Diabetic rats with high levels of endogenous dopamine do not show retinal vascular pathology.

Purpose: Limited research exists on the time course of long-term retinal and cerebral deficits in diabetic rodents. Previously, we examined short term (4-8 weeks) deficits in the Goto-Kakizaki (GK) rat model of Type II diabetes. Here, we investigated the long-term (1-8 months) temporal appearance of functional deficits (retinal, cognitive, and motor), retinal vascular pathology, and retinal dopamine levels in the GK rat. Methods: In GK rats and Wistar controls, retinal neuronal function (electroretinogram), cognitive function (Y-maze), and motor function (rotarod) were measured at 1, 2, 4, 6, and 8 months of age. In addition, we evaluated retinal vascular function (functional hyperemia) and glucose and insulin tolerance. Retinas from rats euthanized at ≥8 months were assessed for vascular pathology. Dopamine and DOPAC levels were measured via HPLC in retinas from rats euthanized at 1, 2, 8, and 12 months. Results: Goto-Kakizaki rats exhibited significant glucose intolerance beginning at 4 weeks and worsening over time (p < 0.001). GK rats also showed significant delays in flicker and oscillatory potential implicit times (p < 0.05 to p < 0.001) beginning at 1 month. Cognitive deficits were observed beginning at 6 months (p < 0.05), but no motor deficits. GK rats showed no deficits in functional hyperemia and no increase in acellular retinal capillaries. Dopamine levels were twice as high in GK vs. Wistar retinas at 1, 2, 8, and 12 months (p < 0.001). Conclusion: As shown previously, retinal deficits were detectable prior to cognitive deficits in GK rats. While retinal neuronal function was compromised, retinal vascular pathology was not observed, even at 12+ months. High endogenous levels of dopamine in the GK rat may be acting as an anti-angiogenic and providing protection against vascular pathology.

Novel Detection and Restorative Levodopa Treatment for Preclinical Diabetic Retinopathy.

Diabetic retinopathy (DR) is diagnosed clinically by directly viewing retinal vascular changes during ophthalmoscopy or through fundus photographs. However, electroretinography (ERG) studies in humans and rodents have revealed that retinal dysfunction is demonstrable prior to the development of visible vascular defects. Specifically, delays in dark-adapted ERG oscillatory potential (OP) implicit times in response to dim-flash stimuli (<-1.8 log cd · s/m2) occur prior to clinically recognized DR. Animal studies suggest that retinal dopamine deficiency underlies these early functional deficits. In this study, we randomized individuals with diabetes, without clinically detectable retinopathy, to treatment with either low- or high-dose Sinemet (levodopa plus carbidopa) for 2 weeks and compared their ERG findings with those of control subjects (no diabetes). We assessed dim-flash-stimulated OP delays using a novel handheld ERG system (RETeval) at baseline and 2 and 4 weeks. RETeval recordings identified significant OP implicit time delays in individuals with diabetes without retinopathy compared with age-matched control subjects (P < 0.001). After 2 weeks of Sinemet treatment, OP implicit times were restored to control values, and these improvements persisted even after a 2-week washout. We conclude that detection of dim-flash OP delays could provide early detection of DR and that Sinemet treatment may reverse retinal dysfunction.

Retinal Deficits Precede Cognitive and Motor Deficits in a Rat Model of Type II Diabetes.

Purpose: To investigate the temporal appearance of retinal, cognitive, and motor deficits in Goto-Kakizaki (GK) rats, a spontaneously occurring, polygenic model of type II diabetes. GK rats develop impaired insulin secretion at 2 weeks and fasting hyperglycemia at 4 weeks. Methods: In male and female GK rats and Wistar controls, glucose tolerance test (hyperglycemia) and electroretinogram (ERG, retinal function) were performed at 4 and 8 weeks of age. Spectral domain-optical coherence tomography (retinal structure) was assessed at 6 weeks. Spatial alternation (cognitive function) and number of entries (exploratory behavior) were assessed via Y-maze at 4, 5, 6, 7, and 8 weeks. Rotarod (motor function) was performed at 4, 6, and 8 weeks. Results: By 4 weeks, the GK rats exhibited significant glucose intolerance (P < 0.001) and retinal deficits, including delays in ERG implicit times (flicker, P < 0.01; oscillatory potentials, P < 0.001). In addition, the GK rats showed greater ERG amplitudes (P < 0.001) and thinner retinas (P < 0.001). At 7 weeks, the GK rats showed deficits in cognitive function (P < 0.001) and exploratory behavior (P < 0.01). However, no motor function deficits were observed by 8 weeks. Interestingly, the male GK rats showed greater hyperglycemia (P < 0.05), but the female rats showed greater ERG delays (P < 0.001). Conclusions: In GK rats, retinal function deficits developed prior to cognitive or motor deficits. Future studies will investigate common mechanistic links, long-term functional and vascular changes, and whether early retinal deficits can predict cognitive dysfunction or late-stage retinal disease.

