Genetic Background of the Polish Primitive Horse (Konik) Coat Color Variation-New Insight into Dun Dilution Phenotypic Effect.

Only the blue dun coat color, produced by the action of the dun allele on the background of a black base coat, is officially permitted in the Polish primitive horse (PPH, Konik) breed, yet the population is not visually homogenous and various coat color shades occur. Herein, the molecular background of PPH coat color was studied based on genotyping of known causative variants in equine coat color-related genes (ASIP, MC1R, TBX3, SLC36A1, SLC45A2, PMEL17, and RALY). Additionally, screening for the new polymorphisms was conducted for the ASIP gene coding sequence and the TBX3 1.6-kb insert (associated with the dun dilution). We did not observe the champagne, silver, or cream color dilution variants in the PPH breed. A significant association (P < 0.01) was recorded for the genotype in TBX3 gene 1.6 kb in/del and the degree of dun coat dilution, demonstrating that the dominant action of the dun mutation is not fully penetrant. In addition to the effect of the 1.6 kb in/del zygosity, variants within the TBX3 insert were significantly associated with PPH coat color variability (P < 0.01), suggesting the presence of an additional allele at this locus. Finally, we identified a high frequency (35%) of genetically bay dun-colored PPH individuals that are officially recorded as blue (black base coat) duns. We propose that the difficulty in distinguishing these 2 phenotypes visually is due to an independent locus upstream of the ASIP gene, which was recently described as darkening the typical bay pigmentation shade.

Dangerous liaisons for pubertal maturation: the impact of alcohol consumption and obesity on the timing of puberty.

Acquisition of reproductive maturity involves one of the most important series of developmental events in an organism's life. The beginning of adolescence is marked by the onset of puberty. Puberty is the continuum of physical changes through which an infantile body matures into an adult capable of reproduction. This is a period of increased brain plasticity, where processes of re-wiring, neuronal proliferation, and pruning are enhanced. The initiation of mammalian puberty requires an increased pulsatile release of gonadotropin-releasing hormone from the hypothalamus. Puberty is regulated by neuroendocrine, genetic, and epigenetic factors. The maturation and function of the reproductive axis are highly sensitive to the energy status of the organism and sophisticated mechanisms exist to inhibit the axis in unfavorable energetic or metabolic conditions.In this review, we will focus on the impact of alcohol and obesity on reproductive outcomes, with emphasis on their effects on the timing of puberty. In the case of obesity, conflictive data are found, and while in females the association of overnutrition with advanced onset of puberty is consistent, in males, discrepant results have been reported. Concerning alcohol exposure, compelling evidence has documented a delay in the onset of puberty. We will present here data from both clinical studies and research involving preclinical models, which do not only delineate the impact of these conditions on the timing of puberty and potential underlying mechanisms, but that may help to define better strategies for the rational management of puberty disorders, especially of metabolic origin.

The paraventricular nucleus of the hypothalamus - the concertmaster of autonomic control. Focus on blood pressure regulation.

The autonomic nervous system regulates internal organs and peripheral circulation, which enables the maintenance of homeostasis in vertebrate species. One of the brain regions involved in autonomic and endocrine homeostasis regulation is the paraventricular nucleus of the hypothalamus (PVN). The PVN is a unique site at which multiple input signals can be assessed and integrated. The regulation of the autonomic system by the PVN and, especially, the sympathetic flow, depends upon the integration of inhibitory and excitatory neurotransmitter action. The excitatory neurotransmitters such as glutamate and angiotensin II, and inhibitory neurotransmitters such as γ­aminobutyric acid and nitric oxide, play a key role in the physiological function of the PVN. Moreover, arginine-vasopressin (AVP) and oxytocin (OXT) are important in the regulation of sympathetic system activity. The PVN is also crucial for maintaining cardiovascular regulation, with its integrity being pivotal for blood pressure regulation. Studies have shown that pre­autonomic sympathetic PVN neurons increase blood pressure and the dysfunction of these neurons is directly related to elevated sympathetic nervous system activity under hypertension. Etiology of hypertension in patients is not fully known. Thus, understanding the role of PVN in the generation of hypertension may help to treat this cardiovascular disease. This review focuses on the PVN's inhibitory and excitatory neurotransmitter interactions that regulate sympathetic system activity in physiological conditions and hypertension.

