New insights into human beta cell biology using human pluripotent stem cells.

Pancreatic ß-cells are responsible for maintaining glucose homeostasis. Therefore, their dysregulation leads to diabetes. Pancreas or islet transplants can be used to treat diabetes but these human tissues remain in short supply. Significant progress has now been made in differentiating human pluripotent stem cells (hPSCs) such as human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs) into pancreatic ß-like cells for potential cell replacement therapy. Additionally, these hPSC-derived ß-like cells represent a new invaluable model for studying diabetes disease mechanisms. Here, we review the use of hPSC-derived ß-like cells as a platform to model various types of defects in human ß-cells in diabetes, comparing them against existing animal models, ex vivo human islets and human ß-cell line. We also discuss how hPSC-derived ß-like cells are being used as a platform for screening novel therapeutic compounds. Last but not least, we evaluate the strengths and limitations of this human cell-based platform as an avenue to study and reveal new insights into human ß-cell biology.

First use of gene therapy to treat growth hormone resistant dwarfism in a mouse model.

The only treatment tested for growth hormone receptor (GHR) defective Laron Syndrome (LS) is injections of recombinant insulin-like-growth factor 1 (rhIGF1). The response is suboptimal and associated with progressive obesity. In this study, we treated 4-5-week-old Laron dwarf mice (GHR-/-) with an adeno-associated virus expressing murine GHR (AAV-GHR) injection at a dose of 4 × 1010 vector genome per mouse. Serum growth hormone (GH) levels decreased, and GH-responsive IGF1, IGF binding protein 3 (IGFBP3) and acid labile subunit (ALS) increased. There was a significant but limited increase in body weight and length, similar to the response to rhIGF1 treatment in LS patients. All the major organs increased in weight except the brain. Our study is the first to use gene therapy to treat GH-receptor deficiency. We propose that gene therapy with AAV-GHR may eventually be useful for the treatment of human LS.

Progressive endoplasmic reticulum stress over time due to human insulin gene mutation contributes to pancreatic beta cell dysfunction.

AIMS/HYPOTHESIS: We studied the effects of heterozygous human INS gene mutations on insulin secretion, endoplasmic reticulum (ER) stress and other mechanisms in both MIN6 and human induced pluripotent stem cells (hiPSC)-derived beta-like cells, as well as the effects of prolonged overexpression of mutant human INS in MIN6 cells. METHODS: We modelled the structure of mutant C109Y and G32V proinsulin computationally to examine the in silico effects. We then overexpressed either wild-type (WT), mutant (C109Y or G32V), or both WT and mutant human preproinsulin in MIN6 cells, both transiently and stably over several weeks. We measured the levels of human and rodent insulin secreted, and examined the transcript and protein levels of several ER stress and apoptotic markers. We also reprogrammed human donor fibroblasts heterozygous for the C109Y mutation into hiPSCs and differentiated these into pancreatic beta-like cells, which were subjected to single-cell RNA-sequencing and transcript and protein analyses for ER stress and apoptotic markers. RESULTS: The computational modelling studies, and short-term and long-term expression studies in beta cells, revealed the presence of ER stress, organelle changes and insulin processing defects, resulting in a decreased amount of insulin secreted but not the ability to secrete insulin. By 9 weeks of expression of mutant human INS, dominant-negative effects of mutant INS were evident and beta cell insulin secretory capacity declined. INS+/C109Y patient-derived beta-like cells and single-cell RNA-sequencing analyses then revealed compensatory upregulation in genes involved in insulin secretion, processing and inflammatory response. CONCLUSIONS/INTERPRETATION: The results provide deeper insights into the mechanisms of beta cell failure during INS mutation-mediated diabetes disease progression. Decreasing spliced X-box binding protein 1 (sXBP1) or inflammatory response could be avenues to restore the function of the remaining WT INS allele.

Immunosuppression overcomes insulin- and vector-specific immune responses that limit efficacy of AAV2/8-mediated insulin gene therapy in NOD mice.