Dopamine Deficiency Mediates Early Rod-Driven Inner Retinal Dysfunction in Diabetic Mice.

Purpose: Electroretinograms (ERGs) are abnormal in diabetic retinas before the appearance of vascular lesions, providing a possible biomarker for diabetic vision loss. Previously, we reported that decreased retinal dopamine (DA) levels in diabetic rodents contributed to early visual and retinal dysfunction. In the current study, we examined whether oscillatory potentials (OPs) could serve as a potential marker for detecting early inner retinal dysfunction due to retinal DA deficiency. Methods: Retinal function was tested with dark-adapted ERGs, taken at 3, 4, and 5 weeks after diabetes induction with streptozotocin. Electrical responses were analyzed and correlations were made with previously reported retinal DA levels. The effect of restoring systemic DA levels or removing DA from the retina in diabetic mice on OPs was assessed using L-3,4-dihydroxyphenylalanine (L-DOPA) treatments and retina-specific tyrosine hydroxylase (Th) knockout mice (rTHKO), respectively. Results: Diabetic animals had significantly delayed OPs compared to control animals in response to dim, but not bright, flash stimuli. L-DOPA treatment preserved OP implicit time in diabetic mice. Diabetic rTHKO mice had further delayed OPs compared to diabetic mice with normal retinal Th, with L-DOPA treatment also providing benefit. Decreasing retinal DA levels significantly correlated with increasing OP delays mediated by rod pathways. Conclusions: Our data suggest that inner retinal dysfunction in early-stage diabetes is mediated by rod-pathway deficits and DA deficiencies. OP delays may be used to determine the earliest functional deficits in diabetic retinopathy and to establish an early treatment window for DA therapies that may prevent progressive vision loss.

mRNA destabilization improves glycemic responsiveness of transcriptionally regulated hepatic insulin gene therapy in vitro and in vivo.

BACKGROUND: Hepatic insulin gene therapy (HIGT) employing a glucose and insulin sensitive promoter to direct insulin transcription can lower blood sugars within 2 h of an intraperitoneal glucose challenge. However, post-challenge blood sugars frequently decline to below baseline. We hypothesize that this 'over-shoot' hypoglycemia results from sustained translation of long-lived transgene message, and that reducing pro-insulin message half-life will ameliorate post-challenge hypoglycemia. METHODS: We compared pro-insulin message content and insulin secretion from primary rat hepatocytes expressing insulin from either a standard construct (2xfur), or a construct producing a destabilized pro-insulin message (InsTail), following exposure to stimulating or inhibitory conditions. RESULTS: Hepatocytes transduced with a 2xfur construct accumulated pro-insulin message, and exhibited increased insulin secretion, under conditions that both inhibit or stimulate transcription. By contrast, pro-insulin message content remained stable in InsTail expressing cells, and insulin secretion increased less than 2xfur during prolonged stimulation. During transitions from stimulatory to inhibitory conditions, or vice versa, amounts of pro-insulin message changed more rapidly in InsTail expressing cells than 2xfur expressing cells. Importantly, insulin secretion increased during the transition from stimulation to inhibition in 2xfur expressing cells, although it remained unchanged in InsTail expressing cells. Use of the InsTail destabilized insulin message tended to more rapidly reduce glucose induced glycemic excursions, and limit post-load hypoglycemia in STZ-diabetic mice in vivo. CONCLUSIONS: The data obtained in the present study suggest that combining transcriptional and post-transcriptional regulatory strategies may reduce undesirable glycemic excursion in models of HIGT.

Hepatic insulin gene therapy prevents diabetic enteropathy in STZ-treated CD-1 mice.

