Clinical islet transplantation has evolved over the last decade and constitutes today an established clinical therapy for subjects with the most severe forms of type 1 diabetes (T1D) in Canada and in many European countries. In the United States, several licensure phase 3 studies are ongoing with an aim to obtain Food and Drug Administration approval and reimbursement for the procedure.
To allow broad application of replacement therapies for T1D, efforts have been directed to develop new and unlimited sources of transplantable insulin producing cells.1,2 Islets obtained from genetically modified pigs provide long-term normalization of glucose metabolism in streptozotocin (STZ)-diabetic nonhuman primates.3,4 Similarly, insulin-producing cells generated from human embryonic stem cells or induced pluripotent stem cells are at present a reality.5,6
Yet, the side effects with the currently available immunosuppressive drugs shift the “cost-benefit” analysis in favor of not performing transplantation for most subjects with T1D. Hence, there is little need for new additional sources of transplantable β cells unless the immune response induced in the recipient can be controlled with novel means with significantly less severe side effects. In principle, the immune system triggered can be controlled using 3 different approaches: (1) Systemic unspecific immunosuppression, (2) induction of antigen-specific immunological nonresponsiveness (tolerance), and (3) physical immune protection encapsulation. As discussed above, the currently available systemic unspecific immunosuppressive regimes do not constitute a valid option for future large-scale β-cell replacement, and even if progression continuously occurs, induction of transplantation tolerance seems far from a clinical reality within the near future.
Techniques for encapsulation present a most attractive strategy to overcome the need for systemic immunosuppression as well as to secure the possibility to safely remove the transplanted cells if needed, for example, infections or formations of teratoma.7 Transplantation of encapsulated insulin-producing cells has been shown to cure diabetes as evidenced by rapid return to normoglycemia and normalization of glucose disposal during an intravenous glucose tolerance test (IVGTT). However, insulin, or c-peptide, release during the IVGTT has in most studies not been determined.2,8‐15 Even so, normalization of glucose dynamics during an IVGTT has been interpreted as evidence of also active insulin secretion even if it has been known since the time of Soskin et al16,17 that glucose could influence its own disposal independent of changes in the plasma insulin level.
We therefore investigated the role of active insulin release in controlling blood glucose disposal during an IVGTT in experimental transplantation. Rats were made diabetic and then implanted subcutaneously with slow-release insulin pellets (Linplant; LinShin Canada Inc, Toronto, Canada) to obtain near normal glucose metabolism but without any active insulin secretion. B-glucose and c-peptide were measured during an IVGTT (0.5 or 1 g of intravenous glucose infusion) in insulin pellet–treated animals and in nondiabetic controls.
MATERIALS AND METHODS
Animal experiments were in accordance with the Swedish Animal Welfare Act (SFS 1988:534) and The Swedish Animal Welfare Ordinance (SFS 1988:539) both in agreement with the Directives 86/609/EEG and 2010/63/EU of the European Parliament and of the Council on the protection of animals used for scientific purposes. The study was approved by the Uppsala Laboratory Animal Ethical Committee (Permit Number: C34/14 and C35/14).
Healthy male Wistar rats weighing 250 to 350 g from Scanbur (Sollentuna, Sweden) were used. The rats were housed 2 by 2 in plastic cages under a 12:12 hour light-dark cycle. Animals were kept under standard laboratory conditions and given water and food ad libitum.
Diabetes was induced by intraperitoneal injection of STZ (55 mg/kg body weight [BW]) (Sigma-Aldrich Sweden AB, Stockholm, Sweden). Only rats with blood glucose levels above 20 mM on 2 consecutive days after STZ administration were classified as diabetic and included in further experiments. A slow-release insulin device, Linplant was inserted subcutaneously under the dorsal skin in isoflurane anesthetized animals (Abbott). Nonfasting blood glucose levels were measured daily after transplantation for 5 days or longer. Only animals with blood glucose below 11.1 mM (200 mg/dL) were used for further experiments.
Glucose tolerance was evaluated during an IVGTT under full anesthesia (thiobutabarbital sodium administered 10 minutes before glucose injection (100 mg/kg BW intraperitoneally) (Inactin; Sigma-Aldrich Sweden AB). Bolus injection of glucose was given within 60 seconds via the tail vein. Blood glucose was measured immediately before and 5, 10, 30, 60, 90, and 120 minutes after glucose injection. Blood glucose was measured with a glucometer (CONTOUR NEXT; Bayer, Solna, Sweden), operating within a range of 0.6 to 33.5 mmol glucose/L. Blood samples (100 μL) from the tail vein were collected immediately before and 10 minutes after glucose injection. C peptide levels were determined using a rat c-peptide enzyme-linked immunosorbent assay (Mercodia, Uppsala, Sweden). Animals were killed via a heart puncture and the pancreas removed and fixed in formalin and processed for immunohistochemical evaluation.
