Cultured Sertoli cells prepared from young rats (13 days old) showed increased incorporation of [3H]thymidine into DNA, increased production of lactate, and increased incorporation of [3H]leucine into protein in response to micromolar concentrations of insulin and nanomolar concentrations of insulin-like growth factor II (IGF-II). The first of these responses was also seen with nanomolar concentrations of IGF-I. Receptor affinity labeling studies using Sertoli cell membranes and whole Sertoli cells showed that these cells possess abundant growth factor receptors of type I (mol wt, 350,000) that show high affinity for IGF-I, moderate affinity for IGF-II, and low affinity for insulin. Sertoli cell membranes also show abundant growth factor receptors of type II (mol wt, 230,000) that show high affinity for IGF-II, moderate affinity for IGF-I, and no detectable affinity for insulin. Moreover, the responses of the Sertoli cell to insulin were observed at concentrations of 100 nM or higher, whereas insulin receptors are known to be saturated by insulin at concentrations of 10 nM or less. It is, therefore, concluded that Sertoli cells possess receptors for IGF-I and that the responses observed to insulin may result from binding of these hormones to receptors for IGF-I.

Diabetic complications such as neuropathy, retinopathy, and renal and cardiovascular disease continue to pose major health risks for diabetic patients. Consequently, much effort has focused on approaches that could replace conventional insulin therapy and provide more precise regulation of blood glucose levels. The biohybrid perfused artificial pancreas was designed to incorporate islet tissue and a selectively permeable membrane that isolates this tissue from the immune system of the recipient. Biohybrid pancreas devices containing canine islet allografts were implanted in ten pancreatectomized dogs requiring 18 to 32 units of injected insulin daily. These implants resulted in good control of fasting glucose levels in six of these animals without further exogenous insulin for periods of up to 5 months.

Permselective acrylic membranes were employed to prevent une rejection of discordant islet xenografts isolated from various large animals. Canine, porcine, and bovine islets were seeded into tubular diffusio abers and transplanted into the peritoneum of 27 nmmunosup pressed streptozotocin-induced diabetic Lewis rats. Si recipients received islet grafts from bovine calves, 7 receIved grafts from pigs, and 14 received grafts from dogs. Four of the latter were removed at 1 month. In the control group of 10 diabetic rats, 4 received nonencapsulted canine islets, 3 received nonencapsulated bovine islets, and 3 received nonencapated porcine islets. Recipients of encapsulated Islets promptly dropped from a pretranpaotation plasma glucose level of 487 ± 36 (mean ± SEM) to 84 ± 2 (canine), 81 ± 4 (bovine), and 81 ± 3 mg/dl (porcine) during the first week. 411 of the animals sustained theqe levels for at least 1 month. One rat taneously reverted to diabetes at 54 days taton; 4 other rats became hyperglycemic (glucose, >600 mg/di) after membrane removal on day 30. The rem 22 rats maintained fasting euglycema for >10 weeks. In contrast, rats that received Encapsulated Islets became hyperglycemic in <7 days. Intravenous glucose tolerance test K values (decline in glucose levels, %/min) at 1 month for the canine and bovine encapsulatedislet tr ant group were 3.5 ± 0.3 and 3.3 ± 0.1 compared with 3.3 ± 0.1 (P = 0.63) and 0.91 ± 0.1 (P < 0.0001) for normal (n = 4) and diabetic (n = 4) control groups. Morphologic studies of long-term functioning grafts (30-130 days) revealed well-preserved a, (3, and 8 (6) and pig (7) could enable the use of readily available supplies of large-animal donor tissue for islet grafting into human diabetics. Unfortunately, there is currently no clinically applicable immunologic method available that prevents the destruction of discordant islet xenografts (8)(9)(10)(11).Encouraged by the results obtained with the encapsulation of allogeneic and xenogeneic rodent islets in capillary hollow fibers (4, 12-14), we sought to determine whether larger tubular membrane diffusion chambers (fabricated from XM-50 acrylic copolymer) can protect xenografts of canine, bovine, and porcine islets from rejection in the absence ofany immunosuppressive drugs. Because the formation of fibrous tissue around the membrane has been a cause offailure in the past, a smooth external skin was used to minimize this host reaction.MATERIALS AND METHODS Animals. Adult male Lewis rats (Charles River Breeding Laboratories) weighing 250-300 g were used'as transplant recipients. Animals were fed ad libitum with a standard pelleted diet (Agway, no. 3000 RHM-Prolab) and allowed free access to water. Diabetes was induced by a single injection of streptozotocin (42 mg/kg of body weight in 0.01 M citrate buffer, pH 4.5; Sigma) into the tail vein 10-14 days prior to surgery. Only rats with plasma glucose levels >400 mg/dl were used in these studies. Fasting plasma glucose concentrations were measured by tail bleedings using -the gl...

