LRP1 regulates food intake and energy balance in GABAergic neurons independently of leptin action.

Low-density lipoprotein receptor-related protein 1 (LRP1) is a member of LDL receptor family that plays a key role in systemic glucose and lipid homeostasis. LRP1 also regulates energy balance in the hypothalamus by mediating leptin's anorexigenic action, although the underlying neurocircuitry involved is still unclear. Because GABAergic neurons are a major mediator of hypothalamic leptin action, we studied the role of GABAergic LRP1 in energy balance and leptin action using mice lacking LRP1 in Vgat- or AgRP-expressing neurons (Vgat-Cre; LRP1loxP/loxP or AgRP-Cre; LRP1loxP/loxP). Here, we show that LRP1 deficiency in GABAergic neurons results in severe obesity in male and female mice fed a normal-chow diet. This effect is most likely due to increased food intake and decreased energy expenditure and locomotor activity. Increased adiposity in GABAergic neuron-specific LRP1-deficient mice is accompanied by hyperleptinemia and hyperinsulinemia. Insulin resistance and glucose intolerance in these mice are occurred without change in body weight. Importantly, LRP1 in GABAergic neurons is not required for leptin action, as evidenced by normal leptin's anorexigenic action and leptin-induced hypothalamic Stat3 phosphorylation. In contrast, LRP1 deficiency in AgRP neurons has no effect on adiposity and caloric intake. In conclusion, our data identify GABAergic neurons as a key neurocircuitry that underpins LRP1-dependent regulation of systemic energy balance and body-weight homeostasis. We further find that the GABAergic LRP1 signaling pathway modulates food intake and energy expenditure independently of leptin signaling and AgRP neurons.

The effect of swimming training on adrenomedullin levels, oxidative stress variables, and gastrocnemius muscle contractile properties in hypertensive rats.

Introduction/Aim: Regular exercise may have beneficial effects on high blood-pressure, as shown in different types of experimental hypertension models in rats. The present study aims to investigate the effects of 6-week swimming training on blood pressure, oxidative stress variables of selected tissues, serum adrenomedullin (ADM) levels, and in situ muscle contraction in rats with hypertension induced by Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME), an inhibitor of endothelial nitric oxide synthases (eNOs). Materials and Methods: Twenty-six male Sprague Dawley, 8 weeks of age, rats were randomly divided into four groups: (I) normotensive (C), (II) normotensive + exercise (E), (III) hypertensive (L), and (IV) hypertensive + exercise (LE). Hypertension was induced by the oral administration of L-NAME (60 mg/kg) for 6 weeks. Exercise was performed 5 times (1-h each) per week for 6 weeks. At the end of the experiment, blood and tissue samples (the gastrocnemius muscle, heart, kidney, and thoracic aorta) were collected following contractile properties of the gastrocnemius muscle in situ weredetermined. In the collected tissues, oxidative stress (e.g., lipid oxidation and antioxidant enzyme activity) and serum ADM levels were measured. 6-week L-NAME administration per se (Group L) led to a significant increase in systolic and diastolic blood pressure compared to other groups.  Results: Importantly, 6-week exercise caused a protective effect of high blood pressure in the rats received L-NAME (Group LE). The level of ADM was lower in the rats received L-NAME than that of the control group. L-NAME increased lipid peroxidation in the thoracic aorta and decreased superoxide dismutase in the heart, kidney and muscle, and decreased catalase and glutathione in the heart. However, the exercise intervention did not have protective effect on the L-NAME-mediated oxidative damage in the collected tissues.  Conclusion: In conclusion, 6-week exercise intervention rescued rats from high blood pressure, but did not have ameliorative effect on the decreased ADM levels.

Effect of dietary probiotic and high stocking density on the performance, carcass yield, gut microflora, and stress indicators of broilers.

