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Department of Medicine (M.R.R., K.L.T.), Division of Endocrinology, Diabetes, and Metabolism, Department of Surgery (A.N.), Division of Transplantation, and the Monell Chemical Senses Center (K.L.T.), University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6149
Address all correspondence and requests for reprints to: Michael R. Rickels, M.D., University of Pennsylvania School of Medicine, Division of Endocrinology, Diabetes, and Metabolism, 778 Clinical Research Building, 415 Curie Boulevard, Philadelphia, Pennsylvania 19104-6149. E-mail: rickels{at}mail.med.upenn.edu.
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Abstract |
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Objective: Our objective was to evaluate insulin sensitivity after islet transplantation performed under tacrolimus-based immunosuppression that used minimal steroids.
Setting: This study was conducted at the University of Pennsylvania General Clinical Research Center.
Participants: Eight islet transplant recipients, six type 1 diabetic (T1D), and 10 nondiabetic control subjects participated.
Intervention: We performed an insulin-modified frequently sampled iv glucose tolerance test to measure insulin sensitivity (SI), glucose effectiveness, and free fatty acid (FFA) dynamics.
Results: SI was significantly greater in the islet transplant and control groups, compared with the T1D group (P < 0.05 for both comparisons). Glucose effectiveness was not significantly different across all three groups but was lower by trend in the T1D and islet transplant groups, compared with the control group (P = 0.07 overall ANOVA). FFA levels suppressed normally in the transplant recipients, but the timing and magnitude of FFA suppression were significantly impaired in the T1D group, compared with the islet transplant and control groups (P < 0.05 for all comparisons). The acute insulin response to glucose and the disposition index (DI = acute insulin response to glucose x SI) were significantly lower in the islet transplant group, compared with the control group (P < 0.05 for all comparisons).
Conclusions: These data suggest that even modest restoration of insulin secretion in islet transplant recipients may result in improved insulin sensitivity and FFA dynamics.
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Introduction |
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In T1D insulin sensitivity is impaired (7, 8, 9), and the development of insulin resistance has been attributed to the sustained hyperglycemia associated with poorly controlled diabetes (10, 11, 12). In islet transplant recipients, insulin sensitivity may be impaired by immunosuppression regimens that use glucocorticoids (13) or with steroid-free regimens containing tacrolimus and rapamycin, which have been implicated in the development of insulin resistance in a rat model of islet transplantation (14). However, the correction of hyperglycemia after islet transplantation may improve insulin sensitivity. To evaluate insulin sensitivity after islet transplantation performed under modern tacrolimus-based immunosuppression, we performed an insulin-modified frequently sampled iv glucose tolerance test (FSIGT) that allows for the derivation of indices of insulin-dependent and insulin-independent glucose disposal, the insulin sensitivity index, and glucose effectiveness, respectively, in islet transplant recipients and compared the results with those derived from T1D and nondiabetic control subjects. Because defects in the ability of insulin to suppress plasma free fatty acid (FFA) levels have also been implicated in the development of impaired insulin-dependent glucose disposal (15), we further compared FFA suppression during the FSIGT in all three groups.
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Subjects and Methods |
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Subjects were recruited from the islet transplantation program at the University of Pennsylvania (Table 1
). The T1D subjects (n = 6) had long-standing C-peptide-negative disease and were on the waiting list for islet transplantation. Three of these six subjects had a well-functioning kidney allograft supported by 5 mg of daily prednisone, tacrolimus, and mycophenolate mofetil. The islet transplant recipients (n = 8) were studied a mean 5 months (range 2–12) after their last islet infusion. Subjects had undergone one or two intraportal islet infusions under daclizumab induction immunotherapy as previously reported (4, 16). Four of these eight subjects had a well-functioning kidney allograft. All received tacrolimus and either rapamycin or mycophenolate mofetil for maintenance immunosuppression, and three subjects with a kidney allograft also received 5 mg of daily prednisone. Both T1D subjects and islet transplant recipients had a comparably long duration of T1D. Islet transplant recipients received 12,664 ± 1,618 islet equivalents per kilogram body weight and required significantly less insulin therapy, compared with the T1D subjects (P < 0.001; Table 1), with three of the eight transplant recipients insulin independent at the time of assessment.
