增龄对大鼠胰岛β细胞功能的影响

目的观察不同月龄大鼠的糖代谢指标变化,了解在基础和糖刺激下增龄对大鼠胰岛β细胞功能的影响。方法以4月龄（青年组,n=15）、14月龄（中年组,n=15）和24月龄（老年组,n=15）健康雄性Wistar大鼠为研究对象,进行口服葡萄糖耐量试验（OGTT）和胰岛素释放试验（IRT）,比较三组间基础血糖和胰岛素水平的差异,计算糖负荷后胰岛素增值与血糖增值的比值（ΔI10/ΔG10）,120min葡萄糖、胰岛素曲线下面积（AUCg,AUCi）,葡萄糖与胰岛素曲线下面积比值（AUCi/g）,胰岛素抵抗指数（HOMA-IR）,胰岛β细胞功能指数（HOMA-β）等,以分析各组间胰岛功能的差异。结果随着月龄的增长,空腹血糖有升高的趋势,但未达到统计学差异。OGTT后老年组大鼠表现为血糖达峰时间延长,血糖峰值增加,OGTT2h血糖及AUCg增加,即老年组大鼠出现糖耐量异常状态,主要表现为餐后高血糖。青年组、中年组和老年组大鼠空腹胰岛素水平分别为（0.59±0.14）、（1.60±0.15）、（2.37±0.04）μg/L（两两相比均P〈0.01）;IRT中,老年组大鼠胰岛素达峰时间延迟,ΔI10/ΔG10降低,AUCi增加（P〈0.05）。使用稳态模型评估,HOMA-β在青年、中年和老年组中呈递增趋势,但未达到统计学差异;而HOMA-IR在青年、中年和老年组分别为3.09±0.80、8.34±0.72、13.14±1.59（两两相比均P〈0.01）。结论正常老龄大鼠存在一定的胰岛素抵抗和代偿性胰岛素分泌增加,但由于胰岛素分泌的早期时相受损,仍然出现糖负荷后血糖增高状态。     

Beta-cell death and dysfunction drives hyperglycaemia in organ donors

Background

Donor hyperglycaemia following brain death has been attributed to reversible insulin resistance. However, our islet and pancreas transplant data suggest that other mechanisms may be predominant. We aimed to determine the relationships between donor insulin use and markers of beta-cell death and beta-cell function in pancreas donors after brain death.

Methods

In pancreas donors after brain death, we compared clinical and biochemical data in ‘insulin-treated’ and ‘not insulin-treated donors’ (IT vs. not-IT). We measured plasma glucose, C-peptide and levels of circulating unmethylated insulin gene promoter cell-free DNA (INS-cfDNA) and microRNA-375 (miR-375), as measures of beta-cell death. Relationships between markers of beta-cell death and islet isolation outcomes and post-transplant function were also evaluated.

Results

Of 92 pancreas donors, 40 (43%) required insulin. Glycaemic control and beta-cell function were significantly poorer in IT donors versus not-IT donors [median (IQR) peak glucose: 8 (7-11) vs. 6 (6-8) mmol/L, p = .016; C-peptide: 3280 (3159-3386) vs. 3195 (2868-3386) pmol/L, p = .046]. IT donors had significantly higher levels of INS-cfDNA [35 (18-52) vs. 30 (8-51) copies/ml, p = .035] and miR-375 [1.050 (0.19-1.95) vs. 0.73 (0.32-1.10) copies/nl, p = .05]. Circulating donor miR-375 was highly predictive of recipient islet graft failure at 3 months [adjusted receiver operator curve (SE) = 0.813 (0.149)].

Conclusions

In pancreas donors, hyperglycaemia requiring IT is strongly associated with beta-cell death. This provides an explanation for the relationship of donor IT with post-transplant beta-cell dysfunction in transplant recipients.     

