Sustained heterologous gene expression in pancreatic islet organoids using adeno-associated virus serotype 8.

Genetic modification of pancreatic islet organoids, assembled in vitro prior to transplantation is an emerging alternative to direct in vivo genetic manipulations for a number of clinical and research applications. We have previously shown that dispersion of islet cells followed by re-aggregation into islet organoids, or pseudoislets, allows for efficient transduction with viral vectors, while maintaining physiological functions of native islets. Among viruses currently used for genetic manipulations, adeno-associated viruses (AAVs) have the most attractive safety profile making them suitable for gene therapy applications. Studies reporting on pseudoislet transduction with AAVs are, however, lacking. Here, we have characterized in detail the performance of AAV serotype 8 in transduction of islet cells during pseudoislet formation in comparison with human adenovirus type 5 (AdV5). We have assessed such parameters as transduction efficiency, expression kinetics, and endocrine cell tropism of AAV8 alone or in combination with AdV5. Data provided within our study may serve as a reference point for future functional studies using AAVs for gene transfer to islet cell organoids and will facilitate further development of engineered pseudoislets of superior quality suitable for clinical transplantation.

Translational assessment of a genetic engineering methodology to improve islet function for transplantation.

BACKGROUND: The functional quality of insulin-secreting islet beta cells is a major factor determining the outcome of clinical transplantations for diabetes. It is therefore of importance to develop methodological strategies aiming at optimizing islet cell function prior to transplantation. In this study we propose a synthetic biology approach to genetically engineer cellular signalling pathways in islet cells. METHODS: We established a novel procedure to modify islet beta cell function by combining adenovirus-mediated transduction with reaggregation of islet cells into pseudoislets. As a proof-of-concept for the genetic engineering of islets prior to transplantation, this methodology was applied to increase the expression of the V1b receptor specifically in insulin-secreting beta cells. The functional outcomes were assessed in vitro and in vivo following transplantation into the anterior chamber of the eye. FINDINGS: Pseudoislets produced from mouse dissociated islet cells displayed basic functions similar to intact native islets in terms of glucose induced intracellular signalling and insulin release, and after transplantation were properly vascularized and contributed to blood glucose homeostasis. The synthetic amplification of the V1b receptor signalling in beta cells successfully modulated pseudoislet function in vitro. Finally, in vivo responses of these pseudoislet grafts to vasopressin allowed evaluation of the potential benefits of this approach in regenerative medicine. INTERPRETATION: These results are promising first steps towards the generation of high-quality islets and suggest synthetic biology as an important tool in future clinical islet transplantations. Moreover, the presented methodology might serve as a useful research strategy to dissect cellular signalling mechanisms of relevance for optimal islet function.

Low-calcium diet prevents fructose-induced hyperinsulinemia and ameliorates the response to glucose load in rats.

BACKGROUND: Consuming a fructose-rich diet leads to hyperinsulinemia, impaired glucose tolerance, and insulin resistance. In humans, the consumption of high levels of refined sugars often coincides with a diet containing suboptimal levels of calcium. Calcium and carbohydrate metabolism interact, so there is potential for fructose to have different health outcomes depending on whether the diet is calcium-rich or calcium-poor. METHODS: We evaluated the metabolic effects of feeding fructose to rats that were maintained on either a calcium-replete diet or a low-calcium diet. Growing male Sprague Dawley rats were fed diets based on the AIN-93G formulation, with the main source of carbohydrate derived either from a mixture of cornstarch and sucrose or from fructose. Half the rats given each carbohydrate source were fed calcium at recommended levels (125 mmol/kg Ca(2+)); the others were fed a diet low in calcium (25 mmol/kg Ca(2+)). At various times, glucose and insulin tolerance tests were conducted to assess glucose metabolism. RESULTS: Rats fed low-calcium diet had lower fasting insulin levels irrespective of the carbohydrate source they ate. They had a normal glycemic response to a glucose load and did not develop hyperinsulinemia under conditions of fructose feeding. The drop in blood glucose levels in response to insulin injection was larger in rats fed low-calcium diet than in those fed calcium-replete diet. CONCLUSIONS: Low-calcium diet prevented fructose-induced hyperinsulinemia and improved glucose handling under conditions of fructose feeding. Potential mechanisms underlying these effects of the low-calcium diet remain to be determined, but possibilities include impairment of insulin release from the pancreas and improved peripheral insulin sensitivity.