We report the restoration of euglycaemia in chemically induced diabetic C57BL/6 mice and spontaneously diabetic Non Obese Diabetic (NOD) mice by intravenous systemic administration of a single-stranded adeno-associated virus (ssAAV2/8) codon optimised (co) vector encoding furin cleavable human proinsulin under a liver-specific promoter. There were no immunological barriers to efficacy of insulin gene therapy in chemically induced C57BL/6 mice, which enjoyed long-lasting correction of hyperglycaemia after therapy, up to 250 days. Euglycaemia was also restored in spontaneously diabetic NOD mice, although these mice required a 7-10-fold higher dose of vector to achieve similar efficacy as the C57BL/6 mice and the immunodeficient NODscid mice. We detected CD8+ T cell reactivity to insulin and mild inflammatory infiltration in the livers of gene therapy recipient NOD mice, neither of which were observed in the treated C57BL/6 mice. Efficacy of the gene therapy in NOD mice was partially improved by targeting the immune system with anti-CD4 antibody treatment, while transfer of NOD mouse AAV2/8-reactive serum to recipients prevented successful restoration of euglycaemia in AAV2/8-HLP-hINSco-treated NODscid mice. Our data indicate that both immune cells and antibodies form a barrier to successful restoration of euglycaemia in autoimmune diabetic recipient mice with insulin gene therapy, but that this barrier can be overcome by increasing the dose of vector and by suppressing immune responses.

Development of a liver-specific Tet-off AAV8 vector for improved safety of insulin gene therapy for diabetes.

BACKGROUND: Diabetes mellitus is caused by a partial or complete lack of insulin production in the body. We have previously shown that a single injection of an adeno-associated virus serotype 8 (AAV8) vector carrying a modified and codon optimized human insulin gene induced hepatic production of insulin and corrected streptozotocin (STZ)-induced diabetes in mice for more than 1 year. Insulin production was constitutive, analogous to long-acting insulin therapy. METHODS: We have developed a single AAV8 vector with a Tet-Off regulatable system as a safety mechanism to turn off insulin secretion should hypoglycaemia develop in vector-treated diabetic mice. We first transfected HepG2 cells or freshly isolated rat hepatocytes in vitro with the Tet-Off system (pAAV-Tetoffbidir -Alb-luc) regulating a luciferase reporter gene. We subsequently incorporated a furin-cleavable codon-optimised human proinsulin cDNA into pAAV-Tetoffbidir backbone to form the doxycycline inducible pAAV-Tetoffbidir -Alb-hINSco. RESULTS: Using STZ-induced diabetic mice, we were able to switch off insulin secretion repeatedly with doxycycline administration, and showed full restoration of insulin secretion on withdrawing doxycycline. CONCLUSIONS: The present study provides proof of concept that, under circumstances when inappropriate basal insulin secretion is a safety concern, insulin secretion from AAV8 gene therapy can be turned off reversibly with doxycycline.

Anti-Cancer Drug HMBA Acts as an Adjuvant during Intracellular Bacterial Infections by Inducing Type I IFN through STING.

The anti-proliferative agent hexamethylene bisacetamide (HMBA) belongs to a class of hybrid bipolar compounds developed more than 30 y ago for their ability to induce terminal differentiation of transformed cells. Recently, HMBA has also been shown to trigger HIV transcription from latently infected cells, via a CDK9/HMBA inducible protein-1 dependent process. However, the effect of HMBA on the immune response has not been explored. We observed that pretreatment of human peripheral blood mononuclear cells with HMBA led to a markedly increased production of IL-12 and IFN-γ, but not of TNF-α, IL-6, and IL-8 upon subsequent infection with Burkholderia pseudomallei and Salmonella enterica HMBA treatment was also associated with better intracellular bacterial control. HMBA significantly improved IL-12p70 production from CD14+ monocytes during infection partly via the induction of type I IFN in these cells, which primed an increased transcription of the p35 subunit of IL-12p70 during infection. HMBA also increased early type I IFN transcription in human monocytic and epithelial cell lines, but this was surprisingly independent of its previously reported effects on positive transcription elongation factor b and HMBA inducible protein-1. Instead, the effect of HMBA was downstream of a calcium influx, and required the pattern recognition receptor and adaptor STING but not cGAS. Our work therefore links the STING-IRF3 axis to enhanced IL-12 production and intracellular bacterial control in primary monocytes. This raises the possibility that HMBA or related small molecules may be explored as therapeutic adjuvants to improve disease outcomes during intracellular bacterial infections.

