Testicular fat deposition attenuates reproductive performance via decreased follicle-stimulating hormone level and sperm meiosis and testosterone synthesis in mouse.

OBJECTIVE: Testicular fat deposition has been reported to affect animal reproduction. However, the underlying mechanism remains poorly understood. The present study explored whether sperm meiosis and testosterone synthesis contribute to mouse testicular fat depositioninduced reproductive performance. METHODS: High fat diet (HFD)-induced obesity CD1 mice (DIO) were used as a testicular fat deposition model. The serum hormone test was performed by agent kit. The quality of sperm was assessed using a Sperm Class Analyzer. Testicular tissue morphology was analyzed by histochemical methods. The expression of spermatocyte marker molecules was monitored by an immuno-fluorescence microscope during meiosis. Analysis of the synthesis of testosterone was performed by real-time polymerase chain reaction and reagent kit. RESULTS: It was found that there was a significant increase in body weight among DIO mice, however, the food intake showed no difference compared to control mice fed a normal diet (CTR). The number of offspring in DIO mice decreased, but there was no significant difference from the CTR group. The levels of follicle-stimulating hormone were lower in DIO mice and their luteinizing hormone levels were similar. The results showed a remarkable decrease in sperm density and motility among DIO mice. We also found that fat accumulation affected the meiosis process, mainly reflected in the cross-exchange of homologous chromosomes. In addition, overweight increased fat deposition in the testis and reduced the expression of testosterone synthesis-related enzymes, thereby affecting the synthesis and secretion of testosterone by testicular Leydig cells. CONCLUSION: Fat accumulation in the testes causes testicular cell dysfunction, which affects testosterone hormone synthesis and ultimately affects sperm formation.

Co-culture of sperm with sertoli cells can improve IVF outcomes by increasing sperm motility in mice.

The micro-environment of spermatogenesis is important for the improvement of in vitro fertilization (IVF). Therefore, developing a co-culture system may be valuable to improve the rate of IVF. In this study, we aimed to investigate the secretions of testicular sertoli cells (SCs) to find whether it can improve the micro-environment of IVF, by which promote the efficiency of fertilization in mice. The results showed that the motility of sperms in CCSCF group (sperms co-culture with SCs) was significantly promoted and the rate of fertilization were significantly increased compared with the CTR group (control group: sperms not co-culture with SCs). Moreover, we found that the estrogen concentrations, the expression of estrogen receptor (ER) and the phosphorylation of AMPK in sperms were higher in the CCSCF group than in CTR group. In all, our results indicated that SCs co-cultured with sperms can improve the motility of sperms, E2 secreted by SCs can increase Ca2+ level in the intracellular and the level of phosphorylation of AMPK through Ca-MKKß in sperms.

Spinophilin-deficient mice are protected from diet-induced obesity and insulin resistance.

Browning of white adipose tissue (WAT) has been shown to reduce obesity and obesity-related complications, suggesting that factors that promote WAT browning may have applications in the development of therapeutic strategies for treating obesity. Here, we show that ablation of spinophilin (SPL), a ubiquitously expressed, multidomain scaffolding protein, increases metabolism and improves energy balance. Male and female SPL knockout (KO) and wild-type (WT) littermate controls were fed a chow diet or a high-fat diet (HFD). Body weight, hepatic steatosis, glucose and insulin tolerance, physical activity, and expression of browning genes in adipose tissues were measured and compared. Male SPL knockout (KO) mice fed a chow diet were significantly leaner, had lower body weights, and exhibited better glucose tolerance and insulin sensitivity than wild-type (WT) littermate controls. When fed an HFD, SPL KO mice were protected from increased body fat, weight gain, hepatic steatosis, hyperinsulinemia, and insulin resistance. Physical activity of SPL KO mice was markedly increased compared with WT controls. Furthermore, expression of the brown adipocyte marker, uncoupling protein-1 (UCP-1), and the mitochondrial activity markers, cd137 and c-idea, were significantly increased in visceral WAT (vWAT) of SPL KO mice, suggesting that SPL knockout protected the mice from HFD-induced obesity and its metabolic complications, at least in part, by promoting the browning of white adipocytes in vWAT. Our data identify a critical role of SPL in regulating glucose homeostasis, obesity, and adipocyte browning. These results suggest SPL may serve as a drug target for obesity and diabetes.