DNA hypermethylation of Kiss1r promoter and reduction of hepatic Kiss1r in female rats with type 2 diabetes.

Kisspeptin, produced from the brain and peripheral tissues, may constitute an important link in metabolic regulation in response to external cues, such as diet. The kisspeptin system is well described in the brain. However, its function and regulation in the peripheral tissues, especially in relation to metabolic disease and sex differences, remain to be elucidated. As Kiss1 and Kiss1r, encoding for kisspeptin and kisspeptin receptors, respectively, are altered by overnutrition/fasting and regulated by DNA methylation during puberty and cancer, epigenetic mechanisms in metabolic disorders are highly probable. In the present study, we experimentally induced type 2 diabetes mellitus (DM2) in female Wistar rats using high-fat diet/streptozocin. We analysed expression and DNA methylation of Kiss1 and Kiss1r in the peripheral tissues, using quantitative-reverse-transcription PCR (qRT-PCR) and pyrosequencing. We discovered differential expression of Kiss1 and Kiss1r in peripheral organs in DM2 females, as compared with healthy controls, and the profile differed from patterns reported earlier in males. DM2 in females was linked to the increased Kiss1 mRNA in the liver and increased Kiss1r mRNA in the liver and adipose tissue. However, Kiss1r promoter was hypermethylated in the liver, suggesting gene silencing. Indeed, the increase in DNA methylation of Kiss1r promoter was accompanied by a reduction in Kiss1r protein, implying epigenetic or translational gene repression. Our results deliver novel evidence for tissue-specific differences in Kiss1 and Kiss1r expression in peripheral organs in DM2 females and suggest DNA methylation as a player in regulation of the hepatic kisspeptin system in DM2.

Animal Foetal Models of Obesity and Diabetes - From Laboratory to Clinical Settings.

The prenatal period, during which a fully formed newborn capable of surviving outside its mother's body is built from a single cell, is critical for human development. It is also the time when the foetus is particularly vulnerable to environmental factors, which may modulate the course of its development. Both epidemiological and animal studies have shown that foetal programming of physiological systems may alter the growth and function of organs and lead to pathology in adulthood. Nutrition is a particularly important environmental factor for the pregnant mother as it affects the condition of offspring. Numerous studies have shown that an unbalanced maternal metabolic status (under- or overnutrition) may cause long-lasting physiological and behavioural alterations, resulting in metabolic disorders, such as obesity and type 2 diabetes (T2DM). Various diets are used in laboratory settings in order to induce maternal obesity and metabolic disorders, and to alter the offspring development. The most popular models are: high-fat, high-sugar, high-fat-high-sugar, and cafeteria diets. Maternal undernutrition models are also used, which results in metabolic problems in offspring. Similarly to animal data, human studies have shown the influence of mothers' diets on the development of children. There is a strong link between the maternal diet and the birth weight, metabolic state, changes in the cardiovascular and central nervous system of the offspring. The mechanisms linking impaired foetal development and adult diseases remain under discussion. Epigenetic mechanisms are believed to play a major role in prenatal programming. Additionally, sexually dimorphic effects on offspring are observed. Therefore, further research on both sexes is necessary.

Two weeks of moderate intensity locomotor training increased corticosterone concentrations but did not alter the number of adropin-immunoreactive cells in the hippocampus of diabetic type 2 and control rats.

Adropin (ADR) plays a role in metabolism regulation and its alterations in obesity and diabetes have been found. Treatment with ADR was beneficial in metabolic diseases, and physical exercise increased ADR concentrations in obese patients. However, data on the distribution of ADR in the brain are sparse. The role of metabolic status and physical exercise on its expression in the brain is undiscovered. We hypothesized that diabetes type 2 (DM2) and/or exercise will alter number of ADR-immunoractive (-ir) cells in the rat brain. Animals were divided into groups: diabetes type 2 (receiving high-fat diet and injections of streptozotocin) and control (fed laboratory chow diet; C). Rats were further divided into: running group (2 weeks of forced exercise on a treadmill) and non-running group. Body mass, metabolic and hormonal profiles were assessed. Immunohistochemistry was run to study ADR-ir cells in the brain. We found that: 1) in DM2 animals, running decreased insulin and increased glucose concentrations; 2) in C rats, running decreased insulin concentrations and had no effect on glucose concentration in blood; 3) running increased corticosterone (CORT) concentrations in DM2 and C rats; 4) ADR-ir cells were detected in the hippocampus and ADR-ir fibers in the arcuate nucleus of the hypothalamus, which is a novel location; 5) metabolic status and running, however, did not change number of these cells. We concluded that 2 weeks of forced moderate intensity locomotor training induced stress response present as increased concentration of CORT and did not influence number of ADR-ir cells in the brain.