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). This study protocol was approved by the Institutional Review Board of the University of Pennsylvania, and all subjects gave their written informed consent to participate. FSIGT
All subjects were admitted to the University of Pennsylvania General Clinical Research Center the afternoon before study and fasted overnight after 2000 h for 12 h before testing. Islet transplant recipients who were not insulin independent held any long-acting insulin for more than 24 h and any rapid-acting insulin for more than 12 h before testing. T1D subjects similarly held injectable insulin and received iv insulin overnight to maintain the blood glucose concentration 100–150 mg/dl (5.5–8.3 mmol/liter); the insulin infusion was discontinued 20 min before testing. All other medications were withheld on the morning of study. By 0700 h in the morning, one catheter was placed in an antecubital vein for injections, and one catheter was placed in a contralateral antecubital vein for blood sampling, with the sampling arm warmed by a heating pad. Patency of the iv catheters was maintained with slow infusions of 0.9% saline.
After baseline blood sampling at –15, –10, and –5 min, 0.3 g/kg of 50% glucose was injected over 1 min starting at t = 0 and 0.03 U/kg of insulin (1 U per 1 ml solution) was injected over 30 sec starting at t = 20 min. Additional blood samples were collected at t = 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, 20, 22, 25, 30, 40, 50, 70, 100, 140, and 180 min after the injection of glucose (18). All samples were collected on ice into chilled tubes containing EDTA, trasylol, and leupeptin; centrifuged at 4 C; separated; and frozen at –80 C for subsequent analysis.
Biochemical analysis
Plasma glucose was measured in duplicate by the glucose oxidase method using an automated glucose analyzer (YSI 2300; Yellow Springs Instruments, Yellow Springs, OH). Plasma immunoreactive insulin was measured in duplicate using a double-antibody RIA (Linco Research, St. Charles, MO). Plasma FFA levels were measured in duplicate using enzymatic colorimetrics (Wako Chemicals, Richmond, VA).
Calculations and statistics
Baseline levels of glucose, insulin, and FFA were calculated from the mean of the –15-, – 10-, and –5-min samples preceding the injection of glucose. The FSIGT parameters insulin sensitivity (SI), glucose effectiveness (SG), acute insulin response to the injection of glucose (AIRg), and disposition index (DI) were derived from Bergman’s minimal model using MINMOD Millennium software (19). The SI index, a measure of the capacity of insulin to promote glucose disposal, is modeled from the relationship of incremental insulin values over baseline to glucose disappearance. SG, a measure of the capacity of glucose to mediate its own disposal, is modeled from the effect of basal insulin levels on glucose kinetics. Good parameter resolution, defined by a fractional SD (FSD) 0.5 or less (20), was achieved for SI and SG in all subjects from all three groups (Table 1). AIRg, a measure of ß-cell function, is calculated as the incremental area under the curve (AUC) for insulin between 0 and 20 min after injection, whereby the AUC is calculated by the trapezoidal rule with the mean of the baseline values subtracted. Because of absent ß-cell function in the T1D group, AIRg resulted in large negative values in this group, and consequently the DI could not be adequately resolved. The DI is a composite measure of ß-cell function that accounts for the relationship between insulin secretion and sensitivity by the product of AIRg and SI and was adequately resolved in the islet transplant recipients and control subjects (Table 2).
