Conditional Deletion of ß-Catenin in the Mediobasal Hypothalamus Impairs Adaptive Energy Expenditure in Response to High-Fat Diet and Exacerbates Diet-Induced Obesity.

ß-Catenin is a bifunctional molecule that is an effector of the wingless-related integration site (Wnt) signaling to control gene expression and contributes to the regulation of cytoskeleton and neurotransmitter vesicle trafficking. In its former role, ß-catenin binds transcription factor 7-like 2 (TCF7L2), which shows strong genetic associations with the pathogenesis of obesity and type-2 diabetes. Here, we sought to determine whether ß-catenin plays a role in the neuroendocrine regulation of body weight and glucose homeostasis. Bilateral injections of adeno-associated virus type-2 (AAV2)-mCherry-Cre were placed into the arcuate nucleus of adult male and female ß-catenin flox mice, to specifically delete ß-catenin expression in the mediobasal hypothalamus (MBH-ß-cat KO). Metabolic parameters were then monitored under conditions of low-fat (LFD) and high-fat diet (HFD). On LFD, MBH-ß-cat KO mice showed minimal metabolic disturbances, but on HFD, despite having only a small difference in weekly caloric intake, the MBH-ß-cat KO mice were significantly heavier than the control mice in both sexes (p < 0.05). This deficit seemed to be due to a failure to show an adaptive increase in energy expenditure seen in controls, which served to offset the increased calories by HFD. Both male and female MBH-ß-cat KO mice were highly glucose intolerant when on HFD and displayed a significant reduction in both leptin and insulin sensitivity compared with controls. This study highlights a critical role for ß-catenin in the hypothalamic circuits regulating body weight and glucose homeostasis and reveals potential mechanisms by which genetic variation in this pathway could impact on development of metabolic disease.

Dietary wheat gluten induces astro- and microgliosis in the hypothalamus of male mice.

Gluten, which is found in cereals such as wheat, rye and barley, makes up a major dietary component in most western nations, and has been shown to promote body mass gain and peripheral inflammation in mice. In the current study, we investigated the impact of gluten on central inflammation that is typically associated with diet-induced obesity. While we found no effect of gluten when added to a low-fat diet (LFD), male mice fed high fat diet (HFD) enriched with gluten increased body mass and adiposity compared with mice fed HFD without gluten. We furthermore found that gluten, when added to the LFD, increases circulating C-reactive protein levels. Gluten regardless of whether it was added to LFD or HFD led to a profound increase in the number of microglia and astrocytes in the arcuate nucleus of the hypothalamus, as detected by immunohistochemistry for ionised calcium binding adaptor molecule 1 (Iba-1) and glial fibrillary acidic protein (GFAP), respectively. In mice fed LFD, gluten mimicked the immunogenic effects of HFD exposure and when added to HFD led to a further increase in the number of immunoreactive cells. Taken together, our results confirm a moderate obesogenic effect of gluten when fed to mice exposed to HFD and for the first-time report gluten-induced astro- and microgliosis suggesting the development of hypothalamic injury in rodents.

Leptin regulates glucose homeostasis via the canonical Wnt pathway in the zebrafish.

Leptin is best known for its role in adipostasis, but it also regulates blood glucose levels. The molecular mechanism by which leptin controls glucose homeostasis remains largely unknown. Here, we use a zebrafish model to show that Wnt signaling mediates the glucoregulatory effects of leptin. Under normal feeding conditions, leptin regulates glucose homeostasis but not adipostasis in zebrafish. In times of nutrient excess, however, we found that leptin also regulates body weight and size. Using a Wnt signaling reporter fish, we show that leptin activates the canonical Wnt pathway in vivo. Utilizing two paradigms for hyperglycemia, it is revealed that leptin regulates glucose homeostasis via the Wnt pathway, as pharmacological inhibition of this pathway impairs the glucoregulatory actions of leptin. Our results may shed new light on the evolution of the physiological function of leptin.

A New Zealand green-lipped mussel oil-enriched high-fat diet exhibits beneficial effects on body weight and metabolism in mice.