Gestational diabetes promotes germ cell cCyst breakdown and primordial follicle formation in newborn mice via the AKT signaling pathway.

Type 1 diabetes (T1D) is a common disease in which pancreatic ß cells are impaired due to auto-immunity, pregnancy in women with it is associated with increased risk of neonatal morbidity, mortality. However, the effects of gestational diabetes on the reproduction of newborn offspring are still poorly understood. Here, we determined the cyst breakdown and primordial follicle formation in neonatal offspring born by streptozotocin (STZ)-induced diabetic or non-diabetic female mice, and found that the germ cell cyst breakdown was promoted in neonatal offspring of STZ -induced diabetic mice at postnatal Day 1, which sequentially accelerated the primordial follicle formation. Further investigation revealed that, the expression level of PI3K and p-AKT were significantly increased in ovaries of offspring born by T1D mice. These results indicated that STZ -induced gestational diabetes promotes germ cell cyst breakdown and primordial follicle formation by regulating the PI3K/AKT signaling pathway in the newborn offspring. In addition, this effect can be rescued by an insulin supplement. Taken together, our results uncover the intergenerational effects of gestational diabetes on neonatal offspring folliculogenesis, and provide an experimental model for treating gestational diabetes and its complications in neonatal offspring.

Adipose stem cells from chronic pancreatitis patients improve mouse and human islet survival and function.

BACKGROUND: Chronic pancreatitis has surgical options including total pancreatectomy to control pain. To avoid surgical diabetes, the explanted pancreas can have islets harvested and transplanted. Immediately following total pancreatectomy with islet autotransplantation (TP-IAT), many islet cells die due to isolation and transplantation stresses. The percentage of patients remaining insulin free after TP-IAT is therefore low. We determined whether cotransplantation of adipose-derived mesenchymal stem cells (ASCs) from chronic pancreatitis patients (CP-ASCs) would protect islets after transplantation. METHODS: In a marginal mass islet transplantation model, islets from C57BL/6 mice were cotransplanted with CP-ASCs into syngeneic streptozotocin-treated diabetic mice. Treatment response was defined by the percentage of recipients reaching normoglycemia, and by the area under the curve for glucose and c-peptide in a glucose tolerance test. Macrophage infiltration, ß-cell apoptosis, and islet graft vasculature were measured in transplanted islet grafts by immunohistochemistry. mRNA expression profiling of 84 apoptosis-related genes in islet grafts transplanted alone or with CP-ASCs was measured by the RT2 Profiler™ Apoptosis PCR Array. The impact of insulin-like growth factor-1 (IGF-1) on islet apoptosis was determined in islets stimulated with cytokines (IL-1ß and IFN-γ) in the presence and absence of CP-ASC conditioned medium. RESULTS: CP-ASC-treated mice were more often normoglycemic compared to mice receiving islets alone. ASC cotransplantation reduced macrophage infiltration, ß-cell death, suppressed expression of TNF-α and Bcl-2 modifying factor (BMF), and upregulated expressions of IGF-1 and TNF Receptor Superfamily Member 11b (TNFRSF11B) in islet grafts. Islets cultured in conditioned medium from CP-ASCs showed reduced cell death. This protective effect was diminished when IGF-1 was blocked in the conditioned medium by the anti-IGF-1 antibody. CONCLUSION: Cotransplantation of islets with ASCs from the adipose of chronic pancreatitis patients improved islet survival and islet function after transplantation. The effects are in part mediated by paracrine secretion of IGF-1, suppression of inflammation, and promotion of angiogenesis. ASCs from chronic pancreatitis patients have the potential to be used as a synergistic therapy to enhance the efficacy of islet transplantation following pancreatectomy.

Construction and Analysis of an Adipose Tissue-Specific and Methylation-Sensitive Promoter of Leptin Gene.

