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.

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.

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.

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.

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.

Bioartificial matrices for therapeutic vascularization.

Therapeutic vascularization remains a significant challenge in regenerative medicine applications. Whether the goal is to induce vascular growth in ischemic tissue or scale up tissue-engineered constructs, the ability to induce the growth of patent, stable vasculature is a critical obstacle. We engineered polyethylene glycol-based bioartificial hydrogel matrices presenting protease-degradable sites, cell-adhesion motifs, and growth factors to induce the growth of vasculature in vivo. Compared to injection of soluble VEGF, these matrices delivered sustained in vivo levels of VEGF over 2 weeks as the matrix degraded. When implanted subcutaneously in rats, degradable constructs containing VEGF and arginine-glycine-aspartic acid tripeptide induced a significant number of vessels to grow into the implant at 2 weeks with increasing vessel density at 4 weeks. The mechanism of enhanced vascularization is likely cell-demanded release of VEGF, as the hydrogels may degrade substantially within 2 weeks. In a mouse model of hind-limb ischemia, delivery of these matrices resulted in significantly increased rate of reperfusion. These results support the application of engineered bioartificial matrices to promote vascularization for directed regenerative therapies.

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.

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.

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.

MRI reveals differential regulation of retinal and choroidal blood volumes in rat retina.

The retina is nourished by two unique (retinal and choroidal) circulations. The lack of depth-resolved blood volume (BV) imaging techniques hampers investigation of vascular-specific regulation of the retina in vivo. This study presents a high-resolution, laminar-specific magnetic resonance imaging (MRI) study to image retinal and choroidal BVs, their responses to physiologic challenges in normal and Royal-College-of-Surgeons (RCS) rats (a model of retinal degeneration). Retinal and choroidal BVs were imaged by MRI (30×30×800 µm) with intravascular administration of monocrystalline iron oxide nanocolloid (MION) contrast agent. Relative baseline BV and BV changes due to physiologic challenges were calculated in normal and RCS rat retinas. BV-MRI revealed two well-resolved retinal and choroidal vascular layers located on either side of the retina and an intervening avascular layer. The ratio of choroidal:retinal BV in normal rats at baseline was 9.8±3.2 in control rat retinas (N=7). Hyperoxia decreased retinal BV (-51±17%, p<0.05) more than choroidal BV (-28±14%), and hypercapnia increased retinal BV (52±11%, p<0.01) more than choroidal BV (12±11%). BV-MRI in degenerated retinas of RCS rats (N=7) revealed thinning of the avascular layer and an increase in relative baseline retinal and choroidal BVs. Only hypercapnia-induced BV changes in the retinal vasculature of RCS rats were significantly different (smaller) from controls (p<0.05). These findings suggest that BV in both retinal vasculatures is regulated. The relative baseline BV in both vasculatures increased in retinal degeneration. BV-MRI provides clinically relevant data that may prove useful for early detection and longitudinal probing of retinal diseases, and could complement optical imaging techniques.

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.

[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.

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.

Hepatic insulin gene therapy diminishes liver glycogen despite insulin responsive transcriptional effects in diabetic CD-1 mice.

BACKGROUND: Hepatic insulin gene therapy (HIGT) produces near-normal glycemia in diabetic rats. Hepatic insulin production is expected to stimulate glycogen storage. However, the effect of HIGT on hepatic glycogen metabolism in vivo is unknown. METHODS: After administration of an adenoviral vector capable of inducing glucose responsive insulin production from hepatocytes, we evaluated circulating hormones, cytokines, hepatic gene expression and hepatic glycogen content in diabetic CD-1 mice receiving intravenous streptozotocin. Nondiabetic mice and diabetic mice treated with empty adenovirus served as controls. RESULTS: Peripheral concentrations of human insulin in HIGT mice were less than concentrations of mouse insulin among controls. However, expression of insulin responsive genes in HIGT livers indicated a significant intra-hepatic insulin effect, with expression changes reflecting appropriate responses to fed-fasting transitions. Transcription factors (hepatocyte nuclear factor-4alpha and peroxisome proliferator-activated receptor gamma co-activator-1alpha), as well as target genes (phospho-enol-pyruvate carboxykinase, glucose-6-phosphatase and glucokinase) exhibited insulin responsive expression. Despite producing near normal glycemia, HIGT diminished hepatic glycogen content in both fasted and fed mice. Serum cytokine responses revealed both vector-related (monocyte chemoatractant protein-1, interleukin-6) and transgene specific (resistin, tumor necrosis factor alpha) effects. CONCLUSIONS: HIGT produces low circulating concentrations of insulin, but produces significant intra-hepatic effects on gene expression. Despite controlling hyperglycemia, HIGT exerts unexpected insulin effects on hepatic carbohydrate metabolism. Although the precise mechanisms remain to be determined, they may relate to vector-induced cytokine effects.

Hepatic insulin gene therapy normalizes diurnal fluctuation of oxidative metabolism in diabetic BB/Wor rats.