Efficacy and safety of pulsed intravenous methylprednisolone in early active thyroid eye disease.

Introduction: The mainstay of therapy for active inflammatory phase of thyroid eye disease (TED) is immunosuppression. Patients in our centre with early active TED are treated with pulsed intravenous methylprednisolone (IVMP). Two different protocols are offered in our centre: High dose (1g/day for 3 days, monthly for 6 months), or EUGOGO protocol (500 mg weekly for six weeks, followed by 250 mg weekly for the next 6 weeks). Methods: A prospective cohort study of patients undergoing the two IVMP protocols was performed from January 2009 to May 2015. Main outcome measures were improvement of Clinical Activity Score (CAS) and International Thyroid Eye Disease (ITEDS) - VISA Inflammatory Index. Results: We had a total of 63 patients. Mean age was 43.1 ± 13.1years, females comprised 49.2% (n = 31), and 31 (49.2%) had a positive smoking history. Following IVMP, 65.0% (n = 41) had good response, 31.7% (n = 20) partial, and 3.3% (n = 2) poor. There were significant differences (p < 0.001) in CAS and ITEDS scores between pre-IVMP and post-IVMP visits, for both protocols. A higher proportion of patients receiving the modified EUGOGO protocol (58.3%) had persistent activity and required additional immunosuppression compared to those who underwent the high dose protocol (33.3%). Mild side effects were experienced by 5 (7.9%) and 3 (4.8%) patients at 3 and 6 months, respectively. There were no severe side effects, cardiovascular events or liver failure. Conclusion: With adequate screening and follow-up, six repeated cycles of high dose pulsed IVMP is an effective treatment for TED and can significantly reduce the morbidity associated with this debilitating condition. None of the 51 patients from the high dose protocol met with any serious side effects.

PGE1 improves diabetic peripheral neuropathy in patients with type 2 diabetes.

BACKGROUND: Diabetic peripheral neuropathy (DPN) is the most common chronic complication of diabetes. We aim to investigate the efficacy of Prostaglandin E1 (PGE1) treatment in addition to intensive insulin therapy on DPN in patients with type 2 diabetes. METHODS: Seventy-seven patients with DPN received daily intravenous injection of Prostaglandin E1 (PGE1) in lipid microspheres (Lipo-PGE1) for 10days as an additional therapy to standard glucose control therapy (PGE1 group). Another 42 patients with DPN receiving only standard glucose control therapy (intensive insulin therapy) acted as a control group. Michigan neuropathy screening instrument (MNSI) score, neurophysiology examination, transcutaneous oxygen sensory threshold, and ankle-brachial index (ABI) were measured to evaluate the efficacy of PGE1 treatment as compared with control group. RESULTS: Standard glucose control therapy decreased plasma glucose to a similar level in both PGE1 and control groups. Compare to control group, PGE1 group displayed improvement in several nerve electrophysiological indexes. MNSI score was significantly improved in patients who received PGE1 treatment compared with the control group (p<0.001) after 10days of PGE1 treatment. Similarly, nerve conduction velocity and foot sensory thresholds (p<0.05 for all) also significantly improved compared with the control group after 10days of PGE1 treatment. However, only intensive insulin therapy did not improve any neural function. CONCLUSIONS: Lipo-PGE1 can effectively improve neural function of patients with DPN.

Thymosin ß4 attenuates early diabetic nephropathy in a mouse model of type 2 diabetes mellitus.

