Volume 08 · Issue 03 · 2016

Results of the EMPA-REG OUTCOME study published in 2015^[1]Its design is in accordance with the requirements of the U.S. Food and Drug Administration (FDA), and the effect of sodium-glucose cotransporter 2 (SGLT-2) inhibitor empagliflozin on cardiovascular events and mortality was observed in people with type 2 diabetes and high cardiovascular risk under standard diabetes treatment. The results were unexpected: the total cardiovascular events, total mortality, cardiovascular death and hospitalization rates of heart failure in diabetic patients in the empagliflozin-added group were significantly lower and better than those in the control group. The results of this study are exciting, not only in the field of endocrinology and metabolism, but also in cardiovascular and other related disciplines, because this study was originally mainly to examine the cardiovascular safety of empagliflozin in the treatment of type 2 diabetes., that is, it meets the requirements of the FDA without increasing cardiovascular events. This research has attracted the common attention of more medical colleagues on the prevention and treatment of diabetic cardiovascular disease.

In recent years, the incidence of abnormal glucose metabolism and cardiovascular disease (CVD) in China has been on the rise, and the relationship between them has been paid more and more attention by clinicians. There is a risk of cardiovascular events in prediabetes, and cardiovascular complications are the main cause of death and disability in patients with type 2 diabetes. Therefore, reducing the occurrence of cardiovascular events is particularly important for patients with abnormal glucose metabolism.

Cardiovascular disease is a common complication and the main cause of death in diabetes mellitus. Although the benefit of intensive hypoglycemia on diabetic microvascular complications has been confirmed by many large evidence-based medicine trials such as the British Prospective Study on Diabetes (UKPDS), its benefit on recent macrovascular complications, especially cardiovascular complications, has not been clearly concluded. The main reason is that diabetic macrovascular disease is closely related to blood pressure and lipid metabolism in addition to glucose metabolism disorder. This article mainly discusses the influence and management of blood lipid disorder on diabetic macrovascular complications.

Oral antidiabetic drugs are the main treatment for type 2 diabetes mellitus (T2DM). While paying attention to their efficacy, the discussion on their cardiovascular safety has never stopped. Back in the 1970s, research from the American University Diabetes Program Working Group (UGDP) found that patients with T2DM treated with the first-generation sulfonylurea, tolbutamide, had a 2.5-fold increase in cardiovascular mortality compared with the placebo group, resulting in early termination in the tolbutamide group^[1]。 Nissen and Wolski in 2007^[2]A meta-analysis of 42 randomized controlled clinical studies (RCTs) on rosiglitazone showed that rosiglitazone increased the risk of myocardial infarction by 43% and the risk of cardiovascular death by 64%. These results have aroused widespread attention in the endocrine community, and also prompted endocrinologists to do more in-depth research and more comprehensive thinking on oral antidiabetic drugs. At present, oral antidiabetic drugs commonly used in clinical practice mainly include the following categories: insulin secretagogues (sulfonylureas and glinides), biguanides, alpha glycosidase inhibitors, thiazolidinediones (TZD) and dipeptide kininase IV inhibitors (DPP-4i). The author will elaborate on the cardiovascular safety of the above drugs to help readers better interpret the results of various clinical studies and guide clinical medication.

To investigate the preventive effect of basic fibroblast growth factor (bFGF) on diabetic cardiomyopathy (DCM) using nanoparticles combined with ultrasound-targeted microbubble destruction technique (UTMD).

