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1

Influence of obesity and type 2 diabetes on the number of kisspeptin-, neurokinin B- and dynorphin A-immunoreactive neurons in the arcuate nucleus of female rats

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Ziarniak K., Kolodziejski P., Pruszynska-Oszmalek E., Matuszewska J., Sliwowska J. H.
Acta Neurobiologiae Experimentalis
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2017
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tom 77
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nr Suppl.1
INTRODUCTION: There is a strong evidence that neurons co-expressing kisspeptin (KP), neurokinin B (NKB) and dynorphin (Dyn), so called KNDy neurons, are important factors governing the hypothalamic-pituitary-gonadal axis. These neurons are present in the arcuate nucleus of the hypothalamus (ARC), which is also a region involved in energy homeostasis. It was shown that expression of KP, NKB and Dyn is dependent on hormonal and metabolic status. We have previous found that type 2 diabetes but not diet-induced obesity increases number of KP-, NKB- and non-pregnant ewes and ewes euthanized at 30, 60, 90, 120 d of pregnancy (3 ewes/group). Real-time PCR was used to measure SOCS-3 mRNA abundance. RESULTS: Results showed that SOCS-3 transcript level increased in MBH at 30, 60 and 90 d of gestation in comparison with non-pregnant ewes (P<0.05). The greatest SOCS-3 transcript abundance was observed at 120 d of pregnancy in ARC and in AP. In ME, SOCS‑3 expression significantly decreased (P<0.05) during early- and mid-pregnancy (at 30 and 60 d of gestation) but during late-pregnancy (120 d of gestation) it increased to a level comparable to that of non-pregnant ewes. In the CP, SOCS-3 mRNA expression in first half of pregnancy was similar to that observed in non-pregnant females, but increased markedly in the second half of pregnancy (P<0.05). Interestingly, SOCS-3 expression decreased throughout pregnancy in the PG (P<0.05). CONCLUSIONS: The pattern of expression of SOCS-3 differs among brain locations and by stage of pregnancy within brain and AP locations and variation in SOCS-3 transcripts may be one of the factors in brain and AP that mediate homeorhetic adjustments in metabolism during gestation. FINANCIAL SUPPORT: Research supported by grant from Polish National Science Centre no 2013/09/B/NZ4/01532.
2

Effects of gonadectomy and testosterone replacement on number of dynorphin-immunoreactive (minusIR) cells in the arcuate nucleus of the hypothalamus in obese and diabetic male rats

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Ziarniak K., Gawalek M., Pruszynska-Oszmalek E., Kolodziejski P. A., Kaczmarek P., Rodak E., Sliwowska J. H.
Acta Neurobiologiae Experimentalis
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2015
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tom 75
|
nr Supl.
BACKGROUND AND AIMS: Dynorphins (Dyn) are involved in the regulation of feeding and kisspeptin together with dynorphin and neurokinin B play a role in reproductive functions. Besides primary metabolic health problems occurring in people with obesity and/or diabetes, there are disruptions of the reproductive system (e.g. hypogonadism, infertility). Moreover, alterations in the hypothalamic dynorphin system in food deprived and diabetic animals were reported. However, there is no study looking at changes in number of Dyn-ir neurons in the arcuate nucleus of the hypothalamus (ARC), where integration of metabolism and reproduction may occur in obese and diabetic rats. There were 2 aims: (1) to assess if obesity induced by high fat diet and/or diabetes induced by injections of streptozotocin (STZ) alters the number of Dyn-ir neurons in the ARC in male rats; (2) to examine if gonadectomy (GDX) and testosterone (T) replacement differentially alters the number of Dyn-ir neurons in the ARC in male rats. METHODS: Rats were fed with high fat diet (HFD) or control (C) diet for 5 weeks. Injections of STZ were performed to induce diabetes type 1 (C/STZ) or diabetes type 2 (HFD/STZ) The following groups were obtained: C, C/STZ, HFD, HFD/STZ. Next, animals were divided into 3 groups: gonadectomy (GDX); gonadectomy and T replacement (GDX+T) and control (Sham). Immunocytochemistry for the Dyn was performed. RESULTS: Dynorphin-ir was found in: paraventricular nucleus of the hypothalamus, supraoptic nucleus, ventromedial nucleus, lateral hypothalamus, ARC and median eminence. Preliminary results indicate that there is a slight increase in number of Dyn-ir cells in C/ STZ and a slight decrease in HFD/STZ group. Currently data from GDX and GDX+T animals is analyzed. CONCLUSIONS: If data is confirmed on the bigger sample (currently analyzed), observed changes may contribute both to metabolic and reproductive deficits observed in these animals. Supported by grant NCN 2011/01/B/NZ4/04992.
3

