Metformin inhibits MAPK signaling and rescues pancreatic aquaporin 7 expression to induce insulin secretion in type 2 diabetes mellitus

Metformin is the first-line antidiabetic agent for type 2 diabetes mellitus (T2DM) treatment. Although accumulated evidence has shed light on the consequences of metformin action, the precise mechanisms of its action, especially in the pancreas, are not fully understood. Aquaporin 7 (AQP7) acts as a critical regulator of intraislet glycerol content, which is necessary for insulin production and secretion. The aim of this study was to investigate the effects of different doses of metformin on AQP7 expression and explore the possible mechanism of its protective effects in the pancreatic islets. We used an in vivo model of high-fat diet in streptozocin-induced diabetic rats and an in vitro model of rat pancreatic β-cells (INS-1 cells) damaged by hyperglycemia and hyperlipidemia. Our data showed that AQP7 expression levels were decreased, whereas p38 and JNK mitogen-activated protein kinases (MAPKs) were activated in vivo and in vitro in response to hyperglycemia and hyperlipidemia. T2DM rats treated with metformin demonstrated a reduction in blood glucose levels and increased regeneration of pancreatic β-cells. In addition, metformin upregulated AQP7 expression as well as inhibited activation of p38 and JNK MAPKs both in vivo and in vitro. Overexpression of AQP7 increased glycerol influx into INS-1 cells, whereas inhibition of AQP7 reduced glycerol influx, thereby decreasing subsequent insulin secretion. Our findings demonstrate a new mechanism by which metformin suppresses the p38 and JNK pathways, thereby upregulating pancreatic AQP7 expression and promoting glycerol influx into pancreatic β-cells and subsequent insulin secretion in T2DM.     

Xenin-25 Potentiates Glucose-dependent Insulinotropic Polypeptide Action via a Novel Cholinergic Relay Mechanism

The intestinal peptides GLP-1 and GIP potentiate glucose-mediated insulin release. Agents that increase GLP-1 action are effective therapies in type 2 diabetes mellitus (T2DM). However, GIP action is blunted in T2DM, and GIP-based therapies have not been developed. Thus, it is important to increase our understanding of the mechanisms of GIP action. We developed mice lacking GIP-producing K cells. Like humans with T2DM, “GIP/DT” animals exhibited a normal insulin secretory response to exogenous GLP-1 but a blunted response to GIP. Pharmacologic doses of xenin-25, another peptide produced by K cells, restored the GIP-mediated insulin secretory response and reduced hyperglycemia in GIP/DT mice. Xenin-25 alone had no effect. Studies with islets, insulin-producing cell lines, and perfused pancreata indicated xenin-25 does not enhance GIP-mediated insulin release by acting directly on the β-cell. The in vivo effects of xenin-25 to potentiate insulin release were inhibited by atropine sulfate and atropine methyl bromide but not by hexamethonium. Consistent with this, carbachol potentiated GIP-mediated insulin release from in situ perfused pancreata of GIP/DT mice. In vivo, xenin-25 did not activate c-fos expression in the hind brain or paraventricular nucleus of the hypothalamus indicating that central nervous system activation is not required. These data suggest that xenin-25 potentiates GIP-mediated insulin release by activating non-ganglionic cholinergic neurons that innervate the islets, presumably part of an enteric-neuronal-pancreatic pathway. Xenin-25, or molecules that increase acetylcholine receptor signaling in β-cells, may represent a novel approach to overcome GIP resistance and therefore treat humans with T2DM.     

