Reduction of Blood Glucose Variability in Type 1 Diabetic Patients Treated By Pancreatic Islet Transplantation: Interest of continuous glucose monitoring

The CGMS (Minimed, Sylmar, CA) consists of a sterile disposable subcutaneous glucose-sensing device connected by a cable to a pager-sized glucose monitor (17). A communication device allows downloading and reviewing of the data on a personal computer. The needle-delivered transcutaneous sensor detects glucose electrochemically in subcutaneous interstitial fluid due to its reaction with glucose oxydase. The system registers the glucose concentration every 10 s and stores an average value every 5 min. A total of 288 data points are collected each day (range 2.2–22 mmol/l). Four capillary blood glucose measurements per day are required for sensor calibration. The correlation between blood and subcutaneous glucose measurements is good (r = 0.95, P < 0.001) (20).

From 1 March 1999 until 1 June 2002, a total of 10 IPII-treated patients consecutively admitted in our diabetologic unit with HbA_1c <7.5% were selected. Nine patients treated with SPK who had normal glycemic profiles during oral glucose tolerance testing and seven patients from the GRAGIL network who were treated with islet transplantation after kidney grafting (IAK) and who had plasma C-peptide levels >0.5 ng/ml were included in the study. All SPK patients and four IAK patients were insulin independent, whereas three IAK patients had partial graft function characterized by ongoing endogenous insulin production but persisting, although reduced, exogenous insulin needs. In these 26 C-peptide–negative type 1 diabetic patients in ambulatory care, the sensor was placed subcutaneously in the abdominal area for 3 days. The characteristics of study subjects are listed in Table 1. Diabetic patients in each group were comparable in terms of age, BMI, and duration of diabetes. HbA_1c was significantly higher in the IPII patients than in the patients who underwent pancreas and islet transplantation. There was no significant difference in HbA_1c between SPK and IAK patients.

In the IPII group, 10 patients received an implantable pump (Minimed Implantable Pump Model 2001; Minimed Technologies, Sylmar, CA), which delivered insulin intraperitoneally for a period of 6.7 ± 2.9 years (5). Pump catheters were made from silicone-coated polysulfone. The infused insulin was Hoechst’s 21 PH neutral semisynthetic human insulin (Hoechst, Frankfurt, Germany) at a concentration of 400 IU/ml and stabilized by a glycol-polyethylene-polypropylene surface-active agent (Genapol; Hoechst). Refilling was performed under aseptic conditions every 6 weeks.

Pancreas and kidney were harvested from heart-beating multiorgan donors. Simultaneous pancreas-kidney transplantation was performed intraperitoneally through a midline incision in nine uremic type 1 diabetic patients as previously described (10); the pancreas and kidney were placed in the right and left iliac fossae, respectively. The recipient’s external iliac artery and vein were used for systemic drainage, whereas exocrine secretions from the pancreas graft were drained into the bladder through a side-to-side duodenocystostomy. Donor pancreas and kidney were selected on the basis of ABO blood group compatibility and a negative cross-match between donor and recipient.

Sequential quadruple immunosuppressive therapy was used for all recipients. This treatment comprised antilymphocyte serum (60 mg/day) for 7 days immediately after transplantation, prednisone (2 mg · kg^–1 · day^–1 decreasing to 0.15 mg · kg^–1 · day^–1 1 year after transplantation), cyclosporin (8 mg · kg^–1 · day^–1 subsequently adjusted to obtain a cyclosporin trough level of 100–200 ng/ml), and azathioprine (1.5 mg · kg^–1 · day^–1). Continuous glucose measurements were performed 6 ± 3.5 years after transplantation.

Pancreata were harvested from heart-beating multiorgan donors. Islet isolation was started within 8 h after cross-clamping and was performed as described previously (21). Islets were counted on the day of injection and only the intact, dithizone-stained islets were taken into consideration. The equivalent islet number (EIN) was calculated by normalizing the islet to a standard islet of 150 μm in diameter. The islet preparation was collected in 50-ml syringes.

Islet transplantation was performed in seven type 1 diabetic patients having a functional kidney graft (creatinine clearance >60 ml/min) for ≥6 months. Before transplantation, islets of five patients were kept in culture for 2–10 days. After intravenous administration of 5,000 units of heparin, islets were injected into the portal vein under radiological control. A mean of 10,560 ± 4,061 EIN/kg was injected per recipient. The first patient was treated with a polyclonal antilymphocyte serum (Atgam; Pharmacia and Upjohn, Dubendorf, Switzerland), cyclosporin (Neoral; Novartis, Basel, Switzerland) with a trough level between 100 and 150 ng/ml, azathioprine (Imurek; Wellcome, Bern, Switzerland), and corticosteroids with rapid tapering to 5 mg of prednisone by the end of the third month after transplantation. Immunosuppression in the four other patients included the anti–interleukin-2-receptor antibody basiliximab (Simulect; Novartis, Basel, Switzerland) 20 mg intravenously at days 0 and 4. The protocol then consisted of oral cyclosporin (trough levels between 150 and 200 ng/ml during the first 3 months and 100 ng/ml thereafter), mycophenolate mofetil (Cellcept; Roche, Basel, Switzerland), and corticosteroids (methylprednisolone at a dose of 1 g intravenously on day 0 and 100 mg intravenously on day +1, followed by oral prednisone at dose of 20 to 0–5 mg/day until the end of the sixth month).

