Arginine plus proline supplementation elicits metabolic adaptation that favors wound healing in diabetic rats.

Diabetic patients with wounds are at risk of protein malnutrition, have low arginine plasma levels, and suffer from delayed wound healing. We sought to determine the efficacy of arginine plus proline supplementation on protein and amino acid metabolism and on wound repair in a model of diabetic rats. Eighteen 11-wk-old Zucker diabetic fatty fa/fa male rats underwent a 7-cm abdominal skin incision with implantation of sponges and daily excision of full-thickness round sections of dorsal skin for 5 days. They were randomized to be fed with either a standard formula (S group, Clinutren Iso), a high-protein and arginine (ARG) plus proline (PRO)-enriched formula (ARG+PRO group, Clinutren Repair), or an isonitrogenous isoenergetic control formula (IC group). Nitrogen balance was calculated daily. The rats were euthanized on day 5, and plasma glucose, insulin, amino acids, skin epithelialization, and angiogenesis were measured. In macrophages, we assessed inducible nitric oxide synthase (iNOS) and arginase expression, production of nitric oxide (NO) and amino acid metabolism. Both the ARG+PRO and IC groups showed improved nitrogen balance. ARG plus PRO supplementation increased proline and branched-chain amino acid plasma concentrations and improved angiogenesis. Arginase and iNOS expressions in macrophages were reduced, together with NO and citrulline production. In diabetic rats, ARG plus PRO supplementation improves wound angiogenesis and favors whole body protein metabolism. Low macrophage iNOS expression at day 5 may reflect a low inflammatory state in the wounds, favoring wound closure.

Dietary and hormonal regulation of aldolase B gene expression.

Aldolase B is an enzyme of the glycolytic pathway whose activity and mRNA levels in the liver fluctuate according to dietary status. Both the enzyme activity and the mRNA concentration decline during fasting and increase four- to eightfold upon refeeding of a carbohydrate-rich diet. The mechanism, however, of the mRNA induction remains unknown. To elucidate the mechanisms that regulate this induction responsive to dietary stimuli, we have studied the roles of hormones and glycolytic substrates on aldolase B gene expression in three tissues that synthesize the enzyme. Using a cDNA probe complementary to rat aldolase B mRNA, we determined the amount of cytoplasmic RNAs in the liver, kidney, and small intestine of normal, adrenalectomized, thyroidectomized, diabetic, and glucagon- or cAMP-treated animals refed either a fructose-rich or a maltose-rich diet. The in vivo hormonal control of gene expression was found to be very different in the three organs tested. In the liver, cortisone and thyroid hormones were required for the induction of the specific mRNA by carbohydrates, while in the kidney none of the hormonal modifications tested altered the level of mRNA induction. In the liver, but not in the kidney, diabetes and glucagon administration abolished the induction of aldolase B mRNAs in animals refed the maltose-rich diets. In the small intestine, only diabetes and thyroidectomy affected the gene expression. Finally, no induction occurred when normal fasted rats were given any of the hormones. Thus, the in vivo hormonal control of liver aldolase B gene expression differs significantly from that of kidney and small intestine. In the liver, the mRNA induction requires the presence of dietary carbohydrates, of permissive hormones, and the cessation of glucagon release, while in the kidney, the induction of the mRNAs by fructose occurs regardless of the hormonal status of the animals. The hormonal control of aldolase B mRNA levels in the small intestine is intermediate.

Effects of an immune-enhancing diet in endotoxemic rats.