Effects of ovariectomy and sex hormone replacement on the number of neurokinin B-immunoreactive neurons in the arcuate nucleus of the hypothalamus of obese and diabetic female rats

86%
Ziarniak K., Kolodziejski P. A., Pruszynska-Oszmalek E., Sassak M., Dudek M., Sliwowska J. H.
Acta Neurobiologiae Experimentalis
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2019
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tom 79
|
nr Suppl.1
INTRODUCTION: The reproductive capacity of mammals is governed by the hypothalamus-pituitary-gonadal (HPG) axis, with gonadotropin-releasing hormone (GnRH) localized on top of it. Neuronal population expressing kisspeptin, neurokinin B (NKB), and dynorphin (KNDy neurons), present in the arcuate nucleus (ARC) of the hypothalamus, are important for regulating GnRH secretion. Both obesity and type 2 diabetes (DM2) are major risk factors for reproductive alterations (e.g., decreased fertility). Because ARC is a site where cross‑talk between metabolism and reproduction occurs, those states may influence KNDy neurons. However, data on the role of metabolic imbalance, gonadectomy, and sex steroid replacement in the regulation of NKB expression are limited, especially in females. AIM(S): The aim of this study was to assess the effects of metabolic disruption (high-fat diet-induced and DM2), ovariectomy, and sex hormone replacement on the number of NKB-immunoreactive (-ir) neurons in the ARC of female rats. METHOD(S): Female rats received a control (C) or high‑fat diet (HFD) for 13 weeks. Streptozotocin injections were performed to induce DM2 in half of the animals from the HFD group. The following groups were obtained: C, HFD, and DM2. Then, animals were divided into three subgroups: ovariectomy (OVX), ovariectomy and estradiol replacement (OVX+E2), and ovariectomy together with estradiol and progesterone replacement (OVX+E2+P4). Metabolic profile was assessed and immunohistochemistry for NKB was performed. RESULTS: There was an effect of operation (p<0.01). In C and DM2, there was a higher number of NKB-ir cells in OVX+E2+P4 vs. OVX. Additionally, in the DM2 group, a higher number of NKB-ir was seen in OVX+E2 vs. OVX. CONCLUSIONS: HFD does not change the response of NKB‑ir neurons to OVX and hormonal replacement. However, in DM2 females, NKB-ir neurons are more sensitive to the OVX+E2 condition. FINANCIAL SUPPORT: Supported by NCN grant 2015/17/B/NZ4/02021.
4

Expression of cannabinoid receptor type-1 in different subpopulations of kisspeptin neurons in the mouse brain