To induce diet-induced obesity (DIO) in rodents, diets high in saturated fat and/or carbohydrates are commonly used. In the laboratory, standardised diets evolved over time without paying particular attention to the effect of fat composition on metabolic alterations. In the present study, customised high-fat diets (HFD) enriched with a combination of lard and different concentrations of New Zealand green-lipped mussel (Perna canaliculus) oil or MSC Hoki (Macruronus novaezelandiae, blue grenadier) liver oil, important sources of n-3 PUFA, in comparison with a solely lard-based diet, were fed to lean and DIO male C57BL/6 mice and their effects on metabolic parameters were monitored. Intriguingly, an isoenergetic HFD containing 63 % of total fat in the form of mussel oil and only 28 % in the form of lard attenuated HFD-induced body weight gain after 1 and 4 weeks, respectively. Consistently, changing a lard-enriched HFD to the mussel oil diet reduced body weight markedly even after mice had been exposed to the former diet for 10 months. The weight-reducing effect of the diet was not caused by altered energy intake or expenditure, but was associated with reduced visceral fat mass. Collectively, these data suggest a novel weight-reducing potential of green-lipped mussel oil.

Hyperleptinemia as a contributing factor for the impairment of glucose intolerance in obesity.

Obesity has emerged as a major risk factor for insulin resistance leading to the development of type 2 diabetes (T2D). The condition is characterized by high circulating levels of the adipose-derived hormone leptin and a state of chronic low-grade inflammation. Pro-inflammatory signaling in the hypothalamus is associated with a decrease of central leptin- and insulin action leading to impaired systemic glucose tolerance. Intriguingly, leptin not only regulates body weight and glucose homeostasis but also acts as a pro-inflammatory cytokine. Here we demonstrate that increasing leptin levels (62,5 µg/kg/d, PEGylated leptin) in mice fed a high-fat diet (HFD) exacerbated body weight gain and aggravated hypothalamic micro- as well as astrogliosis. In contrast, administration of a predetermined dose of a long-acting leptin antagonist (100 µg/kg/d, PESLAN) chosen to block excessive leptin signaling during diet-induced obesity (DIO) showed the opposite effect and significantly improved glucose tolerance as well as decreased the total number of microglia and astrocytes in the hypothalamus of mice fed HFD. These results suggest that high levels of leptin, such as in obesity, worsen HFD-induced micro-and astrogliosis, whereas the partial reduction of hyperleptinemia in DIO mice may have beneficial metabolic effects and improves hypothalamic gliosis.

RFamide-Related Peptide Neurons Modulate Reproductive Function and Stress Responses.

RF-amide related peptide 3 (RFRP-3) is a neuropeptide thought to inhibit central regulation of fertility. We investigated whether alterations in RFRP neuronal activity led to changes in puberty onset, fertility, and stress responses, including stress and glucocorticoid-induced suppression of pulsatile luteinizing hormone secretion. We first validated a novel RFRP-Cre mouse line, which we then used in combination with Cre-dependent neuronal ablation and DREADD technology to selectively ablate, stimulate, and inhibit RFRP neurons to interrogate their physiological roles in the regulation of fertility and stress responses. Chronic RFRP neuronal activation delayed male puberty onset and female reproductive cycle progression, but RFRP-activated and ablated mice exhibited apparently normal fertility. When subjected to either restraint- or glucocorticoid-induced stress paradigms. However, we observed a critical sex-specific role for RFRP neurons in mediating acute and chronic stress-induced reproductive suppression. Female mice exhibiting RFRP neuron ablation or silencing did not exhibit the stress-induced suppression in pulsatile luteinizing hormone secretion observed in control mice. Furthermore, RFRP neuronal activation markedly stimulated glucocorticoid secretion, demonstrating a feedback loop whereby stressful stimuli activate RFRP neurons, which in turn further activate the stress axis. These data provide evidence for a neuronal link between the stress and reproductive axes.

Hypothalamic leptin sensitivity and health benefits of time-restricted feeding are dependent on the time of day in male mice.

Synchronization between biologic clocks and metabolism is crucial for most species. Here, we examined the ability of leptin, important in the control of energy metabolism, to induce leptin signaling at the molecular as well as the behavioral level throughout the 24-h day in mice fed either a control or a high-fat diet (HFD). Furthermore, we investigated the effects of time-restricted feeding (TRF; a limitation of HFD access to 6 h each day) on energy metabolism during different periods throughout the 24-h day. In control mice, molecular leptin sensitivity was highest at zeitgeber time (ZT)0 (lights on), declining during the light phase, and increasing during the dark phase. Surprisingly, leptin resistance in HFD-fed mice was only present from the middle of the dark to the middle of the light period. Specifically, when TRF occurred from ZT21 to ZT3 (when leptin resistance in HFD-fed mice was most profound), it resulted in a disruption of the daily rhythms of locomotor activity and energy expenditure and in increased plasma insulin levels compared with other TRF periods. These data provide evidence that leptin sensitivity is controlled by the circadian rhythm and that TRF periods may be most efficient when aligned with the leptin-sensitive period.-Boucsein, A., Rizwan, M. Z., Tups, A. Hypothalamic leptin sensitivity and health benefits of time-restricted feeding are dependent on the time of day in male mice.