Islets were immediately transplanted after isolation in the two remaining patients. Two intraportal injections were needed to transplant 12,430 EIN/kg per recipient. Immunosuppression in these two patients included the anti–interleukin-2-receptor antibody basiliximab (Simulect; Novartis, Basel, Switzerland) 20 mg intravenously at days 0 and 4. The protocol then consisted of oral cyclosporin (Neoral; Novartis, Basel, Switzerland) with a trough level between 75 and 100 ng/ml and 300–500 ng/ml 2 h after oral administration during the first 3 months and a trough level between 50 and 75 ng/ml thereafter and rapamycine (RAD; Novartis) with trough level between 5 and 15 ng/ml.

In all seven cases, an adjuvant therapy was administered after islet injection: 2 g nicotinamide (Nicobion; Merck), 800 units of vitamin E, and 1,200 mg of pentoxifylline (Trental; Hoechst-Marion-Roussel, Zurich, Switzerland) per day for a period of 1 month. Insulin therapy was maintained unless the patient experienced hypoglycemia. The study started 1.8 + 1.3 years (0.5−5 years) after transplantation.

For each group, a qualitative analysis of the glucose profiles was performed from the graphical data of the CGMS daily analysis. After 3 days, glucose profiles of each group were analyzed quantitatively: the mean glucose concentration and the glucose variability. The latter was calculated as the absolute value of measured glucose minus 5.5 mmol/l. In addition, the number and duration of hypoglycemic events (<3.3 mmol/l) were identified during the night and day periods.

Results are expressed as mean ± SD. A one-way ANOVA followed by a multiple comparison procedure (least significant difference test) was used to compare each parameter between the groups.

The CGMS daily glycemic profiles of the IPII patients are shown in Fig. 1A. A large amplitude of glucose excursion was shown, particularly during the night period. In contrast, after SPK transplantation, glucose values remained stable at ∼5.5 mmol/l (Fig. 1B).

Among the IAK patients, the amplitude of glucose excursion was more important in patients having partial graft function than in insulin-independent patients (Figs. 2A and B).

During the 3-day period, the mean glucose concentration of IPII patients was significantly higher than that observed in SPK and IAK patients: 7.81 ± 1.55 vs. 5.38 ± 1.12 and 5.83 ± 0.81 mmol/l (P < 0.001) (Table 2). There was no significant difference in the mean glucose concentration between SPK and IAK patients.

Glucose variability was comparable between SPK and IAK patients: 1.40 ± 0.42 and 1.32 + 0.53 mmol/l. A significantly higher glucose variability was observed in IPII patients: 3.47 ± 1.66 mmol/l (P < 0.001) (Table 2).

During the 3 days, IPII patients had 4.12 ± 1.66 hypoglycemic events, whereas only 0.66 + 0.57 events were observed in IAK patients with partial graft function (P < 0.001) (Fig. 3A). No hypoglycemia was observed in any of the SPK or insulin-independent IAK patients.

The duration of hypoglycemic events during daytime was significantly longer in IPII patients than in IAK patients with partial graft function: 64 ± 33 vs. 30 ± 15 min (P < 0.001). During the night, hypoglycemia persisted for 130 ± 62 min in IPII-treated patients, whereas in IAK patients with partial graft function, events of hypoglycemia lasted only 30 ± 27 min (Fig. 3B).

By using CGMS in type 1 diabetic patients, our study showed a significant improvement in glucose control after pancreas or islet transplantation as compared with intraperitoneal insulin infusion through an implantable insulin pump. After SPK or IAK transplantation, both the mean glucose concentration and the glucose variability were significantly lower than those observed in IPII patients. Furthermore, these two parameters were comparable between SPK and IAK patients. The number and duration of hypoglycemic events were reduced in IAK patients with partial graft function compared with IPII patients, whereas no hypoglycemia was observed in insulin-independent SPK or IAK patients.

In our study, the CGMS recording was performed in all subjects in ambulatory care over 3 days without any technical problems. The accuracy and reliability of CGMS have been tested under a variety of clinical and ambulatory conditions (17,22). CGMS provides accurate continuous measurement of interstitial glucose levels, correlating closely with blood glucose levels (21). The device collects many data points and detects glucose fluctuations during the day in a more detailed (288 measurements) but less accurate manner than those obtained with glucose self-monitoring. In agreement with several other groups, we found a modest difference between capillary blood and sensor glucose levels. Zung and Zadik (23) reported a total of 1,511 CGMS glucose values collected in a multicenter trial of 62 subjects. Of the 169 sensor readings in the low glucose concentration range, only 30% were confirmed by the capillary blood glucose measurements. In contrast, performance of CGMS was much better in the normal and high glucose concentration range. In addition, in a case report of a patient connected simultaneously with two CGMS devices, a slight difference between the two sensor values was observed, showing the limits of the CGMS system (24). The interpretation of an isolated data point on the CGMS graph must be considered with caution. Despite this limitation, the CGMS system allows better analysis of the duration of hypoglycemic and hyperglycemic events and glucose excursions than capillary blood glucose measurements. Recently, an improved sensor was used by us and other groups and resulted in more accurate glucose concentration measurements.