DNA methylation plays a very important role in the regulation of gene expression. Under general situations, methylation in a gene promoter region is frequently accompanied by transcriptional suppression, and those genes that are highly methylated display the phenomenon of low expression. In contrast, those genes whose methylation level is low display the phenomenon of active expression. In this study, we conducted DNA methylation analysis on the CpG sites within the promoter regions of five adipose tissue-specific transcriptional factors-Adiponectin, Chemerin, Leptin, Smaf-1, and Vaspin-and examined their messenger RNA (mRNA) expression levels in different mouse tissues. We also performed analyses on the correlation between the DNA methylation levels of these genes and their mRNA expression levels in these tissues. The correlation coefficient for Leptin was the highest, and it displayed a high expression in an adipose tissue-specific manner. Thus, we cloned the regulatory region of Leptin gene and incorporated its promoter into the eukaryotic expression vector pEGFP-N1 and constructed a recombinant plasmid named pEGFP-N1-(p-Lep). This recombinant plasmid was first verified by DNA sequencing and then transfected into mouse pre-adipocytes via electroporation. Measurement of the activity of luciferase (reporter) indicated that p-Lep was capable of driving the expression of the reporter gene. This study has paved a solid basis for subsequent studies on generating transgenic animals.

Cell-Permeable Peptide Blocks TLR4 Signaling and Improves Islet Allograft Survival.

Toll-like receptor 4 (TLR4) activation in pancreatic ß cells activates aberrant islet graft cellular pathways and contributes to immune rejection in allogeneic islet transplantation. As an approach to overcoming this problem, we determined the capacity of a 33-amino acid peptide consisting of a protein transduction domain (PTD) from the Hph-1 virus and a fragment of the intracellular domain of TLR4 from the C3H mice (PTD-dnTLR4) to block TLR4 signaling and improve allogeneic islet survival in vitro and after transplantation. The efficacy of PTD-dnTLR4 in blocking TLR4 signaling was assessed in the Raw264.7 macrophage line, in the islets, and the ßTC3 cell line. In Raw264.7 cells, preculture with the peptide reduced LPS-induced NF-κB activation and production of proinflammatory cytokines (IL-1ß, TNF-α, iNOS, and IL-6). In islets and ß cells, preincubation with PTD-dnTLR4 suppressed LPS-induced TNF-α expression via inhibition of NF-κB activation and protected them from stress-induced cell death. In vivo, preincubation of BALB/c (H-2(d)) islets with PTD-dnTLR4 resulted in significantly longer survival than control islets in a streptozotocin-induced diabetes model (two of seven grafts survived long term >100 days). PTD-dnTLR4-treated grafts exhibited reduced expression of TNF-α and iNOS and reduced macrophage infiltration posttransplant. The data indicate that PTD-dnTLR4 blocked TLR4 signaling in both macrophages and ß cells, and prolonged allograft survival at least in part by suppressing inflammation and macrophage infiltration. This strategy for blocking TLR4 activity has potential utilization in the treatment of diseases where excessive TLR4 activation contributes to the pathologic cellular pathways such as islet transplantation.

Adipose-derived mesenchymal stem cells improve glucose homeostasis in high-fat diet-induced obese mice.