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The FFA analysis was performed in all T1D subjects (n = 6) and islet transplant recipients (n = 8) and in a subgroup of the nondiabetic control subjects (n = 6). Incremental AUC for FFA (AUCFFA) was calculated between 0 and 180 min (22). In addition, the FFA profile during the FSIGT was analyzed according to the model of Sumner et al. (23) in which three phases of FFA dynamics have been described during the 180 min after the injection of glucose. The first phase consists of an extension of baseline FFA levels for approximately 10 min and is calculated as the time from t = 0 to the initial sustained decrease in FFA; the second phase is characterized by a suppression of FFA levels until a nadir and is evaluated by the fractional disposal rate of FFA calculated as the slope of log-transformed FFA values between 10 and 40 min using least-squares linear regression; the third phase is defined as the period from the nadir to the time FFA levels have returned to baseline levels. In nondiabetic subjects, the dynamics of these three phases are the same during both a standard and insulin-modified FSIGT, suggesting that a normal endogenous insulin response to the injection of glucose at t = 0 min achieves maximal FFA suppression without any added effect from the exogenous insulin administered at t = 20 min (23).
Results from all three groups were compared by one-way ANOVA, and when significant differences were found, comparisons between groups were performed with two-tailed, unpaired Student’s t tests (24) using Statistica software (Tulsa, OK). Significance was considered at P < 0.05.
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Results |
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). Baseline insulin was significantly higher in the T1D subjects than the transplant recipients or control subjects (P < 0.05 for both comparisons; Table 2), a consequence of the overnight insulin infusion required to maintain normoglycemia in this group.
During the FSIGT, glucose increased comparably in all three groups after injection, but overall glucose disappearance was markedly impaired in the T1D group, compared with the control group, and intermediate in the islet transplant group (Fig. 1A). The glucose disappearance rate was significantly greater in the control group, compared with the T1D and islet transplant groups (P < 0.01 for both comparisons), whereas the AUCglu was significantly greater in the T1D group, compared with the islet recipients and control subjects (P 
0.001 for both comparisons; Table 2). Insulin-dependent glucose disposal, SI, was significantly less in the T1D group, compared with the islet transplant recipients and the control group (P < 0.05 for both comparisons), and SI was less by trend in the transplant recipients, compared with the control group (P = 0.06; Table 2 and Fig. 2A). Insulin-independent glucose disposal, SG, was not significantly different across all three groups but was lower by trend in the T1D and islet transplant groups, compared with the control group (P = 0.07 overall ANOVA; Table 2 and Fig. 2B). The AIRg and DI were significantly less in the islet transplant recipients, compared with the control group (P < 0.05 for both comparisons; Table 2). In Fig. 1B, the inset demonstrates the difference between the groups in endogenous insulin secretion (AIRg). The AUCins and insulin clearance were not different across the three groups (Fig. 1B and Table 2). Final insulin levels were not different across groups, matched baseline insulin levels in the islet transplant recipients and control subjects, but were significantly lower than baseline in the T1D subjects (P < 0.05 paired t test; Table 2).
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). At baseline, FFA levels were comparable among the T1D, islet transplant, and control groups (Table 3); FFA levels subsequently rose in the T1D group until the administration of exogenous insulin at 20 min, likely a result of withdrawal of the overnight insulin infusion. The duration of phase 1 was comparable in the islet transplant and control groups, albeit with a nonsignificant delay occurring in the islet recipients (Fig. 1C), ending before the exogenous administration of insulin at t = 20 min; phase 1 was significantly longer in the T1D group (P < 0.001 for both comparisons; Table 3). Because the suppression of FFA levels that characterizes phase 2 did not occur until after the injection of exogenous insulin in the T1D group, the fractional disposal rate of FFA was evaluated from 10 to 20 min to assess the effect of endogenous insulin secretion (23) and from 25 to 40 min to examine the effect of the exogenous insulin, in addition to the overall disposal from 10 to 40 min. From 10 to 20 min, the suppression of FFA was comparable in the islet transplant recipients and the control group but absent in the T1D group (P < 0.05 for both comparisons; Table 3). From 25 to 40 min, FFA disappeared comparably across all three groups, and overall from 10 to 40 min, the fractional disposal rate of FFA was greater in the transplant recipients than in the T1D and control groups (P < 0.05 for both comparisons; Table 3). The FFA nadir occurred at a similar time in the T1D, islet transplant, and control groups, but the level achieved was significantly lower in the islet transplant and control groups compared with the T1D group (P 0.01 for both comparisons; Table 3). There was a statistical trend for an earlier return to baseline FFA levels in the T1D group, compared with the islet transplant and control groups (P = 0.09 across all groups; Table 3), and subsequent levels increased markedly over baseline in the T1D subjects (Fig. 1C). The resulting AUCFFA was significantly greater in the T1D subjects, compared with the islet recipients and control subjects (P < 0.01 for both comparisons; Table 3).
