Knockdown of regulatory associated protein of TOR (raptor) in hypothalamus-stimulated folliculogenesis and induced ovarian cysts.

CONTEXT: Mammalian target of rapamycin complex 1 (mTORC1) is an essential sensor that regulates fundamental biological processes like cell growth, proliferation and energy metabolism. The treatment of disease by sirolimus, a mTORC1 inhibitor, causes adverse effects, such as female fertility disorders. AIMS: The objective of the study was to decipher the reproductive consequences of a downregulation of mTORC1 in the hypothalamus. METHODS: The reduced expression of mTORC1 was induced after intracerebroventricular injection of lentivirus expressing a short hairpin RNA (shRNA) against regulatory associated protein of TOR (raptor) in adult female mice (ShRaptor mice). KEY RESULTS: The ShRaptor mice were fertile and exhibited a 15% increase in the litter size compared with control mice. The histological analysis showed an increase in antral, preovulatory follicles and ovarian cysts. In the hypothalamus, the GnRH mRNA and FSH levels in ShRaptor mice were significantly elevated. CONCLUSIONS: These results support the hypothesis that mTORC1 in the central nervous system participates in the regulation of female fertility and ovarian function by influencing the GnRH neuronal activity. IMPLICATIONS: These results suggest that a lower mTORC1 activity directly the central nervous system leads to a deregulation in the oestrous cycle and an induction of ovarian cyst development.

Endospanin1 affects oppositely body weight regulation and glucose homeostasis by differentially regulating central leptin signaling.

The hypothalamic arcuate nucleus (ARC) is a major integration center for energy and glucose homeostasis that responds to leptin. Resistance to leptin in the ARC is an important component of the development of obesity and type 2 diabetes. Recently, we showed that Endospanin1 (Endo1) is a negative regulator of the leptin receptor (OBR) that interacts with OBR and retains the receptor inside the cell, leading to a decreased activation of the anorectic STAT3 pathway. Endo1 is up-regulated in the ARC of high fat diet (HFD)-fed mice, and its silencing in the ARC of lean and obese mice prevents and reverses the development of obesity. OBJECTIVE: Herein we investigated whether decreased Endo1 expression in the hypothalamic ARC, associated with reduced obesity, could also ameliorate glucose homeostasis accordingly. METHODS: We studied glucose homeostasis in lean or obese mice silenced for Endo1 in the ARC via stereotactic injection of shRNA-expressing lentiviral vectors. RESULTS: We observed that despite being leaner, Endo1-silenced mice showed impaired glucose homeostasis on HFD. Mechanistically, we show that Endo1 interacts with p85, the regulatory subunit of PI3K, and mediates leptin-induced PI3K activation. CONCLUSIONS: Our results thus define Endo1 as an important hypothalamic integrator of leptin signaling, and its silencing differentially regulates the OBR-dependent functions.

GDH-Dependent Glutamate Oxidation in the Brain Dictates Peripheral Energy Substrate Distribution.

Glucose, the main energy substrate used in the CNS, is continuously supplied by the periphery. Glutamate, the major excitatory neurotransmitter, is foreseen as a complementary energy contributor in the brain. In particular, astrocytes actively take up glutamate and may use it through oxidative glutamate dehydrogenase (GDH) activity. Here, we investigated the significance of glutamate as energy substrate for the brain. Upon glutamate exposure, astrocytes generated ATP in a GDH-dependent way. The observed lack of glutamate oxidation in brain-specific GDH null CnsGlud1(-/-) mice resulted in a central energy-deprivation state with increased ADP/ATP ratios and phospho-AMPK in the hypothalamus. This induced changes in the autonomous nervous system balance, with increased sympathetic activity promoting hepatic glucose production and mobilization of substrates reshaping peripheral energy stores. Our data reveal the importance of glutamate as necessary energy substrate for the brain and the role of central GDH in the regulation of whole-body energy homeostasis.

LKB1 and AMPKα1 are required in pancreatic alpha cells for the normal regulation of glucagon secretion and responses to hypoglycemia.

AIMS/HYPOTHESIS: Glucagon release from pancreatic alpha cells is required for normal glucose homoeostasis and is dysregulated in both Type 1 and Type 2 diabetes. The tumour suppressor LKB1 (STK11) and the downstream kinase AMP-activated protein kinase (AMPK), modulate cellular metabolism and growth, and AMPK is an important target of the anti-hyperglycaemic agent metformin. While LKB1 and AMPK have emerged recently as regulators of beta cell mass and insulin secretion, the role of these enzymes in the control of glucagon production in vivo is unclear. METHODS: Here, we ablated LKB1 (αLKB1KO), or the catalytic alpha subunits of AMPK (αAMPKdKO, -α1KO, -α2KO), selectively in ∼45% of alpha cells in mice by deleting the corresponding flox'd alleles with a preproglucagon promoter (PPG) Cre. RESULTS: Blood glucose levels in male αLKB1KO mice were lower during intraperitoneal glucose, aminoimidazole carboxamide ribonucleotide (AICAR) or arginine tolerance tests, and glucose infusion rates were increased in hypoglycemic clamps (p < 0.01). αLKB1KO mice also displayed impaired hypoglycemia-induced glucagon release. Glucose infusion rates were also elevated (p < 0.001) in αAMPKα1 null mice, and hypoglycemia-induced plasma glucagon increases tended to be lower (p = 0.06). Glucagon secretion from isolated islets was sensitized to the inhibitory action of glucose in αLKB1KO, αAMPKdKO, and -α1KO, but not -α2KO islets. CONCLUSIONS/INTERPRETATION: An LKB1-dependent signalling cassette, involving but not restricted to AMPKα1, is required in pancreatic alpha cells for the control of glucagon release by glucose.

