Volume 07 · Issue 12 · 2015

Diabetes is a devastating disease that endangers the physical and mental health of millions of people worldwide. The latest research results show that the prevalence of diabetes among adults over 18 years old in China is 9.7%^[1]。 Because the improvement of pancreatic islet function in diabetic patients is particularly important to delay the occurrence of complications, the reconstruction of endogenous insulin secretion system in diabetic patients with the help of stem cell transplantation has become a research direction of much attention in the field of regenerative medicine at present.

Stem cell transplantation has been the focus of diabetes treatment since the potential for self-replication and targeted differentiation of stem cells was demonstrated. Previous theoretical understanding of diabetic stem cell therapy is mainly based on the ability of stem cells to differentiate into insulin-secreting cells. However, in recent years, it has been found that the number of donor stem cells migrating into and functionally integrated into the damaged tissue is too small (only 0.1% ~2.0% of the total number of cells in the damaged tissue), which often fails to cause physiological changes^[1,2]； In vivo experiments also revealed that only 1.7% ~3.0% of infused stem cells could differentiate into functional beta cells^[3]It suggests that stem cell transplantation in the treatment of diabetes may not largely depend on the ability of directed differentiation into insulin-secreting cells, and its role in regulating autoimmunity and excessive inflammation has attracted more and more attention by scholars, and is expected to become a new means of diabetic cellular immunotherapy.

Cell replacement therapy (CRT) is a potential hope to reestablish islet beta cell function and cure diabetes. International multicenter clinical trials of pancreatic islet transplantation using the Edmonton protocol have confirmed good near-term safety and efficacy of pancreatic islet transplantation in the treatment of type 1 diabetes. The data of 2-year follow-up showed that islet transplantation could reestablish endogenous insulin secretion function and improve the stability of blood glucose control, but it was difficult to maintain the state of patients independent of insulin therapy for a long time^[1]； Seven of the US subjects were followed up to more than 10 years, confirming the safety and feasibility of islet transplantation^[2]。 However, the lack of donor tissue sources is a huge obstacle that restricts the clinical promotion of islet transplantation. Therefore, inducing stem cells to differentiate into islet β cells in vitro is expected to solve the problem of donor source, which has become a hot spot in the research field of diabetic CRT. Embryonic stem cells (ESCs) are currently the most widely studied and mature stem cell systems. In recent years, the basic research on the induction of directed differentiation of human ESCs in vitro has made promising progress, which has laid a good foundation for the use of pancreatic islet cells derived from human ESCs in the treatment of diabetes.

To investigate the effect and underlying mechanism of miR-375 on regulating differentiation of human embryonic stem (hES) cells into insulin producing cells (IPCs).

hES cells were induced into IPCs in vitro. The dynamic expression of miR-375 and hepatocyte nuclear factor 1β (HNF-1β) the predicted target gene, during the differentiation was analyzed. The stage 2-cells during the differentiation infected with miR-375-overexpression lentiviral vector were divided into 3 groups: control group, GFP control group and miR-375-overexpression group. The expressions of miR-375, HNF-1β and the specific markers related to pancreatic islet differentiation or function were determined by realtime PCR and/or Western blotting. Independent samplet test was used to analyze the difference between two group. ANOVA and Q test were used to analyze the difference among multiple groups.

miR-375 expression in the stage 1-to 5-cells were 100%, (472.25±33.53)%, (768.00±25.65)%, (54.25±5.74)% and (30.75±5.70)%, respectively (F=1137.57, P<0.001). The expression of HNF-1β increased from stage 3 ((279.50±21.30)%), and peaked at stage 4 ((645.00±64.55)%,F=224.86, P<0.001). The dynamic level of HNF-1β was similar as that of 1/miR-375. After the lentivirus infection, the mRNA level of HNF-1β in miR-375-overexpression group was not statistically different from that of both the GFP group and control group (F=1.467, P>0.28), whereas the protein level of HNF-1β decreased to (38.75±9.22)% of the GFP group in the miR-375-overexpression group (F=60.69, P<0.001). miR-375 overexpression upregulated the expression of pancreatic and duodenal homeobox 1 (Pdx-1) in stage 4-cells (F=412.15, P<0.001), and downregulated the expression of the pancreatic islet specific markers, e.g. Nkx6.1, paired box 4 (Pax-4) and insulin in the stage 5-cells (F=105.19-484.05, all P<0.001) when compared with the GFP group.

