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The rats were fed with chow diet and water ad libitum as the initial experiment shown in Fig 2. Fig 3 shows representative Nightscout images of , , and dilutions of Humalog as labeled in the figure.
With more diluted Humalog used, more frequent insulin infusions were observed shown by less gap between light blue boxes. Based on the results of these experiments, we chose to use dilution of Humalog for our subsequent experiments.
Representative Nightscout images of , , and dilutions of Humalog are shown. Each individual experiment was run following the same procedure as described in the Methods section. A total of 20 independent experiments using 5 different diabetic rats and various dilutions of Humalog were performed. To study the effects of different diets on BGL and insulin regulation, we performed closed-loop APS experiments using both healthy and diabetic rats. Fig 4 shows representative Nightscout images of diabetic rats fed with chow diet upper panel or sugar pellets lower panel.
As expected, rats fed with chow diet showed less fluctuations in BG levels than those fed with sucrose pellets. Diabetic rats fed with Sucrose pellets for the duration of closed-loop hours 3 different rats with 5 independent experiments were in euglycemic range for Average insulin infusion rate for diabetic rats fed with chow diet was 0. Representative Nightscout images of diabetic rats fed with chow diet upper panel or sugar pellets lower panel are shown.
A total of closed-loop hours 5 different rats with 10 independent experiments using dilution of Humalog was collected for diabetic rats fed with chow diet. Using the data analysis program described in the Methods section, the percent of time in hyper-, hypo- and normal glycemia as well as insulin infusion rate were calculated as shown in Table 1.
A total of closed-loop hours 3 different rats with 5 independent experiments were collected for diabetic rats fed with sugar pellets lower panel. It is noteworthy how the patterns of OpenAPS-suggested insulin infusion mock insulin infusion based on BGL shown in light blue in Fig 5 upper and lower panels are quite different from those in diabetic rats Fig 4. Suggested insulin infusion rates by OpenAPS were lower in healthy rats than diabetic rats 0.
In addition, suggested insulin infusions by OpenAPS in healthy rats regardless of food type were almost continuous, while those in diabetic rats were intermittent.
Feeding sucrose pellets to healthy rats caused greater range of BGL but still within euglycemic range Representative Nightscout images of healthy rats fed with chow diet upper panel or sugar pellets lower panel are shown.
A total of closed-loop hours 4 different rats with 8 independent experiments was collected for healthy rats fed with chow diet. Using the data analysis program, the percent of time in hyper-, hypo- and normal glycemia as well as OpenAPS-suggested insulin infusion rate were calculated as shown in Table 1.
The red arrow shows the time of injection. Fig 6E shows area under curve for BGL, indicating that healthy rats showed two-fold higher than diabetic rats. Fig 7B shows area under curve for each condition shown in Fig 7A , indicating that healthy rats showed significantly higher overall BGL compared to diabetic rats. Blue lines show suggested insulin infusion by OpenAPS.
B and D BGL determined by Contour Next glucose meter closed circles and Enlite sensor closed inverted triangles for healthy and diabetic rats, respectively. E Area under curve for BGL determined by the glucose meter for healthy and diabetic rats. B Area under curve for BGL determined by Enlite sensor for each condition as indicated on the figure. DAPI was used for nuclear staining blue. Florescent images were obtained using a 40X objective in an Olympus FlowView confocal microscope.
Fig 9A panels a and b show Periodic acid-Schiff PAS staining of frozen liver sections isolated from healthy and diabetic rats, respectively, to assess glycogen storage in the liver. Fig 9A panels c and d show the same conditions as panels a and b but the liver sections were stained with PAS after treatment with amylase which breaks the bonds in glycogen and removes it from the liver sections as a negative control condition. The liver sections treated with amylase panels c and d show much lighter magenta coloring due to removal of glycogen.
Fig 9B shows integrated intensity of the staining, which shows higher values in the conditions treated with amylase due to more light penetration through relatively lighter magenta coloring.
Taken together, these results show that liver function in terms of glycogen storage was not altered in diabetic rats as compared to healthy rats. A Panels a and b show representative images of Periodic acid-Schiff PAS staining of frozen liver sections isolated from healthy and diabetic rats, respectively.
Panels c healthy and d diabetic show the images of PAS staining after amylase treatment as a control. B Integrated intensity of the PAS staining with and without amylase.
Fig 10A panels a and b show lipid accumulation in the liver of healthy and diabetic rats, respectively, determined by Nile Red staining.
Fig 10B shows integrated fluorescent intensity over number of nuclei for each liver section using ImageJ program. Lipid accumulation in the liver of the diabetic rats was significantly lower than that in the healthy rats. We have also measured HbA1c levels of healthy and diabetic rats as shown in Table 3. The diabetic rats showed significantly higher HbA1c levels than healthy rats. A Panels a and b show representative images of Nile red staining of frozen liver sections isolated from healthy and diabetic rats, respectively.
B Integrated fluorescent intensity over number of nuclei for each liver section was determined using ImageJ image processing and analysis program. In this paper, we have established a rat APS as a research tool not only to study alterations of glucose and insulin homeostasis in real-time caused by diet, exercise, hormones or antidiabetic agents, but also to test mathematical and engineering models of blood glucose prediction or new algorithms for closed-loop APS.
One of the major challenges of developing algorithm for BGL prediction or closed-loop APS is a limited selection of validation models that often include virtual patients in silico, retrospective patient data, or a very small number of patients in restrictive settings such as hospital or hotel.
