Studies on the mechanism of testicular dysfunction in the early stage of a streptozotocin induced diabetic rat model

Streptozotocin (STZ) induced diabetic model has been widely used to study the effects of diabetes mellitus (DM) on male infertility, but it remains unclear whether the responses in this model are due to hyperglycemia or STZ per se. This study was designed to investigate the mechanism of STZ on testicular dysfunction. In the present study, sperm characteristics, serum testosterone, steroidogenic enzymes (StAR and 3β-HSD), and the vimentin apical extension of sertoli cells decreased significantly in the STZ group compared with those in the normal controls (p < 0.05), while Johnsen’s score, testicular lipid peroxidation, spermatogenic cell apoptosis, and the expressions of NF-κB and Wnt4 significantly increased (p < 0.05). Insulin replacement mainly restored the decreased serum testosterone and steroidogenic enzymes, but not other parameters. The results indicated that spermatogenic dysfunction in the early stage of STZ-induced diabetic rats was due to direct STZ cytotoxicity to sertoli cells, which could be regulated by Wnt4 and NF-κB, while steroidogenic dysfunction might be a direct or indirect consequence of insulin deficiency. The results suggested that STZ-induced diabetic model, at least in the early stage, is not suitable to study the diabetes-related spermatogenic dysfunction.     

The effect of leptin on adrenal glands in rats with streptozotocin-induced diabetes: an experimental study

OBJECTIVE: To determine the cross-section area of adrenal medulla and the percentage of Ki-67 (a proliferation marker) of the adrenal gland in diabetic rats after leptin injection. STUDY DESIGN: Twenty-nine male Wistar rats were randomly divided into 3 groups: control (C) group (n = 9), diabetes mellitus (DM) group (n = 10) and leptin-injected diabetes mellitus (DM+L) group (n = 10). Experimental DM was induced by a single intraperitoneal dose of streptozotocin (40 mg/kg). After this, leptin (100 microg/kg) was injected subcutaneously for a period of 2 weeks in the diabetic group. RESULTS: An atrophy of adrenal medulla in the DM group was observed, and this atrophy returned to normal morphology after injection of leptin. In addition, an increase in the Ki-67 percentage was demonstrated in the zona reticularis layers in the DM+L group. CONCLUSION: Our study indicated that leptin stimulates the sympathoadrenal system and the androgen producing adrenal cortex in DM rats.     

Add-on therapy with anagliptin in Japanese patients with type-2 diabetes mellitus treated with metformin and miglitol can maintain higher concentrations of biologically active GLP-1/total GIP and a lower concentration of leptin

Metformin, α-glucosidase inhibitors (α-GIs), and dipeptidyl peptidase 4 inhibitors (DPP-4Is) reduce hyperglycemia without excessive insulin secretion, and enhance postprandial plasma concentration of glucagon-like peptide-1 (GLP-1) in type-2 diabetes mellitus (T2DM) patients. We assessed add-on therapeutic effects of DPP-4I anagliptin in Japanese T2DM patients treated with metformin, an α-GI miglitol, or both drugs on postprandial responses of GLP-1 and glucose-dependent insulinotropic polypeptide (GIP), and on plasma concentration of the appetite-suppressing hormone leptin. Forty-two Japanese T2DM patients with inadequately controlled disease (HbA1c: 6.5%–8.0%) treated with metformin (n = 14), miglitol (n = 14) or a combination of the two drugs (n = 14) received additional treatment with anagliptin (100 mg, p.o., b.i.d.) for 52 weeks. We assessed glycemic control, postprandial responses of GLP-1 and glucose-dependent insulinotropic polypeptide (GIP), and on plasma concentration of leptin in those patients. Add-on therapy with anagliptin for 52 weeks improved glycemic control and increased the area under the curve of biologically active GLP-1 concentration without altering obesity indicators. Total GIP concentration at 52 weeks was reduced by add-on therapy in groups treated with miglitol compared with those treated with metformin. Add-on therapy reduced leptin concentrations. Add-on therapy with anagliptin in Japanese T2DM patients treated with metformin and miglitol for 52 weeks improved glycemic control and enhanced postprandial concentrations of active GLP-1/total GIP, and reduce the leptin concentration.     