miR-375 plays a modulatory role in the IPC differentiation from hES cells. That may be mediated via regulating the expression of the transcription factor HNF-1β.

To observe the efficacy of glucagon like peptide-1(GLP-1) receptor agonist liraglutide(LIRA) combined with umbilical cord mesenchymal stem cells(UC-MSCs) on the glucose metabolism and islet β cell function in type 2 diabetic SD rats.

Type 2 diabetic SD rats models were established by giving high-carbonhydrate-fat diet and then intraperitoneal injecting of streptozotocin. The rats models were randomly divided into four groups according the table of random number: type 2 diabetes mellitus(T2DM), T2DM+UC-MSC, T2DM+LIRA, T2DM+LIRA+UC-MSCs, 10 rats in each group. Rats of each group were given the appropriate drug therapy for 8 weeks respectively. Liraglutide was given by subcutaneous injection with 200 μg/kg dosage. UC-MSCs were given by tail vein injection with 5 × 10 ⁶ cells. During the treatment period, the general condition indexs of each group were monitored. The serum glycated hemoglobin A_1c(HbA_1c), peptide C(C-P), glucagon(Glu), GLP-1, gastrin(GAS) and cholecystokinin (CCK) were measured respectively. Mean data between groups was compared with one-way ANOVA.

After 8-week of treatment, compared with T2DM group, HbA_1c of the T2DM + UC-MSCs, T2DM+LIRA and T2DM+LIRA+UC-MSCs group decreased significantly((9.1±0.4)% vs (7.2±0.4)%, (3.9± 0.3)%, (3.1±0.3)%, respectively, F=815.220, P<0.05), and it was lowest in T2DM+LIRA+UC-MSCs group (t=-28.105, -5.918, both P<0.05). Compared with T2DM group, the C-P of T2DM+UC-MSC group increased significantly(t=8.774, P<0.05), while there were no much changes in Glu, GLP-1, GAS and CCK(t=2.168, 1.894, 1.768, 1.516, all P>0.05); but the C-P, GLP-1, GAS and CCK in T2DM+LIRA group and T2DM+ LIRA + UC-MSCs group increased markably(t=- 18.115-35.732, all P<0.05), while the Glu decreased remarkably (t=3.022, 11.859, both P<0.05). Compared with T2DM+LIRA group, the C-P, GLP-1, GAS and CCK in T2DM+LIRA+UC-MSCs group also increased significantly (t=-9.107, 7.635, 9.870, 19.152, all P< 0.05), while the Glu decreased obviously(t=-13.976, P<0.05). Pearson correlation analysis showed that the level of C-P was positively correlated with GLP-1, GAS and CCK(r=0.401, 0.628, 0.619, all P<0.01), whereas it was negatively correlated with Glu(r=-0.603, P<0.01). The level of Glu was negatively correlated with GLP-1, GAS and CCK (r=-0.827, -0.583, -0.516, all P<0.01).

The combined treatment of liraglutide and UC-MSCs is superior to a single treatment of liraglutide or UC-MSCs in improving the glucose metabolish and islet β cell function in type 2 diabetic SD rats models. The effects may be related to the gastrointestinal hormone excretion.

To evaluate cost-effectiveness of switching from human premix insulin to biphasic insulin aspart (BIAsp 30) in patients with type 2 diabetes mellitus (T2DM) in China, in the short term and long term respectively.