The chronic inflammatory processes and endothelial dysfunction play important roles in the development of diabetic nephropathy (DN); the study aims to evaluate the effect of thymosin ß4 (Tß4), which has apparent anti-inflammatory properties and is capable of improving endothelial dysfunction, in early DN in a mouse model of type 2 diabetes mellitus. KK Cg-Ay/J (KK) mice, aged 12-14 weeks, were divided into the following groups: KK control group that was treated with saline; KK Tß4 group that was treated with Tß4 100 ng/10 g of intraperitoneal injection once a day. Nondiabetic age-matched C57BL mice were used as additional normal control and also treated with Tß4. The urinary albumin/creatinine ratio (ACR), plasma urea nitrogen and creatinine, body weight, fasting blood glucose and 2-hour blood glucose during oral glucose tolerance testing, blood hemoglobin A1c, cholesterol, and triglyceride were determined at baseline time and 12 weeks after Tß4 treatment for phenotypic characterizations. The KK Tß4 group had reduced the mean fasting blood glucose, 2-hour blood glucose during oral glucose tolerance testing, hemoglobin A1c, and triglyceride levels compared with that in the KK control group (P < 0.05). Tß4 treatment markedly reduced ACR (KK Tß4 = 328.54 ± 46.14 mg/g vs. KK control = 540.34 ± 50.31 mg/g, P < 0.05). Tß4 also significantly ameliorated renal pathological changes of KK Tß4 mice as compared with that in KK control mice. Tß4 treatment did not affect glucose homeostasis and urinary ACR and glomeruli of C57BL mice. These data in a novel mouse model of DN suggest that Tß4 may ameliorate renal damage. This peptide may be a novel potential alternative agent for treatment of DN.

Matrix metallopeptidase 9 contributes to the beginning of plaque and is a potential biomarker for the early identification of atherosclerosis in asymptomatic patients with diabetes.

Aims: To determine the roles of matrix metallopeptidase-9 (MMP9) on human coronary artery smooth muscle cells (HCASMCs) in vitro, early beginning of atherosclerosis in vivo in diabetic mice, and drug naïve patients with diabetes. Methods: Active human MMP9 (act-hMMP9) was added to HCASMCs and the expressions of MCP-1, ICAM-1, and VCAM-1 were measured. Act-hMMP9 (n=16) or placebo (n=15) was administered to diabetic KK.Cg-Ay/J (KK) mice. Carotid artery inflammation and atherosclerosis measurements were made at 2 and 10 weeks after treatment. An observational study of newly diagnosed drug naïve patients with type 2 diabetes mellitus (T2DM n=234) and healthy matched controls (n=41) was performed and patients had ultrasound of carotid arteries and some had coronary computed tomography angiogram for the assessment of atherosclerosis. Serum MMP9 was measured and its correlation with carotid artery or coronary artery plaques was determined. Results: In vitro, act-hMMP9 increased gene and protein expressions of MCP-1, ICAM-1, VCAM-1, and enhanced macrophage adhesion. Exogenous act-hMMP9 increased inflammation and initiated atherosclerosis in KK mice at 2 and 10 weeks: increased vessel wall thickness, lipid accumulation, and Galectin-3+ macrophage infiltration into the carotid arteries. In newly diagnosed T2DM patients, serum MMP9 correlated with carotid artery plaque size with a possible threshold cutoff point. In addition, serum MMP9 correlated with number of mixed plaques and grade of lumen stenosis in coronary arteries of patients with drug naïve T2DM. Conclusion: MMP9 may contribute to the initiation of atherosclerosis and may be a potential biomarker for the early identification of atherosclerosis in patients with diabetes. Clinical trial registration: https://clinicaltrials.gov, identifier NCT04424706.

Metformin treatment is associated with an increase in bone mineral density in type 2 diabetes mellitus patients in China: A retrospective single center study.

