Hypothalamic Kisspeptin Neurons as a Target for Whole-Cell Patch-Clamp Recordings.

Kisspeptins are essential for the maturation of the hypothalamic-pituitary-gonadal (HPG) axis and fertility. Hypothalamic kisspeptin neurons located in the anteroventral periventricular nucleus and rostral periventricular nucleus, as well as the arcuate nucleus of the hypothalamus, project to gonadotrophin-releasing hormone (GnRH) neurons, among other cells. Previous studies have demonstrated that kisspeptin signaling occurs through the Kiss1 receptor (Kiss1r), ultimately exciting GnRH neuron activity. In humans and experimental animal models, kisspeptins are sufficient for inducing GnRH secretion and, consequently, luteinizing hormone (LH) and follicle stimulant hormone (FSH) release. Since kisspeptins play an essential role in reproductive functions, researchers are working to assess how the intrinsic activity of hypothalamic kisspeptin neurons contributes to reproduction-related actions and identify the primary neurotransmitters/neuromodulators capable of changing these properties. The whole-cell patch-clamp technique has become a valuable tool for investigating kisspeptin neuron activity in rodent cells. This experimental technique allows researchers to record and measure spontaneous excitatory and inhibitory ionic currents, resting membrane potential, action potential firing, and other electrophysiological properties of cell membranes. In the present study, crucial aspects of the whole-cell patch-clamp technique, known as electrophysiological measurements that define hypothalamic kisspeptin neurons, and a discussion of relevant issues about the technique, are reviewed.

Acute effects of somatomammotropin hormones on neuronal components of the hypothalamic-pituitary-gonadal axis.

Growth hormone (GH) and prolactin (PRL) are known as pleiotropic hormones. Accordingly, the distribution of their receptors comprises several organs and tissues, including the central nervous system. The appropriate secretion of both hormones is essential for sexual maturation and maintenance of reproductive functions, while defects in their secretion affect puberty onset and can cause infertility. Conversely, GH therapy at a prepubertal age may accelerate puberty. On the other hand, hyperprolactinemia is a frequent cause of infertility. While the action of PRL in some central components of the Hypothalamic-Pituitary-Gonadal (HPG) axis, such as the kisspeptin neurons, has been well documented, the possible effects of GH in the hypothalamus are still elusive. Thus, the present study was designed to investigate whether somatomammotropin hormones are able to modulate the activity of critical neuronal components of the HPG axis, including kisspeptin neurons and cells of the ventral premammillary nucleus (PMv). Our results revealed that GH effects in kisspeptin neurons of the anteroventral periventricular and rostral periventricular nuclei or in PMv neurons relies predominantly on the recruitment of the signal transducer and activator of transcription 5 (STAT5) rather than through acute changes in resting membrane potential. Importantly, kisspeptin neurons located at the arcuate nucleus were not directly responsive to GH. Additionally, our findings further identified PMv neurons as potential targets of PRL, since PRL induces the phosphorylation of STAT5 and depolarizes PMv neurons. Combined, our data provide evidence that GH and PRL may affect the HPG axis via specific hypothalamic neurons.

Obesity impairs lactation performance in mice by inducing prolactin resistance.

Obesity reduces breastfeeding success and lactation performance in women. However, the mechanisms involved are not entirely understood. In the present study, female C57BL/6 mice were chronically exposed to a high-fat diet to induce obesity and subsequently exhibited impaired offspring viability (only 15% survival rate), milk production (33% reduction), mammopoiesis (one-third of the glandular area compared to control animals) and postpartum maternal behaviors (higher latency to retrieving and grouping the pups). Reproductive experience attenuated these defects. Diet-induced obese mice exhibited high basal pSTAT5 levels in the mammary tissue and hypothalamus, and an acute prolactin stimulus was unable to further increase pSTAT5 levels above basal levels. In contrast, genetically obese leptin-deficient females showed normal prolactin responsiveness. Additionally, we identified the expression of leptin receptors specifically in basal/myoepithelial cells of the mouse mammary gland. Finally, high-fat diet females exhibited altered mRNA levels of ERBB4 and NRG1, suggesting that obesity may involve disturbances to mammary gland paracrine circuits that are critical in the control of luminal progenitor function and lactation. In summary, our findings indicate that high leptin levels are a possible cause of the peripheral and central prolactin resistance observed in obese mice which leads to impaired lactation performance.