Depending on the population examined, from 6 to 83% of people with diabetes mellitus exhibit symptoms of altered gut motility, manifesting as dysphagia, reflux, early satiety, nausea, abdominal pain, diarrhea, or constipation. Hyperglycemia-induced cell loss within the enteric nervous system has been demonstrated in both diabetic rodents and patients with diabetes. Glycemic control is recommended to prevent diabetic gastroenteropathy but is often difficult to achieve with current treatment modalities. We asked if hepatic insulin gene therapy (HIGT) could inhibit the development of diabetic gastroenteropathy in mice. Bowel length, bowel transit, colonic muscle relaxation, and the numbers of both stimulatory and inhibitory neurons in the colonic myenteric plexus were compared in groups of diabetic mice (DM), control nondiabetic mice (Con), and diabetic mice treated with HIGT (HIGT). Delivery of a metabolically responsive insulin transgene to the liver of STZ-diabetic mice with an adeno-associated virus, sero-type 8 (AAV8) produced near-normal blood sugars for over 1 month and prevented anatomic, functional, and neurohistologic changes observed in diabetic mice. We conclude that in addition to normalizing oxidative metabolism in diabetic rodents, HIGT is sufficient to prevent the development of diabetic gastroenteropathy.

In Vivo Imaging of Retinal Oxidative Stress Using a Reactive Oxygen Species-Activated Fluorescent Probe.

PURPOSE: In vivo methods for detecting oxidative stress in the eye would improve screening and monitoring of the leading causes of blindness: diabetic retinopathy, glaucoma, and age-related macular degeneration. METHODS: To develop an in vivo biomarker for oxidative stress in the eye, we tested the efficacy of a reactive oxygen species (ROS)-activated, near-infrared hydrocyanine-800CW (H-800CW) fluorescent probe in light-induced retinal degeneration (LIRD) mouse models. After intravitreal delivery in LIRD rats, fluorescent microscopy was used to confirm that the oxidized H-800CW appeared in the same retinal layers as an established ROS marker (dichlorofluorescein). RESULTS: Dose-response curves of increasing concentrations of intravenously injected H-800CW demonstrated linear increases in both intensity and total area of fundus hyperfluorescence in LIRD mice, as detected by scanning laser ophthalmoscopy. Fundus hyperfluorescence also correlated with the duration of light damage and functional deficits in vision after LIRD. In LIRD rats with intravitreal injections of H-800CW, fluorescent labeling was localized to photoreceptor inner segments, similar to dichlorofluorescein. CONCLUSIONS: Hydrocyanine-800CW detects retinal ROS in vivo and shows potential as a novel biomarker for ROS levels in ophthalmic diseases.

Long-term glycemic control with hepatic insulin gene therapy in streptozotocin-diabetic mice.

BACKGROUND: Insulin self-administration is burdensome and can produce dangerous hypoglycemia. Insulin gene therapy may improve and simplify the treatment of diabetes mellitus. In rats, metabolically responsive hepatic insulin gene therapy (HIGT) delivered by adenovirus normalizes random blood sugars but with a limited duration. To prolong glycemic control, we delivered a metabolically regulated insulin transgene by adeno-associated virus (AAV). METHODS: We administered increasing doses of self-complementary (SC), pseudotyped AAV8 expressing the (GlRE)3 BP1-2xfur insulin transgene to streptozotocin-diabetic CD-1 mice, and monitored blood sugar and body weight. We also compared responses to intraperitoneal glucose and chow withdrawal, assessed for viral genomes in liver by Southern blotting, and measured hepatic glycogen. RESULTS: Glucose lowering required the combination of SC genomes and AAV capsid pseudotyping. HIGT controlled glycemia in diabetic mice (DM) for > 1 year. However, glycemic responses were variable. Approximately 30% of mice succumbed to hypoglycemia, and approximately 30% of mice again became hyperglycemic. During an intraperitoneal glucose tolerance test, blood sugars declined to normal within 180 min in HIGT-treated DM compared to 90 min in control mice. Hypoglycemia was common among HIGT-treated mice during a 24-h fast. However, HIGT mice lost less weight than either diabetic or nondiabetic controls as a result of increased water intake. HIGT treatment reduced the hepatic glycogen content of fed mice. CONCLUSIONS: Our studies demonstrate the possibility for long-term glycemic correction following AAV-mediated HIGT in mice. However, the dose-response relationship is irregular, and metabolic responsiveness may be less than that observed in rats.