The cost-effectiveness analysis was based on clinical data of A₁chieve^®China subgroup and published cost data in China from November 2008 to March 2011. For the short-term analysis, effectiveness derived from quality of life data at baseline and end of study, and total cost includes cost for insulin and hypoglycemic events. For the long-term, CORE diabetes model was used to project life expectancy, quality-adjusted life years (QALYs) and total direct medical costs over 30 years from a societal perspective. Patient characteristics and treatment effects were obtained from Chinese subgroup in the A₁chieve^® study. Total costs included insulin cost, management and complication costs. One-way sensitivity analysis was performed.

After 24-week treatment with BIAsp 30 from human premix insulin, EQ-5D utility value increased for 0.034, and total costs decreased for CNY 749.13. When projecting for 30 years, treatment with BIAsp 30 from human premix insulin increased life expectancy by 0.686 year and improved quality-adjusted life years by 1.958 QALYs per patient, total direct medical cost reduced by CNY 70 252. Sensitivity analyses demonstrated robustness of the results.

Switching to BIAsp 30 from human premix insulin is associated with improvements in life expectancy and QALYs, and is a cost-saving treatment strategy for people with T2DM in China.

To investigate the effects of tanshinone ⅡA (Tan ⅡA) on the intermittent high glucose (IHG)-induced oxidative stress and apoptosis of Schwann cells (SCs) in suckling rat.

Cultured SCs of newborn suckling rats were randomized into several groups including control (con, 5.6 mmol/L glucose), stable high glucose (HG, 50 mmol/L glucose), IHG (5.6 and 50.0 mmol/L glucoseper 8 h alternative), osmotic control, ALA (500 μmol/L) and IHG in the presence of 0.1, 1.0, 10.0 μmol/L Tan Ⅱ A. Apoptosis rate, oxidative stress level, expression of apoptosis related proteins and the mRNA in the cells were detected.

(1) The percentages of apoptotic cells exposed to HG ((34.57± 2.45)%) and IHG ((40.17±2.33)%) were significantly higher than that in control group ((4.74±0.26)%). Furthermore, apoptosis rate of IHG was much higher than that of HG (t=3.70, P<0.01). (2) ROS and 8-OHdG levels increased significantly in the IHG group compared with those in control and HG groups (F=73.44, 194.21, P<0.01). IHG exposure up-regulated expression of bax protein and mRNA (F=66.78, 126.69, both P<0.01), while down-regulating expression of bcl-2 protein and mRNA (F=151.88, 54.99, both P<0.01). In addition, IHG increased activities of caspase-3 and (poly(ADP-ribose) polymerase, PARP) (129% , 18% , 139% , 56% , allP<0.01). (3) Treatment with 1.0 μmol/L and 10.0 μmol/L of Tan IIA inhibited IHG-enhanced ROS production and 8-OHdG levels (t=3.50-6.47, P<0.05). Bax protein and mRNA (t=3.59-5.39, P<0.01), PARP and caspase-3 activation (42%, 61%, 13%, 34%, allP<0.01), and SC apoptosis (t= 12.39, 21.26, P<0.01)were decreased with Tan ⅡA, too.

Tan ⅡA decreases IHG-induced apoptosis via reducing oxidative stressin SCs of newborn suckling rats.

To investigate the molecular mechanism of sterol regulatory element binding protein-1c (SREBP-1c) suppressing insulin receptor substrate-1(IRS-1) in skeletal muscle cells.

Luciferase plasmid for rat IRS-1 promoter, expression plasmid of SREBP-1c and pRL-TK renilla plasmid were cotransfected into L6 cells using Lipofectamine 2000 (Invitrogen). After transfection for 36 h, luciferase activity was measured using the dual-luciferase reporter assay system following the manufacturer's instructions. L6 myotubes were infected with adenoviral vectors expressing SREBP-1c. Adenovirus expressing green fluorescent protein (GFP) was used as control. The cells were harvested for nucleus protein after infection for 48 h. Electrophoretic mobility shift assay (EMSA) was performed using a Light Shift Chemiluminescent EMSA Kit. Chromatin immunoprecipitation assay was performed by CHIP Kit. The interaction between the transcription factor SREBP-1c and the promoter region of IRS-1 was assessed by above experiments. Differences among the groups were determined using two-way ANOVA.