Hyperglycemia induces NF‐κB activation and MCP‐1 expression via downregulating GLP‐1R expression in rat mesangial cells: inhibition by metformin

Hyperglycemia impairs glucagon‐like peptide‐1 receptor (GLP‐1R) signaling in multiple cell types and thereby potentially attenuates the therapeutic effects of GLP‐1R agonists. We hypothesized that the downregulation of GLP‐1R by hyperglycemia might reduce the renal‐protective effects of GLP‐1R agonists in diabetic nephropathy (DN). In this study, we examined the effects of high glucose on the expression of GLP‐1R and its signaling pathways in the HBZY‐1 rat mesangial cell line. We found that high glucose reduced GLP‐1R messenger RNA (mRNA) levels in HBZY‐1 cells and in the renal cortex in db/db mice comparing with control groups. In consistence, GLP‐1R agonist exendin‐4 induced CREB phosphorylation was attenuated by high glucose but not low glucose treatment, which is paralleled with abrogated anti‐inflammatory functions in HBZY‐1 cells linked with nuclear factor‐κB (NF‐κB) activation. In consistence, GLP‐1R inhibition aggravated the high glucose‐induced activation of NF‐κB and MCP‐1 protein levels in cultured HBZY‐1 cells while overexpression of GLP‐1R opposite effects. We further proved that metformin restored high glucose‐inhibited GLP‐1R mRNA expression and decreased high glucose evoked inflammation in HBZY‐1 cells. On the basis of these findings, we conclude that high glucose lowers GLP‐1R expression and leads to inflammatory responses in mesangial cells, which can be reversed by metformin. These data support the rationale of combinative therapy of metformin with GLP‐1R agonists in DN.     

Ellagic acid improved diabetes mellitus-induced testicular damage and sperm abnormalities by activation of Nrf2

Diabetes mellitus induces testicular damage, increases sperm abnormalities, and impairs reproductive dysfunction due to induction of endocrine disturbance and testicular oxidative stress. This study evaluated the reproductive protective effect of ellagic acid (EA) against testicular damage and abnormalities in sperm parameters in Streptozotocin (STZ)-induced diabetic rats (T1DM) and examined some possible mechanisms of protection. Adult male rats were segregated into 5 groups (n = 12 rat/each) as control, control + EA (50 mg/kg/day), T1DM, T1DM + EA, and T1DM + EA + brusatol (an Nrf-2 inhibitor) (2 mg/twice/week). All treatments were conducted for 12 weeks, daily. EA preserved the structure of the seminiferous tubules, prevented the reduction in sperm count, motility, and viability, reduced sperm abnormalities, and downregulated testicular levels of cleaved caspase-3 and Bax in diabetic rats. In the control and diabetic rats, EA significantly increased the circulatory levels of testosterone, reduced serum levels of FSH and LH, and upregulated Bcl-2 and all steroidogenic genes (StAr, 3β-HSD1, and 11β-HSD1). Besides, it reduced levels of ROS and MDA but increased levels of GSH and MnSOD and the transactivation of Nrf2. All these biochemical alterations induced by EA were associated with increased activity and nuclear accumulation of Nrf2. However, all these effects afforded by EA were weakened in the presence of brusatol. In conclusion, EA could be an effective therapy to alleviated DM-induced reproductive toxicity and dysfunction in rats by a potent antioxidant potential mediated by the upregulation of Nrf2.     

The Effects of Metformin and Diet on Plasma Testosterone and Leptin Levels in Obese Men

Objective: The aim of this study was to investigate the effects of combined hypocaloric diet and metformin on circulating testosterone and leptin levels in obese men with or without type 2 diabetes. Research Methods and Procedures: Twenty obese men with type 2 diabetes (mean body mass index [BMI]: 35.5 ± 1.1 kg/m²) and 20 nondiabetic obese men were enrolled in the study. We measured serum follicle‐stimulating hormone, luteinizing hormone (LH), total testosterone (TT), free testosterone (FT), sex‐hormone‐binding globulin (SHBG), dehydroepiandrosterone sulfate (DHEAS), and plasma leptin levels before and 3 months after metformin treatment. Both groups were placed on a hypocaloric diet and 850 mg of metformin taken orally twice daily for 3 months. Results: Metformin and hypocaloric diets led to decreases in BMI and waist and hip circumferences in both groups. A significant decrease in TT levels in the diabetic group and FT levels in the control group was found, whereas follicle‐stimulating hormone, LH, and DHEAS levels were not changed significantly. A significant increase in SHBG levels was observed in the control group but not in the patient group. Leptin levels also decreased after treatment in both groups. Decreased testosterone levels were not correlated to changes in waist and hip circumference, waist‐to‐hip ratio, BMI, and levels of fasting blood glucose, leptin, SHBG, or DHEAS in the diabetic group. However, a decrease in FT was correlated to changes in the levels of SHBG (r = ?0.71, p = 0.001) and LH (r = 0.80, p = 0.001) but not to other parameters. Discussion: We conclude that metformin treatment combined with a hypocaloric diet leads to reduced FT levels in obese nondiabetic men and to reduced TT levels in obese men with type 2 diabetes. Increased SHBG levels may account for the decrease in FT levels in the former group.     