The bFGF-loaded nanoparticles (bFGF-NPs) was prepared by lyophilization technique. Among 75 SD rats, 15 were assigned to control group using random number table after adaptive feeding for 1 week. The rest 60 rats developed type 1 diabetes with intraperitoneal injection of streptozotocin (STZ, 70 mg/kg). The diabetic rats were divided to 4 groups (15 rats per group): diabetic model, bFGF solution treatment, bFGF nanoparticles treatment, bFGF nanoparticles + UTMD treatment. Before and twelve weeks after intervention, all rats underwent the transthoracic echocardiography. The left ventricular end-diastolic diameter (LVIDd), left ventricular posterior wall (LVPW) and left ventricular fraction shortening (LVFS) were measured. At last, the rats were sacrificed and myocardial tissue of all rats were stained with masson staining and CD31 immunohistochemistry staining to quantify myocardial collagen fraction (CVF) and myocardial microvascular density (MVD). One-way ANOVA was used for multiple comparison. LSD-t test were used for comparison between two groups.

bFGF-NPs showed good morphology and high encapsulation efficiency (84.3%±2.8%) with high stability. The animal experiments demonstrated that after 12-week treatment, LVIDd ((7.37±0.43) vs (5.82±0.24), (6.65±0.28), (6.73±0.25) mm;t=8.09, 4.83, 3.51, allP<0.05) and LVFS ((50±4) vs (38±4), (44±4), (43±4) mm,t=6.79, 3.93, 3.54, all P<0.05) increased, and LVPW ((1.59±0.08) vs (1.86±0.09), (1.72±0.11), (1.73±0.08) mm,t=5.42, 3.34, 3.12, all P<0.05) decreased significantly in bFGF-NP + UTMD group, compared with diabetic group, bFGF solution goups and bFGF nanoparticles group. In addition, after 12-week treatment myocardial masson staining and CD31 immunohistochemistry staining showed that CVF (t=23.5, 8.86, 3.34, all P<0.05) in bFGF-NP + UTMD group was lower than diabetic group and other intervene groups; MVD (t=7.30, 5.18, 3.45, all P<0.05) was higher than diabetic group and other intervene groups.

bFGF-NPs combined with UTMD technique can improve myocardial structure and function in diabetes model. It might provide a novel technique for DCM prevention.

To investigate serum levels of pigment epithelium-derived factor (PEDF) in subjects with different glucose tolerance and its influencing factors.

A total of 571 Chinese participants, who underwent 75 g oral glucose tolerance test (OGTT) in the Department of Endocrinology of Nanjing Drum Tower Hospital from June 2010 to February 2013 were divided into three groups: normal glucose tolerance (NGT), impaired glucose regulation (IGR) and type 2 diabetes mellitus (T2DM) group. Circulating PEDF levels and metabolic profiles were assessed in the participants. Serum estradiol levels were determined in females. 1/homeostasis model assessment of insulin resistance (1/HOMA-IR) and homeostasis model assessment of β-cell function(HOMA-β) were used to evaluate insulin sensitivity and β-cell function. Analysis of variance was used for data analysis. The relationship between PEDF and other metabolic parameters was analyzed by using Spearman correlation analysis and multiple stepwise analysis.

There were 186 participants in NGT group, 216 in IGR group and 169 in T2DM group. The mean age was (50±14) years. No significant differences were observed in the male and female ration (χ²=3.138,P= 0.208) and body mass index (BMI) (F=2.410,P=0.091) among the three groups. Compared with that in control group(8.5 (7.1, 10.7) mg/L), serum PEDF level was significantly higher in IGR group(10(8, 11) mg/L) and T2DM group(10(8, 12) mg/L) (F=6.911,P=0.010). Serum PEDF level in males was significantly higher than that in females ((10(8, 12) vs 9(7, 11) mg/L,t=3.001,P=0.003). Spearman correlation analysis showed that PEDF levels was positively associated with BMI, waist circumference, waist-to-hip ratio, fasting and 2 h postprandial glucose (r=0.311, 0.302, 0.124, 0.188, allP<0.001). Multiple stepwise regression analysis revealed that PEDF was associated with 1/HOMA-IR, waist circumference, diastolic blood pressure and systolic blood pressure after the adjustment for age and gender (t=-4.927, 4.532, 3.701, -2.265, allP<0.05). Subjects with a higher tertile of PEDF level exhibited a higher level of BMI, waist circumference, fasting and 2 h postprandial glucose, fasting and 2 h postprandial insulin and HOMA-β than those in patients with the lower tertile of PEDF level(F=8.214-34.461, all P<0.01). Furthermore, serum estradiol level was negatively associated with PEDF (r=- 0.216,P=0.013). Multiple stepwise regression analysis showed that decreasing estradiol was an independent risk factor of increasing PEDF in post-menopause females (β =-0.213,P= 0.009).