Donor selection in heart transplantation

There is increased scrutiny on the quality in health care with particular emphasis on institutional heart transplant survival outcomes. An important aspect of successful transplantation is appropriate donor selection. We review the current guidelines as well as areas of controversy in the selection of appropriate hearts as donor organs to ensure optimal outcomes. This decision is paramount to the success of a transplant program as well as recipient survival and graft function post-transplant.     

How to use image analysis for islet counting

AIM: Assessment of islet mass before islet transplantation requires a reliable technique to enable exact analysis of islet volume. This study aimed to test the applicability of digital image analysis (DIA) for evaluation of samples of purified and non-purified islets. METHODS: Pancreatic islets were isolated from 10 Lewis rats. Samples of purified (n = 10) and non-purified islets (n = 30) were counted conventionally and by using a computerized method. The equipment for the computerized counting consisted of a digital camera installed on a stereomicroscope and connected to a personal computer. Images of 2272x1704 pixels were processed using a previously described non-commercial program originally developed for this purpose. Islets were converted to equivalents using globe and ellipsoid models. The insulin content of purified islets was assessed using radioimmunoassay and was correlated to the absolute and standardized islet number. RESULTS: Mean absolute numbers of purified islets ± SD were 908 ± 130 and 1049 ± 230 (manually and DIA respectively). Mean insulin content ± SD obtained from purified islets was 161 ± 45 mU. The mean equivalents of purified islets (1589 ± 555 for globe and 1219 ± 452 for ellipsoid) significantly correlated with insulin content. However, this correlation was not significant when absolute islet numbers were used, counted using either method. There was no significant difference in absolute non-purified islet numbers assessed by manual and computerized methods (average ± SD in 50 µl samples; 12.6 ± 4.1 and 13.3 ± 5.3 respectively; p = 0.22). The manual method showed a significantly higher yield of islet equivalents (IE; p < 0.001 for both globe and ellipsoid). CONCLUSION: The computer-based system for islet counting correlated better to insulin content than conventional islet estimation and prevented overestimation. Reproducibility and ease of assessment make it potentially applicable to clinical islet transplantation.     

Impact of donor-related variables on islet isolation outcome in dogs

Summary Clinical human islet transplantation programmes are considerably hampered by the variability of islet isolation outcome. The effects of the islet content of the pancreas and other donor-related variables on isolation outcome have not been evaluated systematically so far — either in large animals, or in man. We studied the impact of interindividual differences in age, body weight and pancreatic islet content on the outcome of collagenase isolation of islets from the splenic pancreas of beagle dogs (n=31). The islet volume of the splenic pancreas amounted to a mean (± SEM) 15.7±0.9 l per gramme pancreas, and varied three-fold (from 8.4 to 27.3 l). Isolated islet yield was 7.6±0.7 l/g and varied nine-fold (1.8–16.3 l). Animals also varied in age eight-fold (867 months) and body weight two-fold (8.6–18.3 kg). Differences in body weight and age explained 60% of variance in the fractional islet volume of the pancreas and 50% of the variance in islet yield (p<0.001). Fractional islet volume of the splenic pancreas also explained 50% of the variance in islet yield (p<0.001). We conclude that the outcome of islet isolation may be predictable after controlling for the variable islet content of pancreases, and other donor-related variables, and suggest that similar studies should be done in man.     

Effect of liver histopathology on islet cell engraftment in the model mimicking autologous islet cell transplantation