AIMS: To investigate the association between metformin and bone mineral density (BMD) in a large cohort of Chinese patients with type 2 diabetes mellitus (T2DM). METHODS: A total of 11,458 T2DM patients aged ≥40 years were included. Information on demographic, anthropometric and clinical characteristics was collected from medical records. BMD at lumbar spine (LS), femoral neck (FN), and total hip(TH) was assessed by dual-energy X-ray absorptiometry. RESULTS: Overall prevalence of osteopenia and osteoporosis was 37.4% and 10.3%, and was lower in patients on metformin (34.6% vs 38.3% and 7.1% vs 11.3%, both p < 0.001). Patients who had a lower BMI, older age, and lower estimated glomerular filtration rate (eGFR), had more osteoporosis, lower BMD (osteoporosis or osteopenia), and a lower T-score at LS, FN and TH. Metformin use and male sex was associated with a higher BMD. Metformin treatment was also independently associated with higher T-score at LS, FN and TH (ß values of 0.120, 0.082 and 0.108; all p <0.001), and lower odds ratio of osteoporosis (OR = 0.779, 95%CI: 0.648-0.937, p = 0.008) or low BMD (OR = 0.834, 95%CI: 0.752 - 0.925, p = 0.001). However, when analyzed by sex, this association of a lower odds ratio for osteoporosis with metformin was only significant in women (OR= 0.775, 95% CI: 0.633-0.948; p = 0.013). CONCLUSIONS: Metformin treatment was associated with a higher T-score and a lower odds ratio of osteopenia and osteoporosis, especially in the female population, independent of age, BMI, and eGFR.

Response of blood glucose and GLP-1 to different food temperature in normal subject and patients with type 2 diabetes.

BACKGROUND: Eating behavior is a major factor in type 2 diabetes. We investigated the different responses of glucose-regulating hormones to cold and hot glucose solutions in normal subjects and patients with type 2 diabetes. METHODS: In this crossover, self-controlled study, normal subjects (N = 19) and patients with type 2 diabetes (N = 22) were recruited and randomly assigned to a hot (50 °C) or a cold (8 °C) oral glucose-tolerance test (OGTT). The subsequent day, they were switched to the OGTT at the other temperature. Blood glucose, insulin, GIP, glucagon-like peptide-1 (GLP-1), and cortisol were measured at 0, 5, 10, 30, 60, and 120 min during each OGTT. After the hot OGTT, all subjects ingested hot (>42 °C) food and water for that day, and ingested food and water at room temperature (≤24 °C) for the day after cold OGTT. All participants had continuous glucose monitoring (CGM) throughout the study. RESULTS: Compared to cold OGTT, blood glucose was significantly higher with hot OGTT in both groups (both P < 0.05). However, insulin and GLP-1 levels were significantly higher in hot OGTT in normal subjects only (both P < 0.05). The GIP and cortisol responses did not differ with temperature in both groups. CGM showed that normal subjects had significantly higher 24-h mean glucose (MBG) (6.11 ± 0.13 vs. 5.84 ± 0.11 mmol/L, P = 0.021), and standard deviation of MBG with hot meals (0.59 ± 0.06 vs. 0.48 ± 0.05 mmol/L, P = 0.043), T2DM patients had higher MBG only (8.46 ± 0.38 vs. 8.88 ± 0.39 mmol/L, P = 0.022). CONCLUSIONS: Food temperature is an important factor in glucose absorption and GLP-1 response. These food temperatures elicited differences are lost in type 2 diabetes.

Charting the next century of insulin replacement with cell and gene therapies.

The discovery of insulin a century ago changed the lives of millions of individuals suffering from diabetes, paving the way for long-term survival. While the availability of recombinant insulin for hormone replacement therapy has served extremely well to help control blood glucose in diabetes, there remains significant room for further improvements for an ultimate "cure" for diabetes patients. In this review, we celebrate the 100th anniversary of the discovery of insulin and consolidate the key milestones and advances in the development of recombinant human insulin. We then summarize recent and current technological developments in terms of insulin gene- and cell-replacement therapies that are promising in greater therapeutic potential. We envision that the next era of insulin replacement therapies will effectively treat diabetes and serve our patients even better for the next century to come.