Engineered VEGF-releasing PEG-MAL hydrogel for pancreatic islet vascularization.

Biofunctionalized polyethylene glycol maleimide (PEG-MAL) hydrogels were engineered as a platform to deliver pancreatic islets to the small bowel mesentery and promote graft vascularization. VEGF, a potent stimulator of angiogenesis, was incorporated into the hydrogel to be released in an on-demand manner through enzymatic degradation. PEG-MAL hydrogel enabled extended in vivo release of VEGF. Isolated rat islets encapsulated in PEG-MAL hydrogels remained viable in culture and secreted insulin. Islets encapsulated in PEG-MAL matrix and transplanted to the small bowel mesentery of healthy rats grafted to the host tissue and revascularized by 4 weeks. Addition of VEGF release to the PEG-MAL matrix greatly augmented the vascularization response. These results establish PEG-MAL engineered matrices as a vascular-inductive cell delivery vehicle and warrant their further investigation as islet transplantation vehicles in diabetic animal models.

Rodent Hyperglycemia-Induced Inner Retinal Deficits are Mirrored in Human Diabetes.

PURPOSE: To evaluate the utility of low luminance stimuli to functionally probe inner retinal rod pathways in the context of diabetes mellitus in both rat and human subjects. METHODS: Inner retinal dysfunction was assessed using oscillatory potential (OP) delays in diabetic rats. Scotopic electroretinograms (ERGs) in response to a series of increasing flash luminances were recorded from streptozotocin (STZ)-treated and control Sprague-Dawley rats after 7, 14, 20, and 29 weeks of hyperglycemia. We then evaluated OP delays in human diabetic subjects with (DR) and without (DM) diabetic retinopathy using the International Society for Clinical Electrophysiology in Vision (ISCEV) standard scotopic protocol and two additional dim test flashes. RESULTS: Beginning 7 weeks after STZ, OP implicit times in diabetic rats were progressively delayed in response to dim, but not bright stimuli. In many diabetic subjects the standard ISCEV dim flash failed to illicit measureable OPs. However, OPs became measurable using a brighter, nonstandard dim flash (Test Flash 1, -1.43 log cd s/m2), and exhibited prolonged implicit times in the DM group compared with control subjects (CTRL). CONCLUSIONS: Delays in scotopic OP implicit times are an early response to hyperglycemia in diabetic rats. A similar, inner retinal, rod-driven response was detected in diabetic human subjects without diabetic retinopathy, only when a nonstandard ISCEV flash intensity was employed during ERG testing. TRANSLATIONAL RELEVANCE: The addition of a dim stimulus to standard ISCEV flashes with assessment of OP latency during ERG testing may provide a detection method for early retinal dysfunction in diabetic patients.

Therapeutic effects of a non-ß cell bioartificial pancreas in diabetic mice.

BACKGROUND: Cell-based insulin therapies can potentially improve glycemic regulation in insulin-dependent diabetic patients. Enteroendocrine cells engineered to secrete recombinant insulin have exhibited glycemic efficacy, but have been primarily studied as uncontrollable growth systems in immune incompetent mice. Furthermore, reports suggest that suboptimal insulin secretion remains a barrier to expanded application. METHODS: Genetic and tissue engineering strategies were applied to improve recombinant insulin secretion from intestinal L-cells on both a per-cell and per-graft basis. Transduction of insulin-expressing GLUTag L-cells with lentivirus carrying an additional human insulin gene-enhanced secretion twofold. We infected cells with lentivirus expressing a luciferase reporter gene to track cell survival in vivo. To provide a growth-controlled and immune protective environment without affecting secretory capacity, cells were microencapsulated in barium alginate. Approximately 9×10(7) microencapsulated cells were injected intraperitoneally in immune competent streptozotocin-induced diabetic mice for therapeutic efficacy evaluation. RESULTS: Graft insulin secretion was increased to 16 to 24 mU insulin per day. Transient normoglycemia was achieved in treated mice two days after transplantation, and endogenous insulin was sufficient to sustain body weights of treated mice receiving minimal supplementation. CONCLUSION: Glycemic efficacy of a bioartificial pancreas based on insulin-secreting enteroendocrine cells is insufficient as a standalone therapy, despite enhancement of graft insulin secretion capacity. Supplemental strategies to alleviate secretion limitations should be pursued.