OBJECTIVE: This work compared the nutritional efficiency of a recently available enteral formula enriched with arginine, omega-3 fatty acids, and antioxidants and supplied nitrogen as peptides (Crucial, Nestle Clinical Nutrition) with that of a standard polymeric formula (Sondalis HP, Nestle Clinical Nutrition) in endotoxemic rats. METHODS: Male Wistar rats (209 +/- 2 g) underwent catheter gastrostomy and received Sondalis HP until they recovered their preoperative weight. At that time (day 0), an endotoxemic shock was induced by an intraperitoneal injection of lipopolysaccharide (Escherichia coli, 8 mg/kg) and rats then received 290 kcal x kg(-1) x d(-1) and 3.29 g of nitrogen x kg(-1) x d(-1) in the form of Crucial (IED group, n = 7) or Sondalis HP (S group, n = 6) for 3 d. Another group underwent no treatment and was fed ad libitum (AL group). Rats were killed on day 3. Results are presented as mean +/- standard error of the mean (analysis of variance and Newman-Keuls test). RESULTS: The endotoxemic shock induced a weight loss in group S on days 1 and 2 and a weight gain in group IED (-3.5 +/- 1.3 g in group S versus +6.0 +/- 2.2 g in group IED, P < 0.05). In the same way, atrophy of extensor digitorum longus muscle was observed in group S, whereas wasting was limited in group IED (102 +/- 4 mg in group IED versus 90 +/- 3 mg in group S versus 119 +/- 3 mg in group AL, P < 0.05). Muscular atrophy was associated with muscular glutamine depletion and correlated with hyperphenylalaninemia (R = 0.60), with the latter being blunted in group IED (57 +/- 1 microM/L in group AL versus 77 +/- 4 microM/L in group S versus 66 +/- 2 microM/L in group IED, P < 0.05). No difference was observed between the experimental groups of endotoxemic rats with respect to nitrogen balance, urinary excretion of 3-methyl histidine, or total tissue protein content. CONCLUSION: Crucial counteracts injury-mediated weight loss, extensor digitorum longus muscle atrophy, and hyperphenylalaninemia in endotoxemic rats.

Use of polyethyleneglycol for porcine islet cryopreservation.

The aim of this work was to determine whether polyethylene glycol 20000 Da (PEG) could be used as protective agent in porcine islet cryopreservation. Cryopreservation was performed on 1-wk cultured pig islets and consisted in an overnight storage in liquid nitrogen. In a first set of experiments, we compared the in vitro function of PEG-cryopreserved islets to that of porcine islets cryopreserved under the standard procedure using dimethylsulfoxide (DMSO), by incubating the islets over 45 min in Krebs buffer containing either 2.8 or 10 mmol/L glucose. Insulin secretion of both types of islets reached a maximum at day 10 postthawing and had stimulation indices above 2 up to 3 wk after thawing. PEG-cryopreserved islets secreted more insulin than DMSO-treated islets and showed glucose-dependency insulin secretion in a 0-16.6 mmol/L glucose range. We also established that PEG-cryopreserved islets were as functional in vitro as nonfrozen tissue and that they could reverse experimental diabetes of the mouse for longer periods of time than noncryopreserved islets (p < 0.005 3 wk after transplantation) when implanted in the peritoneal cavity, being immunoprotected in a semipermeable hollow fiber. PEG can, therefore, be considered as a suitable cryoprotective compound for porcine islet storage.

Bioartificial pancreas containing porcine islets of Langerhans implanted in low-dose streptozotocin-induced diabetic mice: effect of encapsulation medium.

Two encapsulation culture media without animal serum were compared for development of a bioartificial pancreas. Porcine islets were suspended in Hams F10 medium supplemented with 2% Ultroser (US) or in Ultraculture medium (UC) and encapsulated in hollow fibres composed of AN69 copolymer. The function of encapsulated islets was assessed by intraperitoneal transplantation of two fibres in streptozotocin-induced diabetic mice. In both groups of transplanted mice (US, n = 26; UC, n = 18), a significant decrease in plasma glucose concentration was observed three days after fibre implantation (from 21.9 +/- to 14.4 +/- 0.8 mmol/l for US fibres and from 22.7 +/- 0.8 to 13.3 +/- 1.3 mmol/l for US fibres and from 22.7 +/- 0.8 to 13.3 +/- 1.3 mmol/l for UC fibres). Graft survival 17 days after implantation was 61% for mice with UC fibres and 35% for those with US fibres (P = 0.0001). Intramuscular glucose tolerance tests were performed in these animals (US, n = 5; UC, n = 10), and a normal glucose pattern was observed in both groups of transplanted mice. The results show that a complete normalisation of blood glucose and glucose tolerance can be achieved by implantation of a bioartificial pancreas. Moreover, UC appears to be a more suitable encapsulation culture medium for porcine islets in vivo.