Mothers' cafeteria diet induced sex-specific changes in fat content, metabolic profiles, and inflammation outcomes in rat offspring.

"Western diet" containing high concentrations of sugar and fat consumed during pregnancy contributes to development of obesity and diabetes type 2 in offspring. To mimic effects of this diet in animals, a cafeteria (CAF) diet is used. We hypothesized that CAF diet given to rats before, and during pregnancy and lactation differently influences fat content, metabolic and inflammation profiles in offspring. Females were exposed to CAF or control diets before pregnancy, during pregnancy and lactation. At postnatal day 25 (PND 25), body composition, fat contents were measured, and blood was collected for assessment of metabolic and inflammation profiles. We have found that CAF diet lead to sex-specific alterations in offspring. At PND25, CAF offspring had: (1) higher percentage of fat content, and were lighter; (2) sex-specific differences in levels of glucose; (3) higher levels of interleukin 6 (IL-6), interleukin-10 (IL-10) and tumor necrosis factor (TNF-α); (4) sex-specific differences in concentration of IL-6 and TNF-α, with an increase in CAF females; (5) higher level of IL-10 in both sexes, with a more pronounced increase in females. We concluded that maternal CAF diet affects fat content, metabolic profiles, and inflammation parameters in offspring. Above effects are sex-specific, with female offspring being more susceptible to the diet.

Expression of type one cannabinoid receptor in different subpopulation of kisspeptin neurons and kisspeptin afferents to GnRH neurons in female mice.

The endocannabinoids have been shown to target the afferents of hypothalamic neurons via cannabinoid 1 receptor (CB1) and thereby to influence their excitability at various physiological and/or pathological processes. Kisspeptin (KP) neurons form afferents of multiple neuroendocrine cells and influence their activity via signaling through a variation of co-expressed classical neurotransmitters and neuropeptides. The differential potency of endocannabinoids to influence the release of classical transmitters or neuropeptides, and the ovarian cycle-dependent functioning of the endocannabinoid signaling in the gonadotropin-releasing hormone (GnRH) neurons initiated us to study whether (a) the different subpopulations of KP neurons express CB1 mRNAs, (b) the expression is influenced by estrogen, and (c) CB1-immunoreactivity is present in the KP afferents to GnRH neurons. The aim of the study was to investigate the site- and cell-specific expression of CB1 in female mice using multiple labeling in situ hybridization and immunofluorescent histochemical techniques. The results support that CB1 mRNAs are expressed by both the GABAergic and glutamatergic subpopulations of KP neurons, the receptor protein is detectable in two-thirds of the KP afferents to GnRH neurons, and the expression of CB1 mRNA shows an estrogen-dependency. The applied estrogen-treatment, known to induce proestrus, reduced the level of CB1 transcripts in the rostral periventricular area of the third ventricle and arcuate nucleus, and differently influenced its co-localization with vesicular GABA transporter or vesicular glutamate transporter-2 in KP neurons. This indicates a gonadal cycle-dependent role of endocannabinoid signaling in the neuronal circuits involving KP neurons.

Effect of Fasting on the Spexin System in Broiler Chickens.

