Time Lag of Glucose From Intravascular to Interstitial Compartment in Type 1 Diabetes

Research Design and Methods

After approval from the Mayo Institutional Review Board and signed informed consent, 6 subjects with T1D were enrolled (4 on insulin pump and 2 on multiple daily insulin injections). Subjects reported to the Clinical Research Unit (CRU) of the Mayo Center for Clinical and Translational Science (CCaTS) in the morning after an overnight fast for the screen visit. Inclusion criteria include age 18-60 years, BMI 19-30 kg/m², HbA1c ≤ 10 %, and serum creatinine ≤1.5 mg/dl. Exclusion criteria included pregnancy, breast-feeding or other comorbidities (eg, nephropathy, neuropathy, macrovascular disease, hypertension) that precluded participation. Those with stable background diabetic retinopathy were included. Medications that could influence glucose tolerance were exclusionary. After history and physical examination, fasting blood samples were drawn to ensure that subjects met enrollment criteria. A negative pregnancy test was obtained in women with child-bearing capabilities before enrollment. A dual-energy X-ray absorptiometry (DXA) scan was performed to measure body composition.⁸ Subjects who met the enrollment criteria were asked to return for the study visit within 2-3 weeks.

Participants were admitted to the CRU at ~1600 hours, consumed a standard 10 kcal/kg evening meal (50% carbohydrate, 20% protein, and 30% fat) at ~1700 hours and remained nil per oral except water for remainder of the study. Subjects administered a premeal insulin bolus according to their customary insulin: carbohydrate ratio adjusted for the prevailing glucose concentrations. Intravenous insulin infusion was started at ~2100 hours and continued throughout the rest of the study as per protocol to maintain euglycemia.⁹ Insulin pump was discontinued for those on the insulin pump when the intravenous insulin infusion was started and those who were on multiple daily insulin did not administer their night-time basal insulin dose. At ~0600 next morning, a cannula was inserted retrogradely into a dorsal hand vein. The heated hand vein method was utilized to periodically draw arterialized venous blood for glucose and tracer concentrations.^8,10 A cannula was also inserted into a forearm vein on the contralateral arm for tracer infusions.

Experimental design was as described earlier.⁸ Briefly, 4 microdialysis catheters (CMA 63, 20 kDa M Dialysis Inc, North Chelmsford, MA) were inserted under local anesthesia and aseptic precautions into subcutaneous abdominal fat, 2 on each side of the abdomen and were infused with CMA perfusion fluid via CMA 107 microdialysis pump at a constant rate of ~1 µl/min for the study period. At periodic intervals, timed-pooled microdialysate effluent and blood samples were collected for glucose and tracer measurements simultaneously. Following insertion of the last microdialysis catheter, at least 1 hour was provided to allow catheters to reach steady state. At ~8 am (0 minutes), an intravenous bolus of [1-¹³C] glucose was administered over 10 seconds. Starting 4 minutes prior to the [1-¹³C] glucose bolus, microdialysate samples were collected every 5 minutes for the next 30 minutes and periodically thereafter until 9:57 am (117 minutes). Subsequent tracer glucose boluses and sequential timing of microdialysate and blood sample collections were as described previously.⁸ The rationale for using multiple glucose tracers was to maximize the timed collection frequency of the microdialysate samples. In preliminary in-vitro studies at different microdialysis pump infusion rates, we had observed that the recovery of the glucose tracer/s were optimal at a pump infusion rate of 1 µl/min. At this optimized low infusion rate, we needed to pool samples from 4 microdialysis catheters, simultaneously every 5 minutes, to obtain the minimum and adequate sample volume for glucose isotope analyses in duplicate. If we had used only 1 glucose tracer, our timed samples would have been restricted (eg, for tracer 1: –4 to 1, 1-6, 6-11, 11-16 minutes, etc). Hence we would not have been able to plot the time course of glucose appearance into ISF at more frequent intervals, for example, at 2 minutes, 3 minutes, and so on. Use of multiple glucose isotopes permitted more flexibility in timed collections (eg, for tracer 2: –3 to 2, 2-7, 7-12, 12-17 minutes and so on). Of course, ideally one would have preferred to use several more (up to 5) glucose tracers with sequential 5 minute collections so that the time course of glucose appearance could have been plotted for every minute from the time of injection, but that would have been impractical both for research subject comfort and convenience and would have added substantially to the study time, resources required for custom labeling such tracers or tags and the cost of the study. Importantly, the plasma sample collections were timed exactly to the end of each microdialysate sample collection times.

