Biomedical subjects

D G Pipeleers

Publications and source records attributed to D G Pipeleers.

At least 73 records · Page 4Linked to original sources

PubMed1993

Rat pancreas preparation for cold storage and subsequent islet cell isolation.

Pancreatic islets can be isolated from cold-preserved organs. They contain 60% more beta cells when prepared from organs that were stored for 24 hr in Collins solution supplemented with albumin and benzamidine (CAB) instead of University of Wisconsin (UW) solution. Recovery from CAB-stored organs was similar when CAB was perfused in situ before organ removal or ex vivo after organ harvesting in UW. In situ flush with cold Ringers before ex vivo replacement by CAB resulted in 25% lower recovery of islet beta cells and in higher contamination with nonendocrine and damaged cells. Recovery of beta cells was 50% reduced when cold storage solution was not chased before collagenase digestion of the organ. It is concluded that the isolation of rat islets from cold-preserved organs can be improved by using UW or CAB instead of Ringers for situ perfusion, by cold storage in CAB, and by adequate chase of cold storage solution with physiologic medium before collagenase digestion. These conditions can be tested in current human islet isolation protocols.

Adenosine↗

PubMed1993

Interaction of interleukin-1 with islet beta-cells. Distinction between indirect, aspecific cytotoxicity and direct, specific functional suppression.

A 5-day culture of adult rat islets with human recombinant IL-1 beta (3 U/ml) resulted in the death of most alpha-cells and 50% of beta-cells. The IL-1--exposed islet tissue contained--in addition to poorly granulated beta-cells--patches of outgrowing monolayers and dispersed activated macrophages. In purified alpha- and beta-cell preparations, no cytodestructive effects of IL-1 (as high as 30 U/ml) were noticed, indicating that the cytokine is in itself not a beta-cell--selective killer. Pure beta-cells were, on the other hand, more sensitive (from 0.3 U/ml on) than intact islets to an IL-1--induced suppression of hormone synthesis. This inhibitory action was reversible and affected predominantly the production of insulin, leading to degranulated cells with modified shape and attachment. Further studies with IL-1 should take into account that isolated islet preparations do not allow distinction between its irreversible, indirect, and aspecific beta-cell toxicity and its reversible, direct, and specific suppression of beta-cell functions. It is not yet known whether IL-1--suppressed beta-cells exhibit an altered sensitivity to beta-cell--toxic conditions.

Animals↗

PubMed1992

Pancreatic beta cell heterogeneity in glucose-induced insulin secretion.

Rat pancreatic beta cells differ in their individual sensitivity to glucose-inducible metabolic changes. The present study examines whether beta cells with a higher metabolic threshold require higher glucose levels for stimulation of their secretory activity. Purified beta cells were distributed according to their metabolic redox state at 7.5 mM glucose; the metabolically responsive (high responsive) and unresponsive (low responsive) subpopulations of comparable size and viability were reaggregated in the presence of [3H]tyrosine and then perfused at 2.8 mM glucose with 10-min pulses of increasing glucose concentration. Glucose elicited first-phase insulin release in both high and low responsive subpopulations from, respectively, 4.2 and 8.3 mM on. The amplitude of both secretory responses increased dose dependently, the rates in the high responsive subpopulation being 2-fold higher than in the low responsive one. At all stimulating glucose levels, fractional release of 3H-labeled insulin was 3- to 4-fold higher than that of immunoreactive insulin. Preferential release of newly formed insulin was already maximally stimulated at 4.2 mM glucose in the high responsive subpopulation, whereas it increased dose-dependently in the low responsive one. These results indicate the existence of intercellular differences in the secretory activity of glucose-exposed beta cells, both in terms of glucose sensitivity and of amplitude. This heterogeneity in beta cell secretory responsiveness parallels that which has been previously described for the cellular metabolic and biosynthetic functions. It is concluded that glucose dose-dependently recruits beta cells into both biosynthetic and secretory activities. Co-existence of inactive and activated cells can explain preferential release of newly synthesized over preformed hormone during glucose stimulation.

Animals↗

PubMed1992

Effect of donor islet mass on metabolic normalization in streptozotocin-diabetic rats.

