Direct actions of adiponectin on changes in reproductive, metabolic, and anti-oxidative enzymes status in the testis of adult mice.

Obesity is a major health problem that is linked to decreased sperm count. It is hypothesized that an obesity-associated reduction in adiponectin secretion may be responsible for impairment of spermatogenesis. Therefore, the aim of the study was to evaluate the direct role of adiponectin in spermatogenesis and steroid synthesis in adult mice. This study showed that adiponectin receptors (AdipoR1 and AdipoR2) were localized in Leydig cells and seminiferous tubules in the testis of adult mice. The result of the in vitro study showed the direct action of adiponectin on spermatogenesis by stimulating cell proliferation (PCNA) and survival (Bcl2) and by suppressing cell apoptosis. Treatment of testis with adiponectin also enhanced transport of the energetic substrates glucose and lactate to protect cells from undergoing apoptosis. Adiponectin treatment further showed a significant reduction in oxidative stress and nitric oxide. Our findings suggest that adiponectin effectively facilitates cell survival and proliferation, as well as protects from apoptosis. Thus, adiponectin treatment may be responsible for enhancing sperm counts. Interestingly, this study showed the stimulatory effect of adiponectin in spermatogenesis but showed an inhibitory effect on testosterone and estradiol synthesis in the testes. Based on the present study, it is hypothesized that systemic adiponectin treatment may be a promising therapeutic strategy for the improvement of spermatogenesis and sperm count.

Direct effects of neuropeptide nesfatin-1 on testicular spermatogenesis and steroidogenesis of the adult mice.

Recent studies have revealed nesfatin-1 as a hypothalamic neuropeptide, regulating food intake, energy expenditure and reproduction primarily by acting on the hypothalamic-pituitary-gonadal axis. Nesfatin-1 is also localized in several peripheral tissues including testes. However, functional significance of nesfatin-1 in testicular activities is not yet well documented in mammals. Therefore, this study was aimed to elucidate the direct effects of nesfatin-1 on testicular markers for steroid productions, spermatogenesis, metabolic changes and oxidative stress. The results revealed the expression of both protein and mRNA of nesfatin-1 in the testes of adult mice. The testes treated in vitro with nesfatin-1 showed significant increase in testosterone production, which correlated significantly with increased expression of steroidogenic markers and insulin receptor proteins in the testes. Furthermore, the in vitro treatment with nesfatin-1 showed stimulatory effects on spermatogenesis by promoting cell proliferation (PCNA) and survival (Bcl2), while inhibiting apoptosis (caspase-3) in the testes. The nesfatin-1 treatment in vitro further increased the expression of insulin receptor and GLUT8 proteins, in parallel with increase in the intra-testicular transport of glucose and production of lactate. This nesfatin-1 induced enhanced transport of energy substrate (glucose and lactate) may be responsible for promoting spermatogenesis and steroidogenesis. Nesfatin-1 significantly reduced oxidative stress and nitric oxide, which may also be responsible for stimulatory effects on testicular activities. The present finding suggests that nesfatin-1 acts via paracrine manner to increase sperm count and fertility, thus promoting the testicular function.

A combinatorial approach for robust transgene delivery and targeted expression in mammary gland for generating biotherapeutics in milk, bypassing germline gene integration.

Protein expression in the milk of transgenic farmed animals offers a cost-effective system for producing therapeutics. However, transgenesis in farmed animals is not only cumbersome but also involves risk of potential hazard by germline gene integration, due to interruptions caused by the transgene in the native genome. Avoiding germline gene integration, we have delivered buffalo ß-casein promoter-driven transgene construct entrapped in virosomes directly in the milk gland through intraductal perfusion delivery. Virosomes were generated from purified Sendai viral membrane, containing hemagglutinin-neuraminidase (HN) and fusion factor (F) proteins on surface (HNF-Virosomes) which initiate membrane fusion, devoid of any viral nucleic acids. Intraductal delivery of HNF-Virosomes predominantly transfected luminal epithelial cells lining the milk duct and buffalo ß-casein promoter of the construct ensured mammary luminal epithelial cell specific expression of the transgene. Mammary epithelial cells expressed EGFP at lactation when egfp was used as a transgene. Similarly, human interferon-γ (hIFN-γ) was expressed in the mammary gland as well as in the milk when hIFN-γ was used as a transgene. This combinatorial approach of using Sendai viral membrane-derived virosomes for entrapment and delivery of the transgene and using buffalo ß-casein promoter for mammary gland specific gene expression provided a better option for generating therapeutic proteins in milk, bypassing germline gene integration avoiding risks associated with animal bioreactor generated through germline gene integration.