Chronic Light Cycle Disruption Alters Central Insulin and Leptin Signaling as well as Metabolic Markers in Male Mice.

Recent evidence suggests that the circadian timing system plays a role in energy and glucose homeostasis, and disruptions to this system are a risk factor for the development of metabolic disorders. We exposed animals to a constantly shifting lighting environment comprised of a 6-hour advance, occurring every 6 days, to chronically disrupt their circadian timing system. This treatment caused a gradual increase in body weight of 12 ± 2% after 12 phase shifts, compared with a 6 ± 1% increase in mice under control lighting conditions. Additionally, after the fifth phase shift, light cycle-disrupted (CD) animals showed a reversal in their diurnal pattern of energy homeostasis and locomotor activity, followed by a subsequent loss of this rhythm. To investigate potential molecular mechanisms mediating these metabolic alterations, we assessed central leptin and insulin sensitivity. We discovered that CD mice had a decrease in central leptin signaling, as indicated by a reduction in the number of phosphorylated signal transducer and activator of transcription 3 immunoreactive cells in the arcuate nucleus of the hypothalamus. Furthermore, CD animals exhibited a marked increase in fasting blood glucose (269.4 ± 21.1 mg/dL) compared with controls (108.8 ± 21.3 mg/dL). This dramatic increase in fasting glucose levels was not associated with an increase in insulin levels, suggesting impairments in pancreatic insulin release. Peripheral hyperglycemia was accompanied by central alterations in insulin signaling at the level of phospho Akt and insulin receptor substrate 1, suggesting that light cycle disruption alters central insulin signaling. These results provide mechanistic insights into the association between light cycle disruption and metabolic disease.

"Insulin-like" effects of palmitate compromise insulin signalling in hypothalamic neurons.

Saturated fatty acids are implicated in the development of metabolic diseases, including obesity and type 2 diabetes. There is evidence, however, that polyunsaturated fatty acids can counteract the pathogenic effects of saturated fatty acids. To gain insight into the early molecular mechanisms by which fatty acids influence hypothalamic inflammation and insulin signalling, we performed time-course experiments in a hypothalamic cell line, using different durations of treatment with the saturated fatty acid palmitate, and the omega-3 polyunsaturated fatty acid, docosahexaenoic acid (DHA). Western blot analysis revealed that palmitate elevated the protein levels of phospho(p)AKT in a time-dependent manner. This effect is involved in the pathogenicity of palmitate, as temporary inhibition of the PI3K/AKT pathway by selective PI3K inhibitors prevented the palmitate-induced attenuation of insulin signalling. Similar to palmitate, DHA also increased levels of pAKT, but to a weaker extent. Co-administration of DHA with palmitate decreased pAKT close to the basal level after 8 h, and prevented the palmitate-induced reduction of insulin signalling after 12 h. The monounsaturated fatty acid oleate had a similar effect on the palmitate-induced attenuation of insulin signalling, the polyunsaturated fatty acid linoleate had no effect. Measurement of the inflammatory markers pJNK and pNFκB-p65 revealed tonic elevation of both markers in the presence of palmitate alone. DHA alone transiently induced elevation of pJNK, returning to basal levels by 12 h treatment. Co-administration of DHA with palmitate prevented palmitate-induced inflammation after 12 h, but not at earlier timepoints.

Leptin Signaling Is Not Required for Anorexigenic Estradiol Effects in Female Mice.