For a rat, the strength of stock solution made a significant difference in the pattern of insulin infusion as well as BGL fluctuation. As anticipated, the higher strength of stock solution Fig 3 , vs and dilution , the longer duration of interval between insulin infusion and the higher frequency of hypoglycemia were observed. The dilution of Humalog caused continuous insulin infusion with no cyclic pattern of BGL shown with or dilution of Humalog.
Of note, the BGL pattern of a healthy rat showed hardly any fluctuation with continuous insulin infusion albeit mock insulin infusion determined by the OpenAPS algorithm Fig 5 , upper panel. These results support the strategy of frequent supermicrobolus injections of insulin as a part of BGL control algorithm [ 12 ].
The effects of different diets on glucose-insulin regulation in healthy and diabetic rats were studied using a rat APS. There are stark differences in terms of insulin infusion and BGL patterns between healthy and diabetic rats Figs 4 and 5. Interpretation of the data, however, should be made cautiously.
For the diabetic rats, the BGL patterns reflect the action of insulin infused externally from the pump in addition to other physiological factors. For the healthy rats, the actual amount or frequency of insulin released from the intact pancreas is unknown and the mock insulin infusion data determined by OpenAPS do not reflect the action of insulin unlike in the case of diabetic rats.
The study, moreover, provided insights how healthy rats respond to challenges of different amounts of glucose. Both the duration and the extent of hyperglycemia area under curve showed non-linear exponential relationship to different glucose amounts i. Recent studies by Zhu et al.
It is not known by which underlying mechanisms regulate insulin release from certain islets but not others. It is understandable that the duration and the extent of hyperglycemia are affected by many factors including insulin release from the pancreas, the rate of glucose uptake, the target cell mass, etc. The significant differences between the healthy and the diabetic rats despite their comparable body weights, however, suggest that there is a temporal or physical barrier in insulin secretion in the healthy rats.
It is of great interest to understand insulin and glucose regulation in normal or disease states, which may help discovering therapeutic agents that enhance insulin release from the dormant islets in the setting of type 2 diabetes mellitus. The rat APS will be a valuable tool in studying alterations of glucose and insulin regulation in both type 1 and type 2 diabetic rats as compared to healthy rats.
In addition, hyperglycemia caused by diabetic condition can also cause damage to liver and kidney. Fig 9A and 9B , however, showed that daily injection of insulin to diabetic rats minimized the liver damage in terms of glycogen storage that allows glucose release from the liver between meals and during sleep.
Liver plays a central role in lipid and glucose hemostasis [ 24 ]. Excess TAG accumulation in the liver occurs in the setting of obesity, insulin resistance, and type 2 diabetes [ 26 , 27 ]. In streptozotocin-induced diabetic rats, a consistent increase in hepatic TAG content was observed only in the ketotic diabetic rats with no insulin treatment [ 28 ]. Our study shows that daily insulin treatment of the diabetic rats prevented hepatic steatosis. In this paper we presented a series of experiments performed to establish a rat APS as a research tool and demonstrated its application.
We are currently using a rat APS to study the effects of SGLT2 inhibitors on streptozotocin-induced diabetic rats to assess its benefits and toxicity especially on diabetic ketoacidosis.
An APS provides data including total insulin requirement to maintain BGL, average BGL, percent of hyperglycemia, target-in-range, or hypoglycemia during the experimental duration of 2—3 days, from which one can infer the general physiological state of a diabetic rat.
A rat APS may be used before, during, or end of drug treatment to assess drug effects. We have previously studied a mathematical model of glucose-insulin homeostasis to estimate insulin secretion, glucose uptake by tissues, and hepatic handling of glucose [ 29 ] and tracked changes of the parameters of glucose-insulin homeostasis during the course of obesity in mice [ 30 ].
A rat APS may provide insights of the progression of both type 1 and type 2 diabetes. In conclusion, a rat APS may be used as a novel tool in addition to a variety of biomedical and engineering approaches to enhance understanding and treatment of diabetes. Browse Subject Areas? Click through the PLOS taxonomy to find articles in your field. Abstract Artificial pancreas system APS is an emerging new treatment for type 1 diabetes mellitus. Data Availability: All relevant data are within the paper.
Introduction The artificial pancreas system APS is an innovative new treatment for type 1 diabetes mellitus T1DM , which reflects how advances in technology are incorporated in developing new treatment strategies for a human disease.
Download: PPT. Analysis of BGL and insulin infusion The default Nightscout reporting view does not have a method allowing users to analyze data in a specific time range. Statistical analysis Statistical analysis was performed using GraphPad Prism 6 statistics software t-tests and Mann-Whitney. Fig 2. Fig 3. Determination of an optimal concentration of Humalog for diabetic rats.
Effects of different diets on BGL and insulin regulation in healthy and diabetic rats To study the effects of different diets on BGL and insulin regulation, we performed closed-loop APS experiments using both healthy and diabetic rats.
Fig 4. Effects of different diets on BGL and insulin infusion in diabetic rats. Table 1. Fig 5. Fig 6. Fig 8. Representative images of islets from healthy and diabetic rats. Table 2. Table 3. Body weight and HbA1c levels of healthy and diabetic rats. Discussion In this paper, we have established a rat APS as a research tool not only to study alterations of glucose and insulin homeostasis in real-time caused by diet, exercise, hormones or antidiabetic agents, but also to test mathematical and engineering models of blood glucose prediction or new algorithms for closed-loop APS.
Conclusions In this paper we presented a series of experiments performed to establish a rat APS as a research tool and demonstrated its application. References 1. Pancreatic islet glucose metabolism and regulation of insulin secretion. Diabetes Metab Rev. Improvement in glycemic excursions with a transcutaneous, real-time continuous glucose sensor: a randomized controlled trial.
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