The effect of donor islet mass on the metabolic normalization in streptozotocin-diabetic rats was examined by comparing normal control rats with two transplant groups receiving 800 or 2,000 islets intraportally, i.e. grafts containing respectively, 0.8 or 2 million Beta cells at 70% purity. After transplantation, basal glycaemia and daily urine volumes normalized in all recipients and remained normal in both groups over the 20-week follow-up period. After intragastric glucose challenge, the 800-islet group exceeded the normal range of glycaemia at all tested time points (0-120 min) whereas the 2,000-islet group exhibited higher glycaemia only at 15 and 30 min; no deterioration in glucose tolerance occurred during the 20-week follow-up. Both transplant groups presented lower serum fructosamine levels than diabetic control rats, normal levels being maintained only in the 2000-islet group. At post-transplantation week 21, both recipient groups exhibited the same body weight as age-matched normal control rats, but their serum triglyceride levels were significantly higher. Hepatic insulin contents were comparable to the insulin contents of the grafts at the time of implantation, representing 16 or 40% of the pancreatic insulin content in the age-matched control rats. Although islet grafts with 0.8 million Beta cells can restore basal glycaemia in adult streptozotocin-diabetic rats, implants of 2 million Beta cells are necessary to correct other parameters of glucose homeostasis. The present data indicate the need of determining the number of grafted Beta cells in studies on the metabolic outcome of islet cell transplantation. DNA content and percentage of Beta cells in the graft are proposed as useful parameters.

Analysis of Variance↗

PubMed1992

Detection of autoantibodies against islet amyloid polypeptide in human serum. Lack of association with type 1 (insulin-dependent) diabetes mellitus, or with conditions favouring amyloid deposition in islets. The Belgian Diabetes Registry.

A radiobinding assay for the detection of autoantibodies against islet amyloid polypeptide was developed, analytically validated, and--in parallel with a similar assay for the detection of autoantibodies against insulin--applied to sera from recent-onset Type 1 (insulin-dependent) diabetic patients and from age- and sex-matched control subjects. There was no difference in islet amyloid polypeptide autoantibody titres between patient groups and matched control subjects, nor within subject groups according to age. At onset of Type 1 diabetes, elevated islet amyloid polypeptide-autoantibody levels (> 97th percentile of control subjects) were only detected in 1 of 30 patients aged 0-19 years and in 2 of 35 patients aged 20-39 years. By contrast, insulin autoantibodies were frequently demonstrated, in particular at onset of diabetes under age 20 (0-19 years: 18 of 30 patients; 20-39 years: 10 of 35 patients; p < 0.01 vs matched control subjects). Islet amyloid polypeptide autoantibodies were not detectable in 3 insulinoma patients nor in 37 patients (aged 33-70 years) with Type 2 diabetes (vs 1 of 40 in matched control subjects). In positive serum, adsorption onto protein A-Sepharose removed islet amyloid polypeptide binding activity, hereby confirming its antibody nature. In conclusion, Type 1 diabetes is associated with an age-dependent autoantibody reaction against insulin but not against islet amyloid polypeptide. Conditions associated with amyloid deposition in islets (Type 2 diabetes, insulinoma and ageing) do not favour the formation of autoantibodies against islet amyloid polypeptide.

Adolescent↗

PubMed1992

Glucose regulation of the autoantigen GAD65 in human pancreatic islets.

Recent data suggest that the enzyme glutamic acid decarboxylase (GAD) may be a primary beta-cell antigen involved in the autoimmune response leading to insulin dependent diabetes mellitus (IDDM). Following three days culture of human islets at different glucose concentrations (5.6, 11 or 28 mM), there was a single 65 kDa/GAD band in the islet extracts, as assessed by immunoprecipitation both with serum from a newly diagnosed IDDM patient and a sheep anti-GAD serum. The synthesis of GAD was strongly stimulated at the higher glucose concentrations. Likewise, Western blot analysis indicated a glucose-induced increase of GAD in the islets. The data suggest that an enhanced function of human islet cells increases expression of GAD65. Possibly, this could exacerbate the autoimmune assault in the early stages of IDDM, and be of practical importance in attempts to ameliorate the autoimmune response towards the beta-cells in IDDM.