Estradiol and leptin are critical hormones in the regulation of body weight. The aim of this study was to determine whether this cross talk between leptin receptor (LepRb) and estrogen receptor-α (ERα) signaling is critical for estradiol's anorexigenic effects. Leprb-Cre mice were crossed with Cre-dependent Tau-green fluorescent protein (GFP) reporter, Stat3-flox or Erα-flox mice to generate female mice with GFP expression, signal transducer and activator of transcription 3 (STAT3) knockout (KO), or ERα KO, specifically in LepRb-expressing cells. The proportion of Leprb-GFP cells colocalizing ERα was high (∼80%) in the preoptic area but low (∼10%) in the mediobasal hypothalamus, suggesting that intracellular cross talk between these receptors is minimal for metabolic regulation. To test whether estradiol enhanced arcuate leptin sensitivity, ovarectomized mice received varying levels of estradiol replacement. Increasing estrogenic states did not increase the degree of leptin-induced STAT3 phosphorylation. LepRb-specific STAT3 KO mice and controls were ovarectomized and given either chronic estradiol or vehicle treatment to test whether STAT3 is required for estrogen-induced body weight suppression. Both groups of estradiol-treated mice showed an equivalent reduction in body weight and fat content compared with vehicle controls. Finally, mice lacking ERα specifically in LepRb-expressing neurons also showed no increase in body weight or impairments in metabolic function compared with controls, indicating that estradiol acts independently of leptin-responsive cells to regulate body weight. However, fecundity was impaired in in Leprb-ERα KO females. Contrary to the current dogma, we report that estradiol has minimal direct actions on LepRb cells in the mediodasal hypothalamus and that its anorexigenic effects can occur entirely independently of LepRb-STAT3 signaling in female mice.

Insulin action on GABA neurons is a critical regulator of energy balance but not fertility in mice.

Insulin signaling in the brain plays an important role in the central regulation of energy homeostasis and fertility, such that mice exhibiting brain-specific deletion of insulin receptors (InsRs) display a diet-sensitive obesogenic phenotype and hypothalamic hypogonadism. However, the specific neurons mediating insulin's central effects on fertility remain largely unidentified. The neurotransmitters γ-aminobutyric acid (GABA) and glutamate are important modulators of fertility and energy homeostasis and are widely distributed in the hypothalamus. We therefore investigated whether insulin signaling via GABAergic or glutamatergic neurons plays an important role in the metabolic regulation of fertility. We used the Cre-loxP system to generate mice with a selective inactivation of the Insr gene from GABAergic (Vgat(+)) or glutamatergic (Vglut2(+)) cells by crossing Insr-flox mice with Vgat-Cre or Vglut2-Cre mice, respectively. Multiple reproductive and metabolic parameters were then compared between male and female Insr-flox/Vgat-Cre(+) (VgatIRKO), Insr-flox/Vglut2-Cre(+) (VglutIRKO), and Insr-flox/Cre-negative control (CON) mice. Female VgatIRKO mice exhibited a significant increase in adult body weight, abdominal fat mass, and fasting plasma insulin and leptin concentrations, but normal fasting glucose concentration and glucose tolerance compared with CON mice. Surprisingly, VgatIRKO and VglutIRKO mice exhibited normal reproductive maturation and function compared with CONs. No differences in the age of puberty onset, estrous cyclicity, or fertility were observed between VgatIRKO, VglutIRKO, and CON mice. However, male VgatIRKO mice exhibited significantly augmented LH concentration and a trend toward reduced seminal vesicle weight compared with CON mice, which may be indicative of primary hypogonadism. Our results therefore demonstrate that insulin signaling via GABAergic and glutamatergic cells is not required for fertility in mice, but show that GABAergic neurons encompass circuitry through which insulin acts to modulate energy homeostasis.

RFamide-related peptide-3 receptor gene expression in GnRH and kisspeptin neurons and GnRH-dependent mechanism of action.

RFamide-related peptide-3 (RFRP-3) is known to inhibit the activity of GnRH neurons. It is not yet clear whether its G protein-coupled receptors, GPR147 and GPR74, are present on GnRH neurons or on afferent inputs of the GnRH neuronal network or whether RFRP-3 can inhibit gonadotropin secretion independently of GnRH. We tested the following: 1) whether GnRH is essential for the effects of RFRP-3 on LH secretion; 2) whether RFRP-3 neurons project to GnRH and rostral periventricular kisspeptin neurons in mice, and 3) whether Gpr147 and Gpr74 are expressed by these neurons. Intravenous treatment with the GPR147 antagonist RF9 increased plasma LH concentration in castrated male rats but was unable to do so in the presence of the GnRH antagonist cetrorelix. Dual-label immunohistochemistry revealed that approximately 26% of GnRH neurons from male and diestrous female mice were apposed by RFRP-3 fibers, and 19% of kisspeptin neurons from proestrous female mice were apposed by RFRP-3 fibers. Using immunomagnetic purification of GnRH and kisspeptin cells, single-cell nested RT-PCR, and in situ hybridization, we showed that 33% of GnRH neurons and 9-16% of rostral periventricular kisspeptin neurons expressed Gpr147, whereas Gpr74 was not expressed in either population. These data reveal that RFRP-3 can act at two levels of the GnRH neuronal network (i.e. the GnRH neurons and the rostral periventricular kisspeptin neurons) to modulate reproduction but is unable to inhibit gonadotropin secretion independently of GnRH.