Hyperglycemia modulates angiotensinogen gene expression

Elevated plasma angiotensinogen (AGT) levels have been demonstrated in insulin-resistant states such as obesity and type 2 diabetes mellitus (DM2), conditions that are directly correlated to hypertension. We examined whether hyperinsulinemia or hyperglycemia may modulate fat and liver AGT gene expression and whether obesity and insulin resistance are associated with abnormal AGT regulation. In addition, because the hexosamine biosynthetic pathway is considered to function as a biochemical sensor of intracellular nutrient availability, we hypothesized that activation of this pathway would acutely mediate in vivo the induction of AGT gene expression in fat and liver. We studied chronically catheterized lean (approximately 300 g) and obese (approximately 450 g) Sprague-Dawley rats in four clamp studies (n = 3/group), creating physiological hyperinsulinemia (approximately 60 microU/ml, by an insulin clamp), hyperglycemia (approximately 18 mM, by a pancreatic clamp using somatostatin to prevent endogenous insulin secretion), or euglycemia with glucosamine infusion (GlcN; 30 micromol. kg(-1). min(-1)) and equivalent saline infusions (as a control). Although insulin infusion suppressed AGT gene expression in fat and liver of lean rats, the obese rats demonstrated resistance to this effect of insulin. In contrast, hyperglycemia at basal insulin levels activated AGT gene expression in fat and liver by approximately threefold in both lean and obese rats (P < 0.001). Finally, GlcN infusion simulated the effects of hyperglycemia on fat and liver AGT gene expression (2-fold increase, P < 0.001). Our results support the hypothesis that physiological nutrient "pulses" may acutely induce AGT gene expression in both adipose tissue and liver through the activation of the hexosamine biosynthetic pathway. Resistance to the suppressive effect of insulin on AGT expression in obese rats may potentiate the effect of nutrients on AGT gene expression. We propose that increased AGT gene expression and possibly its production may provide another link between obesity/insulin resistance and hypertension.     

Morus alba leaves ethanol extract protects pancreatic islet cells against dysfunction and death by inducing autophagy in type 2 diabetes

BackgroundProtection of pancreatic islet cells against dysfunction or death by regulating autophagy is considered to be an effective method for treatment of type 2 diabetes mellitus (T2DM). Morus alba leaves (mulberry leaves), a popular herbal medicine, have been used for prevention of T2DM since ancient times.PurposeThis study aimed to clarify whether Morus alba leaves ethanol extract (MLE) could protect islet cells in vivo and in vitro by regulating autophagy in T2DM, and explore the possible mechanism of action.MethodsThe main chemical constituents in MLE were analyzed by HPLC. The T2DM rat model was induced via high-fat diet combined with peritoneal injection of low-dose streptozotocin, and MLE was administered by oral gavage. Fasting blood glucose (FBG) and plasma insulin were measured, and homeostatic model assessment of β cell function (HOMA-β) and insulin resistance (HOMA-IR) were determined. The histomorphology of pancreas islets was evaluated by haematoxylin and eosin staining. In palmitic acid (PA)-stressed INS-1 rat insulinoma cells, cell viability was assayed by an MTT method. Expression of the autophagy-related proteins LC3 I/II, p62, p-AMPK and p-mTOR in islet tissues and INS-1 cells was evaluated by western blotting or immunohistochemistry analysis.ResultsThe four main chemical constituents in MLE were identified as chlorogenic acid, rutin, isoquercitrin and quercitrin. MLE ameliorated hyperglycemia, insulin resistance and dyslipidemia of T2DM rats with prominent therapeutic effect. Further study indicated that MLE observably improved islet function, alleviated islet injury of T2DM rats, and inhibited PA-induced INS-1 cell death. On the other hand, MLE significantly induced autophagy in islet cells both in vivo and in vitro, and autophagy inhibitors abolished its therapeutic effect on T2DM rats and protective effect on islet cells. Apart from this, MLE markedly activated the AMPK/mTOR pathway in INS-1 cells, and the AMPK inhibitor prevented the autophagy induction ability of MLE.ConclusionTogether, MLE could protect islet cells against dysfunction and death by inducing AMPK/mTOR-mediated autophagy in T2DM, and these findings provide a new perspective for understanding the treatment mechanism of Morus alba leaves against T2DM.     

In Vivo Detection of Extrapancreatic Insulin Gene Expression in Diabetic Mice by Bioluminescence Imaging

Background

Extrapancreatic tissues such as liver may serve as potential sources of tissue for generating insulin-producing cells. The dynamics of insulin gene promoter activity in extrapancreatic tissues may be monitored in vivo by bioluminescence-imaging (BLI) of transgenic mice Tg(RIP-luc) expressing the firefly luciferase (luc) under a rat-insulin gene promoter (RIP).

Methods

The Tg(RIP-luc) mice were made diabetic by a single injection of the pancreatic β-cell toxin streptozotocin. Control mice were treated with saline. Mice were subject to serum glucose measurement and bioluminescence imaging daily. On day eight of the treatment, mice were sacrificed and tissues harvested for quantitative luciferase activity measurement, luciferase protein cellular localization, and insulin gene expression analysis.