PEDF concentration is significantly elevated in patients with IGR. Decreased estradiol level is an independent risk factor for the elevated PEDF, which can contribute to the occurrence of T2DM in post-menopause females.

To investigate protective effects of tripterygium wilfordiipoly glucosides (TWP)on the toll like receptor 4(TLR4)-mediated immune and inflammatory signaling pathway on type 2 diabetes fatty liver disease and elucidate the possible involved mechanisms.

Seventy-five healthy male Sprague Dawley rats, aged 4-5 weeks and weighed (161±9) g, were divided to normal control group (NC,n=15) and high-fat diet group(n=60) by random number table method. Type 2 diabetes model was induced with 8-week high-fat diet followed by low-dose streptozocin(STZ) injection(30 mg/kg). The diabetic rats were randomly assigned to four groups: diabetes mellitus group (DM,n=11), low-dose TWP (1 mg·kg^-1·d^-1, DM+TL,n=10), medium-dose TWP(3 mg·kg^-1·d^-1,DM+TM,n=13), and high-dose TWP (6 mg·kg^-1·d^-1,DM+ TH,n=11). Treatment was given by gavage once daily for 8 weeks.We analyzed the liver histopathological change, and the gene and protein expression of TLR4 and nuclear factor-κB(NF-κB) by real time PCR and Western blotting, and the levels of serum downstream inflammatory factors interleukin 1β(IL-1β) and tumor necrosis factor α(TNF-α) by ELISA.The significance of the data obtained was evaluated using analysis of variance (ANOVA).

(1)Compared to the normal control group,diabetic rats were found to have obvious steatohepatitis and inflammatory cell infiltration. TWP-treated diabetic rats showed reduced hepatic inflammation in different levels. (2)TLR4 and NF-κB mRNA and protein expression in the hepatic tissue in the DM group were significantly increased, compared with those in the NC group(7.02±1.45 vs 1.05±0.18, 8.91±1.59 vs 1.04±0.25, 2.31±0.17 vs 1, 2.84±0.38 vs 1, respectively,t=7.157-16.540, allP<0.05). The expression of TLR4 and NF-κB mRNA and protein showed different significantly between groups (F= 12.992-60.578,P<0.05 all above), and was decreased in TWP treatment groups in a dose-dependent manner (t=2.264-7.223, all P<0.05).(3)Serum IL-1β and TNF-α in different groups showed different significantly (F=57.717,42.992,P<0.05 all above).Serum IL-1β and TNF-α in DM group were significantly increased, compared with those in the NC group(t=14.669,10.327,P<0.05 all above) and were decreased in TM and TH group (t=3.663-4.924,P<0.05 all above).

TWP treatment may delay the progression of diabetic non-alcohol fatty liver disease via inhibition of TLR4/NF-κB signaling cascade pathway, and its downstream inflammatory effectors.

To investigate the mechanism of erythropoietin (EPO) on gluconeogenesis and glycogen synthesis in palmitic acid (PA)-induced HepG2 cells.

HepG2 cells were cultured in medium with 250 μmol/L PA for 24 h , and treated with 5 or 10 U/ml EPO for 24 h. In addition, HepG2 cells were pretreatment with phosphatidylinositol 3-kinase (PI3K)inhibitors LY294002 or wortmannin for 1 h. Glycogen content in HepG2 cells was examined by colorimetric method using glycogen assay kit. The protein expressions of phosphoenolpyruvate carboxykinase (PEPCK),PI3K, protein kinase B (AKT), forkhead box O1(FOXO1), glycogen synthase kinase 3β(GSK-3β) were measured by western blotting. Data analysis between multiple groups was performed by ANOVA method.