A study was carried out to evaluate the effect of dietary probiotic supplementation and stocking density on the performance, relative carcass yield, gut microflora, and stress markers of broilers. One-day-old Ross 308 male broiler chickens (n = 480) were allocated to 4 experimental groups for 42 d. Each treatment had 8 replicates of 15 chicks each. Two groups were subjected to a high stocking density (HSD) of 20 birds/m² and the other 2 groups were kept at low stocking density (LSD) of 10 birds/m². A basal diet supplemented with probiotic 1 and 0.5 g/kg of diet (in starter and finisher diets, respectively) was fed to 2 treatments, one with HSD and the other with LSD, thereby making a 2 × 2 factorial arrangement. There was no interaction between stocking density (LSD and HSD) and dietary probiotic (supplemented and unsupplemented) for all the variables. Feed intake and weight gain were significantly low and feed conversion ratio was poor in broilers at HSD. Dietary probiotic significantly enhanced the feed intake and weight gain in starter phase only. Dietary probiotic supplementation had no effect (P > 0.05) on total aerobs, Salmonella sp., and Lactobacilli populations in the intestines of broilers. However, HSD reduced the Lactobacilli population only (P < 0.05). Relative breast yields were significantly higher in broilers reared at LSD than HSD. Thigh meat yield was higher in broilers in HSD group compared to LSD. Dietary probiotic did not affect the relative carcass yield and weight of lymphoid organs. Serum malondialdehyde, corticosterone, nitric oxide, and plasma heterophil:lymphocyte ratio were not affected either by stocking density or dietary probiotic supplementation. In conclusion, HSD negatively affected the performance and intestinal Lactobacilli population of broilers only, whereas probiotic supplementation enhanced the performance of broilers during the starter phase only. Total aerobes, Salmonella, Lactobacilli carcass yield, and stress indicators of broilers were not affected by the dietary supplementation of probiotic under the conditions of the present study.

Effects of pulsed electromagnetic field and swimming exercise on rats with experimental sciatic nerve injury.

[Purpose] The current study aimed to reveal the therapeutic effects of a pulsed electromagnetic field and swimming exercises on rats with experimental sciatic nerve injury, which was induced with crush-type neuropathy model damage, using electrophysiological methods. [Subjects] In the current study, the sample consisted of 28 adult male Wistar albino rats. [Methods] The rats were randomized into four groups (n=7). Swimming exercise and PEMF (2 Hz and 0.3 MT) were applied one hour a day, five days a week, for four weeks. Electroneuromyographic (ENMG) measurements were taken on day 7. [Results] When the data were evaluated, it was found that the 4 weeks of PEMF and swimming exercises led to an increase in motor conduction rates and a decrease in latency values, but the changes were not significant in comparison with the control and injury groups. The compound muscle action potential (CMAP) values of the left leg were lower in weeks 2, 3, and 4 in the swimming exercise group in comparison with the control group, although for the PEMF group, the CMAP values of the left leg reached the level observed in the control group beginning in week 3. [Conclusion] PEMF and swimming exercise made positive contributions to nerve regeneration after week 1, and regeneration was enhanced.

AgRP neuron cis-regulatory analysis across hunger states reveals that IRF3 mediates leptin's acute effects.

AgRP neurons in the arcuate nucleus of the hypothalamus (ARC) coordinate homeostatic changes in appetite associated with fluctuations in food availability and leptin signaling. Identifying the relevant transcriptional regulatory pathways in these neurons has been a priority, yet such attempts have been stymied due to their low abundance and the rich cellular diversity of the ARC. Here we generated AgRP neuron-specific transcriptomic and chromatin accessibility profiles from male mice during three distinct hunger states of satiety, fasting-induced hunger, and leptin-induced hunger suppression. Cis-regulatory analysis of these integrated datasets enabled the identification of 18 putative hunger-promoting and 29 putative hunger-suppressing transcriptional regulators in AgRP neurons, 16 of which were predicted to be transcriptional effectors of leptin. Within our dataset, Interferon regulatory factor 3 (IRF3) emerged as a leading candidate mediator of leptin-induced hunger-suppression. Measures of IRF3 activation in vitro and in vivo reveal an increase in IRF3 nuclear occupancy following leptin administration. Finally, gain- and loss-of-function experiments in vivo confirm the role of IRF3 in mediating the acute satiety-evoking effects of leptin in AgRP neurons. Thus, our findings identify IRF3 as a key mediator of the acute hunger-suppressing effects of leptin in AgRP neurons.