INTRODUCTION: Effective therapies for obesity and diabetes are still lacking. The aim of this study was to evaluate whether a single intravenous infusion of syngeneic adipose-derived mesenchymal stem cells (ASCs) can reduce obesity, lower insulin resistance, and improve glucose homeostasis in a high-fat diet-induced obese (DIO) mouse model. METHODS: Seven-week-old C57BL/6 mice were fed a high-fat diet for 20 weeks to generate the DIO mouse model. Mice were given a single intravenous infusion of ex vivo expanded syngeneic ASCs at 2 × 10(6) cells per mouse. DIO or CHOW mice injected with saline were used as controls. Body weights, blood glucose levels, glucose, and insulin tolerance test results were obtained before and 2 and 6 weeks after cell infusion. Triglyceride (TG), high-density lipoprotein (HDL), and insulin levels in serum were measured. Expressions of genes related to insulin resistance, including peroxisome proliferator-activated receptor γ (PPARγ) and insulin receptor (InsR), and inflammation (IL-6, F4/80, and nucleotide-binding oligomerization domain containing 2, or NOD2), were measured in livers at mRNA level by real-time-polymerase chain reaction analysis. Beta-cell mass in pancrheases from CHOW, DIO, and DIO + ASC mice was quantified. GFP(+) ASCs were injected, and the presence of GFP(+) cells in livers and pancreases was determined. RESULTS: DIO mice that had received ASCs showed reduced body weights, reduced blood glucose levels, and increased glucose tolerance. ASC treatment was found to reduce TG levels and increase serum HDL levels. In livers, less fat cell deposition was observed, as were increased expression of InsR and PPARγ and reduction in expressions of IL-6 and F4/80. Treated mice showed well-preserved pancreatic ß-cell mass with reduced expression of F4/80 and TNF-α compared with DIO controls. GFP(+) cells were found in liver and pancreas tissues at 1 and 2 weeks after cell injection. CONCLUSIONS: ASC therapy is effective in lowering blood glucose levels and increasing glucose tolerance in DIO mice. The protective effects of ASCs arise at least in part from suppression of inflammation in the liver. In addition, ASCs are associated with better-preserved pancreatic ß-cell mass.

Bilirubin Increases Insulin Sensitivity by Regulating Cholesterol Metabolism, Adipokines and PPARγ Levels.

Obesity can cause insulin resistance and type 2 diabetes. Moderate elevations in bilirubin levels have anti-diabetic effects. This study is aimed at determining the mechanisms by which bilirubin treatment reduces obesity and insulin resistance in a diet-induced obesity (DIO) mouse model. DIO mice were treated with bilirubin or vehicle for 14 days. Body weights, plasma glucose, and insulin tolerance tests were performed prior to, immediately, and 7 weeks post-treatment. Serum lipid, leptin, adiponectin, insulin, total and direct bilirubin levels were measured. Expression of factors involved in adipose metabolism including sterol regulatory element-binding protein (SREBP-1), insulin receptor (IR), and PPARγ in liver were measured by RT-PCR and Western blot. Compared to controls, bilirubin-treated mice exhibited reductions in body weight, blood glucose levels, total cholesterol (TC), leptin, total and direct bilirubin, and increases in adiponectin and expression of SREBP-1, IR, and PPARγ mRNA. The improved metabolic control achieved by bilirubin-treated mice was persistent: at two months after treatment termination, bilirubin-treated DIO mice remained insulin sensitive with lower leptin and higher adiponectin levels, together with increased PPARγ expression. These results indicate that bilirubin regulates cholesterol metabolism, adipokines and PPARγ levels, which likely contribute to increased insulin sensitivity and glucose tolerance in DIO mice.

Bilirubin increases insulin sensitivity in leptin-receptor deficient and diet-induced obese mice through suppression of ER stress and chronic inflammation.

Obesity-induced endoplasmic reticulum (ER) stress causes chronic inflammation in adipose tissue and steatosis in the liver, and eventually leads to insulin resistance and type 2 diabetes (T2D). The goal of this study was to understand the mechanisms by which administration of bilirubin, a powerful antioxidant, reduces hyperglycemia and ameliorates obesity in leptin-receptor-deficient (db/db) and diet-induced obese (DIO) mouse models. db/db or DIO mice were injected with bilirubin or vehicle ip. Blood glucose and body weight were measured. Activation of insulin-signaling pathways, expression of inflammatory cytokines, and ER stress markers were measured in skeletal muscle, adipose tissue, and liver of mice. Bilirubin administration significantly reduced hyperglycemia and increased insulin sensitivity in db/db mice. Bilirubin treatment increased protein kinase B (PKB/Akt) phosphorylation in skeletal muscle and suppressed expression of ER stress markers, including the 78-kDa glucose-regulated protein (GRP78), CCAAT/enhancer-binding protein (C/EBP) homologous protein, X box binding protein (XBP-1), and activating transcription factor 4 in db/db mice. In DIO mice, bilirubin treatment significantly reduced body weight and increased insulin sensitivity. Moreover, bilirubin suppressed macrophage infiltration and proinflammatory cytokine expression, including TNF-α, IL-1ß, and monocyte chemoattractant protein-1, in adipose tissue. In liver and adipose tissue of DIO mice, bilirubin ameliorated hepatic steatosis and reduced expression of GRP78 and C/EBP homologous protein. These results demonstrate that bilirubin administration improves hyperglycemia and obesity by increasing insulin sensitivity in both genetically engineered and DIO mice models. Bilirubin or bilirubin-increasing drugs might be useful as an insulin sensitizer for the treatment of obesity-induced insulin resistance and type 2 diabetes based on its profound anti-ER stress and antiinflammatory properties.