Reversal of diabetes in non-obese diabetic mice by xenografts of porcine islets entrapped in hollow fibres composed of polyacrylonitrile-sodium methallylsulphonate copolymer.

Intraperitoneal xenografting of islets immunoprotected by semipermeable membranes is a potential method of avoiding rejection by reversal of diabetes without immunosuppression. In this preliminary study, a xenograft of porcine islets, immunoprotected in semipermeable hollow fibres composed of a hydrogel of a polyacrylonitrile-sodium methallylsulphonate copolymer (AN 69), was used to reverse autoimmune insulin-dependent diabetes mellitus (IDDM) in the NOD mouse. A diabetic state was maintained in all 46 NOD mice which received transplants of empty fibres. Transplantation of encapsulated islets reversed the diabetic state in 37% (18/54) of the recipients. In these mice, nonfasting blood glucose concentration decreased within 24 h. Glycaemia was kept below the diabetic control range and the initial pretransplant value for 6 weeks. Recipient NOD mice suffered from the severe insulitis characteristic of clinical diabetes, confirming that reversal of the hyperglycaemic state was due solely to the xenografts. Pretransplant glycaemia was slightly (p < 0.05) higher in mice which remained diabetic after grafts of fibre-containing islets than in animals which experienced reversal of hyperglycaemia after transplantation) for the peritoneal cavity of recipients which had returned to normoglycaemia after grafting with islet-containing fibres. In all 4 cases, the islets responded to glucose during a perifusion assay. In 2 out of 4 grafts removed from mice which remained hyperglycaemic after grafting with islet-containing fibres (11, 13, 15 and 27 days after transplantation), no basal or stimulated insulin secretion was detectable. Histological sections of a total of 75 fibres retrieved from the peritoneal cavities of recipient NOD mice showed surrounding inflammation, with adherent cells, neovascularisation and fibrotic reaction. These preliminary results are promising for the continued development of this bioartificial pancreas for xenogeneic islet transplantation since they demonstrate that xenogeneic islets can survive in the autoimmune environment of the NOD mouse with spontaneous diabetes mimicking human IDDM).

Minimisation of microbial contamination for potential islet xenografts using specific pathogen-free pigs and a protected environment during tissue preparation.

One major risk of islet xenotransplantation is transmission of infections. We thus compared microbial contamination during preparation of islets from 4 pigs conventionally breeded and slaughtered or 8 specific pathogen free (SPF) pigs, and different environmental conditions during pancreas excision. Pancreas harvested in a slaughterhouse (for conventional pigs) or in a protected autopsy room (for SPF pigs) were soaked in betadine solution and submitted to enzymatic digestion with collagenase. Islets were purified on histopaque gradient with a COBE 2991 processor. For each step of the process, a 10 ml aliquot was harvested and microbial contamination was analysed. For all animals, contamination of livers, which were not soaked in betadine solution, was also examined. Analysis of livers from the 4 conventional pigs showed polymicrobial contaminations (1,122 +/- 841 CFU/mg) with several species of Staphylococcus, Streptococcus, Bacillus and Enterobacteriaceae. For these conventional pigs, soaking of pancreas in betadine solution and presence of antibiotics in all media decreased the pancreatic contamination compared to hepatic contamination, but were unable to suppress it, as transport solution and crude suspension obtained after the digestion step with collagenase showed persistent contamination (9.7 +/- 2.4 and 10.5 +/- 4 CFU/ml, respectively). After islet purification by histopaque gradient, no medium remained contaminated. During analysis of the 8 SPF pigs, no liver exhibited contamination. Analysis of medium from each preparation step showed complete absence of contamination for 7 pancreases. Only one contamination with Staphylococcus simulans was observed for one pancreas in transport solution (6 CFU/ml), and persisted in digestion medium (16 CFU/ml). Finally, all purified suspensions were completely sterile. In conclusion, breeding conditions of pig islet donors, and controlled environment for pancreas excision, considerably influence the risk of microbial contamination. In order to limit the risk, SPF pigs are a suitable and compulsory source of islets.