The glucose profile was analyzed by calculation of the mean glucose concentration, the glucose variability, and the number and length of hypoglycemic events. Among the different methods to calculate diabetic instability, mean amplitude of glycemic excursion (MAGE) has been widely used in stable and unstable diabetic patients (25). MAGE is calculated by taking the arithmetic mean of blood glucose increases or decreases when both ascending and descending variation exceeds the value of 1 SD of the blood glucose concentration in 24 h. Consequently, the MAGE values are lower in normal subjects than in diabetic patients. However, the MAGE values are only significant for unstable patients with large amplitudes of glucose excursion (26). Because IPII treatment and pancreas and islet transplantation are known to potentially reduce the glucose excursions in diabetic patients, we have chosen to express the glucose variability as the absolute value of measured glucose concentration minus 5.5 mmol/l, instead of MAGE, allowing analysis of smaller amplitudes in glucose excursions and consequently the comparison between the groups in our study.

In terms of glycemic control, our study demonstrates the superiority of SPK and IAK transplantation over IPII. Indeed, both HbA_1c and mean glucose concentrations were comparable between pancreas- and islet-transplanted patients, whereas these values were significantly higher in IPII-treated patients. These results can be explained by the absence of a closed-loop insulin delivery system in IPII treatment. In addition, the glucose profile of three healthy subjects (data not shown) analyzed by CGMS in our unit revealed that both mean glucose concentration and glucose variability were similar between healthy subjects and transplanted patients. Because of the small number of IAK patients, we have regrouped insulin-independent patients and patients with partial graft function. The glucose excursions expressed as MAGE index in the study by Ryan et al. (15) were significantly lower after islet transplantation. However, corticosteroids were included in our immunosuppressive therapy, except for in the latter two patients. Clinical symptoms together with a CGMS glucose value <60 mg/dl confirmed hypoglycemia. However, certain patients did not have symptoms of hypoglycemia. In these cases, a hypoglycemic event was taken into account when the glucose concentration was maintained at <60 mg/dl for >30 min. If capillary blood glucose measurements give only an indication of the number of hypoglycemia events, CGMS also integrates the duration of these events. Hypoglycemia events persisted for 2 h in IPII-treated patients. In our study, both the number and the length of hypoglycemic events were significantly lower in IAK patients than in IPII patients. During the 3 days of CGMS registration, a mean of four hypoglycemic events was observed in IPII patients, whereas SPK and insulin-independent IAK patients did not experience any hypoglycemic events. Some authors reported hypoglycemia together with hyperinsulinism after pancreas transplantation, probably due to the systemic delivery of insulin (27). In our study, a longer CGMS analysis would probably be required to identify hypoglycemic events. The absence of hypoglycemia in insulin-independent islet-transplanted patients has also been reported by other groups (12–15). These results can be explained by the physiological portal delivery of insulin after islet transplantation. In agreement with several studies, islet-transplanted patients with persisting endogenous insulin production exhibited a reduced number and duration of hypoglycemic events (12–14). These results can be explained by the autoregulated endogenous insulin production of grafted islets. Ryan et al. (15) did not report hypoglycemia in patients with partial graft function. However, in their study, the analysis of the glucose profiles was based on the blood glucose auto-monitoring, which does not allow detection of asymptomatic hypoglycemic events.

In summary, CGMS is a useful tool to analyze precisely the glucose profile of type 1 diabetic patients under different investigational treatment modalities. Our results show that islet transplantation can be as efficient as pancreas transplantation in restoring good metabolic control and reducing blood glucose variability. To improve the implantable insulin pump treatment, the closed-loop insulin delivery system is required.

B. Boillot, J. L. Descotes, J. J. Rambeaud, D. Masson, D. Halec, C. Sengel, F. Thony, S. Halimi, J. F. Bayle, L. Guitard-Milliat, P. Vialtel, N. Lefrancois, C. Julien, X. Martin, P. J. Valette, B. Moulin, J. Tongio, M. H. Tongio, M. Greget, V. Fuss, C. Becker, B. Heyd, J. Chabod, J. M. Rebibou, Y. Hudel, I. Carrey, J. Philippe, R. Mage, F. Triponez, C. Goumaz, J. Villard.

This program was supported by grants from France’s Ministry of Health (PHRC), Alfediam Novo-Nordisk, Region Rhône-Alpes (Emergence), and Eli Lilly France.

We thank all the procurement teams of the University Hospitals in Switzerland and France for their continuous effort and expert assistance in this clinical program.