Results

Streptozotocin-induced diabetic Tg(RIP-luc) mice demonstrated a dramatic decline in the BLI signal intensity in the pancreas and a concomitant progressive increase in the signal intensity in the liver. An average of 5.7 fold increase in the liver signal intensity was detected in the mice that were exposed to hyperglycemia for 8 days. Ex vivo quantitative assays demonstrated a 34-fold induction of the enzyme activity in the liver of streptozotocin-treated mice compared to that of the buffer-treated controls. Luciferase-positive cells with oval-cell-like morphology were detected by immunohistochemistry in the liver samples of diabetic mice, but not in that of non-treated control transgenic mice. Gene expression analyses of liver RNA confirmed an elevated expression of insulin genes in the liver tissue exposed to hyperglycemia.

Conclusions

BLI is a sensitive method for monitoring insulin gene expression in extrapancreatic tissues in vivo. The BLI system may be used for in vivo screening of biological events or pharmacologic activators that have the potential of stimulating the generation of extrapancreatic insulin-producing cells.     

Neonatal Exendin-4 Reduces Growth,Fat Deposition and Glucose Tolerance during Treatment in the Intrauterine Growth-Restricted Lamb

Background

IUGR increases the risk of type 2 diabetes mellitus (T2DM) in later life, due to reduced insulin sensitivity and impaired adaptation of insulin secretion. In IUGR rats, development of T2DM can be prevented by neonatal administration of the GLP-1 analogue exendin-4. We therefore investigated effects of neonatal exendin-4 administration on insulin action and β-cell mass and function in the IUGR neonate in the sheep, a species with a more developed pancreas at birth.

Methods

Twin IUGR lambs were injected s.c. daily with vehicle (IUGR+Veh, n = 8) or exendin-4 (1 nmol.kg^-1, IUGR+Ex-4, n = 8), and singleton control lambs were injected with vehicle (CON, n = 7), from d 1 to 16 of age. Glucose-stimulated insulin secretion and insulin sensitivity were measured in vivo during treatment (d 12–14). Body composition, β-cell mass and in vitro insulin secretion of isolated pancreatic islets were measured at d 16.

Principal Findings

IUGR+Veh did not alter in vivo insulin secretion or insulin sensitivity or β-cell mass, but increased glucose-stimulated insulin secretion in vitro. Exendin-4 treatment of the IUGR lamb impaired glucose tolerance in vivo, reflecting reduced insulin sensitivity, and normalised glucose-stimulated insulin secretion in vitro. Exendin-4 also reduced neonatal growth and visceral fat accumulation in IUGR lambs, known risk factors for later T2DM.

Conclusions

Neonatal exendin-4 induces changes in IUGR lambs that might improve later insulin action. Whether these effects of exendin-4 lead to improved insulin action in adult life after IUGR in the sheep, as in the PR rat, requires further investigation.     

A Syntenic Cross Species Aneuploidy Genetic Screen Links RCAN1 Expression to β-Cell Mitochondrial Dysfunction in Type 2 Diabetes

Type 2 diabetes (T2D) is a complex metabolic disease associated with obesity, insulin resistance and hypoinsulinemia due to pancreatic β-cell dysfunction. Reduced mitochondrial function is thought to be central to β-cell dysfunction. Mitochondrial dysfunction and reduced insulin secretion are also observed in β-cells of humans with the most common human genetic disorder, Down syndrome (DS, Trisomy 21). To identify regions of chromosome 21 that may be associated with perturbed glucose homeostasis we profiled the glycaemic status of different DS mouse models. The Ts65Dn and Dp16 DS mouse lines were hyperglycemic, while Tc1 and Ts1Rhr mice were not, providing us with a region of chromosome 21 containing genes that cause hyperglycemia. We then examined whether any of these genes were upregulated in a set of ~5,000 gene expression changes we had identified in a large gene expression analysis of human T2D β-cells. This approach produced a single gene, RCAN1, as a candidate gene linking hyperglycemia and functional changes in T2D β-cells. Further investigations demonstrated that RCAN1 methylation is reduced in human T2D islets at multiple sites, correlating with increased expression. RCAN1 protein expression was also increased in db/db mouse islets and in human and mouse islets exposed to high glucose. Mice overexpressing RCAN1 had reduced in vivo glucose-stimulated insulin secretion and their β-cells displayed mitochondrial dysfunction including hyperpolarised membrane potential, reduced oxidative phosphorylation and low ATP production. This lack of β-cell ATP had functional consequences by negatively affecting both glucose-stimulated membrane depolarisation and ATP-dependent insulin granule exocytosis. Thus, from amongst the myriad of gene expression changes occurring in T2D β-cells where we had little knowledge of which changes cause β-cell dysfunction, we applied a trisomy 21 screening approach which linked RCAN1 to β-cell mitochondrial dysfunction in T2D.     