Long-term culture of free or encapsulated islets isolated from specific pathogen-free (SPF) pigs.

As the risk of recipient contamination is a limiting factor for xenotransplantation, the use of specific pathogen-free (SPF) pigs is mandatory. This study investigated the long-term culture of SPF pig islets and evaluated their insulin production when encapsulated in AN69 hollow fibres. Insulin secretion was studied after 3 weeks (n = 10), 2 months (n = 8) and 3 months (n = 3) by 45-min incubation in the presence of 2.8, 5.5, 11 and 16.5mM glucose. Although a decrease in the amount of secreted insulin occurred (1385 +/- 421 and 4323 +/- 1068 microns U/ml at 3 weeks for 2.8 and 16.5 mM glucose respectively; 702 +/- 261 and 2397 +/- 1047 microU/ml at 2 months; 59 +/- 23 and 154 +/- 34 microU/ml at 3 months), glucose-dependent insulin secretion was observed in all cases, i.e. stimulation indices of 8.1 +/- 3.1 (p < 0.05 vs the presence of 5.5 mM glucose) at 3 weeks, 3.3 +/- 1.1 at 2 months and 3.0 +/- 0.7 at 3 months. The insulin secretion of encapsulated SPF pig islets, cultured for 1 or 3 weeks, was evaluated under perifusion conditions using a stimulus of 10mM glucose plus 5.5 mM theophylline. Glucose stimulation resulted in a significant two-fold increase in insulin secretion (p < 0.05), which was maintained over culture time. These results indicate that SPF-isolated islets remained functional when cultured for several weeks either as free or encapsulated islets, although the magnitude of insulin secretion decreased dramatically after three months of culture.

Perifusion analysis of insulin secretion from specific pathogen-free large-white pig islets shows satisfactory functional characteristics for xenografts in humans.

Though the pig appears to be the islet donor of choice for grafts in diabetic patients, there may be a risk of transmission of infectious agents. In this context, we adopted a strategy of islet isolation from pigs raised and killed in specific pathogen-free (SPF) conditions as a minimum with regard to the concept of quality assurance. Accordingly, the present study investigated the function of SPF pig islets to determine whether they react qualitatively and quantitatively to nutriments, hormones and neuromediators with which they would be confronted in man and could therefore provide effective regulation during physiologic or physiopathologic situations. beta cells from 18 Large-White SPF pigs were functionally intact after 7 days in culture. Insulin stimulation indexes (SI) of 3.1 +/- 0.2, 2.2 +/- 0.1, and 4.4 +/- 0.3 were found respectively for 30 mmol/l K+, 100 mumol/l tolbutamide and 10 mmol/l theophylline. Basal insulin secretion (72.2 +/- 7.6 muU/min) had already increased significantly (p < 0.001) with 5.5 mmol/l glucose (184.2 +/- 25.5 muU/min, SI: 2.5 +/- 0.6), indicating that the threshold stimulatory concentration was comparable to that of human islets. Insulin secretion increased in a glucose dose-dependent manner (p < 0.001): SI: 3.1 +/- 0.3 and 3.6 +/- 0.2 with 11.0 mmol/l and 22.0 mmol/l glucose, which showed a satisfactory magnitude with reference to human islets. Even the subtle phenomenon of "glucose memory" was apparent in these pig islets. Arginine stimulated (p < 0.001) insulin secretion dose-dependently (SI: 2.2 +/- 0.3 with 5 mmol/l and 2.9 +/- 0.2 with 10 mmol/l). The ketone body beta-hydroxybutyrate (10 mmol/l) also induced insulin secretion (SI: 4.3 +/- 0.3). Insulin release was stimulated by 4 mumol/l gastric inhibitory peptide, revealing sensitivity to the hormonal enteroinsular axis, and by 2 mumol/l glucagon. Parasympathetic cholinergic influence was studied using 500 mumol/l carbamylcholine, which increased insulin secretion. The influence of orthosympathetic control and of stress situations was also studied. As in human islet response, epinephrine and the alpha 2-agonist clonidine (50 mumol/l) inhibited insulin secretion. Finally pre-culture of islets may be beneficial for graft outcome, provided that no deterioration in islet function occurs. A prolonged 21-day culture of SPF pig islets showed no decrease in insulin response to glucose, arginine and potassium, even with an unaltered threshold stimulatory glucose concentration. Thus, Large-White SPF pigs and the application of our isolation procedure provided islets with functional characteristics reproducibly compatible with potential utilisation for effective regulation of glycaemia under physiologic and physiopathologic situations in humans.

