Ghrelin-AMPK Signaling Mediates the Neuroprotective Effects of Calorie Restriction in Parkinson's Disease

Jacqeline Bayliss, Romana Stark, Moyra Lemus, Vanessa Santos, Aiysha Thompson, Daniel Rees, Sandra Galic, John Elsworth, Bruce Kemp, Jeffrey Davies, and Zane Andrew

Metformin Prevents Nigrostriatal Dopamine Degeneration Independent of AMPK Activation in Dopamine Neurons

Metformin is a widely prescribed drug used to treat type-2 diabetes, although recent studies show it has wide ranging effects to treat other diseases. Animal and retrospective human studies indicate that Metformin treatment is neuroprotective in Parkinson’s Disease (PD), although the neuroprotective mechanism is unknown, numerous studies suggest the beneficial effects on glucose homeostasis may be through AMPK activation. In this study we tested whether or not AMPK activation in dopamine neurons was required for the neuroprotective effects of Metformin in PD. We generated transgenic mice in which AMPK activity in dopamine neurons was ablated by removing AMPK beta 1 and beta 2 subunits from dopamine transporter expressing neurons. These AMPK WT and KO mice were then chronically exposed to Metformin in the drinking water then exposed to MPTP, the mouse model of PD. Chronic Metformin treatment significantly attenuated the MPTP-induced loss of Tyrosine Hydroxylase (TH) neuronal number and volume and TH protein concentration in the nigrostriatal pathway. Additionally, Metformin treatment prevented the MPTP-induced elevation of the DOPAC:DA ratio regardless of genotype. Metformin also prevented MPTP induced gliosis in the Substantia Nigra. These neuroprotective actions were independent of genotype and occurred in both AMPK WT and AMPK KO mice. Overall, our studies suggest that Metformin’s neuroprotective effects are not due to AMPK activation in dopaminergic neurons and that more research is required to determine how metformin acts to restrict the development of PD

Ghrelin-related peptides exert protective effects in the cerebral circulation of male mice through a nonclassical ghrelin receptor(s)

The ghrelin-related peptides, acylated ghrelin, des-acylated ghrelin, and obestatin, are novel gastrointestinal hormones. We firstly investigated whether the ghrelin gene, ghrelin O-acyltransferase, and the ghrelin receptor (GH secretagogue receptor 1a [GHSR1a]) are expressed in mouse cerebral arteries. Secondly, we assessed the cerebrovascular actions of ghrelin-related peptides by examining their effects on vasodilator nitric oxide (NO) and superoxide production. Using RT-PCR, we found the ghrelin gene and ghrelin O-acyltransferase to be expressed at negligible levels in cerebral arteries from male wild-type mice. mRNA expression of GHSR1a was also found to be low in cerebral arteries, and GHSR protein was undetectable in GHSR-enhanced green fluorescent protein mice. We next found that exogenous acylated ghrelin had no effect on the tone of perfused cerebral arteries or superoxide production. By contrast, exogenous des-acylated ghrelin or obestatin elicited powerful vasodilator responses (EC50 < 10 pmol/L) that were abolished by the NO synthase inhibitor Nω-nitro-L-arginine methyl ester. Furthermore, exogenous des-acylated ghrelin suppressed superoxide production in cerebral arteries. Consistent with our GHSR expression data, vasodilator effects of des-acylated ghrelin or obestatin were sustained in the presence of YIL-781 (GHSR1a antagonist) and in arteries from Ghsr-deficient mice. Using ghrelin-deficient (Ghrl−/−) mice, we also found that endogenous production of ghrelin-related peptides regulates NO bioactivity and superoxide levels in the cerebral circulation. Specifically, we show that NO bioactivity was markedly reduced in Ghrl−/− vs wild-type mice, and superoxide levels were elevated. These findings reveal protective actions of exogenous and endogenous ghrelin-related peptides in the cerebral circulation and show the existence of a novel ghrelin receptor(s) in the cerebral endothelium

Des-acyl ghrelin and ghrelin O-acyltransferase regulate hypothalamic-pituitary-adrenal axis activation and anxiety in response to acute stress