Prior surgery determines islet yield and insulin requirement in patients with chronic pancreatitis.

BACKGROUND: Total pancreatectomy with islet autotransplantation (TP-IAT) is safe and effective in the management of intractable pain associated with chronic pancreatitis (CP). Prevention of pancreatogenic diabetes after TP-IAT is related to islet yield from the diseased pancreas. The purpose of this study is to compare islet yield and insulin requirement in the 76 patients who underwent different surgical procedures before TP-IAT at the Medical University of South Carolina between 2009 and 2011. METHODS: Patients were grouped into four categories based on the operation they had before TP-IAT: transduodenal sphincteroplasty/no prior surgery (n=50), Whipple or Beger procedure (n=14), distal pancreatectomy (n=8), or lateral pancreaticojejunostomy (n=4). Islets were harvested from pancreases of those patients at our current good manufacturing practice facility. Total unpurified islets were transplanted into patients via portal vein infusion. Pancreatic fibrosis, islet yield, cell viability, and insulin requirement were measured. RESULTS: The pancreases of transduodenal sphincteroplasty/no prior surgery and Whipple or Beger procedure patients were less fibrotic and had higher islet yield compared with those who had distal pancreatectomy or lateral pancreaticojejunostomy. Higher islet yield also correlated with a greater diabetes-free rate and a lesser insulin requirement at the following intervals: preoperative, postoperative, and 6 months after TP-IAT. CONCLUSIONS: Prior surgery is strongly correlated with the extent of pancreatic fibrosis, islet yield, and insulin requirements in CP patients undergoing TP-IAT. The history of prior pancreatic resection and drainage procedures may be used to predict postoperative islet function and help to determine the optimal timing for TP-IAT in CP patients.

Immuno-isolation of pancreatic islet allografts using pegylated nanotherapy leads to long-term normoglycemia in full MHC mismatch recipient mice.

Two major hurdles need to be surmounted for cell therapy for diabetes: (i) allo-immune rejection of grafted pancreatic islets, or stem/precursor cell-derived insulin-secreting cells; and (ii) continuing auto-immunity against the diabetogenic endogenous target antigen. Nanotherapeutics offer a novel approach to overcome these problems and here we ask if creation of "stealth" islets encapsulated within a thin cage of pegylated material of 100-200 nanometers thick provides a viable option for islet transplantation. The aims of this study were to test islet viability and functionality following encapsulation within the pegylated cage, and functional efficacy in vivo in terms of graft-derived control of normoglycemia in diabetic mice. We first demonstrated that pegylation of the islet surface, plus or minus nanoparticles, improved long-term islet viability in vitro compared to non-pegylated (naked) control islets. Moreover, pegylation of the islets with nanoparticles was compatible with glucose-stimulated insulin secretion and insulin biogenesis. We next looked for functionality of the created "stealth" DBA/2 (H-2(d)) islets in vivo by comparing glycemic profiles across 4 groups of streptozotozin-induced diabetic C57BL/6 (H-2(b)) recipients of (i) naked islets; (ii) pegylated islets; (iii) pegylated islets with nanoparticles (empty); and (iv) pegylated islets with nanoparticles loaded with a cargo of leukemia inhibitory factor (LIF), a factor both promotes adaptive immune tolerance and regulates pancreatic ß cell mass. Without any other treatment, normoglycemia was lost after 17 d (+/-7.5 d) in control group. In striking contrast, recipients in groups (ii), (iii), and (iv) showed long-term (>100 d) normoglycemia involving 30%; 43%, and 57% of the recipients in each respective group. In conclusion, construction of "stealth" islets by pegylation-based nanotherapeutics not only supports islet structure and function, but also effectively isolates the islets from immune-mediated destruction. The added value of nanoparticles to deliver immune modulators plus growth factors such as LIF expands the potential of this novel therapeutic approach to cell therapy for diabetes.