Preliminary report on cell encapsulation in a hydrogel made of a biocompatible material, AN69, for the development of a bioartificial pancreas.

The occurrence of an inflammatory reaction represents the major obstacle to the development of any implantable system including micro and macroencapsulation. The purpose of this study was to describe an encapsulation method for cells using a membrane made of AN69, a copolymer of acrylonitrile which is considered as a reference in biocompatibility in the field of haemodialysis. The hydrogel of AN69 was obtained after a coagulation step at room temperature followed by a solvent/non-solvent (water) exchange phase. Microcapsules were obtained by co-extrusion of AN69 collodion and saline (with or without cells). The function of encapsulated cells was assessed in vitro, demonstrating cell survival after the microencapsulation procedure. These preliminary data are consistent with the potential interest for the development of the microencapsulation procedure aimed at realising a bioartificial pancreas.

A method for obtaining monodispersed cells from isolated porcine islets of Langerhans.

Micro or macroencapsulation of islets of Langerhans have been proposed as a bioartificial pancreas. Encapsulation of dispersed single cells instead of porcine islets should improve the oxygenation of encapsulated tissue. The aim of this work was, therefore, to develop techniques for dissociating porcine islets and test cell viability and function. After islet isolation and purification, islets were dispersed into single cells with collagenase and DNAse in either an extracellular type ionic solution or a UW solution. After culture, islets or cells were perfused with Krebs buffer. Two consecutive stimulations from 2.8 mM to 20 mM glucose were performed. Viability of cells (trypan blue) was higher than 85% after dispersion in ES or UW solutions. Islets or dispersed cells responded similarly to both stimulations with a return to basal rate between stimulations. No difference was found between cell function cultured during 18 hours or 6 days. However, islet function was improved by a long period of culture. In conclusion, this study demonstrates that dissociated cells performed as well as native islets up to six days culture.

[Use of islet cells in cell therapy]. / Utilisation des cellules des îlots de langerhans en thérapie cellulaire.

Insulin dependent diabetes mellitus is a common disease affecting 1.5 million patients in Europe. It is currently treated by multiple injections of insulin associated with blood glucose self monitoring. The goal of the treatment is to obtain near normal glucose concentration, in order to prevent the later complications (retinopathy, nephropathy, neuropathy, macroangiopathy) of the disease--which may be severe--while avoiding severe hypoglycaemia. Although diabetes therapy has improved enormously in the last few decades, intensive research is currently aimed at replacing not only the missing hormone but also the cells which normally produce insulin in the pancreas. Transplantation of insulin secreting cells as a treatment of diabetes mellitus therefore has a special significance among other applications of cell therapy, since it deals with a disease which already has an efficient therapy. The aim of this article is the discussion of the objectives and the hopes in this field.

Dose-ranging effects of citrulline administration on plasma amino acids and hormonal patterns in healthy subjects: the Citrudose pharmacokinetic study.