Spexin (SPX) is a highly conservative peptide hormone containing 14 amino acids and was discovered in 2007 by bioinformatics methods. However, nothing is yet known about its role in the metabolism of birds, including broilers. The aim of this study was to investigate the effect of short-term fasting (2, 4, and 8 h) on the concentration of SPX in blood serum and the expression levels of the genes encoding this peptide (SPX1) and its receptors, GALR2 and GALR3, in the tissues involved in carbohydrate and lipid metabolism (muscles, adipose tissue, and liver). We also analyzed the mRNA expression of these genes in various chicken tissues. Moreover, we studied the correlation between the serum level of SPX and other metabolic parameters (insulin, glucagon, glucose, triglycerides, and cholesterol). Using RT-qPCR, we found that SPX1, GALR2, and GALR3 are expressed in all investigated tissues in broiler chicken. Moreover, using a commercially available radio-immunoassay, we noted an increase of the SPX level in blood serum after 4 and 8 h of fasting compared to nonfasted animals (p < 0.05). This increase was positively correlated with glucagon concentration (r = 0.341; p < 0.05) and negatively with glucose concentration (r = -0.484; p < 0.01). Additionally, we discovered that in the short term, food deprivation leads to the expression regulation of SPX1, GALR2, and GLAR3 in tissues associated with metabolism of carbohydrates and lipids. The obtained results indicate that SPX is involved in the regulation of metabolism in broiler chickens.

Effects of short-term exposure to high-fat diet on histology of male and female gonads in rats.

Obesity, which reaches an epidemic, is characterized by alterations in metabolic and hormonal profiles. Moreover, uncontrolled obesity may lead to development of diabetes type 2, which accounts for about 90% of all diabetic cases. In obesity, besides changes in metabolism, numerous co-morbidities are reported, e.g. disruptions of reproductive functions. Additionally, sex differences in development of this disease occur. We hypothesized that short-term exposure to high-fat diet (HFD; containing 50% of total energy from fat) would alter histology of testes and ovaries, and thus contribute to reproductive disruptions in male and female rats. Adult rats were fed ad libitum with HFD for 6-7 weeks and its effects on histology of testes and ovaries (n = 4/sex and treatment group) were studied using hematoxylin-eosin staining followed by microscopic analysis and compared to control (laboratory chow fed) group. We have found that in male rats fed with HFD there were: 1) decrease in diameter of seminiferous tubules due to smaller luminar diameter, and no change in epithelium height; 2) decrease in number of Sertoli cells; 3) no changes in number of spermatogonia and in percentage of semen in seminiferous tubules. In female rats exposed to HFD we have seen: 1) decrease in diameters of corpora lutea; 2) decrease in diameter of ovarian follicles types 7 and 8, but no changes in their number; 3) no changes in number of early primary follicles, primary follicles, and secondary follicles. We concluded that relatively short-term exposure to HFD in rats leads to changes in histology of both testes and ovaries, thus affecting reproductive functions.

Effects of Ovariectomy and Sex Hormone Replacement on Numbers of Kisspeptin-, Neurokinin B- and Dynorphin A-immunoreactive Neurons in the Arcuate Nucleus of the Hypothalamus in Obese and Diabetic Rats.

KNDy neurons co-expressing kisspeptin (KP), neurokinin B (NKB) and dynorphin A (DYN A) in the arcuate nucleus of the hypothalamus (ARC) are key regulators of reproduction. Their activity is influenced by metabolic and hormonal signals. Previously, we have shown that orchidectomy alters the KP-, NKB-, and DYN A-immunoreactivity in the high-fat diet-induced (HFD) obesity and diabetes type 2 (DM2) models. Considering the potential sex difference in the response of KNDy neurons, we have hypothesized that ovariectomy (OVX) and post-ovariectomy replacement with estradiol (OVX+E2) or estradiol and progesterone (OVX+E2+P4) will also affect these neurons in HFD and DM2 females. Thus, each of these treatment protocols were employed for control, HFD, and DM2 groups of rats leading to nine experimental conditions within which we have determined the number of KP-, NKB-, or DYN-immunoreactive (-ir) neurons and assessed the metabolic and hormonal profiles of the animals. Accordingly: (1) no effects of group and surgery were observed on the number of KP-ir neurons; (2) the overall number of NKB-ir neurons was higher in the OVX+E2+P4 and OVX+E2 animals compared to OVX; (3) overall, the number of DYN A-ir neurons was higher in DM2 vs. control group, and surgery had an effect on the number of DYN A-ir neurons; (4) the metabolic and hormonal profiles were altered in HFD and DM2 animals compared to controls. Current data together with our previously published results indicate sex-specific differences in the response of KNDy neurons to DM2.