Macronutrient selection by seven inbred mouse strains and three taste-related knockout strains.

Many animals thrive when given a choice of separate sources of macronutrients. How they do this is unknown. Here, we report some studies comparing the spontaneous choices between carbohydrate- and fat-containing food sources of seven inbred mouse strains (B6, BTBR, CBA, JF1, NZW, PWD and PWK) and three mouse models with genetic ablation of taste transduction components (T1R3, ITPR3 and CALHM1). For 8days, each mouse could choose between sources of carbohydrate (CHO-P; sucrose-cornstarch) and fat (Fat-P; vegetable shortening) with each source also containing protein (casein). We found that the B6 and PWK strains markedly preferred the CHO-P diet to the Fat-P diet, the BTBR and JF1 strains markedly preferred the Fat-P diet to the CHO-P diet, and the CBA, NZW and PWD strains showed equal intakes of the two diets (by weight). Relative to their WT littermates, ITPR3 and CALHM1 KO mice had elevated Fat-P preferences but T1R3 KO mice did not. There were differences among strains in adaption to the diet choice and there were differences in response between males and females on some days. These results demonstrate the diverse responses to macronutrients of inbred mice and they point to the involvement of chemosensory detectors (but not sweetness) as contributors to macronutrient selection.

Influence of cross-fostering on preference for calcium chloride in C57BL/6J and PWK/PhJ mice.

We investigated whether maternal influences during the suckling period alter the avidity for calcium, using as models mice from the calcium-preferring PWK/PhJ strain and the calcium-avoiding C57BL/6J strain. We found that milk collected from PWK/PhJ dams had higher calcium concentrations than did milk collected from C57BL/6J dams. Despite this, cross-strain fostering had no effect on adult calcium preferences relative to mice of the same strain that were within-strain fostered or not fostered. Our results indicate that calcium avoidance by C57BL/6J mice and acceptance by PWK/PhJ mice are unaffected by maternal environment during the suckling period.

Influence of estrous and circadian cycles on calcium intake of the rat.

The food, water and sodium intake of laboratory rats fluctuates over the circadian and estrous cycles. Blood calcium and calcium-regulating hormones also wax and wane in response to these cycles, raising the possibility that the same might be true of calcium intake. To investigate this, we monitored the fluid intakes of female Long-Evans rats given a choice between water and 10mM CaCl2 solution for two consecutive estrous cycles. We found that calcium solution intake changed over the circadian cycle in a similar manner to water intake; the preference scores for CaCl2 solution remained stable. We did not detect any changes in calcium solution intake or preference scores during the estrous cycle despite a decrease in fluid intake at estrus. Thus, fluctuations in intake of calcium solution during the circadian cycle appear to be nonspecific and probably the result of changes in fluid balance. Estrous changes either do not influence calcium intake or their effects are masked by other factors, resulting in stable levels of calcium intake.

Chorda tympani nerve modulates the rat's avoidance of calcium chloride.

Calcium intake depends on orosensory factors, implying the presence of a mechanism for calcium detection in the mouth. To better understand how information about oral calcium is conveyed to the brain, we examined the effects of chorda tympani nerve transection on calcium chloride (CaCl(2)) taste preferences and thresholds in male Wistar rats. The rats were given bilateral transections of the chorda tympani nerve (CTX) or control surgery. After recovery, they received 48-h two-bottle tests with an ascending concentration series of CaCl(2). Whereas control rats avoided CaCl(2) at concentrations of 0.1mM and higher, rats with CTX were indifferent to CaCl(2) concentrations up to 10mM. Rats with CTX had significantly higher preference scores for 0.316 and 3.16 mM CaCl(2) than did control rats. The results imply that the chorda tympani nerve is required for the normal avoidance of CaCl(2) solution.