Previous experimental studies have highlighted that citrulline (CIT) could be a promising pharmaconutrient. However, its pharmacokinetic characteristics and tolerance to loading have not been studied to date. The objective was to characterise the plasma kinetics of CIT in a multiple-dosing study design and to assess the effect of CIT intake on the concentrations of other plasma amino acids (AA). The effects of CIT loading on anabolic hormones were also determined. Eight fasting healthy males underwent four separate oral loading tests (2, 5, 10 or 15 g CIT) in random order. Blood was drawn ten times over an 8 h period for measurement of plasma AA, insulin and growth hormone (Gh). Urine samples were collected before CIT administration and over the next 24 h. None of the subjects experienced side effects whatever the CIT dose. Concerning AA, only CIT, ornithine (ORN) and arginine (ARG) plasma concentrations were affected (maximum concentration 146 (sem 8) to 303 (sem 11) micromol/l (ARG) and 81 (sem 4) to 179 (sem 10) micromol/l (ORN); time to reach maximum concentration 1.17 (sem 0.26) to 2.29 (sem 0.20) h (ARG) and 1.38 (sem 0.25) to 1.79 (sem 0.11) h (ORN) according to CIT dose). Even at high doses, urinary excretion of CIT remained low ( < 5 %). Plasma insulin and Gh were not affected by CIT administration. Short-term CIT administration is safe and well-tolerated. CIT is a potent precursor of ARG. However, at the highest doses, CIT accumulated in plasma while plasma ARG levels increased less than expected. This may be due to saturation of the renal conversion of CIT into ARG.

The equivocal metabolic response to endotoxaemia in type 2 diabetic and obese ZDF rats.

AIMS/HYPOTHESIS: The metabolic and endocrine disturbances associated with obesity and type 2 diabetes may impair the normal metabolic response to injury. Our objective was to investigate amino acid metabolism in endotoxaemic type 2 diabetic obese rats. MATERIALS AND METHODS: A metabolic study was performed over 4 days using male Zucker diabetic fatty (ZDF) rats (fa/fa) and lean littermates (fa/+) divided into three groups: ad libitum-fed groups which underwent no treatment, lipopolysaccharide (LPS)-treated groups receiving E. coli LPS by i.p. injection, and pair-fed groups to the respective LPS groups. We evaluated the effect of endotoxaemia on body weight, food intake and tissue weights. Nitrogen loss and muscular proteolysis were measured daily by determination of urinary 3-methylhistidine (3-MH) excretion. Plasma, intestine and muscle amino acid levels were measured. RESULTS: The data showed that ad libitum-fed ZDF rats had lower plasma arginine and glutamine levels than ad libitum-fed control rats. Compared with control rats, the LPS-treated ZDF rats presented lower thymic involution, a lower 3-MH:creatinine ratio and higher cumulative nitrogen balance. CONCLUSIONS/INTERPRETATION: Against our working hypothesis, ZDF rats did not show an impaired metabolic response, and even appeared to be less sensitive to the stress.

Immunoisolation of pancreatic B cells by microencapsulation. An in vitro study.

The selective permeability of alginate microcapsules, containing isolated rat islets of Langerhans or insulin secreting RINm5F cells, was investigated in vitro. An increase in insulin release was observed when microencapsulated islets were stimulated by glucose+theophylline, and when microencapsulated RINm5F cells were stimulated by arginine+theophylline. These findings demonstrate the permeability of the microcapsule membrane to these B-cell secretagogues and to insulin. Immunoisolation of RINm5F cells by microencapsulation was assessed using a 51chromium cytotoxicity test. Significant 51Cr release was observed when nonencapsulated cells were incubated with complement and either the serum of a rabbit immunized with RIN cells or the sera of two patients with recently diagnosed Type 1 (insulin-dependent) diabetes. This effect was not observed with encapsulated cells. Both free and encapsulated cells released 80% of their initial radioactivity when incubated in the presence of HC1. These results clearly demonstrate pancreatic cell immunoisolation by microencapsulation. They also provide a method for the in vitro evaluation of the functional characteristics of microcapsules, in terms of both insulin permeability and immunoprotection.

[Clinical perspectives of xenotransplantation of islets of Langerhans]. / Perspectives cliniques de la x¿enogreffe d'¿ilots de Langerhans.

This paper describes the rationale and different approaches for developing islets of Langerhans xenotransplantation. Implementing this therapeutic strategy in the treatment of human diabetes mellitus requires careful consideration of its potential risks and benefits, taking into account the current status of the treatment of this disease.