Autoantigens↗

PubMed1992

Cold storage of rat pancreas before purification of islet beta-cells.

Allotransplantations in rats have demonstrated that aggregates of purified islet endocrine cells are less immunogenic than isolated islets. If purified islet cells are to be tested in humans, their preparation should be feasible from cold-preserved organs. We compared the yield in purified beta-cells from freshly harvested rat pancreases with that from cold-stored organs. After 24 h of preservation in Collins' solution or in University of Wisconsin (UW) solution, the number of purified beta-cells per pancreas was 40% lower (P less than 0.05) than that obtained from nonpreserved controls. Addition of 5 mM benzamidine/4% bovine serum albumin to Collins' solution resulted in similar recoveries as with freshly harvested organs; this addition did not increase the yield from UW solution-stored organs. When compared to preparations from fresh pancreases, islets and islet cells isolated from pancreases preserved in Collins' solution-bovine serum albumin-benzamidine were comparable in structural integrity, viability in culture, secretory responsiveness in vitro, and capability of correcting hyperglycemia in streptozocin-induced diabetic rats. We conclude that addition of benzamidine to Collins' preservation solution allows purification of islet beta-cells from 24-h-preserved rat pancreases in the same yield and quality as from freshly harvested organs. These results indicate that cold-preserved pancreases can be used for the preparation of purified islet cell grafts.

Adenosine↗

PubMed1992

Heterogeneity in pancreatic beta-cell population.

All pancreatic beta-cells are identified by specific morphological characteristics. Similarity in microscopic features is not necessarily associated with identity in functional properties. In vitro studies on isolated rat beta-cells have indicated intercellular differences in the threshold for glucose-induced shifts in metabolic redox state. The cellular heterogeneity in glucose sensitivity results in a dose-dependent recruitment of glucose-exposed beta-cells into biosynthetic and secretory activities. The molecular basis of this diversity is not known. Indirect evidence supports the concept that the in situ pancreatic beta-cell population is also composed of functionally diverse subpopulations. The heterogeneity in glucose responsiveness is expected to create subpopulations of beta-cells with either constant, fluctuating, or occasional glucose-dependent functions; whether any subpopulation is preferentially responsive to other regulatory factors and/or committed to other activities is unknown. Morphological markers may help identify beta-cell subpopulations in situ and quantify their size in conditions known to affect total beta-cell mass or function. The concept of a functionally heterogeneous beta-cell population influences views on the role of pancreatic beta-cells in health and disease.

Animals↗

PubMed1991

Measuring the balance between insulin synthesis and insulin release.

The absolute rates of hormone synthesis and release were determined in purified pancreatic B cells. Newly synthesized proteins were labeled with L-[3,5-3H]tyrosine or L-[2,5-3H]histidine. When medium glucose was less than or equal to 10 mM, the production of insulin exceeded or equaled its release. Raising the glucose levels above 10 mM did not further increase the rate of insulin synthesis (67 +/- 10 fmol/10(3) cells/2 hour) but elevated that of insulin release up to 3-fold the production rates (181 +/- 10 fmol/10(3) cells/2 hour). In the presence of glucagon or of the phorbol ester 12-O-tetradecanoylphorbol 13-acetate the cells also released 3-fold more hormone that they synthesized; release was however reduced to 25% of the rate of production in the presence of epinephrine. It is concluded that glucose as well as hormonal regulators of islet B cells can influence, bi-directionally, the balance between the rates of insulin synthesis and release.

Animals↗

PubMed1991

Transplantation of purified islet cells in diabetic rats. I. Standardization of islet cell grafts.