Author Correction: Apolipoprotein J is a hepatokine regulating muscle glucose metabolism and insulin sensitivity.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Apolipoprotein J is a hepatokine regulating muscle glucose metabolism and insulin sensitivity.

Crosstalk between liver and skeletal muscle is vital for glucose homeostasis. Hepatokines, liver-derived proteins that play an important role in regulating muscle metabolism, are important to this communication. Here we identify apolipoprotein J (ApoJ) as a novel hepatokine targeting muscle glucose metabolism and insulin sensitivity through a low-density lipoprotein receptor-related protein-2 (LRP2)-dependent mechanism, coupled with the insulin receptor (IR) signaling cascade. In muscle, LRP2 is necessary for insulin-dependent IR internalization, an initial trigger for insulin signaling, that is crucial in regulating downstream signaling and glucose uptake. Of physiologic significance, deletion of hepatic ApoJ or muscle LRP2 causes insulin resistance and glucose intolerance. In patients with polycystic ovary syndrome and insulin resistance, pioglitazone-induced improvement of insulin action is associated with an increase in muscle ApoJ and LRP2 expression. Thus, the ApoJ-LRP2 axis is a novel endocrine circuit that is central to the maintenance of normal glucose homeostasis and insulin sensitivity.

Role of POMC and AgRP neuronal activities on glycaemia in mice.

Leptin regulates both feeding and glycaemia primarily through its receptors expressed on agouti-related peptide (AgRP) and pro-opiomelanocortin-expressing (POMC) neurons; however, it is unknown whether activity of these neuronal populations mediates the regulation of these processes. To determine this, we injected Cre-dependent designer receptors exclusively activated by designer drugs (DREADD) viruses into the hypothalamus of normoglycaemic and diabetic AgRP-ires-cre and POMC-cre mice to chemogenetically activate or inhibit these neuronal populations. Despite robust changes in food intake, activation or inhibition of AgRP neurons did not affect glycaemia, while activation caused significant (P = 0.014) impairment in insulin sensitivity. Stimulation of AgRP neurons in diabetic mice reversed leptin's ability to inhibit feeding but did not counter leptin's ability to lower blood glucose levels. Notably, the inhibition of POMC neurons stimulated feeding while decreasing glucose levels in normoglycaemic mice. The findings suggest that leptin's effects on feeding by AgRP neurons are mediated by changes in neuronal firing, while the control of glucose balance by these cells is independent of chemogenetic activation or inhibition. The firing-dependent glucose lowering mechanism within POMC neurons is a potential target for the development of novel anti-diabetic medicines.

Mice with diet-induced obesity demonstrate a relative prothrombotic factor profile and a thicker aorta with reduced ex-vivo function.

: Classical risk factors such as cholesterol and lipoproteins are currently not sufficient to explain all physiopathological processes of obesity-related vascular dysfunction as well as atherosclerosis and arteriosclerosis. Therefore, the discovery of potential markers involved in vascular dysfunction in the obese state is still needed. Disturbances in hemostatic factors may be involved in the developmental processes associated with obesity-related cardiovascular disorders. We hypothesized that alterations of several hemostatic factors in the obese state could correlate with the function and morphology of the aorta and it could play an important role in the development of vascular dysfunction. To test this, we fed mice with a high-fat diet for 18 weeks and investigated the relationships between selected hemostatic factors (in either plasma or in the liver), metabolic hormones and morphology, and ex-vivo function of the aorta. Here, we show that 18-week exposure to a high-fat diet results in a higher plasma fibrinogen and prolonged prothrombin time in diet-induced obese mice compared to the controls. In addition, liver levels or activities of FII, FX, activated protein C, AT-III, and protein S are significantly different in diet-induced obese mice as compared to the controls. Curiously, FII, FVIII, FX, activated protein C, PTT, and protein S are correlated with both the aorta histology (aortic thickness and diameter) and ex-vivo aortic function. Notably, ex-vivo studies revealed that diet-induced obese mice show a marked attenuation in the functions of the aorta. Taken together, aforementioned hemostatic factors may be considered as critical markers for obesity-related vascular dysfunction and they could play important roles in diagnosing of the dysfunction.