The expression of p-GSK-3β(PA 0.74±0.11, EPO 5 group 1.02±0.03, EPO 10 group 1.06±0.02,t=4.236, 4.996,P<0.05) and p-FOXO1(PA 0.73±0.09, EPO 5 group 0.99±0.05, EPO 10 group 1.13±0.06,t=4.798, 6.757,P<0.05), down stream molecular of AKT, was enhanced by EPO treatment. Importantly, specific PI3K inhibitors markedly blocked EPO-mediated increase in p-AKT(EPO 1.25±0.08, LY294002 0.74±0.14, wortmannin 0.63±0.03,t=-5.403,-12.255,P< 0.05), p-GSK-3β(EPO 1.12±0.14, LY294002 0.68±0.09, wortmannin 0.76±0.10,t=-4.632, -3.655,P<0.05) and p-FOXO1(EPO 1.15±0.06, LY294002 0.92±0.02, wortmannin 0.72±0.07,t=-6.532, -7.984,P<0.05).

The findings suggest that EPO treatment improves glucose metabolism potentially through PI3K/AKT/FOXO1, GSK-3β signaling pathway in PA-induced HepG2 cells.

To clarify the mechanism of calcium alginate dressings for improving the express of wound healing by its effects on the expression of matrix metallopeptidase 9(MMP-9) in the wound tissue in diabetes rats.

The thirty-six male diabetic SD rats, wounded on the back(with a diameter of 20 mm) with sterile scissors, were randomized divided into two groups according to random number table: the experimental group (n=18, treated with calcium alginate dressing for 14 days) and the control group (n=18, treated with conventional gauze dressing). The wound healing rates in the two groups were obtained, HE and Masson staining methods were used to evaluate the thickness and inflammatory response of granulation tissue and level of collagen in it, meanwhile Western blotting and realtime polymerase chain reaction(RT-PCR) methods were applied to evaluate the expression of mRNA and protein of MMP-9 during the process of wound closure. The t-test and chi-square were used to test the differences between the data of the two groups.

The wound healing rate in the experimental group was faster than that in the control group at day 8, 10,12 and 14 (χ²=8.333, 5.124, 14.047, 16.547, all P<0.05). The inflammatory response in experimental group was milder than that in control group at different periods post-operation. Significant statistical difference in thickness of granulation were found in the two groups at day 3((1476±118) vs (812±136)μm,t=6.384,P=0.003) and day 7((1369±86) vs (480±105)μm,t=11.307,P= 0.000). The level of collagen in control group were much lower than those in in experimental group at day 7 and 14 (15%±5% vs 51%±5%, 28%±5% vs 61%±5%,t=9.308, 8.196,bothP<0.05). And the mRNA expression of MMP-9 in the experimental group decreased significantly when compared with those in the control group at day 3, 7 and 14 (0.028±0.003 vs 0.037±0.004, 0.026±0.003 vs 0.033±0.003, 0.021±0.003 vs 0.030±0.002,t=3.118, 2.858, 7.003, allP<0.05). Meanwhile the protein levels of MMP-9 in the experimental group were significantly lower than those in the control group at day 3, 7, 14(t=10.595, 10.400, 12.359, all P<0.05).

Calcium alginate dressing promotes skin ulcer healing by inhibiting the expression of MMP-9 so as to reduce the degradation of collagen in diabetic SD rats.

To observe the changes of oxidative stress and the expression of p22phox,a membrane subunit of Nicotinamide Adenine Dinucleotide Phosphate-oxidase(NADPH), and effects of the antioxidant N-acetylcysteine(NAC) in the lung tissue of diabetic rats.

A total of 18 male SD rats (220-250 g) were randomly divided into normal control group (group C,n=6), diabetic group (group D,n=6), and diabetes with NAC treated group (group (D+N),n=6) by random digital table method. After 4 weeks, the plasma and lung tissue of rats were collected for detection of the total antioxidant, free 15-F2t-isoprostane, total superoxide dismutase(SOD) activity and Catalase(CAT) activity by kits, and p22phox protein expression by Western blotting.