A standardized procedure was developed for the preparation of rat islet cell grafts with selected cell number and composition. After collagenase digestion of pancreases and elutriation of tissue fragments, islets were isolated and dissociated, and cells were purified by autofluorescence-activated cell sorting. Approximately 30% of the initial beta-cell mass was lost during digestion and elimination of small mostly exocrine particles. Fifty percent was recovered in isolated islet preparations and 30% in the purified beta-cell suspensions of greater than 95% purity and viability. Sorting according to cellular flavin adenine dinucleotide content discriminated islet beta-cells from islet endocrine non-beta-cells, fibroblasts, leukocytes, and exocrine cells. Purified endocrine islet cell grafts were prepared by aggregating 10(6) pure beta-cells with or without 8 x 10(5) pure endocrine non-beta-cells. In contrast to intact islets, the purified aggregates were devoid of nonendocrine and damaged cells. Intraportal implantation of a pure beta-cell graft rapidly and permanently normalized the diabetic state of streptozocin-administered animals. The standardized preparation of purified beta-cell grafts allows us to address several metabolic and immunological questions concerning islet cell transplantation in diabetes.

Animals↗

PubMed1991

Transplantation of purified islet cells in diabetic rats. II. Immunogenicity of allografted islet beta-cells.

This study examines whether the survival of allografted rat islet beta-cells is influenced by the presence of other pancreatic donor cells. Grafts (RT1u/l) of different cellular composition were intraportally transplanted in streptozocin-induced diabetic rats (RT1n/n). All grafts corrected the diabetic state within 3 days. Implants of freshly isolated islets contained various endocrine and nonendocrine cell types; they became diffusely infiltrated within 1 wk and were completely destroyed within 2 wk. A 4-day culture period did not lead to major changes in the cellular composition of the islets or in their survival as allograft. Islet cell aggregates prepared after islet dissociation and cell purification were less acutely infiltrated and less rapidly rejected. Aggregates composed of sorted MHC class II-negative cells maintained basal normoglycemia in 3 of 5 recipients for 5 wk but only in 1 of 5 for 20 wk. Aggregates of purified islet beta-cells remained relatively free of diffuse infiltrations during the 1st wk and preserved the normalized state in 7 of 13 recipients for 5 wk; after 20 wk, 6 of 13 were still aglucosuric, but 40% of the implants were diffusely infiltrated and depleted of insulin. Reaggregation of purified islet beta-cells with purified islet endocrine non-beta-cells promoted their long-term survival as allograft: 11 of 13 recipients of mixed islet endocrine cells maintained normal basal glycemia over 20 wk; their implants contained relatively constant insulin reserves and remained virtually devoid of diffuse infiltrations. These results demonstrate that techniques aiming at the elimination of surface MHC class II-positive cells are less successful in preparing rat islet allografts of low immunogenicity than methods of positive cell selection. Pure islet beta-cells are immunogenic as an allograft but illicit a milder and less-acute immune attack than undissociated islet tissue. Nonendocrine and damaged islet cells are suspected of enhancing the rapidity and intensity of the cytotoxic reaction. Survival of allografted beta-cells is markedly prolonged by the presence of islet endocrine non-beta-cells within the graft. The mechanisms underlying this effect have not yet been elucidated; they may involve immune and metabolic interactions of the endocrine non-beta-cells. We conclude that purification of islet endocrine cells represents a new and powerful method for preparing insulin-producing allografts that can survive in hosts without pharmacological immunosuppression.

Animals↗

PubMed1991

Transplantation of purified islet cells in diabetic rats. III. Immunosuppressive effect of cyclosporin.

This study examined the effect of cyclosporin on the survival of islet beta-cell allografts in streptozocin (STZ)-induced diabetic rats. At a daily oral dose of 5 mg/kg, the agent prevented the rejection of isolated islets, provided they were little contaminated by other pancreatic tissue. The immunosuppressive effect rapidly disappeared after discontinuation of the drug, except when the donor tissue had been pretreated to reduce its nonendocrine content. All recipients of cultured and selected islets maintained a normalized state for greater than 15 wk beyond the 5-wk drug course; this was not the case for shorter periods of treatment. A long-term beneficial effect was also observed in all recipients of purified islet beta-cell grafts, which reversed without treatment in half of the cases. Cyclosporin markedly reduced the mononuclear cell infiltration in each type of islet beta-cell allograft; aggregates of mixed endocrine islet cells were kept virtually infiltration free. Conditions with minimal initial infiltration were associated with long-term graft survival without the need for continuous pharmacological immunosuppression. We conclude that a short-term cyclosporin treatment can induce long-term survival of allografted islet beta-cells, provided the grafts are only slightly contaminated by nonendocrine elements. In rodents, sufficient immunosuppression was achieved by circulating cyclosporin levels of 100-400 ng/ml. Higher concentrations were cytotoxic for cultured islet beta-cells and islet non-beta-cells. A 5-wk treatment with the immunomodulator ciamexone also resulted in long-term survival of purified beta-cell allografts but not of cultured islets.(ABSTRACT TRUNCATED AT 250 WORDS)