Molecular and morphometric analysis of the rat ventral prostate injected with leptin

The aim of this study was to evaluate the effect of leptin administration on the ventral prostate lobe of adult rat. Twenty adult male rats were divided into 2 groups: L-animals were daily injected with 50 μL of leptin (8 μg/100 g BW, subcutaneous) for four days and C-animals received the same volume of saline solution. Lipid profile and testosterone serum levels were evaluated. The prostate ventral lobe was processed for histomorphometric analysis. Gene expression of aromatase, androgen, leptin and estrogen receptors isoforms was evaluated by real-time PCR. Cell proliferation was evaluated by PCNA immunohistochemistry. Data were expressed as mean±standard error and analyzed by student's t-test. Serum levels of cholesterol (C=39.7±4.2;L=55.2±4.2, mg/dL; P<0.02) increased and testosterone (C=1.6±0.43;L=0.6±0.15, ng/dL; P<0.03) decreased in L group. The histomorphometric analysis showed a reduction in cell density (C=8868±242; L=8211±210, mm(2); P<0.04), in total (C=0.24±0.026; L=0.10±0.009, mm(2); P<0.001) and in the internal acini areas (C=0.16±0.009; L=0.08±0.006, mm(2); P<0.0002). On the other hand, there was an increase in the epithelial height (C=17.3±0.3; L=22.8±0.2, μm; P<0.0001) and in the number of acini (C=7.0±0.2; L=8.7±0.1, mm(2); P<0.0002). The histomorphometric analyses together with PCNA immunohistochemistry results suggest that leptin increases cell proliferation. In relation to the gene expression, leptin treatment increased the expression of all genes, but ER-α, in more than 200 times compared to the expression in C group. In conclusion, in this paper we showed that leptin has a direct effect on the prostate gland of adult rats leading to an increase in proliferation and in the gene expression of aromatase, androgen, leptin and estrogen receptors isoforms that are important for the physiology of the prostate gland.     

Proinsulin C-peptide as an alternative or combined treatment with insulin for management of testicular dysfunction and fertility impairments in streptozotocin-induced type 1 diabetic male rats

Diabetes mellitus (DM) is closely associated with male infertility and sexual dysfunction. Recent data indicate that the proinsulin C-peptide (CP) exerts important physiological effects and shows the characteristics of an endogenous peptide hormone. So, this study was done to investigate the effect of C-peptide with or without insulin treatment on testicular function and architecture in diabetic rats. Rats were divided into the following groups: control, diabetic, and diabetic groups treated with either CP alone or combined with insulin. Tested parameters included, estimation of serum follicle-stimulating hormone (FSH), luteinizing hormone (LH), testosterone, and glucose levels, testicular samples for histopathology and estimation of malondialdehyde (MDA), total antioxidant capacity (TAC), and B-cell leukemia/lymphoma-2 (BCL-2) levels as well as sperm count and motility. Results showed that DM caused a severe alteration in hormonal profile and reduced sperm parameters along with increased MDA and decrease in both TAC and BCL-2 levels. CP alone or with insulin treatment efficiently reversed all the negative effects of DM on rat testes, with maximum improvement in the combined regimen. Proposed mechanisms may involve its hypoglycemic, antioxidant, and antiapoptotic properties. Thus, CP could substitute for or better combined with insulin to prevent or retard diabetic-induced testicular dysfunction.     

Efficacy of Photobiomodulation and Metformin on Diabetic Cell Line of Human Periodontal Ligament Stem Cells through Keap1/Nrf2/Ho-1 Pathway

Background:Diabetes mellitus (DM) is a metabolic disorder resulting from hyperglycemia. Hyperglycemia contributes to oxidative stress, and the release of advanced glycation end products (AGEs) further promotes disease pathogenesis. Uncontrolled diabetes reflects great oral complications and affects human oral health. So, the present study aimed to assess the effects of photobiomodulation therapy (PBMT) and Metformin on proliferation and viability of human periodontal ligament stem cells (HPDLSCs) cultured in high glucose medium.Methods:HPDLSCs were collected, isolated, and characterized and then divided into eight groups. Addition of extra glucose to diabetic groups 24 hours before cell irradiations. Metformin was added to half of the diabetic groups. Cells were irradiated with 808 nm diode laser 24, 48 hours. Cell viability was analyzed with MTT assay 24 hours post-irradiation to detect cell viability in each group. Real-time (PCR) was used to evaluate gene expression of Nrf2, Keap1, PIK3, and HO-1 and the effect of PBMT on Keap1/Nrf2/Ho-1 Pathway. ELISA reader was used to evaluating cell viability through (ROS, TNF-α, IL-10) protein levels after cell irradiation.Results:Photobiomodulation at 1, 2, and 3 J/cm2 combined with metformin significantly promoted diabetic cell lines of HPDLSCs viability (in MTT assay and ELISA reader of ROS, TNF-α, IL-10 results) and gene expression of Nrf2, Keap1, PIK3, and HO-1 levels (p< 0.05).Conclusion:photobiomodulation with 3 J/cm² combined with metformin enhanced proliferation and viability of diabetic cell lines of HPDLSCs and thus could improve differentiation and function of diabetic cell lines of HPDLSCs with minimum side effects.Key Words: Diabetes Mellitus, Metformin, Periodontal Ligament Stem Cells, Photobiomodulation     