Background: The inflammatory milieu in the liver as determined by histopathology is different in individual patients undergoing autologous islet cell transplantation. We hypothesized that inflammation related to fatty-liver adversely impacts islet survival. To test this hypothesis, we used a mouse model of fatty-liver to determine the outcome of syngeneic islet transplantation after chemical pancreatectomy. Methods: Mice (C57BL/6) were fed a high-fat-diet from 6 weeks of age until attaining a weight of ≥28 grams (6–8 weeks) to produce a fatty liver (histologically > 30% fat);steatosis was confirmed with lipidomic profile of liver tissue. Islets were infused via the intra-portal route in fatty-liver and control mice after streptozotocin induction of diabetes. Outcomes were assessed by the rate of euglycemia, liver histopathology, evaluation of liver inflammation by measuring tissue cytokines IL-1β and TNF-α by RT-PCR and CD31 expression by immunohistochemistry. Results: The difference in the euglycemic fraction between the normal liver group (90%, 9/10) and the fatty-liver group (37.5%, 3/8) was statistically significant at the 18^th day post- transplant and was maintained to the end of the study (day 28) (p = 0.019, X² = 5.51). Levels of TNF–α and IL-1β were elevated in fatty-liver mice (p = 0.042, p = 0.037). Compared to controls cytokine levels were elevated after islet cell transplantation and in transplanted fatty-liver mice as compared to either fatty- or islet transplant group alone (p = NS). A difference in the histochemical pattern of CD31 could not be determined. Conclusion: Fatty-liver creates an inflammatory state which adversely affects the outcome of autologous islet cell transplantation.     

Pancreatic islet cell transplantation using non-heart-beating donors (NHBDs)

Recent dramatic improvements in clinical islet cell transplantation demonstrated by the Edmonton group have increased the demand for this treatment, and donor shortage could become a major problem. Utilization of marginal donors could alleviate the donor shortage, and non-heart-beating donors (NHBDs) might be good resources. The University of Pennsylvania group demonstrated that it was possible to isolate islets from NHBDs, and the group actually transplanted islets from NHBDs, for the first time. The patient became insulin-independent; however, there had been no more cases using NHBDs until our group initiated islet transplantations from NHBDs in Japan. In order to utilize NHBDs effectively, we modified the standard islet isolation method. These modifications included minimizing the warm ischemic time, the use of trypsin inhibition during isolation, carrying out density measurement before purification and the use of a less toxic islet purification solution. With these modifications we were able to transplant nine of ten islet preparations from ten NHBDs (90%), into five type-1 diabetic patients. The first transplantation was performed on April 7, 2004 (the first time in Japan), and this patient became insulin-independent after the second islet transplantation (first time in Japan). All patients showed improved glycemic control and reduced insulin requirements, without hypoglycemic events. We also performed living-donor islet transplantation, with our modified islet isolation protocol, on January 19, 2005. The improved islet isolation protocol enabled us to perform effective islet transplantations from NHBDs, and it also enabled us to perform the living-donor islet transplantation.     

Strategy for clinical setting in intramuscular and subcutaneous islet transplantation

Intraportal islet transplantation has a long history as a procedure for clinical islet transplantation. However, many recent studies revealed that the intraportal procedure has some disadvantages in transplant efficiency and safety. Many candidates as an optimal transplant site for islets have been assessed, but further studies and clinical trials are still necessary. Intramuscular and subcutaneous spaces are important candidates, because the transplant and biopsy procedures are simple approaches with minimal invasion and few complications. Although they are sites with hypovascularity and hypoxia, which contribute to the poor transplant efficiency, many experimental trials for improving the outcome in intramuscular and subcutaneous islet transplantations have been performed, focusing on early angiogenesis and scaffolds for engrafting transplanted islets. We review current progress in intramuscular and subcutaneous islet transplantations and discuss ways to develop them as optimal transplant sites for islets. Copyright © 2013 John Wiley & Sons, Ltd.     

Islet-after-failed-pancreas and pancreas-after-failed islet transplantation: Two complementary rescue strategies to control diabetes