Emerging Role of Adiponectin/AdipoRs Signaling in Choroidal Neovascularization, Age-Related Macular Degeneration, and Diabetic Retinopathy.

Age-related macular degeneration (AMD), a leading cause of irreversible blindness in adults, may result in poor central vision, making it difficult to see, read, and drive. AMD is generally classified in either dry or wet types. Milder cases of dry AMD may progress to geographic atrophy (GA), leading to significant visual disability; wet, or neovascular AMD, which involves choroidal neovascularization (CNV), can lead to complete loss of central vision. Adiponectin (APN) discovery in the mid-1990's and, subsequently, its two cognate receptors (AdipoRs) in the early 2000s have led to a remarkable progress in better understanding metabolic disorders, as well as metabolism-associated ocular pathology. APN/AdipoRs signaling plays a central role in a variety of molecular and cellular physiological events, including glucose and lipid metabolism, whole-body energy regulation, immune and inflammation responses, insulin sensitivity and retinal cell biological functions. This review is an amalgamation of recent information related to APN/AdipoRs in the pathophysiology of retinal diseases and furthers its association with AMD and diabetic retinopathy. Additionally, we present our original research, where we designed control peptide and CNV inhibitory peptide from the globular region of APN to see the effect of these peptides on the mouse model of laser-induced CNV. The inhibitory peptide (APN1) inhibited CNV by more than 75% while the control peptide did not inhibit CNV.

Linking Adiponectin and Its Receptors to Age-Related Macular Degeneration (AMD).

In recent years, there has been a captivating focus of interest in elucidating the intricate crosstalk between adiponectin (APN), a versatile fat-associated adipokine and ocular pathologies. Unveiling the intricate relationship between adipocytokine APN and its receptors (AdipoRs) with aging eye disorders has emerged as a fascinating frontier in medical research. This review article delves into this connection, illuminating the hidden influence of APN on retinal health. This comprehensive review critically examines the latest findings and breakthroughs that underscore the pivotal roles of APN/AdipoRs signaling in maintaining ocular homeostasis and protecting against eye ailments. Here, we meticulously explore the intriguing mechanisms by which APN protein influences retinal function and overall visual acuity. Drawing from an extensive array of cutting-edge studies, the article highlights APN's multifaceted functions, ranging from anti-inflammatory properties and oxidative stress reduction to angiogenic regulation within retinal and macula tissues. The involvement of APN/AdipoRs in mediating these effects opens up novel avenues for potential therapeutic interventions targeting prevalent aging eye conditions. Moreover, this review unravels the interplay between APN signaling pathways and age-related macular degeneration (AMD). The single-cell RNA-seq results validate the expression of both the receptor isoforms (AdipoR1/R2) in retinal cells. The transcriptomic analysis showed lower expression of AdipoR1/2 in dry AMD pathogenesis compared to healthy subjects. The inhibitory adiponectin peptide (APN1) demonstrated over 75% suppression of CNV, whereas the control peptide did not exert any inhibitory effect on choroidal neovascularization (CNV). The elucidation of these relationships fosters a deeper understanding of adipose tissue's profound influence on ocular health, presenting new prospects for personalized treatments and preventative measures. Because APN1 inhibits CNV and leakage, it can be used to treat human AMD, although the possibility to treat human AMD is in the early stage and more clinical research is needed. In conclusion, this review provides a captivating journey into the enthralling world of APN, intertwining the realms of adipose biology and ophthalmology in aging.