Unsaturated fatty acids disrupt Smad signaling in gonadotrope cells leading to inhibition of FSHß gene expression.

Reproductive function is highly dependent on nutritional input. We recently provided evidence that the unsaturated ω6 fatty acid (FA), linoleic acid (linoleic), interferes with transcription and secretion of the gonadotropin LH, highlighting the existence of a lipid sensing in pituitary gonadotropes. Here, we show, using a combination of in vivo and in vitro models, that linoleic differentially regulates Lhb and Fshb expression. Central exposure of rats to linoleic over 7 days was associated with increase of Lhb but not Fshb transcript levels. Consistently, exposure of rat pituitary cells or LßT2 cells to linoleic increased Lhb, whereas it dramatically decreased Fshb transcript levels without affecting its stability. This effect was also induced by ω9 and ω3-polyunsaturated FA but not by saturated palmitic acid. Analysis of the underlying mechanisms in LßT2 cells using small interfering RNA revealed that early growth response protein 1 mediates linoleic stimulation of Lhb expression. Furthermore, we demonstrated that linoleic counteracts activin and bone morphogenetic protein-2 stimulation of Fshb expression. Using Western blotting and Smad-responsive reporter gene assays, linoleic was shown to decrease basal Smad2/3 phosphorylation levels as well as activin- and bone morphogenetic protein-2-dependent activation of Smad, uncovering a new FA-sensitive signaling cascade. Finally, the protein phosphatase magnesium-dependent 1A was shown to mediate linoleic inhibition of basal Smad phosphorylation and Fshb expression, identifying protein phosphatase magnesium-dependent 1A as a new target of FA in gonadotropes. Altogether, this study provides a novel mechanism by which FAs target gene expression and underlines the relevant role of pituitary gonadotropes in mediating the effects of nutritional FA on reproductive function.

Unsaturated fatty acids stimulate LH secretion via novel PKCepsilon and -theta in gonadotrope cells and inhibit GnRH-induced LH release.

The activity of pituitary gonadotrope cells, crucial for reproductive function, is regulated by numerous factors including signals related to nutritional status. In this work, we demonstrated, for the first time, that in vivo central exposure of rats to lipids intracarotid infusion of a heparinized triglyceride emulsion selectively increases the expression of pituitary LH subunit genes without any alteration of pituitary GnRH receptor and hypothalamic GnRH or Kiss-1 transcript levels. Furthermore, we showed that unsaturated fatty acids (UFA), oleate and linoleate, increase LH release in a dose-dependent manner as well as LHß mRNA levels in both immortalized LßT2 gonadotrope cell line and rat primary cell cultures. In contrast, the saturated palmitate was ineffective. ACTH or TSH secretion was unaffected by UFA treatment. We demonstrated in LßT2 cells that linoleate effect is mediated neither by activation of membrane fatty acid (FA) receptors GPR40 or GPR120 although we characterized these receptors in LßT2 cells, nor through nuclear peroxisome proliferator-activated receptors. Furthermore, linoleate ß-oxidation is not required for its action on LH secretion. In contrast, pharmacological inhibition of protein kinase C (PKC) or ERK pathways significantly prevented linoleate-stimulated LH release. Accordingly, linoleate was shown to activate novel PKC isoforms, PKCε and -θ, as well as ERK1/2 in LßT2 cells. Lastly, unsaturated, but not saturated, FA inhibited GnRH-induced LH secretion in LßT2 cells as well as in pituitary cell cultures. Altogether, these results suggest that the pituitary is a relevant site of FA action and that UFA may influence reproduction by directly interfering with basal and GnRH-dependent gonadotrope activity.

Intestinal gluconeogenesis is a key factor for early metabolic changes after gastric bypass but not after gastric lap-band in mice.

Unlike the adjustable gastric banding procedure (AGB), Roux-en-Y gastric bypass surgery (RYGBP) in humans has an intriguing effect: a rapid and substantial control of type 2 diabetes mellitus (T2DM). We performed gastric lap-band (GLB) and entero-gastro anastomosis (EGA) procedures in C57Bl6 mice that were fed a high-fat diet. The EGA procedure specifically reduced food intake and increased insulin sensitivity as measured by endogenous glucose production. Intestinal gluconeogenesis increased after the EGA procedure, but not after gastric banding. All EGA effects were abolished in GLUT-2 knockout mice and in mice with portal vein denervation. We thus provide mechanistic evidence that the beneficial effects of the EGA procedure on food intake and glucose homeostasis involve intestinal gluconeogenesis and its detection via a GLUT-2 and hepatoportal sensor pathway.