At the end of the study at ~4 pm, all infusions were discontinued, catheters and cannulae removed and subjects provided a meal. Those on insulin pump resumed their insulin rates at the customary settings and those on multiple dose insulin returned to their dosing schedule. They were then discharged from the CRU after ensuring that their fingerstick glucose values were clinically in the safe range.

Results

Discussion

Applying a combination of glucose isotope analyses and microdialysis techniques, we have demonstrated that the maximum physiological time lag of glucose transport from the intravascular to subcutaneous interstitial fluid compartment in those with T1D is <10 minutes during resting overnight fasted steady-state conditions. There is good evidence that at least half of the participants had detectable values in as little as 6.8 minutes. As described recently⁸ in healthy subjects, we employed the same stringent criteria to account for isotope assay noise based on the limit of detectability of the isotopes by mass spectrometry. Plasma glucose concentrations were held at a steady state of ~114 mg/dl (~6.3 mM) with an intravenous insulin infusion throughout the study. It is noteworthy that at steady-state, the lag time cannot be estimated by simply super-posing ISF glucose to venous glucose, hence the need for glucose tracers. Furthermore, the effects of non-steady-state conditions and stimuli, that is, exercise and meals on time lag and equilibration times are currently being conducted to advance our knowledge in this area. We chose to administer insulin intravenously as opposed to subcutaneously to minimize confounding of local insulin delivery into the subcutaneous space affecting subcutaneous glucose transport and hence time lag by influencing glucose uptake by local adipose tissue. We did so to develop and establish a “clean” model of subcutaneous glucose kinetics in T1D subjects. Furthermore, to determine equilibration times of glucose in the ISF, sophisticated modeling of the data is currently being performed and will be reported in a separate manuscript. Needless to say, further studies are also necessary to determine the effects of subcutaneous insulin delivery, either continuously through an insulin pump or intermittently via insulin bolus injections, on time lag of glucose appearance in T1D subjects.

It is indeed surprising that the physiological time lag described here in T1D subjects was similar to our recent report in healthy subjects.⁸ Given well-established clinical factors of local anatomical abnormalities that exist in patients with T1D that include changes in micro-vasculature and lipo-hypertrophy^13-15 which has been linked to poor glycemic control, trauma from repeated insulin injections, resultant fibrosis, and so on, we had anticipated that the time lag would be considerably greater than observed in healthy subjects. This is because, in theory, glucose molecules will have to traverse more barriers as they move across from the intravascular to the interstitial space. Microvascular involvement of subcutaneous fat could also pose an additional barrier to the time lag. In contrast, insulin induced lipoatrophy, an immune mediated process that continues to be observed with insulin analogs^16,17 could influence glucose transport and hence the time lag. Although none of our T1D subjects had clinically obvious lipoatrophy or lipo-hypertrophy, it is highly likely that the anatomical structure of the abdominal subcutaneous space was different compared to those without diabetes due to insulin related changes in local adipogenesis and/or injection related trauma. Furthermore, the effects of duration of diabetes and degree of glycemic control on time lag will need to be evaluated in future studies.

The study had similar limitations as discussed in our recent report in healthy subjects.⁸ In particular, this related both to the unreliability of using [2-¹³C] glucose data due to interference from the [1-¹³C] glucose moiety infused earlier. However, the robustness of measurement of both [1-¹³C] and [6,6-²H₂] glucose isotopes and the congruence of the results with both isotopes, strengthen our ultimate observations.

To conclude, we have demonstrated that the time lag of glucose appearance into the ISF from the intravascular compartment is between 7 and 8 minutes in overnight fasted T1D subjects, which is considerably shorter than many have hypothesized. Taken together, such information is critical to further refine and develop next generation algorithms for the CGM especially in the context of the closed-loop artificial pancreas systems currently being developed for T1D.