Rapid Changes in Serum Testosterone in Men With Newly Diagnosed Type 2 Diabetes With Intensive Insulin and Metformin.

OBJECTIVE: To investigate the effect of metformin on testosterone levels in men with type 2 diabetes mellitus (T2DM). RESEARCH DESIGN AND METHODS: Seventy men with newly diagnosed drug-naive T2DM and HbA1c >9.0% (75 mmol/mol) were treated with intensive insulin pump therapy for 5 days to achieve glucose normalization. They were randomized to control (continued on intensive insulin only) and metformin (plus metformin) groups (1:1) for 1 month. Testosterone was measured at baseline, randomization, and after 1-month treatment. RESULTS: Total, free, and bioavailable testosterone increased significantly within 5 days (all P < 0.001). After 1 month, compared with the control group, the metformin group had lower total (12.7 vs. 15.3 nmol/L), free (0.20 vs. 0.24 nmol/L), and bioavailable (4.56 vs. 5.31 nmol/L) testosterone (all P < 0.05). CONCLUSIONS: In men with T2DM, 1-month oral metformin may decrease serum testosterone levels independent of blood glucose control. The effects of long-term metformin on testosterone in men need further study.

Flash Glucose Monitoring Improves Glucose Control in People with Type 2 Diabetes Mellitus Receiving Anti-diabetic Drug Medication.

OBJECTIVE: To investigate the effects of Flash Glucose Monitoring (FGM) on glucose profile in people with Type 2 Diabetes Mellitus (T2DM) receiving anti-diabetic drug medication. METHODS: This is a prospective non-randomized uncontrolled study. 111 people with T2DM were enrolled and received FGM for 14 days. There was no change of anti-diabetic medication during the 14 days. The plasma glucose concentration on day 2 was used as baseline and the day 13 was considered as study end point. The parameters to compare were mean plasma glucose (MPG), glucose variations, and incidence of hypoglycemia during the FGM period. The multivariate linear stepwise regression analysis was applied to determine the independent factors that affect MPG difference. RESULTS: This study analyzed the data of a total of 111 people with T2DM (male 60 and female 51). The general clinical data of these patients were as follows: age: 65.0±6.7 years old; duration of diabetes: 11.6±6.8 years; HbA1c: 61.2±13.3 mmol/mol; body mass index (BMI): 25.2±3.2 kg/m². Using FGM, people with T2DM were able to change daily diet and exercise through which significant reductions in MPG on days 12 or 13 were achieved as compared with that of day 2 (P=0.04 or P=0.003, respectively). The glucose variations, such as standard deviation (SD) of plasma glucose, coefficient of variation (CV), and mean amplitude of glycemic excursion (MAGE), progressively declined starting from day 6 as compared with baseline (P=0.016, P=0.003, or P=0.012, respectively). The incremental area over the curve (AOC) of the hypoglycemia (<3.9 mmol/L) had a significant reduction starting from the day 3 (P=0.001). When people with T2DM were divided into 3 groups based on the tertile of HbA1c (high, middle, and low concentrations), the reduction of MPG in patients with high concentration of HbA1c were much larger than that in middle and low concentration group patients (P=0.001 for both). The incidence of hypoglycemia was improved in the low concentration group (P=0.017). The optimal frequency of scanning time required to maintain euglycemia was 11.7 times/day as calculated by the receiver operating characteristic (ROC) analysis. CONCLUSION: Using FGM to monitor glucose concentration at 11.7 times/day, people with T2DM can achieve a better glucose control in addition to anti-diabetic drug medication through changing daily diet and exercise, especially in patients with high concentration of HbA1c (>66.1 mmol/mol).

Association of decreased sperm motility and increased seminal plasma IGF-I, IGF-II, IGFBP-2, and PSA levels in infertile men.

