islets in a dose- and glucose-dependent manner, and was as potent as GLP-1. In contrast, N-PEG/GLP-1 showed extended metabolic stability but had significantly lower biological activity. The administration of Lys-PEG/GLP-1 (9 nmol/kg i.p.) to non-fasted db/db mice stabilised glycaemia (p<0.001), whereas GLP-1 (9 nmol/kg) only caused small changes in glucose level. During OGTT in fasted db/db mice, Lys-PEG/GLP-1 administered at 1, 3 and 9 nmol/kg (i.p.) reduced the glucose AUC_0–3h by 48.7±9.4, 55.0±2.9 and 63.4±2.5%, respectively, compared with placebo (p<0.01), whereas GLP-1 (9 nmol/kg) lowered the glucose level by 39.5±12.9% (p<0.01).Conclusions/interpretation This study demonstrates that site-specific PEGylated GLP-1 analogues are resistant to degradation. The enhanced biological potencies of these analogues highlight their potential as new, GLP-1-like glucose-lowering agents.     

Combination treatment with alogliptin and voglibose increases active GLP‐1 circulation,prevents the development of diabetes and preserves pancreatic beta‐cells in prediabetic db/db mice

Aim: Alogliptin, a dipeptidyl peptidase‐4 (DPP‐4) inhibitor, and voglibose, an alpha‐glucosidase inhibitor, have different but complementary mechanisms of action on glucagon‐like peptide‐1 (GLP‐1) regulation and glucose‐lowering effects. The present study evaluated the chronic effects of combination treatment with alogliptin and voglibose in prediabetic db/db mice. Methods: Alogliptin (0.03%) and voglibose (0.001%) alone or in combination were administered in the diet to prediabetic db/db mice. Results: After 3 weeks, voglibose treatment increased GLP‐1 secretion (voglibose alone, 1.6‐fold; alogliptin plus voglibose, 1.5‐fold), while it decreased plasma glucose‐dependent insulinotropic polypeptide (GIP) (voglibose alone, ?30%; alogliptin plus voglibose, ?29%). Alogliptin, voglibose and combination treatment decreased plasma DPP‐4 activity by 72, 15 and additively by 80%, respectively, and increased plasma active GLP‐1 levels by 4.5‐, 1.8‐ and synergistically by 9.1‐fold respectively. Combination treatment increased plasma insulin by 3.6‐fold (alogliptin alone, 1.3‐fold; voglibose alone, 1.8‐fold), decreased plasma glucagon by 30% (alogliptin alone, 11%; voglibose alone, 8%), and prevented the development of diabetes, much more effectively than either agent alone. After 4 weeks, alogliptin, voglibose and combination treatment increased pancreatic insulin content by 1.6‐, 3.4‐ and synergistically by 8.5‐fold respectively. Furthermore, combination treatment resulted in an increased expression of insulin, pancreatic and duodenal homeobox 1 (PDX1) and glucose transporter 2 (GLUT2), and maintenance of normal beta/alpha‐cell distribution in the pancreatic islet. Conclusions: Chronic treatment with alogliptin in combination with voglibose concurrently increased active GLP‐1 circulation, increased insulin secretion, decreased glucagon secretion, prevented the onset of the disease, and preserved pancreatic beta‐cells and islet structure in prediabetic db/db mice.     

Beneficial effects of phlorizin on diabetic nephropathy in diabetic db/db mice

AimsThis study observes the effects of phlorizin on diabetic nephrology in db/db diabetic mice and explores possible underlying mechanisms.MethodsSixteen diabetic db/db mice and eight age-matched db/m mice were divided into three groups: vehicle-treated diabetic group (DM group), diabetic group treated with phlorizin (DMT group) and normal control group (CC group). Phlorizin was given in normal saline solution by intragastric administration for 10 weeks. Differentially expressed proteins in three groups were identified using iTRAQ quantitative proteomics and the data were further analyzed with ingenuity pathway analysis.ResultsThe body weight and serum concentrations of fasting blood glucose (FBG), advanced glycation end products (AGEs), total cholesterol, triglycerides, blood urea nitrogen, creatinine and 24-h urine albumin were increased in the DM group compared to those of the CC group (P < 0.05), and they were decreased by treatment with phlorizin (P < 0.05). Morphologic observations showed phlorizin markedly attenuated renal injury. Phlorizin prevented diabetic nephropathy by regulating the expression of a series of proteins involved in renal and urological disease, molecular transport, free radical scavenging, and lipid metabolism.ConclusionsPhlorizin protects mice from diabetic nephrology and thus may be a novel therapeutic approach for the treatment of diabetic nephrology.     