Ghrelin exists in two forms in circulation, acyl ghrelin and des-acyl ghrelin, both of which have distinct and fundamental roles in a variety of physiological functions. Despite this fact, a large proportion of papers simply measure and refer to plasma ghrelin without specifying the acylation status. It is therefore critical to assess and state the acylation status of plasma ghrelin in all studies. In this study we tested the effect of des-acyl ghrelin administration onthe hypothalamic-pituitaryadrenal axis and on anxiety-like behavior of mice lacking endogenous ghrelin and in ghrelin-O-acyltransferase (GOAT) knockout (KO) mice that have no endogenous acyl ghrelin and high endogenous des-acyl ghrelin. Our results show des-acyl ghrelin produces an anxiogenic effect under nonstressed conditions, but this switches to an anxiolytic effect under stress. Des-acyl ghrelin influences plasma corticosterone under both nonstressed and stressed conditions, although c-fos activation in the paraventricular nucleus of the hypothalamus is not different. By contrast, GOAT KO are anxious under both nonstressed and stressed conditions, although this is not due to corticosterone release from the adrenals but rather from impaired feedback actions in the paraventricular nucleus of the hypothalamus, as assessed by c-fos activation. These results reveal des-acyl ghrelin treatment and GOAT deletion have differential effects on the hypothalamic-pituitaryadrenal axis and anxiety-like behavior, suggesting that anxiety-like behavior in GOAT KO mice is not due to high plasma des-acyl ghrelin.Fil: Stark, Romana. Monash University; AustraliaFil: Santos, Vanessa V.. Monash University; AustraliaFil: Geenen, Bram. Radboud University Medical Center; Países BajosFil: Cabral, Agustina Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; ArgentinaFil: Dinan, Tara. RMIT University; AustraliaFil: Bayliss, Jacqueline A.. Monash University; AustraliaFil: Lockie, Sarah H.. Monash University; AustraliaFil: Reichenbach, Alex. Monash University; AustraliaFil: Lemus, Moyra B.. Monash University; AustraliaFil: Perello, Mario. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; ArgentinaFil: Spencer, Sarah J.. RMIT University; AustraliaFil: Kozicz, Tamas. Radboud University Medical Center; Países Bajos. University of Tulane; Estados UnidosFil: Andrews, Zane B.. Monash University; Australi

Metformin is neuroprotective in AMPK WT and KO mice.

<p>Plasma Triglycerides (<b>A & B</b>), NEFA (<b>C & D</b>) and corticosterone (<b>E & F</b>) are elevated in response to MPTP. Representative Western Blot images of pAMPK/AMPK in the SN (<b>G</b>) and Striatum (<b>H</b>). Protein analysis of the pAMPK/AMPK ratio showing no elevation in response to MPTP in the SN (<b>J</b>) and Striatum (<b>L</b>) in AMPK KO but an elevation in both the SN and Striatum of AMPK WT mice (<b>I & K</b>). Representative images of TH levels in the SN (<b>M</b>) and Striatum (<b>N</b>). In the SN there is a significant reduction in TH levels in both AMPK WT (<b>O</b>) and KO (<b>P</b>) in response to MPTP. In the Striatum Metformin elicits a neuroprotective effect in MPTP treated AMPK WT (<b>Q</b>) and KO (<b>R</b>) mice. MPTP reduced dopamine and DOPAC in both AMPK WT (<b>S & T</b>) and AMPK KO (<b>V & W</b>) mice. Metformin reduced the elevation of the DOPAC:DA ratio in MPTP treated mice compared to water alone, in AMPK WT and KO mice (<b>U & X</b>) a, significant compared to water/saline treated mice and b, significant compared to water/MPTP treated mice. Data are represented as mean ± SEM (n = 6–9, two-way ANOVA, p<0.05).</p

Metformin preserves cell number and volume after MPTP exposure, independent of genotype.

<p>Stereological quantification of TH levels in the SN shows a protective effect of Metformin after MPTP exposure in both AMPK WT (<b>A</b>) and KO (<b>B</b>) mice. Overall cell volume shows a significant reduction after MPTP exposure in water treatment but not with Metformin treatment in AMPK WT (<b>C</b>) and KO (<b>D</b>). When TH cells were separated and plotted based on volume distribution, mice treated with MPTP and Metformin had a significant effect on smaller volume (1000–2000μm³) cells compared to those not treated with Metformin in both AMPK WT (<b>E</b>) and KO (<b>F</b>) mice. <b>G</b>, Representative image showing MPTP induced microglial activation in the SN (green = TH, red = IBA1). Stereological quantification of IBA1 (<b>H & I</b>) and GFAP (<b>J & K</b>) showing increased numbers after MPTP with a significant protective effect of Metformin in both AMPK WT and KO. <b>L</b>, Representative images showing MPTP induced astrocytic activation in the SN (green = TH and red = GFAP). a, significant compared to Water/saline treated mice and b, significant compared to Water/MPTP treated mice. * = p<0.05. Data are represented as mean ± SEM (n = 7–10, two-way ANOVA, p<0.05).</p

Body Weight and blood glucose measurements in AMPK WT and KO mice.