PURPOSE: Previous studies have suggested the involvement of serum insulin-like growth factors (IGFs) and IGF binding proteins (IGFBPs) in the regulation of the female reproductive system. Little is known of these peptides in the seminal plasma (SP) of men and their potential effects on fertility. We assessed SP levels of these peptides in infertile men with low sperm motility (asthenozoospermic; AZ) and low sperm counts (oligozoospermic; OZ), its effects on in vivo sperm motility, and whether there is a correlation with aging. METHODS: Twenty-eight infertile men (AZ; n = 18 and OZ; n = 10) and 20 fertile normozoospermic (NZ) men were studied. Seminal plasma IGF-I, IGF-II, IGFBP-2, IGFBP-3, and prostate-specific antigen (PSA) levels were measured, and spermatozoa mRNA transcript patterns were examined. RESULTS: Asthenozoospermic men had higher SP IGF-I, IGF-II, IGFBP-2, and PSA levels than NZ and OZ men, whereas SP IGFBP-3 levels were similar between the three groups. Sperm count positively correlated with SP IGF-I, IGF-II, and IGFBP-2; sperm motility negatively correlated with SP IGF-II and IGFBP-2; and age correlated positively with SP IGF-II. The expression of IGF-I and IGF-II mRNA and mRNA receptors was detectable, but no variations in transcript levels were noted. CONCLUSION: Decreased sperm motility, but not sperm count, in infertile AZ men is associated with increased SP IGF-I, IGF-II, IGFBP-2, and PSA levels. Changes in SP IGFs and their interactions with IGFBPs and IGF receptors, and PSA levels suggest a role of these SP peptides in modulating sperm motility and possibly prostate disease development in aging men.

Increased thalamo-cortical functional connectivity in patients with diabetic painful neuropathy: A resting-state functional MRI study.

Functional changes in the brain of patients with painful diabetic neuropathy (PDN) have remained largely elusive. The aim of the present study was to explore changes in thalamo-cortical functional connectivity (FC) of patients with PDN using resting-state functional MRI. A total of 20 patients with type 2 diabetes mellitus (T2DM) with non-painful diabetic neuropathy (Group NDN), 19 patients with T2DM with PDN (Group-PDN) and 13 age-, sex- and education-matched healthy controls were recruited. The differences in thalamo-cortical FC among the three groups were compared. Patients in Group PDN had increased FC in the left thalamus, the right angular gyrus and the occipital gyrus as compared to those in Group NDN. Furthermore, patients in Group PDN had increased FC in the right thalamus and angular gyrus as compared to those in Group NDN. In conclusion, the present results suggested that the thalamo-cortical FC is increased in patients with T2DM and PDN. Furthermore, the increased FC in the thalamic-parietal-occipital connectivity may be a central pathophysiological mechanism for PDN. The study was retrospectively registered at ClinicalTrials.gov on 3 October 2018 (identifier no. NCT03700502).

Long-term ethanol exposure inhibits glucose transporter 4 expression via an AMPK-dependent pathway in adipocytes.

AIM: The roles of AMP-activated protein kinase (AMPK) and myocyte enhancer factor 2 isoforms (MEF2A, D) as mediators of the effects of ethanol on glucose transporter 4 (GLUT4) expression are unclear. We studied the effects of ethanol in adipocytes in vivo and in vitro. METHODS: Thirty-six male Wistar rats were divided into three groups and given ethanol in a single daily dose of 0, 0.5, or 5 g/kg for 22 weeks. The expression of AMPK, MEF2 isoforms A and D, and GLUT4 was measured and compared in the three groups. The existence of the AMPK/MEF2/GLUT4 pathway in adipocytes and the effects of ethanol on this pathway were studied in (a) epididymal adipose tissue from six male Wistar rats subcutaneously injected with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR, an AMPK activator) or with 0.9% NaCl (control); and (b) isolated rat and human adipocytes treated with or without ethanol, AICAR, and compound C (a selective AMPK inhibitor). Expression of AMPK, MEF2, and GLUT4 was measured by RT-PCR and Western blotting. RESULTS: (1) Long-term ethanol exposure decreased activated AMPK, MEF2A, MEF2D, and GLUT4 expression in rat adipose tissue. (2) In rat and human adipocytes, AICAR-induced AMPK activation, with subsequent elevation of MEF2 and GLUT4 expression, was inhibited by compound C. (3) In vitro ethanol-treatment suppressed the AMPK/MEF2/GLUT4 pathway. CONCLUSION: The AMPK/MEF2/GLUT4 pathway exists in both rat and human adipocytes, and activated AMPK may positively regulate MEF2 and GLUT4 expression. Ethanol inhibition of this pathway leads to decreased GLUT4 expression, thus reducing insulin sensitivity and glucose tolerance.