No abstract

Spermatogenic cells have been previously shown to be a major site of testicular proenkephalin gene expression. Using RNA gel-blot analysis of purified mouse and hamster germ cells and of testes from prepuberal and germ cell-deficient mutant mice, we now have demonstrated that, in addition to its previously described expression by somatic (Leydig) cells, the gene for a second opioid peptide precursor, pro-opiomelanocortin (POMC), is also expressed by spermatogenic cells. Of particular significance is the finding that the RNAs for proenkephalin and POMC are differentially regulated during spermatogenesis. Two forms of POMC RNA were detected in mouse testis, a larger component 675-to 750-nucleotides (nt) in size common to somatic and spermatogenic cells and a smaller 625-nt RNA found only in pachytene spermatocytes. Two distinct, cell-specific proenkephalin RNAs were also shown to be present in mouse testis: a 1700-nt transcript previously shown to be expressed by spermatogenic cells and a 1450-nt form associated with somatic cells. These data suggest that (i) proenkephalin-and PONMC-derived peptides are produced by both somatic cells and germ cells in the testis and (ii) in germ cells these two families of opioid peptides may function at different stages of spermatogenesis.Paracrine mechanisms play an important role in the maintenance and regulation of spermatogenesis within the testis (1-3). Several factors appear to mediate interactions between different testicular cell types, including P Mod-S, a protein produced by peritubular cells that modifies Sertoli cell secretion (4); transport proteins and mitogenic factors produced by Sertoli cells that seem important for germ cell proliferation (3-5); and testosterone produced by Leydig cells and required for normal spermatogenesis (2). The recent demonstration that each of the three opioid peptide precursors, pro-opiomelanocortin (POMC), proenkephalin, and prodynorphin, are expressed in the testis (6-9) suggests that their peptide products also locally regulate testicular function. We have previously shown that spermatogenic cells are a major site of proenkephalin RNA expression in the mouse testis (10), results suggesting that proenkephalin-derived peptides function as germ cell-associated paracrine factQrs. The gene for the opioid peptide precursor POMC has already been shown to be expressed by Leydig cells (6, 7). In this report the POMC gene is demonstrated to be expressed also by testicular germ cells and in a distinct manner during spermatogenesis from that described for the proenkephalin gene. Multiple proenkephalin and POMC transcripts are further shown to be produced in the rodent testis in a cell-type-specific manner. MATERIALS AND METHODSGuanidine thiocyanate and N-lauroylsarcosine were from Fluka Chemical (Happauge, NY), 32P-labeled deoxynucleotides (3000 Ci/mmol; 1 Ci = 37 GBq) were purchased from New England Nuclear, cesium chloride was from Bethesda Research Laboratories, collagenase was obtained from Worthington, and trypsin (pancreatic) and D...

Previously the authors reported on a Hybrid Artificial Pancreas device that maintained patent vascular anastomoses in normal dogs and, when seeded with allogeneic canine islets, maintained normal fasting blood sugars (FBS) in diabetic pancreatectomized dogs. Eventual failure of these devices was believed to be related to loss of islet viability and/or insufficient islet mass. The current study evaluates the effect of increased islet mass produced by implantation of two islet-seeded devices in pancreatectomized dogs and compares the results with those from dogs that received a single device. Twelve of fifteen dogs receiving single devices showed initial function as determined by elimination or reduction of exogenous insulin requirement; four showed initial function and seven showed extended function (100 to 284 days). Excessive weight loss (more than 20%), despite normal FBS and insulin dependence, required that four animals in this latter group be killed. Devices seeded with xenogeneic islets have met with limited success. One dog that received two bovine islet-seeded devices achieved function for more than 100 days; the remaining bovine-seeded devices (n = 8) functioned for only 3 to 16 days. Porcine islet-seeded devices were assessed by intravenous glucose tolerance tests (IVGTT). Recipients of two devices seeded with allogeneic islets demonstrated improved IVGTT results when compared to those from pancreatectomized dogs and recipients of single devices but were abnormal when compared to intact animals. Histologic examination of device and autopsy material from all failed experiments was performed and showed no mononuclear cell infiltration of the islet chamber or vascular graft material, only a few incidence of device thrombosis, and varying degrees of islet viability as judged by morphologic and immunohistochemical evaluation. The authors believe they have demonstrated progress toward the development and clinical applicability of the Hybrid Artificial Pancreas.

No abstract

Long-term survival of dog islet allografts implanted in diabetic pancreatectomized dogs was achieved by islet encapsulation inside cylindrical chambers fabricated from permselective acrylic membranes (nominal M r exclusion of 50,000-80,000). Dog islets were isolated from the pancreases of outbred mongrel dogs by collagenase digestion. Chambers containing mean ± SE 316 ± 63K islet equivalents (mean islet volume, 558 ± 1 1 1 mm 3 , purify 90-95%) were peritoneally implanted into six totally pancreatectomized dogs. The dogs were monitored for glycemic control by fasting and postprandial blood glucose determinations, and responses to both intravenous glucose (intravenous glucose tolerance test 0.5 g/kg) and oral glucose (oral glucose tolerance test 1 g/kg). All of the dogs required appreciably lower dosages of exogenous insulin therapy for control of fasting blood glucose levels, with the mean daily insulin dose dropping from 38 ± 7 to 5 ± 1 U/day during the 1st wk. Three recipients required no insulin for >82, >68, and 51 days. Intravenous glucose tolerance test K values (decline in glucose levels, %/min) at 1 and 2 mo postimplantation were 2.7 ± 0.4 and 2.0 ± 0.5, respectively compared with 3.5 ± 0.5 before pancreatectomy. The glucose values during oral glucose tolerance tests at 2 wk, although returning to <125 mg/dl (<7.0 mM) by 2 h, exceeded the normal range, with peak values of 174 to 202 mg/dl (9.7 to 11.3 mM). These preliminary results are encouraging, and represent an important step in determining the feasibility of using this type of diffusion-based hybrid artificial pancreas as treatment for diabetes mellitus in humans.