Stem Cell Therapy in Diabetic Polyneuropathy: Recent Advancements and Future Directions.

Diabetic polyneuropathy (DPN) is the most frequent, although neglected, complication of long-term diabetes. Nearly 30% of hospitalized and 20% of community-dwelling patients with diabetes suffer from DPN; the incidence rate is approximately 2% annually. To date, there has been no curable therapy for DPN. Under these circumstances, cell therapy may be a vital candidate for the treatment of DPN. The epidemiology, classification, and treatment options for DPN are disclosed in the current review. Cell-based therapies using bone marrow-derived cells, embryonic stem cells, pluripotent stem cells, endothelial progenitor cells, mesenchymal stem cells, or dental pulp stem cells are our primary concern, which may be a useful treatment option to ease or to stop the progression of DPN. The importance of cryotherapies for treating DPN has been observed in several studies. These findings may help for the future researchers to establish more focused, accurate, effective, alternative, and safe therapy to reduce DPN. Cell-based therapy might be a permanent solution in the treatment and management of diabetes-induced neuropathy.

Adiponectin: A Promising Target for the Treatment of Diabetes and Its Complications.

Diabetes mellitus, a chronic metabolic disorder characterized by hyperglycemia, presents a formidable global health challenge with its associated complications. Adiponectin, an adipocyte-derived hormone, has emerged as a significant player in glucose metabolism and insulin sensitivity. Beyond its metabolic effects, adiponectin exerts anti-inflammatory, anti-oxidative, and vasoprotective properties, making it an appealing therapeutic target for mitigating diabetic complications. The molecular mechanisms by which adiponectin impacts critical pathways implicated in diabetic nephropathy, retinopathy, neuropathy, and cardiovascular problems are thoroughly examined in this study. In addition, we explore possible treatment options for increasing adiponectin levels or improving its downstream signaling. The multifaceted protective roles of adiponectin in diabetic complications suggest its potential as a novel therapeutic avenue. However, further translational studies and clinical trials are warranted to fully harness the therapeutic potential of adiponectin in the management of diabetic complications. This review highlights adiponectin as a promising target for the treatment of diverse diabetic complications and encourages continued research in this pivotal area of diabetes therapeutics.

Adiponectin/AdipoRs signaling as a key player in testicular aging and associated metabolic disorders.

Aging undergoes serious worsening of peripheral organs and vital physiological processes including reproductive performances. Altered white adipose tissue and adipocyte functioning during aging results in ectopic lipid storage/obesity or metabolic derangements, leading to insulin resistance state. Eventually, accelerating cellular senescence thereby enhancing the high risk of age-associated metabolic alterations. Such alterations may cause derangement of numerous physiologically active obesity hormones, known as "adipokines." Specifically, adiponectin exhibits insulin sensitizing action causing anti-aging and anti-obesity effects via activation of adiponectin receptors (AdipoRs). The male reproductive physiology from reproductive mature stage to advanced senescent stage undergoes insidious detrimental changes. The mechanisms by which testicular functions decline with aging remain largely speculative. Adiponectin has also recently been shown to regulate metabolism and longevity signaling thus prolonging lifespan. Therefore, the strategy for activating adiponectin/AdipoRs signaling pathways are expected to provide a solid basis for the prevention and treatment of aging and obesity-associated reproductive dysfunctions, as well as for ensuring healthy reproductive longevity in humans.

Adipocyte CAMK2 deficiency improves obesity-associated glucose intolerance.