For selected patients with type 1 diabetes, β-cell replacement is the treatment of choice, either by islet transplantation (ITX) or whole pancreas transplantation (PTX). When either modality fails, current practice is to consider retransplantation, or return to exogenous insulin. We investigate outcomes with PTX after failed ITX (PAI), and ITX after failed PTX (IAP). All patients receiving PAI or IAP at a single institution were identified. Donor and recipient variables were documented, including transplant outcomes analyzed for insulin requirement and metabolic control. Five subjects were listed for PAI, and 2 received transplants. Of the 4 listed for IAP, 3 have received transplants. The mean waitlist time was 4.5 ± 4.1 y for PAI and 0.35 ±0 .4 y for IAP (p = 0.08). Metabolic control was excellent after PAI, with 2/2 insulin-independent. After IAP, 1/2 achieved insulin independence and good metabolic control after 2 islet infusions. The third could not receive 2^nd infusion and presented c-peptide levels < 0.1 nmol/L. Both strategies are feasible. The outcomes after PAI in our center must be offset by much longer waitlist time due to the sensitization status of these patients. Data from multicentre experience will allow more robust comparative outcomes to be made, the current observations being restricted to a limited patient set.     

Islet-after-failed-pancreas and pancreas-after-failed islet transplantation: Two complementary rescue strategies to control diabetes

For selected patients with type 1 diabetes, β-cell replacement is the treatment of choice, either by islet transplantation (ITX) or whole pancreas transplantation (PTX). When either modality fails, current practice is to consider retransplantation, or return to exogenous insulin. We investigate outcomes with PTX after failed ITX (PAI), and ITX after failed PTX (IAP). All patients receiving PAI or IAP at a single institution were identified. Donor and recipient variables were documented, including transplant outcomes analyzed for insulin requirement and metabolic control. Five subjects were listed for PAI, and 2 received transplants. Of the 4 listed for IAP, 3 have received transplants. The mean waitlist time was 4.5 ± 4.1 y for PAI and 0.35 ±0 .4 y for IAP (p = 0.08). Metabolic control was excellent after PAI, with 2/2 insulin-independent. After IAP, 1/2 achieved insulin independence and good metabolic control after 2 islet infusions. The third could not receive 2^nd infusion and presented c-peptide levels < 0.1 nmol/L. Both strategies are feasible. The outcomes after PAI in our center must be offset by much longer waitlist time due to the sensitization status of these patients. Data from multicentre experience will allow more robust comparative outcomes to be made, the current observations being restricted to a limited patient set.     

Quantification of islet size and architecture

Human islets exhibit distinct islet architecture particularly in large islets that comprise of a relatively abundant fraction of α-cells intermingled with β-cells, whereas mouse islets show largely similar architecture of a β-cell core with α-cells in the periphery. In humans, islet architecture is islet-size dependent. Changes in endocrine cell mass preferentially occurred in large islets as demonstrated in our recent study on pathological changes of the pancreas in patients with type 2 diabetes. ( 1) The size dependency of human islets in morphological changes prompted us to develop a method to capture the representative islet distribution in the whole pancreas section combined with a semi-automated analysis to quantify changes in islet architecture. The computer-assisted quantification allows detailed examination of endocrine cell composition in individual islets and minimizes sampling bias. The standard immunohistochemistry based method is widely applicable to various specimens, which is particularly useful for large animal studies but is also applied to a large-scale analysis of the whole organ section from mice. In this article, we describe the method of image capture, parameters measured, data analysis and interpretation of the data.     

Correction of insulin sensitivity and glucose disposal after pancreatic islet transplantation: preliminary results