Modification of a Constitutive to Glucose-Responsive Liver-Specific Promoter Resulted in Increased Efficacy of Adeno-Associated Virus Serotype 8-Insulin Gene Therapy of Diabetic Mice.

We have previously used a hepatotropic adeno-associated viral (AAV) vector with a modified human insulin gene to treat diabetic mice. The HLP (hybrid liver-specific promoter) used was constitutively active and non-responsive to glucose. In this study, we examined the effects of addition of glucose responsive elements (R3G) and incorporation of a 3' albumin enhancer (3'iALB) on insulin expression. In comparison with the original promoter, glucose responsiveness was only observed in the modified promoters in vitro with a 36 h lag time before the peak expression. A 50% decrease in the number of viral particles at 5 × 109 vector genome (vg)/mouse was required by AAV8-R3GHLP-hINSco to reduce the blood sugar level to near normoglycemia when compared to the original AAV8-HLP-hINSco that needed 1 × 1010 vg/mouse. The further inclusion of an 860 base-pairs 3'iALB enhancer component in the 3' untranslated region increased the in vitro gene expression significantly but this increase was not observed when the packaged virus was systemically injected in vivo. The addition of R3G to the HLP promoter in the AAV8-human insulin vector increased the insulin expression and secretion, thereby lowering the required dosage for basal insulin treatment. This in turn reduces the risk of liver toxicity and cost of vector production.

Contributions of Fasting and Postprandial Glucose Concentrations to Haemoglobin A1c in Drug-Naïve Mal-Glucose Metabolism in Chinese Population Using Continuous Glucose Monitoring System.

AIM: To clarify the contributions of fasting glucose (FG) and postprandial glucose (PG) to HbA1c in drug-naïve patients with type 2 diabetes (T2D) and impaired glucose tolerate (IGT)/impaired fasting glucose (IFG). METHODS: Continuous glucose monitoring (CGM) was performed in 305 drug-naïve Chinese patients with T2D or IGT/IFG. The incremental area under the curve (AUC) above a glucose value of 6.1 mmol/L or FG glucose levels were calculated to evaluate the contributions of PG or FG to HbA1c values. RESULTS: According to quintiles of HbA1c, T2D patients were divided into five groups (group 1 to 5), and patients with IGT/IFG were assigned into group 0. PG was the predominant contributor in the lower groups with HbA1c 4.9∼6.0% and 6.1∼7.8%. The relative contributions of FG and PG to HbA1c had no significance in the middle groups of HbA1c (7.9∼8.7% and 8.8∼9.5%). FG contributed significantly more than PG in the higher groups of HbA1c (9.6∼10.9% and 11.0∼14.6%). Regression analyses indicate that the contributions of FG and PG were equal (both 50%) when the level of HbA1c was 8.5%. CONCLUSIONS: In drug-naïve patients with T2D or IGT/IFG, PG contributed more in patients with HbA1c < 8.5%, whereas FG became the predominant contributor in the poorly controlled patients with HbA1c ≥ 8.5%. These results may help the health-care provider set appropriate plasma glucose testing goals with the expectation of achieving specific HbA1c values.