The following cohorts of STZ-diabetic rats were used: Cohort 1 inserted with a single Linplant implant and challenged with a bolus of 0.5 g glucose/kg BW; Cohort 2 inserted with a single Linplant implant and challenged a bolus of 1.0 glucose/kg BW; Cohort 3 inserted with 2 Linplant implants and challenged a bolus of 0.5 glucose/kg BW. Non-STZ–treated rats without any Linplant implants served as controls. Each Linplant implanted rat had a corresponding control rat examined in parallel.
Consecutive 5-μm pancreas sections were processed and stained using a standard immunoperoxidase technique for paraffin section, as previously described.18 In short, primary antibody was A0564 Guinea pig anti-insulin (DAKO, Glostrup, Denmark). Bound antibodies were visualized using DAKO EnVision (DAKO) and DAB (diaminobenzidine-based substrate, DAKO). Sections were counterstained with hematoxylin and analyzed by light microscopy by an investigator unaware of the origin of the sections.
Data are presented as means ± SEM. The statistical significance of the differences between groups was analyzed by the Kruskal-Wallis test followed by Dunn test for multiple comparisons.
Implantation of a single Linplant implant resulted in normalization of nonfasting blood glucose values in the period (≥5 days) before the IVGTT (Table 1).
There were no significant differences in peek glucose values and subsequent glucose disposal between the STZ diabetic rats in cohort 1 with 1 Linplant implant and challenged with a bolus of 0.5 g glucose/kg BW (open triangles) and corresponding nondiabetic controls (open circles: P > 0.05; Figure 1A).
In the animals, we aimed to overload the capacity of glucose effectiveness by injecting a bolus of 1 g glucose/kg BW, the initial blood glucose rose to 24.8 ± 0.6 mmol/L, with no significant difference between the STZ diabetic rats with a Linplant implant and controls (P > 0.05). Again, no main differences in glucose disposal were found between diabetic rats treated with a Linplant (open diamonds) and nondiabetic controls (open squares: Figure 1A).
The STZ diabetic rats treated with Linplant implant had circulating c-peptide levels below or close to the detection limit of the enzyme-linked immunosorbent assay both before and during the IVGGT. In comparison nondiabetic rats had markedly higher c-peptide levels under basal conditions (P < 0.005), which was further increased during the IVGTT (Figure 1B).
Nondiabetic rats had an abundant number of insulin-producing cells in their pancreases, whereas almost no such cells could be found in the pancreases of the STZ diabetic animals treated with Linplants (Figure 1C and D).
The cohort of rats that received 2 Linplant pellets displayed hypoglycemia in the period (≥5 days) before IVGTT (Table 1). When challenged with a bolus of glucose (0.5 g/kg), blood glucose in these rats peeked at 10.2 mM, significantly less than their corresponding control rats (17.1 mM; P < 0.05). However, there were no difference in glucose disposal rate between the Linplant-treated and the control rats in returning to their initial glucose values.
The results presented demonstrate that glucose disposal during an IVGTT in rats with only basal and nonglucose-regulated plasma insulin levels are mainly due to insulin independent mechanisms. In light of this, it is not possible to draw any firm conclusions on active insulin secretion based on findings of normal blood glucose values during an IVGTT.