Aims: Pancreatic islet transplantation (PIT) represents a potential curative treatment for patients with type 1 diabetes, but only 10–15% of patients remain insulin independent 5 years post‐transplant. It is not known whether intrinsic insulin resistance exacerbated by immunosuppression plays a pivotal role in low graft survival. The study objective was to understand the changes in insulin resistance, glucose effectiveness (S_g) and free fatty acid dynamics (FFAd) before and after PIT. Methods: Insulin sensitivity index (S_i), S_g and FFAd were measured before and after PIT in 10 lean patients, 8 of whom reached insulin independence. Modified Bergman minimal model of frequently sampled intravenous glucose tolerance tests were performed pretransplant and at 12 months post‐transplant. Nine non‐diabetic control (NDC) subjects matched by age, gender and BMI were used. Results: Pretransplant S_i and S_g were 3.5 ± 0.8 × 10^?5/min/(pmol/l) and 0.74 ± 0.24 × 10^?2/min, respectively. S_i was significantly lower than matched NDCs [10.8 ± 0.6 × 10^?5/min/(pmol/l), p < 0.004]; S_g did not reach statistical significance (1.27 ± 0.22 × 10^?2/min, p = 0.25). Compared to pretransplant values, mean post‐transplant S_i and S_g were 9.6 ± 1.3 × 10^?5/min/(pmol/l)and 1.28 ± 0.22 ×10^?2/min, respectively, indicating significant improvement for S_i but not S_g (p = 0.008and p = 0.06). Twelve‐month post‐PIT compared to NDC values were not significantly different (p = 0.58 and 0.97, respectively). In addition, fractional disposal rate for FFA which directly depends on the endogenous insulin release (10–20 min) nearly normalized after PIT (p = 0.06). Conclusion: These preliminary findings demonstrate that PIT can restore glucose disposal and insulin sensitivity and partially correct glucose effectiveness and FFAd.     

Quantification of islet size and architecture

Human islets exhibit distinct islet architecture particularly in large islets that comprise of a relatively abundant fraction of α-cells intermingled with β-cells, whereas mouse islets show largely similar architecture of a β-cell core with α-cells in the periphery. In humans, islet architecture is islet-size dependent. Changes in endocrine cell mass preferentially occurred in large islets as demonstrated in our recent study on pathological changes of the pancreas in patients with type 2 diabetes.¹ Kilimnik G, Zhao B, Jo J, Periwal V, Witkowski P, Misawa R, et al. Altered islet composition and disproportionate loss of large islets in patients with type 2 diabetes. PLoS One 2011; 6:e27445; http://dx.doi.org/10.1371/journal.pone.0027445; PMID: 22102895 [Crossref], [PubMed], [Web of Science ®] , [Google Scholar] The size dependency of human islets in morphological changes prompted us to develop a method to capture the representative islet distribution in the whole pancreas section combined with a semi-automated analysis to quantify changes in islet architecture. The computer-assisted quantification allows detailed examination of endocrine cell composition in individual islets and minimizes sampling bias. The standard immunohistochemistry based method is widely applicable to various specimens, which is particularly useful for large animal studies but is also applied to a large-scale analysis of the whole organ section from mice. In this article, we describe the method of image capture, parameters measured, data analysis and interpretation of the data.     

Transgenic mice overproducing islet amyloid polypeptide have increased insulin storage and secretion in vitro

Summary To determine whether chronic overproduction of islet amyloid polypeptide alters beta-cell function, we studied a line of transgenic mice which overexpress islet amyloid polypeptide in their beta-cells. At 3 months of age, these transgenic mice had greater pancreatic content of both islet amyloid polypeptide and insulin. Further, basal and glucose-stimulated secretion of both islet amyloid polypeptide and insulin were also elevated in the perfused pancreas of the transgenic animals. These findings demonstrate that chronic overproduction and secretion of islet amyloid polypeptide are associated with increased insulin storage and enhanced secretion of insulin in vitro. This increase in insulin storage and secretion may be due to a direct effect of islet amyloid polypeptide on the beta-cell or a betacell adaptation to islet amyloid polypeptide-induced insulin resistance.Abbreviations IAPP Islet amyloid polypeptide - bp base pair - TFA trifluoroacetic acid - IRI immunoreactive insulin - SLI somatostatin-like immunoreactivity - IAPP-LI IAPP-like immunoreactivity     

Improved outcome of islet transplantation in insulin-treated diabetic mice: effects on beta-cell mass and function

