Among Other Tissues, Short-Term Garlic Oral Treatment Incrementally Improves Indicants of Only Pancreatic Islets of Langerhans Histology and Insulin mRNA Transcription and Synthesis in Diabetic Rats.

BACKGROUND: The source, mRNA transcription, and synthesis of insulin in the pancreas, in addition to the bile duct and liver, in streptozotocin (STZ)-induced diabetic rats (DR) in response to garlic oral treatment are not yet clear. OBJECTIVE: This study investigated the accumulative effects of continued garlic oral treatment on changes in the pancreas, bile duct, and liver with regards to: 1-Insulin mRNA transcription, synthesis, and concentration in relation to changes in serum insulin (SI); 2-Insulinogenic cells insulin intensity and distribution, proliferation, and morphology. METHOD: Fasting blood glucose (FBG) and insulin concentration in serum and pancreas (PI) and sources and mRNA transcription in the pancreas, bile duct, and liver in normal rats given normal saline (NR-NS) and DR given either NS (DR-NS) or garlic extract (DR-GE) before and after 1, 4, and 8 weeks of oral treatment were examined. RESULTS: Compared to NR-NS, DR-NS showed a significant increase in FBG and reductions in SI and PI and deterioration in islets histology, associated pancreatic insulin numerical intensities, and mRNA transcription. However, compared to DR-NS, the targeted biochemical, histological, and genetic variables of DR-GE were significantly and incrementally improved as garlic treatment continued. Insulin or its indicators were not detected either in the bile duct or the liver in DR-GE. CONCLUSIONS: 8 weeks of garlic oral treatment is enough to incrementally restore only pancreatic islets of Langerhans insulin intensity and insulinogenic cells proliferation, morphology, and distribution. These indices were associated with enhanced pancreatic insulin mRNA transcription and synthesis. Eight weeks of garlic treatment were not enough to stimulate insulinogenesis in either the bile duct or the liver.

Chronic heat stress induces the expression of HSP genes in the retina of chickens (Gallus gallus).

Introduction: Chronic heat stress during summer is a major challenge imposed by global warming. Chickens are more sensitive to heat stress than mammals because they lack sweat glands. Thus, chickens are more susceptible to heat stress during summer than other seasons. Induction of heat shock protein (HSP) genes is one of the primary defense mechanisms against heat stress. Tissue-specific responses exhibited by different classes of HSPs upon exposure to heat stress have been reported previously in different tissues including the heart, kidney, intestine, blood, and muscle, but not in the retina. Therefore, this study aimed to investigate the expression levels of HSP27, HSP40, HSP60, HSP70, and HSP90 in the retina under chronic heat stress. Methods: This study was conducted during the summers of 2020 and 2021 in Kuwait. Chickens (Gallus gallus) were divided into control and heat-treated groups and sacrificed at different developmental stages. Retinas were extracted and analyzed by using Real Time quantitative Polymerase Chain Reaction (RT-qPCR). Results: Our results from the summer of 2021 were similar to that from the summer of 2020, regardless of whether GAPDH or RPL5 was used as a gene normalizer. All five HSP genes were upregulated in the retina of 21-day-old heat-treated chickens and stayed upregulated until 35 days of age, with the exception of HSP40, which was downregulated. The addition of two more developmental stages in the summer of 2021 showed that at 14 days, all HSP genes were upregulated in the retina of heat-treated chickens. In contrast, at 28 days, HSP27 and HSP40 were downregulated, whereas HSP60, HSP70, and HSP90 were upregulated. Furthermore, our results showed that under chronic heat stress, the highest upregulation of HSP genes was seen at the earliest developmental stages. Discussion: To the best of our knowledge, this is the first study to report the expression levels of HSP27, HSP40, HSP60, HSP70, and HSP90 in the retina under chronic heat stress. Some of our results match the previously reported expression levels of some HSPs in other tissues under heat stress. These results suggest that HSP gene expression can be used as a biomarker for chronic heat stress in the retina.

Expression Profiling of Pdx1, Ngn3, and MafA in the Liver and Pancreas of Recovering Streptozotocin-Induced Diabetic Rats.