In vivo therapeutic exploring for Mori folium extract against type 2 diabetes mellitus in rats

Background: The study was aimed to investigate the potential therapeutic effect of Mori folium aqueous extracts (MFAE) on type 2 diabetes mellitus (T2DM) in vivo.Methods and results: A rat model of T2DM was established with the combination of high sugar and high-fat diet (HSFD) and streptozotocin (STZ). The T2DM rats were administrated with low (2 g.kg⁻¹) and high (5 g.kg⁻¹) doses of MFAE for 60 consecutive days. The biochemical indices of glucose metabolism disorders, insulin resistance and oxidative stress were observed. The results indicated that MFAE significantly promoted the synthesis of hepatic glycogen, reduced the levels of fasting blood glucose and fasting blood insulin, and improved the insulin sensitivity index (ISI). MFAE administration also remarkably increased the levels of superoxide dismutase (SOD) and reduced the levels of malondialdehyde (MDA).Conclusion: MFAE showed a therapeutic effect on T2DM with the bioative effect of improve glucose metabolism disorders, decrease insulin resistance, and ameliorate the antioxidative ability.     

In Vivo Electroporation of Minicircle DNA as a Novel Method of Vaccine Delivery To Enhance HIV-1-Specific Immune Responses

DNA vaccines offer advantage over conventional vaccines, as they are safer to use, easier to produce, and able to induce humoral as well cellular immune responses. Unfortunately, no DNA vaccines have been licensed for human use for the difficulties in developing an efficient and safe in vivo gene delivery system. In vivo electroporation (EP)-based DNA delivery has attracted great attention for its potency to enhance cellular uptake of DNA vaccines and function as an adjuvant. Minicircle DNA (a new form of DNA containing only a gene expression cassette and lacking a backbone of bacterial plasmid DNA) is a powerful candidate of gene delivery in terms of improving the levels and the duration of transgene expression in vivo. In this study, as a novel vaccine delivery system, we combined in vivo EP and the minicircle DNA carrying a codon-optimized HIV-1 gag gene (minicircle-gag) to evaluate the immunogenicity of this system. We found that minicircle-gag conferred persistent and high levels of gag expression in vitro and in vivo. The use of EP delivery further increased minicircle-based gene expression. Moreover, when delivered by EP, minicircle-gag vaccination elicited a 2- to 3-fold increase in cellular immune response and a 1.5- to 3-fold augmentation of humoral immune responses compared with those elicited by a pVAX1-gag positive control. Increased immunogenicity of EP-assisted minicircle-gag may benefit from increasing local antigen expression, upregulating inflammatory genes, and recruiting immune cells. Collectively, in vivo EP of minicircle DNA functions as a novel vaccine platform that can enhance efficacy and immunogenicity of DNA vaccines.     

3,5 Diiodo-L-Thyronine (T2) Does Not Prevent Hepatic Steatosis or Insulin Resistance in Fat-Fed Sprague Dawley Rats

Thyroid hormone mimetics are alluring potential therapies for diseases like dyslipidemia, nonalcoholic fatty liver disease (NAFLD), and insulin resistance. Though diiodothyronines are thought inactive, pharmacologic treatment with 3,5- Diiodo-L-Thyronine (T2) reportedly reduces hepatic lipid content and improves glucose tolerance in fat-fed male rats. To test this, male Sprague Dawley rats fed a safflower-oil based high-fat diet were treated with T2 (0.25 mg/kg-d) or vehicle. Neither 10 nor 30 days of T2 treatment had an effect on weight, adiposity, plasma fatty acids, or hepatic steatosis. Insulin action was quantified in vivo by a hyperinsulinemic-euglycemic clamp. T2 did not alter fasting plasma glucose or insulin concentration. Basal endogenous glucose production (EGP) rate was unchanged. During the clamp, there was no difference in insulin stimulated whole body glucose disposal. Insulin suppressed EGP by 60% ± 10 in T2-treated rats as compared with 47% ± 4 suppression in the vehicle group (p = 0.32). This was associated with an improvement in hepatic insulin signaling; insulin stimulated Akt phosphorylation was ~2.5 fold greater in the T2-treated group as compared with the vehicle-treated group (p = 0.003). There was no change in expression of genes thought to mediate the effect of T2 on hepatic metabolism, including genes that regulate hepatic lipid oxidation (ppara, carnitine palmitoyltransferase 1a), genes that regulate hepatic fatty acid synthesis (srebp1c, acetyl coa carboxylase, fatty acid synthase), and genes involved in glycolysis and gluconeogenesis (L-pyruvate kinase, glucose 6 phosphatase). Therefore, in contrast with previous reports, in Sprague Dawley rats fed an unsaturated fat diet, T2 administration failed to improve NAFLD or whole body insulin sensitivity. Though there was a modest improvement in hepatic insulin signaling, this was not associated with significant differences in hepatic insulin action. Further study will be necessary before diiodothyronines can be considered an effective treatment for NAFLD and dyslipidemia.     