The luciferase deletion studies suggested the region from -450 to -210 bp on the IRS-1 promoter was a potential target region for SREBP-1c. Sequence analysis showed that the target region of the rat IRS-1 promoter gene contained a potential binding site (GCCTCCCGAG), which was located between -302 and -292 bp. PCR site-directed mutagenesis (TGTTAAATTA) was generated and analyzed using a luciferase assay. The transcriptional activity of wild-type IRS-1 promoter was dramatically down-regulated by SREBP-1c, whereas SREBP-1c had no effect on the IRS-1 promoter bearing mutation of SREBP-1c binding site (7.03±1.28 vs 19.09±2.45, 3.55±1.68 vs 3.96±1.09, F=114.437, 0.251, all P>0.05). The EMSA results showed that the labeled wild-type probe successfully formed a complex with nuclear proteins. But the unlabelled mutant probe did not interfere the complex. The results from CHIP assay showed that SREBP-1c could bind directly to the IRS-1 promoter region. The quantity of SREBP-1c protein binding to the IRS-1 promoter was two times of control under PA conditions in L6 cells or five times of control when SREBP-1c was over-expressed (2.15±0.03 vs 1.07±0.24, 5.48±1.28 vs 0.86±0.19,t=6.877, 5.495, all P<0.05).

SREBP-1c could directly bind to the atypical SRE sequence in the promoter region of IRS-1, suppressing IRS-1 expression and the subsequent insulin signaling pathway.

To examine whether metformin promotes glucose metabolism in the absence of adenosine monophosphate-activated protein kinase (AMPK) activation.

Glucose consumption and lactate production were used to evaluate the glucose-lowering and anaerobic glycolysis effects of metformin on HepG2 hepatocytes and C2C12 myotubes after treated with different concentration of metformin(2 mmol/L, 5 mmol/L) for 24 h. C2C12 cells were divided into 6 groups as blow: group 1(control group), group 2(2 mmol/L metformin treated for 24 h), group 3 (5 mmol/L metformin treated for 24 h), group 4 (10 μmol/L Compound C treated for 24 h), group 5 (10 μmol/L Compound C treated for 30 min then 2 mmol/L metformin treated for 24 h) and group 6(10 μmol/L Compound C treated for 30 min then 5 mmol/L metformin treated for 24 h). HepG2 cells were also divided into 6 groups as blow: group 1 (Ad-GFP transfected ), group 2(Ad-GFP transfected + 2 mmol/L metformin treated fro 24 h), group 3(Ad-GFP transfected + 5 mmol/L metformin treated fro 24 h), group 4(Ad-DN-AMPK transfected), group 5(Ad-DN-AMPK transfected + 2 mmol/L metformin treated for 24 h) and group 6 (Ad-DN-AMPK transfected + 5 mmol/L metformin treated for 24 h). AMPK phosphorylation and ACC phosphorylation were measured with Western blotting to assess the activity of AMPK pathway, and detected the glucose consumption and lactate production. Oxygen consumption rate of C2C12 myotubes was determined in Seahorse XF24 analyzer to estimate the production of ATP and function of respiratory chain complex I. Variance analysis was used to compare among multi-groups, and t test was used to compare two groups.