硫辛酰胺对db/db小鼠肝损伤的保护作用研究

目的观察硫辛酰胺(ALM)对db/db小鼠肝损伤的保护作用及可能机制。方法db/db小鼠随机分为糖尿病组(DM)和ALM组,C57BL/6J小鼠为正常对照组(NC),每组各6只。ALM组于第9周时予ALM[100 mg/(kg·d)]进行灌胃干预,干预8周后处死小鼠,检测生化指标及肝组织谷丙转氨酶(ALT)、谷草转氨酶(AST)、过氧化氢酶(CAT)活性及丙二醇(MDA)表达量,油红O、HE染色观察肝脏病理学改变,Western blot法检测肝组织中核因子E2相关因子2(Nrf2)、血红素氧合酶1(HO-1)蛋白水平。结果与NC组比较,DM组体重、TG、TC、FBG升高,MDA含量升高[(0.73±0.04)vs(0.92±0.17)nmol/mg,P<0.05],CAT活性降低[(1.08±0.18)vs(0.52±0.14)U/mg,P<0.05],ALT、AST活性升高[(16.85±3.84)vs(22.42±4.56)U/g,(6.07±1.91)vs(8.19±1.51)U/g,P<0.05],Nrf2、HO-1的表达升高[(0.33±0.25)vs(1.81±0.34),(0.29±0.13)vs(1.25±0.19),P<0.05]。与DM组比较,ALM组体重、TG、TC降低,MDA降低[(0.92±0.17)vs(0.56±0.11)nmol/mg,P<0.05],CAT活性升高[(0.52±0.14)vs(0.91±0.20)U/mg,P<0.05],ALT、AST活性降低[(22.42±4.56)vs(17.08±5.08)U/g,(8.19±1.51)vs(5.10±0.46)U/g,P<0.05],Nrf2、HO-1表达降低[(1.81±0.34)vs(1.01±0.30),(1.25±0.19)vs(0.52±0.17),P<0.05]。结论ALM可抑制T2DM小鼠肝损伤的发生发展,其机制可能是通过改善肝组织脂质沉积、抑制氧化应激,调节Nrf2、HO-1蛋白表达以实现。     

2型糖尿病模型db/db小鼠海马超微结构观察

目的：观察2型糖尿病动物模型－C57BL／KsJ(db/db)小鼠海马超微结构变化,方法：糖尿病组：6周龄C57BL／KsJ(db/db)小鼠5只,尾静脉空腹血糖高于11．1mmol/L且肥胖,对照组;非糖尿病小鼠C57BL／KsJ5只,尾静脉空腹血糖低于6．0mmol/L,体重正常,于30周龄（成模第6个月要）时,灌注固定取脑,透射电镜下观察海马CAI区,结果：糖尿病小鼠海马超微结构发生显著改变包括锥体细胞退变,髓鞘崩解,突触变性等。结论：糖尿病所致海马超微结构改变可能与糖尿病学习记忆功能减退有关。     

2‐Methoxyestradiol ameliorates glucose tolerance with the increase in β‐cell mass in db/db mice

Aims/Introduction: 2‐Methoxyestradiol (2ME) is an estradiol metabolite with little estrogenic activity. Previous data identified its anti‐carcinogenic properties and possible cardiovascular benefits. However, its effect on diabetes mellitus has not been fully elucidated. The aim of the present study was to determine the effects of 2ME on glucose metabolism in the diabetic state. Materials and Methods: To evaluate the effects of 2ME, pellets of two different doses of the drug were implanted into female db/db mice at the age of 5 weeks. Intraperitoneal glucose tolerance test and insulin tolerance test were carried out at the age of 8 weeks. The pancreas was harvested for morphological analysis and β‐cell function at the age of 9 weeks. Results: 2ME improved random blood glucose levels and glucose tolerance with increases in insulin levels during an intraperitoneal glucose tolerance test. Insulin sensitivity judged by an insulin tolerance test was comparable in the low‐ and high‐dose 2ME groups and the control group. Although glucose‐stimulated insulin secretion in isolated islets was comparable among the three groups, β‐cell mass in 2ME‐treated groups was higher than the control group. In the 2ME‐treated groups, the number of Ki67‐positive cells in islets was higher, whereas the number of cleaved caspase‐3‐positive cells was comparable with the control. Conclusions: 2ME ameliorates glucose tolerance by promoting the proliferation of β‐cell mass in db/db mice. Our data suggests its potential clinical usefulness as a disease‐modifying drug for type 2 diabetes mellitus. (J Diabetes Invest, doi: 10.1111/j.2040‐1124.2010.00087.x, 2011)     

Restoration of gut motility in Kit-deficient mice by bone marrow transplantation

Purpose  Interstitial cells of Cajal (ICC) play important roles in autonomic gut motility as electrical pacemakers and mediators of neural regulation of smooth muscle functions. Insufficiency of ICC has been reported in a wide range of gut dysmotilities. Thus, restoration of ICC may be a therapeutic modality in these diseases. Here we provide evidence that transplanted bone marrow (BM) cells can restore gut dysmotility in part via transdifferentiation to ICC. Methods  Bone marrow cells obtained from Kit insufficient W/W ^v mice or syngeneic GFP-transgenic mice with wild-type Kit were transferred to W/W ^v recipients. Whole gut transit time and gastric emptying were examined 5 and 6 weeks after BM transplantation, respectively, and ICCs were identified in whole mounts, frozen sections and transmission electron immunomicroscopy of the gut smooth muscle layers using specific antibodies. Results  Transplantation of wild-type BM into W/W ^v mice significantly improved whole gut transit time and gastric emptying. Fluorescent immunohistochemistry revealed GFP⁺Kit⁺ cells in the myenteric plexus, deep muscular plexus, and submucosal plexus smooth muscle layers of the stomach, small intestine, and colon, respectively. In the whole mounts, GFP⁺Kit⁺ cells were bipolar and spindle shaped, and transmission electron immunomicroscopy showed GFP⁺ cells rich in mitochondria and endoplasmic reticulum between gut smooth muscle layers, suggesting the presence of GFP⁺ cells with morphological characteristics of ICC. Conclusions  These results suggest that BM contains cells that may incorporate into ICC networks and improve dysmotility in W/W ^v mice. Thus, BM transplantation may become to a new therapeutic modality for gut dysmotilities due to ICC insufficiency.