<p><b>A–D</b>, throughout the experiment there was no difference in body weight or volume of water consumed comparing genotype or treatment. Arrows indicate MPTP injections. <b>E–H</b>, Plasma analysis of insulin and glucose levels in trunk blood show no differences between genotype and treatment. During an oGTT Metformin treatment does not alter glucose clearance in AMPK WT (<b>I & J</b>) or AMPK KO mice (<b>K & L)</b>. Insulin sensitivity is not altered during an ITT in AMPK WT (<b>M</b>) or KO (<b>N</b>) mice treated with Metformin. * = p<0.05. Data are represented as mean ± SEM (n = 8–10, two-way ANOVA, p<0.05).</p

Mechanisms underlying the efficacy of a rodent model of vertical sleeve gastrectomy — A focus on energy expenditure

Objective: Bariatric surgery remains the only effective and durable treatment option for morbid obesity. Vertical Sleeve Gastrectomy (VSG) is currently the most widely performed of these surgeries primarily because of its proven efficacy in generating rapid onset weight loss, improved glucose regulation and reduced mortality compared with other invasive procedures. VSG is associated with reduced appetite, however, the relative importance of energy expenditure to VSG-induced weight loss and changes in glucose regulation, particularly that in brown adipose tissue (BAT), remains unclear. The aim of this study was to investigate the role of BAT thermogenesis in the efficacy of VSG in a rodent model. Methods: Diet-induced obese male Sprague–Dawley rats were either sham-operated, underwent VSG surgery or were pair-fed to the food consumed by the VSG group. Rats were also implanted with biotelemetry devices between the interscapular lobes of BAT to assess local changes in BAT temperature as a surrogate measure of thermogenic activity. Metabolic parameters including food intake, body weight and changes in body composition were assessed. To further elucidate the contribution of energy expenditure via BAT thermogenesis to VSG-induced weight loss, a separate cohort of chow-fed rats underwent complete excision of the interscapular BAT (iBAT lipectomy) or chemical denervation using 6-hydroxydopamine (6-OHDA). To localize glucose uptake in specific tissues, an oral glucose tolerance test was combined with an intraperitoneal injection of 14C-2-deoxy-D-glucose (14C-2DG). Transneuronal viral tracing was used to identify 1) sensory neurons directed to the stomach or small intestine (H129-RFP) or 2) chains of polysynaptically linked neurons directed to BAT (PRV-GFP) in the same animals. Results: Following VSG, there was a rapid reduction in body weight that was associated with reduced food intake, elevated BAT temperature and improved glucose regulation. Rats that underwent VSG had elevated glucose uptake into BAT compared to sham operated animals as well as elevated gene markers related to increased BAT activity (Ucp1, Dio2, Cpt1b, Cox8b, Ppargc) and markers of increased browning of white fat (Ucp1, Dio2, Cited1, Tbx1, Tnfrs9). Both iBAT lipectomy and 6-OHDA treatment significantly attenuated the impact of VSG on changes in body weight and adiposity in chow-fed animals. In addition, surgical excision of iBAT following VSG significantly reversed VSG-mediated improvements in glucose tolerance, an effect that was independent of circulating insulin levels. Viral tracing studies highlighted a patent neural link between the gut and BAT that included groups of premotor BAT-directed neurons in the dorsal raphe and raphe pallidus. Conclusions: Collectively, these data support a role for BAT in mediating the metabolic sequelae following VSG surgery, particularly the improvement in glucose regulation, and highlight the need to better understand the contribution from this tissue in human patients.ISSN:2212-877

AgRP Neurons Require Carnitine Acetyltransferase to Regulate Metabolic Flexibility and Peripheral Nutrient Partitioning

Summary: AgRP neurons control peripheral substrate utilization and nutrient partitioning during conditions of energy deficit and nutrient replenishment, although the molecular mechanism is unknown. We examined whether carnitine acetyltransferase (Crat) in AgRP neurons affects peripheral nutrient partitioning. Crat deletion in AgRP neurons reduced food intake and feeding behavior and increased glycerol supply to the liver during fasting, as a gluconeogenic substrate, which was mediated by changes to sympathetic output and peripheral fatty acid metabolism in the liver. Crat deletion in AgRP neurons increased peripheral fatty acid substrate utilization and attenuated the switch to glucose utilization after refeeding, indicating altered nutrient partitioning. Proteomic analysis in AgRP neurons shows that Crat regulates protein acetylation and metabolic processing. Collectively, our studies highlight that AgRP neurons require Crat to provide the metabolic flexibility to optimize nutrient partitioning and regulate peripheral substrate utilization, particularly during fasting and refeeding