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Kallo I., Mahendravarman M., Nagy K., Ziarniak K., Sliwowska J. K., Wilheim T.
Acta Neurobiologiae Experimentalis
|
2019
|
tom 79
|
nr Suppl.1
INTRODUCTION: Kisspeptin (KP) is involved in multiple hypothalamic regulatory processes such as estrogenic feedback to GnRH neurons via synaptic connections. We have reported previously that GnRH neurons send endocannabinoid retrograde signals to regulate their GABAergic input. KP‑producing neurons co‑synthesize GABA or glutamate both in the preoptic (POA) and arcuate (ARC) subpopulations. Moreover, we found the cannabinoid receptor type one (CB1) mRNA in these areas. However, it is unknown whether any subpopulation of KP neurons is under the influence of endocannabinoids. AIM(S): 1) To identify the GABAergic and glutamatergic subpopulations of KP neurons in female mice. 2) To determinate whether CB1 transcripts can be detected in any of the subpopulation of POA or ARC KP neurons. METHOD(S): The transcripts of KP, CB1, and the GABAergic and glutamatergic marker proteins, i.e., vesicular inhibitory amino acid transporter (VIAAT), and vesicular glutamate transporter-2 (VGLUT2) have been detected by RNAscope in situ hybridization technique. The signal was analyzed by confocal microscopy and quantified using our in‑house developed program. RESULTS: We found a significantly increased KP mRNA expression in the POA of ovariectomised mice after estradiol replacement. CB1 mRNAs were found in GABAergic and glutamatergic KP neurons of the POA and ARC. About 67% (AVPV) – 43% (ARC) of the GABAergic KP neurons expressed CB1 mRNA in the OVX+OIL mice, and about 31% (AVPV) – 36% (ARC) of the GABAergic KP neurons expressed CB1 mRNA in the OVX+EB mice. Concerning the glutamatergic KP neurons, about 19% (AVPV) ‑14% (ARC) expressed CB1 mRNA in the OVX- +OIL mice and about 24% (AVPV) – 43% (ARC) of them expressed CB1 mRNA in the OVX+EB mice. CONCLUSIONS: The hormonal status of mice influences the number of KP neurons expressing CB1 and endocannabinoids likely regulate the electrical activity of different KP subpopulations involved in the regulation of various hypothalamic processes.
5

Effects of gonadectomy and testosterone replacement on NKB-ir cell number in the arcute nucleus of the hypothalamus in obese and diabet male rats

86%
Gawalek M., Kolodziejski P., Pruszynska-Oszmalek E., Rodak E., Ziarniak K., Kaczmarek P., Sliwowska J. H.
Acta Neurobiologiae Experimentalis
|
2015
|
tom 75
|
nr Supl.
BACKGROUND AND AIMS: Reproduction is governed by the hypothalamus-pituitary-gonadal (HPG) axis, with the gonadotropin releasing hormone being on the top of the axis. In the arcute nucleus (ARC) of hypothalamus, population of neurons expressing kisspeptin, neurokinin B (NKB) and dynorphin (KNDy neurons) is present. Those neurons are important in regulation of GnRH secretion. Beside metabolic problems obesity and diabetes are a major risk factors for reproductive dysfunctions (e.g. steroid imbalance and hypogonadism). Moreover, in hypogonadotropic hypogonadism patients mutation in NKB gene (TAC3) and its receptor – TAC3R was reported. In animals data on the role of gonadectomy (GDX) and sex steroids replacement in regulation of NKB expression is spare. We hypothesized that: (1) diet-induced obese (DIO), and/or streptozotocin (STZ)-induced diabetic (type 1 and 2) male rats would have altered number of NKB-ir neurons in the ARC; (2) gonadectomy and testosterone (T) replacement would differentially altered number of NKB-ir neurons. METHODS: Rats were fed with high fat diet (HFD) or control (C) diet for 5 weeks. Injections of STZ were performed to induce diabetes type 1 (C/STZ) or diabetes type 2 (HFD/STZ). The following groups were obtain: C, C/STZ, HFD, HFD/STZ. Next, animals were divided into 3 groups: gonadectomy (GDX); gonadectomy and T replacement (GDX+T) and  (Sham). Immunocytochemistry for the NKB was performed. RESULTS: We found that in C group there was no difference in number of NKB-ir neurons in the ARC between Sham and GDX. In contrast, in all experimental groups a decrease in NKB-ir cell number after GDX was shown. T replacement caused a decrease in NKB-ir cell number in C, HFD and HFD/STZ groups compare to Sham, respectively. CONCLUSION: Obesity and diabetes type 1 and type 2 leads to alter response of NKB-ir cells in response to GDX. Supported by NCN grant 2011/01/B/NZ4/04992.
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