Adjuvants, Immunologic↗

PubMed1990

Cellular and subcellular localization of an Mr 64,000 protein autoantigen in insulin-dependent diabetes.

Antibodies to an Mr 64,000 protein from human or rat islets have been detected at high frequency in newly diagnosed insulin-dependent diabetic patients. In this study, we show that the antigenic and amphiphilic properties of the rat islet Mr 64,000 protein resemble that of the human protein. We have analyzed the expression of the Mr 64,000 protein in populations of pancreatic beta and non-beta cells and in selected rat tissues by immunoprecipitation of [35S]methionine-radiolabeled proteins with sera from diabetic patients or from healthy control individuals. When islet cell populations enriched in beta or non-beta cells were tested for the expression of the Mr 64,000 antigen, the protein was primarily observed in the beta cells. On analyzing preparations of islets, liver, kidney, thyroid, adrenal, pituitary, spleen, and thymus, the protein could only be detected in islets. The protein was also characterized in terms of its subcellular localization by Percoll density gradient centrifugation and was recovered in a fraction enriched in the plasma membrane marker, 5'-nucleotidase. These results are consistent with a beta cell-restricted plasma membrane expression of the protein and support the hypothesis that this protein is a target antigen of beta cell-specific autoimmunity in insulin-dependent diabetes.

Animals↗

PubMed1990

Direct effect of insulin and insulin-like growth factor-I on the secretory activity of rat pancreatic beta cells.

Purified pancreatic Beta cells were labelled with 3H-tyrosine before studying their secretory activity in perifusion. At 1.4 mmol/l glucose, the cells released similar fractions (0.01% per min) of their contents in preformed and in newly formed insulin. At 20 mmol/l glucose plus 10(-8) mol/l glucagon, these fractional release rates increased by 16 and 40-fold respectively. The preferential release of newly synthesized as compared to stored insulin is attributable to a heterogeneity in individual cell responses. The secretory responsiveness to glucose plus glucagon was completely suppressed by 10(-7) mol/l clonidine. Insulin induced a 20% reduction at 10(-6) mol/l, but remained without effect at 10(-7) mol/l. Insulin-like growth factor-I provoked a 30% decrease at 5.10(-9) mol/l. It is concluded that the type-I insulin-like growth factor receptors on pancreatic Beta cells mediate a suppressive action on the insulin release process. Their high affinity for insulin-like growth factor-I allows physiologic levels of this peptide to participate in the regulation of insulin release. Their low affinity for insulin provides the basis for a minor feedback action by this hormone at concentrations exceeding the normal circulating levels.

Animals↗

PubMed1990

Deoxyribonucleic acid synthesis in cultured adult rat pancreatic B cells.

Previous studies in rodent islets have suggested the existence of a small number of proliferating islet cells. Since islet tissue is composed of endocrine as well as nonendocrine cells, we examined whether the DNA synthesis that is detectable in intact islets in vitro corresponds to an activity of islet B cells, islet endocrine non-B cells and/or nonendocrine islet cells. DNA synthesis was quantified by [3H]thymidine incorporation in trichloracetic acid precipitable material, by nuclear thymidine labeling in autoradiographs, and by nuclear bromodeoxyuridine fluorescence. Adult islet endocrine purified B cells, as well as other endocrine islet cells, incorporated 1 to 2 fmol thymidine/1000 cells, which is 3 times lower than intact islet tissue and 30 times lower than nonendocrine islet cells. Addition of 10% fetal calf serum did not increase DNA synthesis in purified endocrine islet cells but doubled it in intact islets and enhanced it 8-fold in nonendocrine islet cells. The higher thymidine incorporation in intact islets was due to the presence of nonendocrine cells. An increase in medium glucose concentration from 100 to 200 mg/100 ml doubled the thymidine incorporation in purified islet B cells, but not in other endocrine islet cells; no concomitant increase in the number of thymidine or bromodeoxyuridine-labeled nuclei was observed. A phenomenon of glucose-stimulated DNA repair was not excluded. Using three different methods, we have found no evidence for a proliferating activity of adult rat islet B cells under the selected in vitro conditions of this study.