Diabetes Type 2 and Kisspeptin: Central and Peripheral Sex-Specific Actions.

Kisspeptin (KP) plays a major role in the regulation of reproduction governed by the hypothalamic-pituitary-gonadal (HPG) axis. However, recent findings suggest that the KP system is present not only centrally (at the level of the hypothalamus), but also in the peripheral organs crucial for the control of metabolism. The KP system is sexually differentiated in the hypothalamus, and it is of particular interest to study whether sex-specific responses to type 2 diabetes (DM2) exist centrally and peripherally. As collection of data is limited in humans, animal models of DM2 are useful to understand crosstalk between metabolism and reproduction. Sex-specific variations in the KP system reported in animals suggest a need for the development of gender specific therapeutic strategies to treat DM2.

Kisspeptin and Metabolism: The Brain and Beyond.

Apart from the well-established role of kisspeptin (Kp) in the regulation of reproductive functions, recent data described its action in the control of metabolism. Of particular interest for the review is the population of Kp neurons localized in the arcuate nucleus (ARC) of the hypothalamus, the site of the brain where reproductive and metabolic cross talk occurs. However, within the hypothalamus Kp does not work alone, but rather interacts with other neuropeptides, e.g., neurokinin B, dynorphin A, proopiomelanocortin, the cocaine- and amphetamine-regulated transcript, agouti-related peptide, and neuropeptide Y. Beyond the brain, Kp is expressed in peripheral tissues involved in metabolic functions. In this review, we will mainly focus on the local action of this peptide in peripheral organs such as the pancreas, liver, and the adipose tissue. We will concentrate on dysregulation of the Kp system in cases of metabolic imbalance, e.g., obesity and diabetes. Importantly, these patients besides metabolic health problems often suffer from disruptions of the reproductive system, manifested by abnormalities in menstrual cycles, premature child birth, miscarriages in women, decreased testosterone levels and spermatogenesis in men, hypogonadism, and infertility. We will review the evidence from animal models and clinical data indicating that Kp could serve as a promising agent with clinical applications in regulation of reproductive problems in individuals with obesity and diabetes. Finally, emerging data indicate a role of Kp in regulation of insulin secretion, potentially leading to development of further therapeutic uses of this peptide to treat metabolic problems in patients with these lifestyle diseases.

High-fat diet and type 2 diabetes induced disruption of the oestrous cycle and alteration of hormonal profiles, but did not affect subpopulations of KNDy neurones in female rats.

Apart from the primary metabolic symptoms of obesity and/or diabetes, there are numerous secondary problems, including disruptions of the reproductive system. The KNDy neurones, which express kisspeptin, neurokinin B and dynorphin A and are located in the arcuate nucleus of the hypothalamus (ARC), are important regulators of reproduction. Their functions are highly influenced by metabolic and hormonal status. We have previously shown that, in male rats with experimentally-induced diabetes type 2 (but not with high-fat diet-induced obesity), there are alterations in the number of these cells. In the present study, we hypothesised that a high-fat diet (HFD) and/or diabetes type 2 (DM2) in female rats affect the oestrous cycle, hormonal profiles and the number of kisspeptin-immunoreactive, neurokinin B-immunoreactive and/or dynorphin A-immunoreactive neurones in the ARC. Rats were assigned to one of three groups: a control group fed a regular chow diet, a high-fat diet group (HFD) and a diabetic group (DM2), with both of the latter two groups receiving a high calorie diet (50% of energy from lard). The third group was additionally treated with streptozotocin to induce DM2. Their oestrous cycles was monitored and their metabolic and hormonal status were assessed. We found that HFD and DM2 female rats, despite having significant alterations in their metabolic and hormonal profiles, as well as disruptions of the oestrous cycle, showed no changes in the number of the kisspeptin-immunoreactive, neurokinin B-immunoreactive and/or dynorphin A-immunoreactive neurones in the ARC. However, slight differences in the rostrocaudal distribution of these neurones among groups were reported. In conclusion, the data from the present study, together with our previously published results in males, indicate sex differences in the response of KNDy neurones to DM2 but not to HFD conditions.