Engineered insulin secretion from neuroendocrine cells isolated from human thyroid.

BACKGROUND: Insulin-secreting beta-like cells are vulnerable to diabetic autoimmunity. We hypothesized that human thyroid neuroendocrine (NE) cells could be engineered to secrete human insulin, be glucose-responsive, and avoid autoimmunity. METHODS: Collagenase-digested thyroid tissue was cultured and subjected to size-based fluorescence-activated cell sorting. Insulin secretion and storage in NE cells transduced with viral vectors carrying an insulin sequence was assessed by enzyme-linked immunosorbent assay (ELISA) and immunogold transmission electron microscopy (TEM). Baseline mRNA expression was assessed by Illumina expression array analysis. Transduction with retrovirus expressing transcription factors PDX1, NGN3, MAFA, or HNF6 altered mRNA expression in a custom polymerase chain reaction (PCR) array. Gastrin-releasing peptide (GRP) in conditioned medium and cell lysates was determined by reverse transcription (RT)-PCR, ELISA, and immunohistochemistry. RESULTS: Isolation yielded an average of 2.2 × 10(6) cells/g thyroid tissue, which stained for calcitonin/calcitonin gene-related protein, expressed genes consistent with NE origins, and secreted GRP. Transduced cells secreted 56 % and retained 48 % of total insulin produced. Immunogold TEM revealed insulin in secretory vesicles. PDX1, NGN3, and MAFA overexpression increased expression of genes typical for hepatocytes and beta cells. Overexpression of HNF6 also increased the message of genes critical for glucose sensing. CONCLUSIONS: Human thyroid NE cells can produce human insulin, fractions of which are both secreted and retained in secretory granules. Overexpression of HNF6, PDX1, or NGN3 enhances expression of both hepatocyte and beta cell typical mRNAs, including the message of proteins critical for glucose sensing. These data suggest that reimplantation of engineered autologous NE cells may develop as a viable treatment for diabetes mellitus type 1.

Sulfonylureas: a new look at old therapy.

Sulfonylurea compounds were the first available oral antidiabetic agents and they remain an important tool in our quest for optimal glucose control. The sulfonylureas stimulate the release of insulin from pancreatic ß-cells and have a number of extrapancreatic effects, including decreasing hepatic insulin clearance and reducing glucagon secretion in patients with type 2 diabetes. Although these agents have been the mainstay of pharmacotherapy for patients with type 2 diabetes mellitus (T2DM), their safety and clinical utility has been a matter of active debate in recent years, as their use is associated with risks of hypoglycemia and weight gain. We review the discovery and mechanisms of action of sulfonylureas, and the results of clinical trials to provide practical information on the pros and cons of their use in clinical practice. This review addresses advances in our understanding of mechanisms of action of sulfonylurea agents, their efficacy in T2DM, side effects, and impact on cardiovascular disease outcomes.

Dopamine deficiency contributes to early visual dysfunction in a rodent model of type 1 diabetes.

Dopamine (DA) functions as an essential neuromodulator in the brain and retina such that disruptions in the dopaminergic system are associated with common neurologic disorders such as Parkinson's disease. Although a reduction in DA content has been observed in diabetes, its effects in the development of diabetes-induced neuropathy remains unknown. Because the retina is rich in DA and has a well known diabetes-induced pathology (diabetic retinopathy or DR), this study was designed to examine the role of retinal DA deficiency in early visual defects in DR. Using rodent models of type 1 diabetes mellitus, we investigated whether diabetes caused a reduction in retinal DA content in both rats and mice and determined whether restoring DA levels or activating specific DA receptor pathways could improve visual function (evaluated with optokinetic tracking response) of diabetic mice, potentially via improvement of retinal function (assessed with electroretinography). We found that diabetes significantly reduced DA levels by 4 weeks in rats and by 5 weeks in mice, coincident with the initial detection of visual deficits. Treatment with l-DOPA, a DA precursor, improved overall retinal and visual functions in diabetic mice and acute treatment with DA D1 or D4 receptor agonists improved spatial frequency threshold or contrast sensitivity, respectively. Together, our results indicate that retinal DA deficiency is an underlying mechanism for early, diabetes-induced visual dysfunction and suggest that therapies targeting the retinal dopaminergic system may be beneficial in early-stage DR.