Compared with C group rats,the levels of p22phox expression in lung tissues in diabetic rats significantly increased (1.25±0.18 vs 1.00±0.00,t=3.39,P<0.05).The levels of 15-F2tisoprostane in plasma and lung tissue and total antioxidant concentration in diabetic rats significantly increased(t=14.39, 8.48, 14.72,allP<0.05) as compared with that in C rats, but the total antioxidant concentration in lung tissue, and total SOD activity and CAT activity in plasma and lung tissue significantly decreased(t=22.70, 12.64, 11.39, all P<0.05).The levels of p22phox, total antioxidant, free 15-F2tisoprostane,total superoxide dismutasein lung tissue by NAC treatment for 4 weeks were significantly improved than that of D group (1.01±0.14 vs 1.25±0.18, (0.69±0.06) vs (0.41±0.03) mmol/g, (390±21) vs (505±40)pg/mg, (2.22±0.12)vs (1.84±0.05)U/mg;t=10.54,3.19,2.56,6.26,allP<0.05). All these changes were reversed or attenuated (t=8.90,6.23,2.58,6.26,10.54,1.81,7.19,all P<0.05)remarkably by NAC treatment for 4 weeks, while the CAT activity in lung tissue unexpectedly declined (t=8.65, all P<0.05).

NAC can attenuate oxidative stress in plasma and lung tissue of diabetic rats, while decrease p22phox expression levels in lung tissue.

To explore the changes and related mechanism of the autophagy of pancreatic islet cells during the neonatal period in mice.

Mice islets at 1-, 3-, 8-week after birth were isolated by collagenase. The morphological structure of autophagosome was observed by electron microscope. Double immunofluorescence staining was performed for insulin with microtubule-associ ated protein 1 light chain 3(LC3). The expression of autophgy-related genes(Atg) were detected by real-time quantitative PCR. The expression of the targeted proteins was detected by Western blotting. One-way analysis of variance was used for data analysis.

(1) The typical doubled-membrane autophagosome appeared at 3 w. Compared with 1 w and 8 w, the cytoplasmic fluorescence intensity of LC3 at 3 w was significantly increased (57.6±2.3 vs 24.4±1.3, 28.1±2.3,t=-21.44、-15.66, both P<0.01). (2) The mRNA level of p53 was higher at 3 w than that at 1 w and 8 w (P<0.05), while mammalian target of rapamycin (mTOR) was lower than that at 1 w and 8 w (P<0.01). The mRNA expression of the autophagy-related genes dynamically changed. Compared with 1 w and 8 w, the levels of ULK-1, Beclin1 and Map1lc3b markedly increased at 3 w (P<0.05). Compared with 1 w, the levels of PI3Kc3, Atg5, Map1lc3a and Atg16l1 increased at 3 w (P<0.05); the level of Atg4d was significantly increased at 3 w and 8 w (P<0.05). At 3 w, the expression of Atg4c was higher than that at 8 w (P<0.05). (3) Western blotting showed that compared with 1 w and 8 w, the protein levels of p53, phosphorylated adenosine monophosphate-activated protein kinase(pAMPK), Beclin1 and LC3-II were markedly increased at 3 w (F=29.14-56.05, all P<0.01), while mTOR protein significantly decreased at 3 w (0.61±0.06 vs 1.00±0.02, 0.89±0.09,F=29.41, P<0.01).

The p53/pAMPK/mTOR/Beclin1 mediated autophagy signaling pathway maybe involved in the regulation of autophagy in neonatal mice islet cells.