Fatness rather than leptin sensitivity determines the timing of puberty in female mice.

Leptin is a permissive factor for the onset of puberty. However, changes in adiposity frequently influence leptin sensitivity. Thus, the objective of the present study was to investigate how changes in body weight, fatness, leptin levels and leptin sensitivity interact to control the timing of puberty in female mice. Pre-pubertal obesity, induced by raising C57BL/6 mice in small litters, led to an early puberty onset. Inactivation of Socs3 gene in the brain or exclusively in leptin receptor-expressing cells reduced the body weight and leptin levels at pubertal onset, and increased leptin sensitivity. Notably, these female mice exhibited significant delays in vaginal opening, first estrus and onset of estrus cyclicity. In conclusion, our findings suggest that increased leptin sensitivity did not play an important role in favoring pubertal onset in female mice. Rather, changes in pubertal body weight, fatness and/or leptin levels were more important in influencing the timing of puberty.

Reviewing the Effects of L-Leucine Supplementation in the Regulation of Food Intake, Energy Balance, and Glucose Homeostasis.

Leucine is a well-known activator of the mammalian target of rapamycin (mTOR). Because mTOR signaling regulates several aspects of metabolism, the potential of leucine as a dietary supplement for treating obesity and diabetes mellitus has been investigated. The objective of the present review was to summarize and discuss the available evidence regarding the mechanisms and the effects of leucine supplementation on the regulation of food intake, energy balance, and glucose homeostasis. Based on the available evidence, we conclude that although central leucine injection decreases food intake, this effect is not well reproduced when leucine is provided as a dietary supplement. Consequently, no robust evidence indicates that oral leucine supplementation significantly affects food intake, although several studies have shown that leucine supplementation may help to decrease body adiposity in specific conditions. However, more studies are necessary to assess the effects of leucine supplementation in already-obese subjects. Finally, although several studies have found that leucine supplementation improves glucose homeostasis, the underlying mechanisms involved in these potential beneficial effects remain unknown and may be partially dependent on weight loss.

L-leucine supplementation worsens the adiposity of already obese rats by promoting a hypothalamic pattern of gene expression that favors fat accumulation.

Several studies showed that l-leucine supplementation reduces adiposity when provided before the onset of obesity. We studied rats that were exposed to a high-fat diet (HFD) for 10 weeks before they started to receive l-leucine supplementation. Fat mass was increased in l-leucine-supplemented rats consuming the HFD. Accordingly, l-leucine produced a hypothalamic pattern of gene expression that favors fat accumulation. In conclusion, l-leucine supplementation worsened the adiposity of rats previously exposed to HFD possibly by central mechanisms.

Oral leucine supplementation is sensed by the brain but neither reduces food intake nor induces an anorectic pattern of gene expression in the hypothalamus.

Leucine activates the intracellular mammalian target of the rapamycin (mTOR) pathway, and hypothalamic mTOR signaling regulates food intake. Although central infusion of leucine reduces food intake, it is still uncertain whether oral leucine supplementation is able to affect the hypothalamic circuits that control energy balance. We observed increased phosphorylation of p70s6k in the mouse hypothalamus after an acute oral gavage of leucine. We then assessed whether acute oral gavage of leucine induces the activation of neurons in several hypothalamic nuclei and in the brainstem. Leucine did not induce the expression of Fos in hypothalamic nuclei, but it increased the number of Fos-immunoreactive neurons in the area postrema. In addition, oral gavage of leucine acutely increased the 24 h food intake of mice. Nonetheless, chronic leucine supplementation in the drinking water did not change the food intake and the weight gain of ob/ob mice and of wild-type mice consuming a low- or a high-fat diet. We assessed the hypothalamic gene expression and observed that leucine supplementation increased the expression of enzymes (BCAT1, BCAT2 and BCKDK) that metabolize branched-chain amino acids. Despite these effects, leucine supplementation did not induce an anorectic pattern of gene expression in the hypothalamus. In conclusion, our data show that the brain is able to sense oral leucine intake. However, the food intake is not modified by chronic oral leucine supplementation. These results question the possible efficacy of leucine supplementation as an appetite suppressant to treat obesity.