The role of GluN2A and GluN2B NMDA receptor subunits in AgRP and POMC neurons on body weight and glucose homeostasis.

OBJECTIVE: Hypothalamic agouti-related peptide (AgRP) and pro-opiomelanocortin (POMC) expressing neurons play critical roles in control of energy balance. Glutamatergic input via n-methyl-d-aspartate receptors (NMDARs) is pivotal for regulation of neuronal activity and is required in AgRP neurons for normal body weight homeostasis. NMDARs typically consist of the obligatory GluN1 subunit and different GluN2 subunits, the latter exerting crucial differential effects on channel activity and neuronal function. Currently, the role of specific GluN2 subunits in AgRP and POMC neurons on whole body energy and glucose balance is unknown. METHODS: We used the cre-lox system to genetically delete GluN2A or GluN2B only from AgRP or POMC neurons in mice. Mice were then subjected to metabolic analyses and assessment of AgRP and POMC neuronal function through morphological studies. RESULTS: We show that loss of GluN2B from AgRP neurons reduces body weight, fat mass, and food intake, whereas GluN2B in POMC neurons is not required for normal energy balance control. GluN2A subunits in either AgRP or POMC neurons are not required for regulation of body weight. Deletion of GluN2B reduces the number of AgRP neurons and decreases their dendritic length. In addition, loss of GluN2B in AgRP neurons of the morbidly obese and severely diabetic leptin-deficient Lep (ob/ob) mice does not affect body weight and food intake but, remarkably, leads to full correction of hyperglycemia. Lep (ob/ob) mice lacking GluN2B in AgRP neurons are also more sensitive to leptin's anti-obesity actions. CONCLUSIONS: GluN2B-containing NMDA receptors in AgRP neurons play a critical role in central control of body weight homeostasis and blood glucose balance via mechanisms that likely involve regulation of AgRP neuronal survival and structure, and modulation of hypothalamic leptin action.

In vivo effects of leptin on lymphocyte subpopulations in mice.

Leptin, a hormone-cytokine mainly produced by the adipose tissue, has pleitropic effects on many biological system including metabolic, endocrine, and immune system. Although it is well known that leptin controls food intake on hypothalamic regions of brain, the role of leptin in hematopoietic and immune processes has been mainly investigated with in vitro and transgenic mouse studies. The aim of this study was to investigate the effects of peripheral leptin on lymphocyte subpopulation. Initially forty male Swiss albino mice were divided into five groups. Mice in group I (Control) were given serum physiologic (SP) and group L100, group L250, group L500, and group L1000 were given 100, 250, 500 and 1000 µg/kg/day recombinant mouse leptin, respectively. Leptin or SP was injected subcutaneously for the next 6 days. Daily food/water intake was recorded for each group. At the end of the study, whole blood samples (500 µl) were obtained via intracardiac punction in anesthetized mice. Leptin levels and lymphocyte subpopulations in blood samples were analyzed. We show that no in vivo dose-dependent effect of leptin is existed on lymphocyte subpopulations count in mice. Treatment of mice with high-dose leptin led to increase only CD4+ cells (P<0.05). In addition, high-dose leptin slightly increased CD3+ cells but this was not statistically confirmed (P=0.08). Notably, it was found that leptin caused insignificant changes on body weight and food intake in normal body weight mice. The data support that high-dose leptin has proliferative effect on CD4+ cells in vivo. However, more in vivo study needs to be examined to clarify how leptin affect lymphocyte subpopulations.