IntroductionWith the shortage of donor organs for islet transplantation, insulin-producing cells have been generated from different types of stem cell. Human fetal pancreatic stem cells have a better self-renewal capacity than adult stem cells and can readily differentiate into pancreatic endocrine cells, making them a potential source for islets in diabetes treatment. In the present study, the functions of pancreatic islets derived from human fetal pancreatic progenitor cells were evaluated in vitro and in vivo.MethodsHuman pancreatic progenitor cells isolated from the fetal pancreas were expanded and differentiated into islet endocrine cells in culture. Markers for endocrine and exocrine functions as well as those for alpha and beta cells were analyzed by immunofluorescent staining and enzyme-linked immunosorbent assay (ELISA). To evaluate the functions of these islets in vivo, the islet-like structures were transplanted into renal capsules of diabetic nude mice. Immunohistochemical staining for human C-peptide and human mitochondrion antigen was applied to confirm the human origin and the survival of grafted islets.ResultsHuman fetal pancreatic progenitor cells were able to expand in medium containing basic fibroblast growth factor (bFGF) and leukemia inhibitor factor (LIF), and to differentiate into pancreatic endocrine cells with high efficiency upon the actions of glucagon-like peptide-1 and activin-A. The differentiated cells expressed insulin, glucagon, glucose transporter-1 (GLUT1), GLUT2 and voltage-dependent calcium channel (VDCC), and were able to aggregate into islet-like structures containing alpha and beta cells upon suspension. These structures expressed and released a higher level of insulin than adhesion cultured cells, and helped to maintain normoglycemia in diabetic nude mice after transplantation.ConclusionsHuman fetal pancreatic progenitor cells have good capacity for generating insulin producing cells and provide a promising potential source for diabetes treatment.
Aims/Introduction
Blockade or reversal the progression of diabetic nephropathy is a clinical challenge. The aim of the present study was to examine whether recombinant human glucagon‐like peptide‐1 (rh
GLP
‐1) has an effect on alleviating urinary protein and urinary albumin levels in diabetic rats.
Materials and Methods
Streptozotocin‐induced diabetes rats were treated with rh
GLP
‐1 insulin and saline. Using immunostaining, hematoxylin–eosin, electron microscopy and periodic acid–Schiff staining to study the pathology of diabetic nephropathy, and we carried out quantitative reverse transcription polymerase chain reaction, western blot and immunohistochemistry to identify the differentially expressed proteins. The mechanism was studied through advanced glycation end‐products‐induced tubular epithelial cells.
Results
rh
GLP
‐1 inhibits protein kinase C (PKC)‐β, but increases protein kinase A
(PKA)
, which reduces oxidative stress in glomeruli and in cultured glomerular microvascular endothelial cells. In tubules, rh
GLP
‐1 increased the expression of two key proteins related to re‐absorption – megalin and cubilin – which was accompanied by downregulation of
PKC
‐β and upregulation of
PKA
. On human proximal tubular epithelial cells, rh
GLP
‐1 enhanced the absorption of albumin, and this was blocked by a
PKC
activator or
PKA
inhibitor.
Conclusions
These findings suggest that rh
GLP
‐1 can reverse diabetic nephropathy by protecting both glomeruli and tubules by inhibiting
PKC
and activating
PKA
.
Microvasculopathy is the most serious and predictable threat to the health of diabetic patients, which often results in end-stage renal disease, blindness, and limb amputations. Up to the present, the underlying mechanisms have remained elusive. Here, it was found that the differential activations of PKC/PKA were involved in diabetic microvasculopathy in diabetic GK rats. By real-time PCR, Western blot, immunohistochemistry, and enzyme activity assay, upregulation of PKC was prominent in kidney but was not significant in liver and brain. The expression and activity of PKA were lowered in kidney but comparable in brain and liver during diabetic nephropathy. Furthermore, the generation of reactive oxygen species, production of nitric oxide, and expression of inducible nitric oxide synthase induced by advanced glycation end products were inhibited by PKCβ inhibitor LY-333531 or a PKA agonist in rat glomerular microvascular endothelial cells. Finally, albuminuria was significantly lowered by a PKA agonist and boosted by a PKA antagonist. It suggested that the differential activations of PKC/PKA related to microvasculopathy in diabetes and that activation of PKA may protect the diabetic microvasculature.
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