Glucose tolerance in mammals depends on a complex interaction among both insulin-dependent and -independent mechanisms. The former being determined by the interaction of insulin secretion from the pancreatic β cells and the action of insulin to: (1) enhance uptake of glucose in peripheral tissues, for example, adipose and muscle tissue; and (2) to suppress endogenous glucose production from the liver.19 Less recognized is the function of the insulin independent mechanisms, that is, the capacity of glucose per se, independent of changes in insulin concentration, to stimulate its own uptake and suppress the endogenous glucose production. When plasma insulin is at basal levels, this effect of plasma glucose, termed glucose effectiveness is the primary determinant of glucose disposal in rodents.16,17,20‐24 As illustrated by the results herein, in rodents with only baseline insulin secretion delivered by a slow-release device perfectly normal dynamics in blood glucose during an IVGTT is obtained even though no active insulin secretion is possible. An inherent limitation of the experimental model is the potential influence of anesthesia on glucose metabolism in rats. In the present study, thiobutabarbital sodium was used to minimize the influence of anesthesia because this drug have been shown to induce only marginal effects on glucose-induced insulin secretion, glucose disposal, and islet blood flow.25
The role of glucose effectiveness during an IVGTT is more prominent in rodents when compared with humans.22 In humans, tolerance to an intravenous glucose load, quantified as the disappearance rate for glucose (Kg), was decreased by 50% when the endogenous insulin response was suppressed.20,22,26 Based on this and other studies, it is assumed that in healthy humans, glucose effectiveness, and insulin-dependent mechanisms contribute approximately equally to glucose disposal during an IVGTT.22 However, in individuals with insulin resistance and impaired glucose tolerance, insulin sensitivity is decreased, and glucose effectiveness becomes more important.21,27
The importance of glucose effectiveness for glucose disposal during an IVGTT in rodents has implications for the interpretation of in vivo studies conducted in the development of novel sources of insulin-producing cells from human embryonic stem cells. In the studies published so far, a gradual improvement of glucose metabolism over a period of weeks occurs until blood glucose levels are normalized. After establishment of normoglycemia, the transplanted animals are usually subjected to an IVGTT to demonstrate maturation of the transplanted cells and establishment of glucose-induced insulin secretion; however, as shown herein, normal glucose disposal during an IVGTT could equally well be achieved with immature insulin-producing cells only able to provide constitutive baseline insulin secretion, blind to increases in blood glucose. Surprisingly, several studies within the area do not include insulin, or c peptide, measurements during the IVGTT,28,29 whereas other studies show only moderate increases in insulin or c-peptide after 30 minutes or more,5,6 that is, at a time too late to provide evidence of normal and prompt glucose-stimulated insulin secretion.
Our findings have perhaps even more relevance for strategies to develop islet encapsulation to avoid systemic immunosuppression and thereby allowing more widespread application of replacement therapies for the treatment of subjects with T1D. Here, allogeneic or xenogeneic insulin-producing cells are positioned within an immune isolation device preventing direct cellular communication with the transplanted cells. Hence, glucose transportation is solely dependent on diffusion from the blood stream to the interstitial space, over the immune barrier, and to the insulin-producing cells. Often, these encapsulation devices have a considerable “dead space” which must be saturated with the ambient glucose level before insulin secretion is initiated. Released insulin molecules accumulate within the capsule until a sufficient concentration gradient has been created to drive diffusion out from the chamber.30 Again, only diffusion, based on differences in concentration gradients between various tissue compartments, drives insulin released in the surrounding interstitial space to be delivered over the capillary walls and into the blood stream. Taken together, these processes should cause a significant delay in the response to a glucose challenge. Even so, most experimental studies conducted in this area of research show correction of diabetes and normal glucose disposal during an IVGTT. Again, obtained findings have been interpreted to show normal insulin release dynamics, however, without accurate insulin or c-peptide determinations in conjunction with glucose infusion.2,8‐15,31 In contrast, in vitro studies show a significant delay in insulin release from encapsulated when compared with nonencapsulated islets.30 A finding in agreement with the results presented herein demonstrate that the reported normal glucose disposal during an IVGTT could equally well be achieved with an encapsulation device with poor glucose and insulin diffusion dynamics resulting in baseline insulin release comparable with that from a slow-release device, such as the Linplant.
The degree of revascularization of transplanted islets remains an important question. Numerous, experimental studies demonstrate that islets transplanted do not reach same level of vascularization as naive islets, and assessments of oxygen tension show pO2 of 10 or less with the graft.32 In vitro islet hypoxia rapidly causes loss of active insulin secretion, that is, a decrease in pO2 to 10 mm Hg results in loss of 90% of glucose-induced insulin secretion.33 Again, the contradiction of a normal response to a glucose challenge reported in experimental islet transplantation34 and the severe impairment of glucose stimulated insulin release imposed by hypoxia and can likely be explained by the dominant role of glucose effectiveness in the rodent models. Little data exist on the degree of revascularization after clinical islet transplantation. Still, the few studies that do exist show a considerable revascularization of the transplanted islets.35 Also, insulin secretion is promptly induced during an IVGTT performed on a person with a well-functional islet transplant.36
In conclusion, functional assessments of transplanted β cells remain challenging, and the predominant effect of glucose effectiveness in rodents is commonly overlooked. Based on the results presented herein, normalization of glucose disposal during an IVGTT should not be interpreted as evidence for active glucose-stimulated insulin release unless substantiated by insulin or c peptide measurements in immediate conjunction with glucose administration. In light of the here present results, it seems important to reassess conclusions based on these experimental studies before translation into clinical trials are considered.
The authors thank Karin Fonnaland for excellent technical assistance.
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