OBJECTIVE: Obesity-related adipose tissue dysfunction has been linked to the development of insulin resistance, type 2 diabetes, and cardiovascular disease. Impaired calcium homeostasis is associated with altered adipose tissue metabolism; however, the molecular mechanisms that link disrupted calcium signaling to metabolic regulation are largely unknown. Here, we investigated the contribution of a calcium-sensing enzyme, calcium/calmodulin-dependent protein kinase II (CAMK2), to adipocyte function, obesity-associated insulin resistance, and glucose intolerance. METHODS: To determine the impact of adipocyte CAMK2 deficiency on metabolic regulation, we generated a conditional knockout mouse model and acutely deleted CAMK2 in mature adipocytes. We further used in vitro differentiated adipocytes to dissect the mechanisms by which CAMK2 regulates adipocyte function. RESULTS: CAMK2 activity was increased in obese adipose tissue, and depletion of adipocyte CAMK2 in adult mice improved glucose intolerance and insulin resistance without an effect on body weight. Mechanistically, we found that activation of CAMK2 disrupted adipocyte insulin signaling and lowered the amount of insulin receptor. Further, our results revealed that CAMK2 contributed to adipocyte lipolysis, tumor necrosis factor alpha (TNFα)-induced inflammation, and insulin resistance. CONCLUSIONS: These results identify a new link between adipocyte CAMK2 activity, metabolic regulation, and whole-body glucose homeostasis.

Protective role of adiponectin against testicular impairment in high-fat diet/streptozotocin-induced type 2 diabetic mice.

Type 2 diabetes (T2D) is the most common endocrine and metabolic disorder, leading to reproductive impairments and infertility in male. Our recent study showed crucial role of adiponectin in the regulation of testicular functions, and the circulating level of adiponectin declines in diabetes. The current study thus aimed to examine the efficacy of adiponectin in improving testicular dysfunction in high-fat diet/streptozotocin-induced T2D mice. T2D was induced in pre-pubertal mice fed with high-fat diet for ∼10 weeks followed by a single dose of streptozotocin. T2D mice showed presence of increased body mass, hyperglycemia, hyperinsulinemia, insulin resistance, increased oxidative stress, and declined serum testosterone compared to vehicle-treated control mice. The spermatogenic, steroidogenic, metabolic, and antioxidative parameters were evaluated in T2D mice treated with adiponectin for both two and four weeks. The exogenous administration of adiponectin to T2D mice showed enhanced serum testosterone and expression of testicular steroidogenic markers proteins, insulin receptor and GLUT8 proteins, increase in intra-testicular concentrations of glucose and lactate and activity of LDH and antioxidant enzymes compared to the levels in untreated T2D mice. This suggests that treatment of adiponectin effectively improves testicular functions by increasing expression of insulin receptor-mediated increased transport of energy substrate (glucose and lactate) and a marked reduction in oxidative stress are the possible mechanism by which adiponectin effectively improves testicular function in T2D mice.

Immunohistochemical localization and possible functions of nesfatin-1 in the testis of mice during pubertal development and sexual maturation.

The study was aimed to address the role of nesfatin-1 on the sexual maturation of testis during the pubertal transition. The immunostaining of testis suggested nesfatin-1 is expressed in Leydig cells with pubertal maturation. The pre-pubertal mice for in vivo study were randomly divided in three groups; (a) control-saline (b) treated with low (0.25 nM) dose of nesfatin-1/gbw/day and (c) treated with high (1.25 nM) dose nesfatin-1/gbw/day. Histological analysis showed that nesfatin-1 loaded mice showed facilitated maturation of testis. Western blot analysis on various protein expressions upon injection of nesfatin-1 into pre-pubertal mice suggested that expressions of proteins involving steroid hormone production, spermatogenic markers (PCNA, Bcl2, AR), glucose uptake-related proteins (GLUT8 and insulin receptor) and GnRH-R and GPR-54 proteins were facilitated. Both of lactose dehydrogenase activity and lactate levels were increased. The treatment with nesfatin-1 also reduced oxidative stress, which further facilitates testicular functions during puberty. The treatment of nesfatin-1 on cultured testis also supports in vivo findings as evident by the increased testosterone production and StAR protein expression as well as increased glucose and lactate production. In sum, our data report for the first time the accelerative role of nesfatin-1 on spermatogenesis and steroidogenesis of pre-pubertal male mice by directly acting on the testis coupled with the advancement of puberty.

Role of adiponectin as a modulator of testicular function during aging in mice.