Western diet given to healthy rats mimics the human phenotype of diabetic cardiomyopathy

Diabetes mellitus (DM) is a major problem worldwide. Within this patient group, cardiovascular diseases are the biggest cause of morbidity and mortality. Diabetic cardiomyopathy (DCM) is defined as diabetes-associated structural and functional changes in the myocardium, not directly attributable to other confounding factors such as coronary artery disease or hypertension. Pathophysiology of DCM remains unclear due to a lack of adequate animal models reflecting the current pandemic of diabetes, associated with a high increased sugar intake and the ‘Western’ lifestyle. The aim of this study was to develop an animal model mimicking this ‘Western’ lifestyle causing a human-like phenotype of DCM. Twenty-four Sprague–Dawley rats were randomly assigned into a normal or a ‘Western’ diet group for 18 weeks. Glucose and insulin levels were measured with an OGTT. Heart function was assessed by echocardiography and hemodynamic measurements in vivo. Cardiac fibrosis and inflammation were investigated in vitro. ‘Western’ diet given to healthy rats for 18 weeks induced hyperglycemia together with increased AGEs levels, insulin levels and hypertriglyceridemia. Heart function was altered with increased end-diastolic pressure, left ventricle hypertrophy. Changes in vivo were associated with increased collagen deposition and increased PAI-1 levels in the heart. High-sugar diet or ‘Western’ diet causes T2DM and the hallmarks of DCM in rats, reflecting the phenotype of the disease seen in patients. Using this new model of T2DM with DCM might open new insight in understanding the pathophysiology of DCM and on a long term, test targeted therapies for T2DM with DCM patients.     

Circulating Level of CTRP1 in Patients with Nonalcoholic Fatty Liver Disease (NAFLD): Is It through Insulin Resistance?

Nonalcoholic fatty liver disease (NAFLD) is considered as one of the most common liver diseases. It is robustly linked to obesity and insulin resistance and is regarded as hepatic manifestation of metabolic syndrome (MetS). Adipokines are involved in the pathophysiology of liver diseases. The aim of this study was to evaluate the plasma concentrations of CTRP1 (complement-C1q TNF-related protein 1) in 22 patients with NAFLD, 22 patients with type 2 diabetes mellitus (T2DM), 22 patients with NAFLD+T2DM and 21 healthy controls, as well as their correlation with the level of metabolic and hepatic parameters. Plasma concentration of CTRP1 was measured with ELISA method. Plasma concentration of CTRP1 in patients with NAFLD, T2DM and NAFLD+T2DM were significantly higher than healthy subjects (p<0.0001). Moreover, we observed significant positive correlations between plasma level of CTRP1 and fasting blood glucose (FBG) (p<0.001), homeostasis model assessment of insulin resistance (HOMA-IR) (p<0.001), body mass index (BMI) (p = 0.001), alanine amino transferase (ALT) (p = 0.002), gamma glutamyl transferase (γ-GT) (p<0.001) and liver stiffness (LS) (p<0.001). Our results indicate the strong association of CTRP1 with insulin resistance in NAFLD. Also, it seems that CTRP1 can be considered as an emerging biomarker for NAFLD, however, more studies are necessary to unravel the role of CTRP1 in NAFLD pathogenesis.     