Animals↗

PubMed1989

Sensitivity of rat pancreatic A and B cells to somatostatin.

Islet A and B cells were purified from the rat pancreas and examined for their respective sensitivity to somatostatin. Both somatostatin-14 (S14) and -28 (S28) inhibited glucagon and insulin release through direct interactions with the corresponding cell types. A dose-dependent suppression of the secretory activities was paralleled by a reduction in cellular cyclic AMP formation with similar ED50 values for both actions. The somatostatin effects on pancreatic hormone release may thus be mediated via an inhibition of adenylate cyclase activity. In pancreatic A cells, S14 and S28 were equally potent inhibitors with ED50 values ranging from 2 x 10(-12) to 2 x 10(-11) mol/l. Pancreatic B cells exhibited a similar sensitivity to S28 as the A cells (ED50 of 2 to 5 x 10(-11) mol/l), but not to S14 (ED50 of 2 x 10(-9) mol/l). Extrapolation of these in vitro sensitivities of islet A and B cells to the in vivo situation suggests that both cell types can respond to circulating S28 levels and that A cells are sensitive to both locally and distally released S14. Islet B cells appear insensitive to the normal peripheral S14 levels but could respond to locally released somatostatin. The marked difference in the sensitivities of islet A and B cells to S14 suggest that these cell types are equipped with different somatostatin receptors. This notion was further supported by the cell-selective actions of the synthetic S14 analogues [D-Trp8, D-Cys14]S14 and desAsn5[D-Trp8, D-Ser13]S14.

Animals↗

PubMed1988

Glucose stimulates proinsulin biosynthesis by a dose-dependent recruitment of pancreatic beta cells.

Glucose is a well-known stimulus of proinsulin biosynthesis. In purified beta cells, the sugar induces a 25-fold increase in the synthesis of insulin immunoreactive material over 60-min incubation. Autoradiographic analysis of the individual cells shows that this effect is achieved via dose-dependent recruitment of pancreatic beta cells to biosynthetic activity. Recruitment of beta cells is also seen in isolated islets exposed to glucose. The sigmoidal dose-response curve for glucose-induced proinsulin biosynthesis thus reflects a heterogeneous responsiveness of pancreatic beta cells rather than a progressively increasing activity of functionally homogeneous cells. Dose-dependent recruitment of functionally diverse cells may be a ubiquitous mechanism in tissue function.

Animals↗

PubMed1988

In situ analysis of pancreatic islets in rats developing diabetes. Appearance of nonendocrine cells with surface MHC class II antigens and cytoplasmic insulin immunoreactivity.

Aberrant expression of MHC class II molecules on endocrine cells has been proposed to induce autoimmune reactions in thyroid and endocrine pancreas. The present study examines whether MHC class II positive insulin-containing islet cells occur at the onset of diabetes in rats, in analogy to the findings in man. At the onset of diabetes, both streptozotocin-treated and diabetes-prone BB rats exhibited numerous class II positive islet cells that presented ultrastructural features of monocytes and were surrounded by class II negative islet B cells. These class II positive cells were characterized by vacuoles that contained insulin immunoreactive granules and disrupted membranes. Similar cells also appeared positive for the monocyte marker OX-42. The presence of class II positive monocytes with insulin-containing vacuoles may indicate a removal of damage B cells by infiltrating leukocytes. A similar electron microscopical study in man will be necessary to distinguish the putative endocrine pancreatic B cells with aberrant class II expression from infiltrating nonendocrine class II positive cells with insulin-containing phagosomes.

Animals↗