The mechanisms by which testicular functions decline with aging remain largely speculative. Our recent finding showed the importance of adiponectin in the regulation of testicular functions, whereas its concentration declines during male infertility. Thus, the aim of present study was to explore the potential role of adiponectin during aging. The changes in adiponectin, adiponectin-receptors, and insulin receptor proteins expression in the testis were evaluated and compared with the testicular parameters, mass, and testosterone level in the mice from early post-natal to late senescence period. Further, the current study has examined the effect of exogenous adiponectin treatment on testicular functions in aged mice. The results showed a significant decline in adiponectin/adiponectin-receptors expression simultaneously with a significant decline in testicular mass, insulin receptor expression and testosterone synthesis in the testis of aged mice. Exogenous treatment of adiponectin to aged mice resulted in marked improvements in testicular mass, histological features (cells proliferation), insulin receptor expression, testicular glucose uptake, anti-oxidative enzymes activity and testosterone synthesis as compared with the control. Based on these findings, it may be concluded that a marked decline in adiponectin synthesis and action results in decreased insulin sensitivity (development of insulin resistance) and increased oxidative stress which consequently suppresses testicular functions during aging. This study further showed that treatment with adiponectin ameliorates reduced testicular functions by enhanced expression of insulin receptor in the testis of senescent mice. It is thus hypothesized that systemic adiponectin treatment could be a promising therapeutic strategy for improvement of testosterone production and sperm counts during aging.

Adiponectin and Chemerin: Contrary Adipokines in Regulating Reproduction and Metabolic Disorders.

Metabolic disorders such as obesity and type 2 diabetes are one of the most familiar risk factors in the present time among every age-group. It is associated with altered levels of adipokines such as adiponectin, chemerin, leptin, resistin, visfatin, and so on. Adiponectin is one of the adipocyte-specific protein with novel applications pertaining to metabolism by promoting insulin sensitivity and regulating glucose and fatty acid catabolism, while chemerin is considered as an inhibitor of insulin signaling and glucose catabolism. Other than these established functions, both the adipokines are intimately involved in coordinating reproductive activities, but they exhibit contrary functions. This review is an amalgamation of recent information related to adiponectin and chemerin in male and female reproduction and further its association with metabolism-related reproductive disorders. The direct effect of adiponectin and chemerin on various reproductive parameters has been investigated, but there was a rampant failure to account for in vivo data which gives a broad outlook on the regulatory mechanism of both adiponectin and chemerin related to male and female reproductive functions. Adiponectin is known to promote gonadal activities, while chemerin exerts antigonadal actions. Recent research suggests that high chemerin/low adiponectin ratio plays a vital role in causing dyslipidemia and metabolic syndrome in patients. The dysregulated ratio of adiponectin to chemerin during various metabolic disorders makes it really worthy in relation to an application for therapeutics. Still, a lot regarding both the adipokines has to be explored and brought forward in order to deal with therapeutics of metabolism-related reproductive disorders.

Robust generation of transgenic mice by simple hypotonic solution mediated delivery of transgene in testicular germ cells.

Our ability to decipher gene sequences has increased enormously with the advent of modern sequencing tools, but the ability to divulge functions of new genes have not increased correspondingly. This has caused a remarkable delay in functional interpretation of several newly found genes in tissue and age specific manner, limiting the pace of biological research. This is mainly due to lack of advancements in methodological tools for transgenesis. Predominantly practiced method of transgenesis by pronuclear DNA-microinjection is time consuming, tedious, and requires highly skilled persons for embryo-manipulation. Testicular electroporation mediated transgenesis requires use of electric current to testis. To this end, we have now developed an innovative technique for making transgenic mice by giving hypotonic shock to male germ cells for the gene delivery. Desired transgene was suspended in hypotonic Tris-HCl solution (pH 7.0) and simply injected in testis. This resulted in internalization of the transgene in dividing germ-cells residing at basal compartment of tubules leading to its integration in native genome of mice. Such males generated transgenic progeny by natural mating. Several transgenic animals can be generated with minimum skill within short span of time by this easily adaptable novel technique.