Vasculogenic bio-synthetic hydrogel for enhancement of pancreatic islet engraftment and function in type 1 diabetes.

Type 1 diabetes (T1DM) affects one in every 400 children and adolescents in the US. Due to the limitations of exogenous insulin therapy and whole pancreas transplantation, pancreatic islet transplantation has emerged as a promising therapy for T1DM. However, this therapy is severely limited by donor islet availability and poor islet engraftment and function. We engineered an injectable bio-synthetic, polyethylene glycol-maleimide hydrogel to enhance vascularization and engraftment of transplanted pancreatic islets in a mouse model of T1DM. Controlled presentation of VEGF-A and cell-adhesive peptides within this engineered material significantly improved the vascularization and function of islets delivered to the small bowel mesentery, a metabolically relevant site for insulin release. Diabetic mice receiving islets transplanted in proteolytically degradable hydrogels incorporating VEGF-A exhibited complete reversal of diabetic hyperglycemia with a 40% reduction in the number of islets required. Furthermore, hydrogel-delivered islets significantly improved weight gain, regulation of a glucose challenge, and intra-islet vascularization and engraftment compared to the clinical standard of islet infusion through the hepatic portal vein. This study establishes a simple biomaterial strategy for islet transplantation to promote enhanced islet engraftment and function.

Early visual deficits in streptozotocin-induced diabetic long evans rats.

PURPOSE: Although diabetic retinopathy (DR) is clinically diagnosed based on vascular pathology, diabetic patients with angiographically normal retinas have been found to exhibit subtle defects in vision. This has led to the theory that diabetes-associated metabolic abnormalities directly impair neural retinal function before the development of vasculopathy, thereby resulting in visual deficits. In this study, we sought to delineate the temporal relationship between retinal dysfunction and visual deficits in a rat model of Type 1 diabetes. Moreover, we investigated the relative contribution of retinal dysfunction versus diabetes-induced lens opacity, to the visual deficits found in early-stage DR. METHODS: Pigmented Long Evans rats were rendered diabetic with streptozotocin (STZ). Control and diabetic rats were assessed across 12 weeks of hyperglycemia for visual function with optokinetic tracking weekly visual acuity and monthly contrast sensitivity, retinal function with dark-adapted electroretinograms (monthly electroretinograms [ERGs]), and cataract formation with slit lamp exam (biweekly). RESULTS: Diabetic rats exhibited significantly reduced visual function and delayed ERG responses by 1 month post-STZ. Significant cataracts did not develop until 6 weeks post-STZ. Moreover, increases in lens opacity (r = -0.728) and ERG implicit times (r = -0.615 for rod-dominated response and r = -0.322 for rod/cone mixed response) showed significant correlations with reductions in visual acuity in diabetic rats. CONCLUSIONS: STZ-induced hyperglycemia reduces visual function, affecting both visual acuity and contrast sensitivity. The data suggest that visual defects found in early-stage DR may initially involve abnormalities of the neural retina and worsen with later development of cataracts.

Mechanisms of current therapies for diabetes mellitus type 2.

The array of medications available for the treatment of hyperglycemia has increased rapidly in the previous decade, and recent investigations have clarified novel mechanisms underlying the antihyperglycemic efficacy of these drugs. This article reviews the mechanisms of action for medications currently approved to treat diabetes mellitus in the United States, with the exception of insulin and its analogs. Finally, it attempts to integrate these mechanisms into the schema of pathophysiological factors that combine to produce hyperglycemia in patients with diabetes mellitus.

Combinatorial insulin secretion dynamics of recombinant hepatic and enteroendocrine cells.