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). Glucose tolerance and FFA suppression were also significantly associated with SI (Table 4).
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Discussion |
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0.5) in all subjects, indicating that an insulin-modified FSIGT can be used to evaluate glucose disposal in islet transplant recipients, as has previously been demonstrated for T1D subjects (20, 25). In addition, normal suppression of plasma FFA levels occurred in the islet transplant group, in contrast to the T1D subjects, implicating an additional mechanism in addition to correction of hyperglycemia, which could contribute to improved insulin sensitivity after transplant. Whereas these data suggest a possible benefit of islet transplantation on insulin sensitivity, confirmation by prospectively conducted FSIGTs in T1D subjects before and after transplantation is required.
The insulin sensitivity estimates reported here are consistent with a prior euglycemic clamp study that demonstrated decreased insulin sensitivity in islet transplant recipients, compared with normal, but greater insulin sensitivity in recipients with functional vs. nonfunctional islet grafts (13). In this earlier study, islet recipients were immunosuppressed with cyclosporine, azathioprine, and prednisone. The average prednisone dose was less in the group with functional vs. nonfunctional islets (9 vs. 16 mg daily), possibly contributing to the better insulin sensitivity in the group with functional islets (13). In the present study, islet recipients received tacrolimus, either rapamycin or mycophenolate mofetil, and little or no prednisone (5 mg daily). The T1D subjects received either the same immunosuppression to support a kidney graft or no immunosuppression, so the superior insulin sensitivity in the islet transplant recipients in this study is likely a consequence of the islet transplant and resultant improvement in metabolic control and not a difference in the immunosuppression.
Whereas insulin sensitivity in the islet transplant recipients was not significantly different from normal, there was a trend toward lower sensitivity in the islet recipients. The younger age of the control subjects might result in a greater SI than expected for a control group comparably aged to the islet recipients and T1D subjects. Nevertheless, the possibly reduced SI in the transplant recipients may be functionally important given the markedly reduced reserve capacity for insulin secretion after transplant (3). When assessed as the AIRg, ß-cell function in the islet transplant recipients was 39% of normal; however, when assessed in relation to SI, the DI was only 18% of normal, suggesting inadequate compensation for insulin resistance by the already reduced insulin secretion. Interestingly, the low SG present in T1D (20) may persist in islet transplant recipients. Similar results were reported from islet autografted dogs (26, 27) and T1D subjects in clinical remission (25) who had normal SI but impaired SG. Both islet transplant recipients and T1D subjects in clinical remission share poor insulin secretory function, and the greater AIRg in normal subjects may lead to an overestimation of SG by the minimal model approach (28). However, subjects with very early T1D identified through the Diabetes Prevention Trial, type 1, exhibited normal SG despite a markedly reduced AIRg (29). In the present study, SG appears comparable in both T1D subjects and islet transplant recipients, but differences from normal require confirmation by direct measurement (28, 30).
Whereas insulin secretory function is impaired after islet transplantation, there are several metabolic benefits consequent to the partially restored insulin secretion in islet transplant recipients. It is clear that functioning islet grafts lead to stabilization of glycemic lability and elimination of severe hypoglycemic episodes (31), an effect of both endogenous insulin secretion in response to hyperglycemia (2, 3) and appropriate inhibition of endogenous insulin secretion in response to hypoglycemia (32). Here the superior insulin sensitivity in islet transplant recipients, compared with T1D subjects, may be another metabolic benefit of restored insulin secretion. Indeed, first-phase insulin secretion is important in the restraint of hepatic glucose production (33). In the present study, the significant correlation of AIRg with measures of both glucose tolerance and FFA suppression supports an important role for endogenous insulin secretion on glucose and FFA disappearance, in particular during the first 20 min before exogenous insulin administration.