Cells expressing RFamide-related peptide-1/3, the mammalian gonadotropin-inhibitory hormone orthologs, are not hypophysiotropic neuroendocrine neurons in the rat.

An RFamide peptide named gonadotropin-inhibitory hormone, which directly inhibits gonadotropin synthesis and secretion from the anterior pituitary gland, has recently been discovered in the avian hypothalamus. It is not known whether the mammalian orthologs of gonadotropin-inhibitory hormone and RFamide-related peptide (RFRP)-1 and -3 act in the same way. We used a newly generated antibody against the rat RFRP precursor combined with retrograde tract tracing to characterize the cell body distribution and fiber projections of RFRP-1 and -3 neurons in rats. RFRP-1/3-immunoreactive cell bodies were found exclusively within the dorsomedial hypothalamus. Immunoreactive fibers were observed in the septal-preoptic area, hypothalamus, midbrain, brainstem, and hippocampus but not in the external zone of the median eminence. Intraperitoneal injection of the retrograde tracer Fluoro-Gold in rats resulted in the labeling of the majority of GnRH neurons but essentially no RFRP-1/3 neurons. In contrast, intracerebral injections of Fluoro-Gold into the rostral preoptic area and CA2/CA3 hippocampus resulted in the labeling of 75 +/- 5% and 21 +/- 8% of RFRP-1/3 cell bodies, respectively. To assess actions at the pituitary in vivo, RFRP-3 was administered as an iv bolus to ovariectomized rats and plasma LH concentration measured at 0, 2.5, 5, 10, and 30 min. RFRP-3 had no effects on basal secretion, but GnRH-stimulated LH release was reduced by about 25% at 5 min. Together these observations suggest that RFRP-3 is not a hypophysiotropic neuroendocrine hormone in rats.

Central and peripheral effects of RFamide-related peptide-3 on luteinizing hormone and prolactin secretion in rats.

Hypothalamic RFamide-related peptide-3 (RFRP-3) neurons inhibit LH secretion via a central action. A direct hypophysiotropic action on the gonadotropes has also been suggested. To assess central RFRP-3 effects on the GnRH/LH surge that induces ovulation, ovariectomized rats were subjected to an estradiol plus progesterone surge-induction protocol. Chronic infusion of RFRP-3 (2.5 or 25 ng/h, intracerebroventricularly) caused a dose-dependent 50-60% inhibition of GnRH neuronal activation (assessed by colocalization with the immediate early gene c-Fos) at the surge peak compared with vehicle-treated controls. RFRP-3 also suppressed neuronal activation in the anteroventral periventricular region, which provides stimulatory input to GnRH neurons, by 50-80% compared with control values. To test whether centrally administered RFRP-3 inhibits pulsatile GnRH/LH secretion, chronically ovariectomized, low-level estradiol-treated rats without surge induction were blood sampled every 10 min for 4 h. Bolus injection of RFRP-3 (0, 2.5, or 25 microg, intracerebroventricularly) after 1.5 h did not affect subsequent LH pulse frequency, pulse amplitude, or the mean concentrations of LH or prolactin. RFRP-3 treatment of isolated anterior pituitary cells at moderate doses of up to 10(-7) m did not significantly inhibit LH release, either with or without GnRH cotreatment. These data reveal a central inhibitory effect of RFRP-3 on the hypothalamo-pituitary gonadal axis specifically during the estradiol-induced GnRH/LH surge. This effect may include actions of RFRP-3 on GnRH neurons and/or their anteroventral periventricular afferent inputs but is unlikely to involve direct inhibition of LH secretion at the level of the gonadotrope.