The main manifestations of diabetic foot are limb paresthesia, foot ulcer, and in severe cases, limb gangrene^[1]。 The multi-center survey data in China confirmed that about 70% of patients with diabetic foot ulcer were co-infected. At the same time, these patients often have more than three chronic complications of diabetes, especially peripheral neuropathy, lower extremity vascular disease and kidney disease, and malnutrition^[2]。 The causes of diabetic foot infection are complex, and the treatment often requires multiple methods. Pancreatic kininogenase can degrade kininogen into peptides, which can dilate blood vessels, regulate blood pressure, improve microcirculation, prevent platelet aggregation, prevent hemagglutination and antithrombosis, etc., while recombinant human epidermal growth factor can promote epithelial cells, neutrophils, fibroblasts and other cells to move to the wound surface, which can promote wound healing. We added pancreatic kininogenase combined with recombinant human epidermal growth factor in the treatment of diabetic foot patients on the basis of conventional therapy, and attempted to explore the possible mechanism of the combined application of the two.

The number of diabetic patients is increasing year by year. According to the statistics of the International Diabetes Federation, the number of diabetic patients in the world has reached 380 million in 2014, and it is estimated that the number of diabetic patients in the world will increase to 550 million by 2030. In recent years, studies have shown that the prevalence of anemia in diabetic patients (20% ~45%) is also increasing, and it has been paid more and more attention by clinicians^[1,2,3]。 Anemia is an independent risk factor for cardiovascular events in diabetic patients, and is closely related to the occurrence and development of diabetic microvascular complications. It is also an independent high risk factor for predicting the prognosis of diabetes^[4]And patients with diabetes combined with anemia are more likely to develop cardiovascular and cerebrovascular diseases, hypertension, and chronic kidney disease^[2], but no studies have demonstrated that correcting anemia improves the prognosis of diabetic patients. However, identifying the anemia associated with diabetes as early as possible and clarifying the cause of anemia will help clinicians choose appropriate solutions to correct anemia. At present, diabetic nephropathy is the most common cause of diabetes combined with anemia, but more and more studies have found that many diabetic anemias have appeared in the early stage of diabetic nephropathy or even before the decline of renal function, suggesting that there are other factors in addition to diabetic nephropathy that require further analysis^[5]。 This article summarizes the causes of anemia associated with diabetes mellitus as follows, in order to provide ideas for prevention and treatment in clinical work.

At present, many studies have been made on the hypoglycemic mechanism of pancreatic islet β cells. Recently, researchers have gained a new understanding of the conversion between pancreatic islet α, δ cells and β cells, which may provide a new idea for improving the function of pancreatic islet β cells. Therefore, this paper reviews the transition between islet cells and its molecular mechanism in recent years.

long non-coding RNAs (lncRNAs) are one of the most popular research fields at present, and their abnormal expression or loss of function is related to psoriasis, coronary heart disease, diabetes, tumor and other diseases. Diabetes mellitus is a metabolic disease caused by genetic and immune dysfunction, which induces insulin resistance and insufficient insulin secretion, and is accompanied by many serious complications. With the continuous development of diabetes research, a large number of evidences show that lncRNAs are widely involved in the development of diabetes, which provides new strategies for the prevention, early diagnosis and treatment of diabetes.

Patients with type 2 diabetes mellitus (T2DM) who present with early phase insulin secretion disorders have a significant post-load hyperglycemia as a consequence. Chinese patients with T2DM have a significant feature of postprandial blood glucose increase. Investigation on diabetes mellitus in Shanghai^[1]It shows that among patients with diabetes and pre-diabetes, the proportion of patients with postprandial blood glucose increase is as high as 88%. Among them, the proportion of simple fasting blood glucose increased was 12%, while the proportion of simple postprandial blood glucose increased was as high as 49%. It is worth noting that even among healthy people, the blood glucose response of Chinese people after eating the same amount of carbohydrates is significantly higher than that of European people: a recent study showed that after eating the same amount of rice (five kinds of rice), the blood glucose increase in healthy Chinese people at 2 h after a meal was significantly higher than that of European healthy people. All these studies suggest that for Chinese patients with T2DM, we should pay attention to the management of postprandial hyperglycemia, start with its pathophysiological mechanism and characteristics when choosing treatment plan, emphasize early insulin secretion, and restore and protect β cell function.