Insulin Resistance Is Not Associated with an Impaired Mitochondrial Function in Contracting Gastrocnemius Muscle of Goto-Kakizaki Diabetic Rats In Vivo

Insulin resistance, altered lipid metabolism and mitochondrial dysfunction in skeletal muscle would play a major role in type 2 diabetes mellitus (T2DM) development, but the causal relationships between these events remain conflicting. To clarify this issue, gastrocnemius muscle function and energetics were investigated throughout a multidisciplinary approach combining in vivo and in vitro measurements in Goto-Kakizaki (GK) rats, a non-obese T2DM model developing peripheral insulin resistant without abnormal level of plasma non-esterified fatty acids (NEFA). Wistar rats were used as controls. Mechanical performance and energy metabolism were assessed strictly non-invasively using magnetic resonance (MR) imaging and 31-phosphorus MR spectroscopy (³¹P-MRS). Compared with control group, plasma insulin and glucose were respectively lower and higher in GK rats, but plasma NEFA level was normal. In resting GK muscle, phosphocreatine content was reduced whereas glucose content and intracellular pH were both higher. However, there were not differences between both groups for basal oxidative ATP synthesis rate, citrate synthase activity, and intramyocellular contents for lipids, glycogen, ATP and ADP (an important in vivo mitochondrial regulator). During a standardized fatiguing protocol (6 min of maximal repeated isometric contractions electrically induced at a frequency of 1.7 Hz), mechanical performance and glycolytic ATP production rate were reduced in diabetic animals whereas oxidative ATP production rate, maximal mitochondrial capacity and ATP cost of contraction were not changed. These findings provide in vivo evidence that insulin resistance is not caused by an impairment of mitochondrial function in this diabetic model.     

Hyperglycaemia Enhances Nitric Oxide Production in Diabetes: A Study from South Indian Patients

Background

We have previously reported that increased glucose levels were associated with higher serum nitric oxide (NO) levels in fructose-fed insulin resistant rats. However, the relationship between hyperglycemia and serum NO level was not clear. Therefore, the present study was designed to find the association between hyperglycemia and serum NO levels in Type 2 diabetic (T2DM) patients and T2DM with cardiovascular complication.

Methods

Endothelial cells (HUVEC) were treated with of D-glucose (10-100mM), and NO levels and NOS gene expression was measured. Hyperglycaemia was induced in Sprague-Dawley rats, and serum NO levels were measured after 8 weeks. For clinical evaluation, five groups of patients were recruited: Control (CT, n=48), Type 2 diabetes (T2DM, n=26), T2DM with hypertension (DMHT, n=46), Coronary artery diseases (CAD, n=29) and T2DM with coronary artery diseases (DMCD, n=38). NO (nitrite + nitrate) levels were measured from human serum.

Results

We found a significant (p<0.05) and dose-dependent increase in NO levels in HUVEC cells after 4 hours of high glucose exposure. eNOS and iNOS gene expression was increased in HUVEC cells after different concentrations and time periods of glucose treatment. We also observed significant (149.1±25μM, p<0.01) increase in serum NO levels in hyperglycaemic rats compared to control (76.6±13.2μM). Serum NO level was significantly higher in T2DM (111.8 μM (81.7-122.4), p<0.001) and DMCD patients ((129.4 μM (121.2-143.5), p <0.001) but not in CAD patients (76.4 μM (70.5-87)), as compared to control (68.2 μM (56.4-82.3)). We found significantly lower NO levels (83.5 μM (60.5-122.9)) in subjects suffering from diabetes since more than 5 years, compared to subjects (115.3 μM (75.2-127.1), p<0.001) with less than 5 years.

Conclusion

In conclusion, high NO levels were observed in South Indian diabetic patients. Higher glucose levels in serum might be responsible for activation of endothelial cells to enhance NO levels.     

Induction of Tolerogenic Dendritic Cells by a PEGylated TLR7 Ligand for Treatment of Type 1 Diabetes