One of the most promising cell-based therapies for combating insulin-dependent diabetes entails the use of genetically engineered non-ß cells that secrete insulin in response to physiologic stimuli. A normal pancreatic ß cell secretes insulin in a biphasic manner in response to glucose. The first phase is characterized by a transient stimulation of insulin to rapidly lower the blood glucose levels, which is followed by a second phase of insulin secretion to sustain the lowered blood glucose levels over a longer period of time. Previous studies have demonstrated hepatic and enteroendocrine cells to be appropriate hosts for recombinant insulin expression. Due to different insulin secretion kinetics from these cells, we hypothesized that a combination of the two cell types would mimic the biphasic insulin secretion of normal ß cells with higher fidelity than either cell type alone. In this study, insulin secretion experiments were conducted with two hepatic cell lines (HepG2 and H4IIE) transduced with 1 of 3 adenoviruses expressing the insulin transgene and with a stably transfected recombinant intestinal cell line (GLUTag-INS). Insulin secretion was stimulated by exposing the cells to glucose only (hepatic cells), meat hydrolysate only (GLUTag-INS), or to a cocktail of the two secretagogues. It was found experimentally that the recombinant hepatic cells secreted insulin in a more sustained manner, whereas the recombinant intestinal cell line exhibited rapid insulin secretion kinetics upon stimulation. The insulin secretion profiles were computationally combined at different cell ratios to arrive at the combinatorial kinetics. Results indicate that combinations of these two cell types allow for tuning the first and second phase of insulin secretion better than either cell type alone. This work provides the basic framework in understanding the secretion kinetics of the combined system and advances it towards preclinical studies.

[Effects of hepatic insulin gene therapy on enteric neuropathy in STZ-diabetic mice].

OBJECTIVE: To evaluate the effect of hepatic insulin gene therapy on diabetic enteric neuropathy. METHODS: Mice were randomly allocated into 3 groups: a normal control group, a diabetic group, and a diabetic gene therapy group. Diabetes were induced by penial vein injection of streptozocin (STZ). The gene therapy group received hepatic insulin gene therapy while the other 2 groups only received an empty virus expressing green fluorescent protein. Random blood glucose, body weight growth, gastric emptying, total bowel length, absolute and relative bowel transit, electric field stimulation of colon smooth muscle, colon nuclei staining and counting were measured. RESULTS: We successully established a mouse model of diabetic enteric neuropathy which manifests as: 8 weeks of continuous hyperglycemia,increased total bowel length, decreased relative bowel transit, impaired colon smooth muscle relaxation and loss of inhibitory neurons in colon. Through gene therapy, the above indexes were normalized or ameliorated, suggesting hepatic insulin gene therapy is capable of preventing diabetic enteric neuropathy. CONCLUSION: Hepatic insulin gene therapy can prevent STZ induced diabetic enteric neuropathy.

Effects of common anesthetics on eye movement and electroretinogram.

High-resolution magnetic resonance imaging (MRI) provides non-invasive images of retinal anatomy, physiology, and function with depth-resolved laminar resolution. Eye movement and drift, however, could limit high spatial resolution imaging, and anesthetics that minimize eye movement could significantly attenuate retinal function. The aim of this study was to determine the optimal anesthetic preparations to minimize eye movement and maximize visual-evoked retinal response in rats. Eye movements were examined by imaging of the cornea with a charge-coupled device (CCD) camera under isoflurane, urethane, ketamine/xylazine, and propofol anesthesia at typical dosages in rats. Combination of the paralytic pancuronium bromide with isoflurane or ketamine/xylazine anesthesia was also examined for the eye movement studies. Visual-evoked retinal responses were evaluated using full-field electroretinography (ERG) under isoflurane, ketamine/xylazine, urethane, and ketamine/xylazine + pancuronium anesthesia in rats. The degree of eye movement, measured as displacement per unit time, was the smallest under 1% isoflurane + pancuronium anesthesia. The ketamine/xylazine groups showed larger dark-adapted ERG a- and b-waves than other anesthetics tested. The isoflurane group showed the shortest b-wave implicit times. Photopic ERGs in the ketamine/xylazine groups showed the largest b-waves with the isoflurane group showing slightly shorter implicit times at the higher flash intensities. Oscillatory potentials revealed an early peak in the isoflurane group compared with ketamine/xylazine and urethane groups. Pancuronium did not affect the a- and b-wave, but did increase oscillatory potential amplitudes. Compared with the other anesthetics tested here, ketamine/xylazine + pancuronium was the best combination to minimize eye movement and maximize retinal function. These findings should set the stage for further development and application of high-resolution functional imaging techniques, such as MRI, to study retinal anatomy, physiology, and function in anesthetized rats.