Specific neural phase relation of serotonin and dopamine modulate the testicular activity in Japanese quail

Specific phase relation of serotonin and dopamine modulate the hypothalamo–hypophyseal–gonadal axis as well as photosexual responses in Japanese quail, but the effect of these specific phase relations on testicular activity and steroidogenesis is not yet been investigated. We hypothesized that temporal phase relation induced alteration in local testicular gonadotropin-releasing hormone (GnRH)–Gonadotropin-inhibitory hormone (GnIH) and their receptor system may modulate the testicular activity and steroidogenesis through local (paracrine and autocrine) action. To validate this hypothesis, we have checked the alterations in the expression of gonadotropin-releasing hormone receptor (GnRH-R), gonadotropin-inhibitory hormone receptor (GnIH-R) messenger RNA (mRNA), growth hormone receptor (GH-R), proliferating cell nuclear antigen (PCNA), cell communication and gap junctional proteins (14-3-3 and connexin-43 [Cnx-43]), steroidogenic factor-1 (SF-1), steroidogenic acute regulatory (StAR) protein, steroidogenic enzyme (3β-hydroxysteroid dehydrogenase [3β-HSD]) in testis as well as androgen receptor (AR) in testis and epididymis of control, 8-, and 12-hr quail. Experimental findings clearly indicate the increased expression of GnIH-R mRNA and suppression of GnRH-R, GH-R, PCNA, 14-3-3, Cnx-43, SF-1, StAR, 3β-HSD in testis as well as AR in testis and epididymis in 8-hr quail, while 12-hr quail exhibited the opposite results that is significantly decreased expression of GnIH-R mRNA and increased expression of GnRH-R, GH-R, PCNA, 14-3-3, Cnx-43, SF-1, StAR, 3β-HSD in testis as well as AR in testis and epididymis. The significantly increased intratesticular testosterone has been observed in the 12-hr quail while, 8-hr quail showed opposite result. Hence, it can be concluded that 12-hr quail showed significantly increased testicular activity and steroidogenesis while opposite pattern was observed in 8-hr quail.     

Is it time to test biguanide metformin in the treatment of melanoma?

Metformin is the most widely used antidiabetic drug that belongs to the biguanide class. It is very well tolerated and has the major clinical advantage of not inducing hypoglycemia. Metformin decreases hepatic glucose production via a mechanism requiring liver kinase B1, which controls the metabolic checkpoint, AMP‐activated protein kinase‐mammalian target of rapamycin and neoglucogenic genes. The effects of metformin on this pathway results in reduced protein synthesis and cell proliferation. These observations have given the impetus for many investigations on the role of metformin in the regulation of tumor cell proliferation, cell‐cycle regulation, apoptosis, and autophagy. Encouraging results from these studies have shown that metformin could potentially be used as an efficient anticancer drug in various neoplasms such as prostate, breast, lung, pancreas cancers, and melanoma. These findings are strengthened by retrospective epidemiological studies that have found a decrease in cancer risk in diabetic patients treated with metformin. In this review, we have focused our discussion on recent molecular mechanisms of metformin that have been described in various solid tumors in general and in melanoma in particular.     

Effects of diethylstilboestrol on testicular function and luteinizing hormone receptors

Adult male Fisher-344 rats were implanted with DES-filled or empty Silastic capsules. After 14 weeks, capsules were removed and a second group of rats received DES capsules. Seven weeks later, all the rats were sacrificed. DES treatment decreased body, testes and seminal vesicle weights, and removal of the capsules partially restored the weight of these organs. The concentration of testicular LH receptors was increased by DES treatment. Circulating PRL levels were increased and gonadotropin levels were reduced in all animals having received DES at anytime. Plasma testosterone (T) levels were similar in all groups, but testicular T levels were reversibly decreased by DES. Similarly, whereas basal incubation media T levels were unchanged by DES treatment, the steroidogenic response in vitro to hCG was abolished by the presence of DES, and removal of the capsules restored this response. It appears that in this animal model DES and PRL exert opposing effects on testicular LH receptor.