Studies in animal diabetic models have demonstrated the possibility of islet regeneration through treatment with natural extracts, such as Allium sativum (garlic). This study aimed to investigate the effect of garlic extract (GE) on the expression of three genes (Ngn3, Pdx1, and MafA) in the pancreas and liver of diabetic rats. Thirty-two rats were divided into two groups, streptozotocin (STZ)-induced diabetic rats (n = 16) and healthy rats (n = 16). Both groups were subdivided into GE-treated (n = 8), and those administered 0.9% normal saline (NS) (n = 8) for 1 week (n = 4) and 8 weeks (n = 4). In the pancreas of diabetic rats treated with GE for 1 week, all three genes, Ngn3, Pdx1, and MafA, were significantly upregulated (p ≤ 0.01, p ≤ 0.05, and p ≤ 0.001, respectively) when compared to diabetic rats treated with NS only. However, after eight weeks of GE treatment, the expression of all three genes decreased as blood insulin increased. In the liver, only Pdx1 expression significantly (p ≤ 0.05) increased after 8 weeks. The significant expression of Ngn3, Pdx1, and MafA in the pancreas by week 1 may have induced the maturation of juvenile ß-cells, which escaped the effects of STZ and caused an increase in serum insulin.

Comparing and Optimizing RNA Extraction from the Pancreas of Diabetic and Healthy Rats for Gene Expression Analyses.

Advanced differential gene expression analysis requires high-quality RNA. However, isolating intact pancreatic RNA is challenging due to abundant pancreatic ribonucleases, which limits efficient downstream gene expression analysis. RNAlater treatment reduces endogenous ribonucleases effects through either pre-organ excision via organ mass or bile duct direct injection or organ mass injection post-isolation. We compared RNA extraction protocols to establish a reproducible and effective pancreatic RNA extraction method to obtain high RNA integrity number (RIN) values from healthy and streptozotocin (STZ)-induced diabetic rats for gene expression analyses. Different methods were tested focusing on RNase activity inhibition using RNAlater (Qiagen) pre-harvest of the pancreatic tissue, and extracted RNA quality and concentration were analyzed using NanoDrop spectrophotometer, Agilent Bioanalyzer, and RT-PCR. Inclusion of several pre- and post-excision modifications in the RNeasy Mini Kit (Qiagen) protocol resulted in RIN values more than two-fold higher compared to those using the standard protocol. Additionally, RT-PCR amplification of the housekeeping gene, ß-actin, revealed no differences in extracted RNA quality from healthy and STZ-induced diabetic rats. We compared and developed a more effective and reproducible pancreatic RNA extraction method from healthy and diabetic rats, which resulted in RNA of superior quality and integrity and is suitable for complex molecular investigations.

Correction: Dexamethasone Treatment Induces the Reprogramming of Pancreatic Acinar Cells to Hepatocytes and Ductal Cells.

[This corrects the article DOI: 10.1371/journal.pone.0013650.].

Dexamethasone treatment induces the reprogramming of pancreatic acinar cells to hepatocytes and ductal cells.

BACKGROUND: The pancreatic exocrine cell line AR42J-B13 can be reprogrammed to hepatocytes following treatment with dexamethasone. The question arises whether dexamethasone also has the capacity to induce ductal cells as well as hepatocytes. METHODOLOGY/PRINCIPAL FINDINGS: AR42J-B13 cells were treated with and without dexamethasone and analyzed for the expression of pancreatic exocrine, hepatocyte and ductal markers. Addition of dexamethasone inhibited pancreatic amylase expression, induced expression of the hepatocyte marker transferrin as well as markers typical of ductal cells: cytokeratin 7 and 19 and the lectin peanut agglutinin. However, the number of ductal cells was low compared to hepatocytes. The proportion of ductal cells was enhanced by culture with dexamethasone and epidermal growth factor (EGF). We established several features of the mechanism underlying the transdifferentiation of pancreatic exocrine cells to ductal cells. Using a CK19 promoter reporter, we show that a proportion of the ductal cells arise from differentiated pancreatic exocrine-like cells. We also examined whether C/EBPß (a transcription factor important in the conversion of pancreatic cells to hepatocytes) could alter the conversion from acinar cells to a ductal phenotype. Overexpression of an activated form of C/EBPß in dexamethasone/EGF-treated cells provoked the expression of hepatocyte markers and inhibited the expression of ductal markers. Conversely, ectopic expression of a dominant-negative form of C/EBPß, liver inhibitory protein, inhibited hepatocyte formation in dexamethasone-treated cultures and enhanced the ductal phenotype. CONCLUSIONS/SIGNIFICANCE: These results indicate that hepatocytes and ductal cells may be induced from pancreatic exocrine AR42J-B13 cells following treatment with dexamethasone. The conversion from pancreatic to hepatocyte or ductal cells is dependent upon the expression of C/EBPß.