The maintenance of close-to-normal blood glucose levels slows the onset and progression of long-term microvascular, and possibly macrovascular, complications in patients with type 1 diabetes. Artificial pancreas or integrated CLC \[a CLC algorithm which takes into account continuous glucose monitoring (CGM) readings and the effects of previous insulin infusions to continuously compute the amount of insulin dose to be administered\] aims to minimize, in real time, glucose variability and prevent extreme glucose excursions (hypoglycemia and hyperglycemia). Despite important developments in sensor and pump technology, the artificial pancreas system must cope with anatomical and functional barriers other than the ones encountered by the normally functioning glucose-insulin system in physiology. For instance , CGM devices measure glucose concentration in the interstitium, not in the blood compartment, introducing a time lag due to glucose diffusion from plasma to the interstitial fluid. Furthermore, insulin (actually, one of current fast insulin analogues) is delivered into the subcutaneous adipose tissue, not in blood, and directly accesses the systemic, not the portal circulation. Owing to these hindrances, developing a smart and suitable CLC algorithm still is an ongoing process. This research aims to alleviate these shortcomings by carrying out clinical physiology study aiming at improving development and optimization of a suitable control algorithm.

Twenty adults patients with type 1 diabetes, regularly attending the Division of Endocrinology and Metabolic Diseases of University of Verona School of Medicine, using continuous subcutaneous fast insulin analogue infusion (CSII) through a permanent pump and on subcutaneous glucose sensing will be enrolled.

Inclusion criteria (see below for more details) are diagnosis of type 1 diabetes as defined by WHO for at least 12 months or confirmed C-peptide negative. Exclusion criteria are recurrent severe hypoglycemia unawareness or clinically significant nephropathy, neuropathy, or proliferative retinopathy.

Standard clinical parameters will be assessed in all patients. Metabolic tests will be carried out at the Division of Endocrinology and Metabolic Diseases of University of Verona School, starting at 08:00 a.m. on two separate days in random order and after a 10-12-h overnight fast. Two metabolic tests will be performed: a euglycemic insulin clamp and a standardized mixed meal test.

A. Euglycemic insulin clamp. A standard euglycemic insulin clamp will be carried out to assess insulin sensitivity, as previously described (1). Subjects will be instructed to use their usual nocturnal fast insulin analogue basal rate, to be left unchanged for at least five hours before the beginning of the test. A CGM device will be inserted two days before the test and calibrated with capillary blood glucose (measured by glucometer) at pre-established hours of the day. Human insulin concentration will be raised with an i.v. prime (0.8 U/m\^2 BSA) and maintained constant by an i.v. infusion (40 mU/min·m\^2 BSA). Plasma glucose will be allowed to fall until it reaches the physiologic range (i.e. \< 5.6 mmol/L), after which it will be clamped at 5.0 mmol/L for at least 60 min by appropriately changing an intravenous infusion of 20% dextrose. Glucose will be measured at bedside by a YSI Glucose Analyzer. Blood samples will be collected at timed intervals to measure plasma insulin (both fast analogue and human insulin), free fatty acids, and glucagon. Continuous glucose (interstitial glucose) monitoring will be performed throughout the insulin clamp.

B. Mixed meal test. This test will be performed to assess the pathophysiology of glucose control during a standardized physiologic challenge. Subjects will be on an Indian corn free and cane sugar free diet for at least one week before study and will be instructed to use the usual nocturnal fast insulin analogue basal rate, to be left unchanged for at least five hours before the beginning of the test. A CGM device will be in place and properly working throughout the test. All participants will ingest a standardized mixed meal (292 Kcal, 38,9 g carbohydrates, 8,9 g fats and 14 g proteins), under the form of maize polenta plus seasoned Italian parmesan cheese, and will be monitored for 300 minutes thereafter. Right before meal ingestion, a s.c. fast insulin analogue bolus will be administered by the pump, according to individual insulin-to-carbohydrate ratio and correction dose. The 13C/12C ratio of maze starch is different from other carbohydrate sources and allows the detection of polenta-derived glucose in the circulation. Plasma glucose, free fatty acid, amino acid, insulin, glucagon, and incretin hormone concentrations will be assessed at baseline and during the entire test. The plasma 13C/12C glucose ratio throughout the test will be monitored by isotope ratio mass spectrometry. CGM data will be collected. This test will determine the time courses of plasma glucose, 13C/12C glucose ratio (hence, meal-derived and endogenous glucose), insulin, free fatty acids, aminoacids, glucagon, and incretin hormones during a mixed meal.

In both studies, blood samples will be put in ice and quickly spun at 1500 g at +4°C. Plasma/serum will be collected and stored at -80°C. Both tests (insulin clamp and mixed meal) will form the biological biobank of this study.

The combined analysis of insulin clamp and mixed meal test data will allow to build a comprehensive model of the glucose insulin system during a mixed meal test in each subject, thereby resulting into an in silico virtual patient, according to a well established methodology developed in our laboratory. The collection of virtual patients with type 1 diabetes undergoing a mixed meal test will form the in silico biobank derived form this study. Virtual patients will be used to carry out Metabolic Control Analysis (MCA), i.e. to compute glucose control coefficients (CCs), which quantify the role played by each component (e.g.: absorption rate of the fast insulin analogue, carbohydrate absorption through the gut, etc.) of the system in determining glucose concentration at each time point during the mixed meal in patients with type 1 diabetes, in close parallelism to the analysis previously carried out by us in patients with type 2 diabetes undergoing an intravenous glucose tolerance test (2). Furthermore, the incretin hormone, glucagon and substrate response to a mixed meal will be quantified, allowing the putative identification of further modifiers of the glucose-insulin system (one for all: glucagon). This database will be instrumental in devising a control algorithm able to guarantee a normal glucose regulation during a mixed meal in patients with type 1 diabetes with CGM on insulin pump therapy.