(1) 2 mmol/L and 5 mmol/L metformin significantly increased glucose consumption(F=104.7, P<0.05) and lactate production (F=280.1, P<0.05) in HepG2 hepatocyte and also increased glucose consumption (F=38.53, P<0.05) and lactate production (F=172.90, P<0.05) in C2C12 myotubes in a dose-dependent manner. (2)AMPK and ACC phosphorylation were stimulated in group 2 and group 3 of HepG2 and C2C12 cells, but Compound C or Ad-DN-AMPK could inhibited the activity of AMPK. However, compare with the group 4, the group 5 and group 6 still had the ability to enhanced glucose consumption and lactate production by 39.5%, 62.6% and 39.0%, 61.0% in C2C12 cells(t=8.727, 21.38, 12.69, 27.31, all P<0.05) and 29.0%, 39.3% and 49.3%, 67.3% in HepG2 cells(t=9.96, 15.61, 23.32, 30.24, all P<0.05). (3)Metformin significantly inhibited the activity of mitochondrial respiratory chain complex I(t=36.08, P<0.05) and reduced ATP production(t=73.32, P<0.05). The inhibition of AMPK activity by Compound C failed to diminish the inhibition of activity of mitochondrial respiratory chain complex I(t=53.18, P<0.05)and the reduction of ATP production(t=246.10, P<0.05) which was stimulated by metformin.

Metformin promotes glucose metabolism by stimulation of glycolysis, which is probably resulted from inhibition of mitochondrial respiratory chain complex I, independent of AMPK activation.

To observe the effect of endoplasmaic reticulum stress (ERS) inhibitor 4-phenylbutyric acid (4-PBA) on lipid deposition and hepatic insulin signal transduction in obese rats induced by high-fat-diet.

Forty-three male Wistar rats were randomly divided into 3 groups by random number table (body weight: 240-270 g): control group (n=14), obesity group (n=14) and 4-PBA group (n= 15) (4-PBA was administered to rats after 4 weeks of high fat feeding, 0.35 g/kg · d). Hyperinsulinemic-euglycemic clamp study was performed to detect glucose infusion rate (GIR) after 8 weeks of feeding. After rats were sacrificed, liver triglyceride content was measured. Protein expressions of ERS stress markers and hepatic insulin signaling transduction factors were detected by western blotting.

(1) GIR in 3 groups were respectively (17.1±1.5), (10.2±1.4), (14.2±2.3) mU/(kg·min) (F=16.92, P<0.05). GIR was significantly decreased in obesity group((10.2±1.4) mU/(kg·min) ) compared with control group ((17.1±1.5) mU/(kg·min),t=33.12, P<0.05). GIR was significantly increased in 4-PBA group((14.2±2.3)mU/(kg·min) ) compared with obesity group (t=21.69, P<0.05). (2) Hepatic TG was significantly increased in obesity group ((29.2 ± 3.3) μmol/g) compared with control group((11.6 ± 0.5) μmol/g,t=24.31, P<0.05) while was significantly decreased in 4-PBA group((16.9±2.8) μmol/g) compared with obesity group (t=61.22, P<0.05). (3) Compared with control group, the protein expression of phosphorylated eukaryotic translation initiation factor 2α (p-eIF2α) and splice X-box binding protein (s-XBP-1) were significantly increased in obesity group (bothP<0.05). ERS markers were significantly down-regulated in 4-PBA group compared with obesity group (bothP<0.05). (4) Compared with control group, the protein expression of phosphorylated-IRS-1, phosphorylated-Akt and phosphorylated-GSK-3α/β were significantly decreased in obesity group (t=16.80, 24.66, 16.43; all P<0.05). The protein expression of p-IRS-1, p-Akt and p-GSK-3α/β were significantly increased in 4-PBA group compared with obesity group (t=19.12, 39.52, 23.72; all P<0.05).

4-PBA may improve insulin sensitivity and fatty liver in obese rats induced by high-fat-diet through improving hepatic insulin signal transduction by suppressing ERS.

Bardet-Biedl syndrome (BBS) is a rare autosomal recessive genetic disease, which is characterized by mental retardation, retinopathy pigmentosa, polydactyly (toe), obesity, and gonadal dysplasia. BBS combined with metabolic syndrome is rare. At present, there is no special treatment for BBS, and it mainly treats the components of metabolic syndrome.