In T1D, impaired FFA suppression by insulin has been demonstrated using the euglycemic clamp technique (34), and impaired sensitivity of lipolysis to inhibition by insulin has been shown using labeled palmitate during a pancreatic clamp (35). To our knowledge, the FFA response to insulin has not been evaluated in T1D subjects using the FSIGT. The data here are consistent with impaired inhibition of lipolysis by insulin in T1D as indicated by the incomplete suppression of FFA levels despite comparable insulin levels from 20 to 180 min. Significantly higher peripheral insulin levels at baseline were required in the T1D group to restrain FFAs. Whereas final insulin levels were comparable (even slightly higher) in the T1D, compared with the other groups, the final insulin levels were significantly lower than baseline within the T1D group. This occurred by 100 min when FFA first exceeded baseline levels in the T1D group. Thus, peripheral insulin levels matched to the islet transplant recipients and control subjects were insufficient to normally suppress FFA levels in the T1D subjects. The portal insulin delivery present in the islet transplant recipients and control subjects, but not in the T1D group, might contribute to more potent FFA suppression than seen in T1D subjects dependent on systemic insulin delivery alone.
In the islet transplant recipients, the endogenous insulin response, albeit impaired, to the injection of glucose, initiated suppression of FFA levels well before the exogenous insulin bolus. This initiation of FFA suppression was nonsignificantly delayed in the islet recipients, likely an effect of only partial restoration of endogenous insulin secretion (AIRg). These data suggest that even modest increments in AIRg can potently lower plasma FFA levels, an effect that may enhance insulin-dependent glucose disposal. Indeed, AIRg correlated significantly to the fractional disposal rate for FFA between 10 and 20 min. Furthermore, after the exogenous insulin bolus, complete suppression of FFA levels occurred in the islet transplant and control groups but not in the T1D group despite achieving comparable insulin levels after 20 min. Thus, the presence of some insulin response during the first 20 min of the FSIGT may be necessary for normal FFA disposal. Moreover, in women with gestational diabetes and reduced endogenous insulin secretion during a FSIGT performed without exogenous insulin administration, FFA suppression was impaired (36). Whether the endogenous insulin response after islet transplantation is sufficient to maximally suppress FFA levels without the exogenous administration of insulin requires further study, such as in response to a more physiological stimulus such as meal ingestion.
In conclusion, minimal model parameters derived from an insulin-modified FSIGT are estimated with good resolution in islet transplant recipients, and suggest a possible improvement in insulin sensitivity after transplant. Improved insulin sensitivity may be explained by a combination of correction of hyperglycemia and restored suppression of FFA because both measures of glucose tolerance and FFA suppression correlated significantly with SI. Nevertheless, SI may not be fully restored to normal after islet transplantation, likely because of current immunosuppressive drugs or incomplete normalization of glycemia. This may impact graft function, reflected in the markedly impaired DI, in which a low engrafted ß-cell mass may not adequately compensate for an increased insulin secretory demand. Nevertheless, strategies directed at increasing the mass of engrafted ß-cells should further augment endogenous insulin secretion, which may enhance both glucose and FFA disposal, leading to improved SI and glucose tolerance for islet transplant recipients.
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Acknowledgments |
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Footnotes |
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First Published Online March 28, 2006
Abbreviations: AIRg, Acute insulin response to the injection of glucose; AUC, area under the curve; AUCFFA, AUC for FFA; AUCglu, AUC for glucose; AUCins, AUC for insulin; DI, disposition index; FFA, free fatty acid; FSD, fractional SD; FSIGT, frequently sampled iv glucose tolerance test; Kg, glucose disappearance rate; SG, glucose effectiveness; SI, insulin sensitivity; T1D, type 1 diabetes.
Received November 21, 2005.
Accepted March 22, 2006.
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References |
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