Summary Insulin treatment may improve the outcome of islet transplantation. To determine the effects of insulin treatment on transplanted islets, 4 groups of streptozotocin-diabetic C57BL/6 mice were transplanted with 100 islets, an insufficient beta-cell mass to restore normoglycaemia. Groups 1 (n = 12) and 2 (n = 12), were kept normoglycaemic with insulin treatment from day 10 before transplantation to day 14 after transplantation; groups 3 (n = 12) and 4 (n = 18), were not treated with insulin. Grafts were harvested 14 (groups 1 and 3) or 60 (groups 2 and 4) days after transplantation and beta-cell mass and replication were measured. When insulin was discontinued all mice maintained normoglycaemia; in contrast, non-insulin-treated groups remained hyperglycaemic throughout the study. Fourteen days after transplantation the beta-cell mass was reduced both in group 1 (0.09 ± 0.01 mg) and group 3 (0.14 ± 0.02 mg) compared to the initially transplanted mass (0.22 ± 0.02 mg, p < 0.01); beta-cell replication and area did not change in group 1, but were increased in group 3. Insulin content, expressed as a function of beta-cell mass, was maintained in group 1 grafts (12.5 ± 2.0 μg/mg), but was severely reduced in group 3 (1.0 ± 0.2 μg/mg) compared to non-transplanted islets (20.4 ± 3.3 μm/mg). In group 2, beta-cell mass increased when insulin was discontinued; 60 days after transplantation beta-cell mass was similar to the initially transplanted mass (0.23 ± 0.04 mg), glucose levels after an intraperitoneal glucose challenge were normal, and insulin content was preserved (19.6 ± 2.7 μg/mg). In contrast, beta-cell mass was progressively reduced in group 4 (0.08 ± 0.02 mg, p < 0.001). In summary, insulin treatment reduced the beta-cell mass needed to achieve normoglycaemia in islet transplantation. Islets transplanted to insulin-treated mice showed better beta-cell function, preserved insulin content, and were able to increase their beta-cell mass to meet an increased functional demand. [Diabetologia (1997) 40: 1004–1010] Received: 7 January 1997 and in revised form: 17 April 1997     

妊娠期母体胰岛的适应性变化

妊娠时随着胎儿对母体物质需求的增加,孕妇出现了糖代谢的改变以及生理性胰岛素抵抗。为了适应胰岛素需求的增加,妊娠期母体胰岛在结构和功能上都会发生相应的改变,包括葡萄糖刺激下胰岛素分泌能力的增强,以及伴随的葡萄糖刺激阈值的下降。此外,还出现母体胰岛素合成增多,胰岛细胞增殖以及胰岛容积的增加。这些变化与妊娠期激素特别是胎盘生乳素(HPL)、催乳素(PRL)及生长激素(GH)等密切相关。不仅如此,脂肪细胞因子、炎症因子、细胞因子信号传递抑制体(SOCS)、胰岛血管内皮细胞等因素对妊娠期母体胰岛的适应性变化也会产生影响。     

Human islet cell transplantation--future prospects.

BACKGROUND: Islet transplantation has the potential to cure diabetes mellitus. Nevertheless despite successful reversal of diabetes in many small animal models, the clinical situation has been far more challenging. The aim of this review is to discuss why insulin-independence after islet allotransplantation has been so difficult to achieve. METHODS: A literature review was undertaken using Medline from 1975 to July 2000. Results reported to the International Islet Transplant Registry (ITR) up to December 1998 were also analysed. RESULTS: Up to December 1998, 405 islet allotransplants have been reported the ITR. Of those accurately documented between 1990 and 1998 (n = 267) only 12% have achieved insulin-independence (greater than 7 days). However with refined peri-transplant protocols insulin independence at 1 year can reach 20%. CONCLUSIONS: There are many factors which can explain the failure of achieving insulin-independence after islet allotransplantation. These include the use of diabetogenic immunosuppressive agents to abrogate both islet allo-immunity and auto-immunity, the critical islet mass to achieve insulin-independence and the detrimental effects of transplanting islets in an ectopic site. However recent evidence most notably from the Edmonton group demonstrates that islet allotransplantation still has great potential to become an established treatment option for diabetic patients.