Diabetes mellitus is a common metabolic disease, and its pathogenesis includes insulin secretion dysfunction and insulin resistance. Islet transplantation has been proven to reestablish the islet function of patients, but due to insufficient donor sources, immune rejection and other problems, it cannot meet the treatment needs of a large number of patients. Using new technologies related to somatic cell reprogramming, patient-specific islet cells can be prepared in vitro or the total amount of β cells can be directly supplemented in vivo, which is expected to provide new solutions for diabetes therapy. This article mainly focuses on the research progress of new stem cell-related technologies in the field of diabetes, such as induced pluripotent stem cells (iPSCs), direct lineage reprogramming between different somatic cells, and blastocyst complementation technology.

Adult occult autoimmune diabetes mellitus (LADA) is a subtype of type 1 diabetes mellitus (T1DM) mediated by T lymphocytes^[1]Its early clinical manifestations are similar to type 2 diabetes mellitus (T2DM), but due to the slowly progressing autoimmune damage of pancreatic islet β cells, pancreatic islet function decreases progressively, and it ultimately depends on insulin therapy. Therefore, early diagnosis and intervention of LADA is of great significance for preserving the residual islet β cell function and delaying the occurrence and development of chronic complications. However, the clinical phenotype of LADA varies widely, and can be presented as classical T1DM with absolute insulin deficiency or classical T2DM with obesity and insulin resistance^[2]。 It was found that the titer, epitope, multiple antibody positivity and genetic susceptibility of glutamate decarboxylase antibody (GADA) were related to LADA heterogeneity. The author will review the research progress of LADA heterogeneity in recent years.

Congenital hyperinsulinemia (CHI) is a genetically heterogeneous disease. Due to excessive spontaneous insulin secretion, blood insulin concentration increases, which leads to intractable and persistent hypoglycemia after birth. Eight genetic types have been identified so far, and molecular genetic studies confirm that: K_ATP-HI is the most common type of CHI (caused by mutations in ABCC8 and KCNJ11 genes), accounting for 40% to 45% of CHI, and the other seven rare types of CHI account for 5% to 10%^[1]。 Due to different pathogenic genes, the age of onset, clinical manifestations, severity of hypoglycemia, drug efficacy and prognosis of children with rare types of CHI are also different. This article reviews the genetic pathogenesis and progress in diagnosis and treatment of rare types of CHI, in order to improve clinicians' understanding of rare types of CHI.

Large-scale clinical trials in the field of diabetes, the UK Prospective Diabetes Study (UKPDS) and the Diabetes Control and Complications Trial (DCCT), etc.^[1,2]Both suggest that enhanced glycemic control can reduce early diabetic microvascular disease. At the same time, some studies suggest that enhanced blood sugar control does not reduce the risk of cardiovascular disease and death in people with long course of diabetes, older age, multiple cardiovascular risk factors or previous cardiovascular disease^[3,4,5]。 Although some findings support enhanced glycemic control, the additional benefits that enhanced glycemic control can bring to different patients vary, and these studies^[3,4,5]The problems in current treatment strategies for type 2 diabetes are highlighted: namely, for patients at risk of severe hypoglycemia and weight gain, the advantages of intensive therapy on cardiovascular events may be very limited and must be evaluated carefully. British and Italian scholars have proposed the "ABCD principle" of selecting appropriate treatment goals and treatment options according to the baseline characteristics of patients, that is, the age (A), body weight (B), complication (C) and duration (D) of patients at baseline are related to the treatment outcome of patients. This is in line with the individualized treatment recommended by current guidelines^[6]。 According to the comprehensive consideration of patients' ABCD characteristics, this paper "tailored clothes" for patients, fully weighed the risks and benefits of diabetes treatment, formulated appropriate blood sugar control targets, and selected appropriate treatment plans.