[Expression of GFP gene in mouse early embryo produced by sperm-mediated gene transfer].

The efficiency of the exogenous DNA transfecting mouse sperm was studied by the DIG end labeled and immunohistochemistry technology. The results suggested that: the efficiency of transfecting positive rate of individual mouse sperm was distinct difference (P < 0.01), and the average rate was 13%. The acrosomal reaction was evaluated using the technology of Coomassie brilliant blue stained, and the appropriate in vitro fertilization (IVF) medium TYH was elected. Mouse sperms were transferred with GFP gene in vitro, and the mature oocytes were fertilized using IVF, and then the zygotes were cultured in vitro. The embryos were observed using the fluorescence microscopy, and the transgenic rate was 4.7%. The results suggested that sperm mediated gene transfer (SMGT) was an effective and feasible method.

Human lactoferrin transgenic rabbits produced efficiently using dimethylsulfoxide-sperm-mediated gene transfer.

Transgenic animal mammary gland bioreactors are used to produce recombinant proteins. However, it is difficult to validate whether these transgenic domestic animals are able to express the recombinant protein efficiently in their mammary glands before the birth of transgenic offspring. In the present study, a simple and efficient method was established to evaluate the functionality of animal mammary gland tissue-expressed cassettes. The gene transfer vector pGBC2LF was constructed, and the expression of human lactoferrin (LF) gene was controlled by the goat beta-casein gene 5' flanking sequence. To obtain the most efficient transfection, the influence of DNA concentration, dimethylsulfoxide (DMSO) concentration, and the ratio of linear-to-circular DNA required for associating DNA with spermatozoa were evaluated. Transfection of exogenous DNA into rabbit spermatozoa was found to be efficient using 30 microg mL(-1) DNA, DMSO at a final concentration of 3%, and a 3 : 1 ratio of linear-to-circular DNA, with 29 of 85 (34.1%) in vitro-fertilised embryos being transgenic. Using DMSO-sperm-mediated gene transfer (DMSO-SMGT), 89 rabbit offspring were produced, with 46 of these (57.1%) being transgenic. As mammary gland bioreactor models, 17 of 21 (81%) transgenic female rabbits could express human LF protein in their glands. During lactation of the transgenic rabbits, the highest level of human LF protein expressed was 153 +/- 31 microg mL(-1), and the mean expression level in all of the transgenic rabbits was 103 +/- 20 microg mL(-1) in the third week, declining gradually after this time. Our results demonstrate that transgenic rabbits produced by DMSO-SMGT were able to express human LF protein in the correct tissue.

Efficient and simple production of transgenic mice and rabbits using the new DMSO-sperm mediated exogenous DNA transfer method.

A high efficient and simple transgenic technology on mice and rabbits to transfect spermatozoa with exogenous DNA/DMSO complex to obtain transgenic offspring, which is namely called DMSO-sperm mediated gene transfer (SMGT). Mouse sperm could be either directly transfected via injection into testis or cultured in vitro with the plasmed DNA containing the enhanced green fluorescent protein (EGFP) that could be expressed in the embryos and offspring. Then, 36 living transgenic rabbits were produced using the same technology, and the transgenic ratio of 56.3% was detected using PCR and Southern blot. As the controls, the transgenic ratios of 39.6% and 47.8% have also been tested using the liposomes mediated technology of Tfx-50 Reagent or Lipefectamin-2000, respectively. The results show that the female transgenic rabbits, as the mammary gland bioreactor models, could express the human tissue plasminogen activator mutant (htPAm) in their mammary cells when they are adult.