Autoimmune diabetes mellitus (DM) results from the destruction of pancreatic islet cells by activated T lymphocytes, which have been primed by activated dendritic cells (DC). Individualized therapy with ex vivo DC manipulation and reinfusion has been proposed as a treatment for DM, but this treatment is limited by cost, and requires specialized facilities. A means of in situ modulation of the DC phenotype in the host would be more accessible. Here we report a novel innate immune modulator, 1Z1, generated by conjugating a TLR7 ligand to six units of polyethylene glycol (PEG), which skews DC phenotype in vivo. 1Z1 was less potent in inducing cytokine production by DC than the parent ligand in vitro and in vivo. In addition, this drug only modestly increased DC surface expression of activation markers such as MHC class II, CD80, and CD86; however, the expression of negative regulatory molecules, such as programmed death ligand 1 (PD-L1), and interleukin-1 receptor-associated kinase M (IRAK-M) were markedly increased. In vivo transfer of 1Z1 treated DC into prediabetic NOD mice delayed pancreatic insulitis. Daily administration of 1Z1 effectively prevented the clinical onset of hyperglycemia and reduced histologic islet inflammation. Daily treatment with 1Z1 increased PD-L1 expression in the CD11c⁺ population in peri-pancreatic lymph nodes; however, it did not induce an increase in regulatory T cells. Pharmaceutical modulation of DC maturation and function in situ, thus represents an opportunity to treat autoimmune disease.     

Human-IAPP disrupts the autophagy/lysosomal pathway in pancreatic β-cells: protective role of p62-positive cytoplasmic inclusions

In type II diabetes (T2DM), there is a deficit in β-cells, increased β-cell apoptosis and formation of intracellular membrane-permeant oligomers of islet amyloid polypeptide (IAPP). Human-IAPP (h-IAPP) is an amyloidogenic protein co-expressed with insulin by β-cells. IAPP expression is increased with obesity, the major risk factor for T2DM. In this study we report that increased expression of human-IAPP led to impaired autophagy, due at least in part to the disruption of lysosome-dependant degradation. This action of IAPP to alter lysosomal clearance in vivo depends on its propensity to form toxic oligomers and is independent of the confounding effect of hyperglycemia. We report that the scaffold protein p62 that delivers polyubiquitinated proteins to autophagy may have a protective role against human-IAPP-induced apoptosis, apparently by sequestrating protein targets for degradation. Finally, we found that inhibition of lysosomal degradation increases vulnerability of β-cells to h-IAPP-induced toxicity and, conversely, stimulation of autophagy protects β-cells from h-IAPP-induced apoptosis. Collectively, these data imply an important role for the p62/autophagy/lysosomal degradation system in protection against toxic oligomer-induced apoptosis.     

Protective effect of aldehyde dehydrogenase 2 against rat corneal dysfunction caused by streptozotocin-induced type I diabetes

Aldehyde dehydrogenase 2 plays a pivotal role in detoxifying aldehydes, and our previous study revealed that aldehyde dehydrogenase 2 could alleviate diabetic retinopathy-associated damage. We aimed to characterize the potential role of aldehyde dehydrogenase 2 in diabetic keratopathy. Twenty-four rats with streptozotocin-induced (60 mg/kg, single intraperitoneal injection) type 1 diabetes mellitus (T1DM) were divided the T1DM group and the T1DM + Alda1 (an activator of aldehyde dehydrogenase 2) group (5 mg/kg/d, intraperitoneal injection, 1/2/3 months), while an additional 12 healthy rats served as the control group. Corneal morphology was examined in vivo and in vitro at one, two, and three months after T1DM induction. Additionally, serum inflammatory factors were measured by ELISA, and the expression of corneal vascular endothelial growth factor A (VEGF-A) and aldehyde dehydrogenase 2 was measured by immunofluorescence staining. Corneal cell death was evaluated by terminal-deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) staining. Slit lamp analysis showed that the area of corneal epithelial cell injury in the T1DM + Alda1 group was significantly smaller than that in the T1DM group at one and two months after T1DM induction (all P < 0.05). OCT analysis and HE staining showed that the central corneal thickness (indication of corneal edema) and the epithelial keratinization level in the T1DM + Alda1 group was evidently decreased compared with those in the T1DM group (all P < 0.05). The serum inflammatory factors interleukin-1 and interleukin-6 were significantly upregulated in the T1DM group compared with the T1DM + Alda1 group at three months after T1DM induction (all P < 0.05), while there were no differences in SOD or TNF-α levels among all groups. Furthermore, corneal VEGF-A expression and corneal cell death in the T1DM + Alda1 group were dramatically reduced compared to those in the T1DM group (all P < 0.05). In conclusion, the aldehyde dehydrogenase 2 agonist Alda1 attenuated rat corneal dysfunction induced by T1DM by alleviating corneal edema, decreasing corneal cell death, and downregulating corneal VEGF-A expression.