Previous studies of hepatic insulin gene therapy (HIGT) focused on glycemic effects of insulin produced from hepatocytes. In this study, we extend the observations of glycemic control with metabolically regulated HIGT to include systemic responses and whole-body metabolism. An insulin transgene was administered with an adenoviral vector [Ad/(GlRE)(3)BP1-2xfur] to livers of BB/Wor rats made diabetic with polyinosinic polycytidilic acid (poly-I:C) (HIGT group), and results compared with nondiabetic controls (non-DM), and diabetic rats receiving different doses of continuous-release insulin implants (DM-low BG and DM-high BG). Blood glucose and growth normalized in HIGT, with lower systemic insulin levels, elevated glucagon, and increased heat production compared with non-DM. Minimal regulation of systemic insulin levels were observed with HIGT, yet the animals maintained normal switching from carbohydrate to lipid metabolism determined by respiratory quotients (RQs), and tolerated 24-hour fasts without severe hypoglycemia. HIGT did not restore serum lipids as we observed increased triglycerides (TGs) and increased free fatty acids, but reduced weight of visceral fat pads despite normal total body fat content and retroperitoneal fat depots. HIGT favorably affects blood glucose, normalizes metabolic switching in diabetic rats, and reduces intra-abdominal fat deposition.

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.

Gene transfer to induce insulin production for the treatment of diabetes mellitus.

BACKGROUND: Gene transfer can induce insulin production from non-beta-cells. Multiple gene transfer protocols have demonstrated efficacy correcting diabetes-associated hyperglycemia and growth abnormalities in vivo. OBJECTIVES: To review the literature reporting induction of insulin secretion from non-beta-cells by gene transfer. METHODS: Database search of literature in Ovid Medline. RESULTS/CONCLUSIONS: Gene transfer for the treatment of diabetes mellitus has advanced significantly, but remains premature for clinical translation. Approaches inducing metaplasia produce beta-like-cells that normalize glycemia in diabetic rodents. Insulin gene transfer strategies provide somewhat inferior glycemic control, but avoid the overproduction of counter-regulatory hormones. Both approaches will require extensive investigations into their effects on host cells and tissues, and the efficacy of neither has been satisfactorily verified in a large animal model.

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.

Adeno-associated viral delivery of a metabolically regulated insulin transgene to hepatocytes.

Transduction with a liver specific, metabolically responsive insulin transgene produces near-normal blood sugars in STZ-diabetic rats. To overcome the limited duration of hepatic transgene expression induced by E1A-deleted adenoviral vectors, we evaluated recombinant adeno-associated virus (rAAV2) for cell type specificity and glucose responsiveness in vitro. Co-infection of AAV2 containing the glucose responsive, liver-specific (GlRE)(3)BP-1 promoter with an empty adenovirus enhanced transduction efficiency, and shortened the duration of transgene expression in HepG2 hepatoma cells, but not primary hepatocytes. However, in the context of rAAV2, (GlRE)(3)BP-1 promoter activity remained confined to cells of hepatocyte lineage, and retained glucose responsiveness. While isolated infection with an insulin expressing rAAV2 failed to attenuate blood sugars in diabetic mice, adenoviral co-administration with the same rAAV2 induced transient, near-normal random blood sugars in a diabetic animal. We conclude that rAAV2 can induce metabolically responsive insulin secretion from hepatocytes in vitro and in vivo. However, alternative AAV serotypes will likely be required to efficiently deliver therapeutic genes to the liver for the treatment of diabetes mellitus.

The PPARgamma ligand, rosiglitazone, reduces vascular oxidative stress and NADPH oxidase expression in diabetic mice.

Oxidative stress plays an important role in diabetic vascular dysfunction. The sources and regulation of reactive oxygen species production in diabetic vasculature continue to be defined. Because peroxisome proliferator-activated receptor gamma (PPARgamma) ligands reduced superoxide anion (O(2)(-.)) generation in vascular endothelial cells in vitro by reducing NADPH oxidase and increasing Cu/Zn superoxide dismutase (SOD) expression, the current study examined the effect of PPARgamma ligands on vascular NADPH oxidase and O(2)(-.) generation in vivo. Lean control (db(+)/db(-)) and obese, diabetic, leptin receptor-deficient (db(-)/db(-)) mice were treated with either vehicle or rosiglitazone (3 mg/kg/day) by gavage for 7-days. Compared to controls, db(-)/db(-) mice weighed more and had metabolic derangements that were not corrected by treatment with rosiglitazone for 1-week. Aortic O(2)(-.) generation and mRNA levels of the NADPH oxidase subunits, Nox-1, Nox-2, and Nox-4 as well as Nox-4 protein expression were elevated in db(-)/db(-) compared to db(+)/db(-) mice, whereas aortic Cu/Zn SOD protein and PPARgamma mRNA levels were reduced in db(-)/db(-) mice. Treatment with rosiglitazone for 1-week significantly reduced aortic O(2)(-.) production and the expression of Nox-1, 2, and 4 but failed to increase Cu/Zn SOD or PPARgamma in aortic tissue from db(-)/db(-) mice. These data demonstrate that the vascular expression of Nox-1, 2, and 4 subunits of NADPH oxidase is increased in db(-)/db(-) mice and that short-term treatment with the PPARgamma agonist, rosiglitazone, has the potential to rapidly suppress vascular NADPH oxidase expression and O(2)(-.) production through mechanisms that do not appear to depend on correction of diabetic metabolic derangements.