Leptin boosts cellular metabolism by activating AMPK and the sirtuins to reduce tau phosphorylation and β-amyloid in neurons

https://doi.org/10.1016/j.bbrc.2011.09.050Get rights and content

Abstract

Leptin is a pleiotropic hormone primarily secreted by adipocytes. A high density of functional Leptin receptors has been reported to be expressed in the hippocampus and other cortical regions of the brain, the physiological significance of which has not been explored extensively. Alzheimer’s disease (AD) is marked by impaired brain metabolism with decreased glucose utilization in those regions which often precede pathological changes. Recent epidemiological studies suggest that plasma Leptin is protective against AD. Specifically, elderly with plasma Leptin levels in the lowest quartile were found to be four times more likely to develop AD than those in the highest quartile. We have previously reported that Leptin modulates AD pathological pathways in vitro through a mechanism involving the energy sensor, AMP-activated protein kinase (AMPK). To this end, we investigated the extent to which activation of AMPK as well as another class of sensors linking energy availability to cellular metabolism, the sirtuins (SIRT), mediate Leptin’s biological activity. Leptin directly activated neuronal AMPK and SIRT in cell lines. Additionally, the ability of Leptin to reduce tau phosphorylation and β-amyloid production was sensitive to the AMPK and sirtuin inhibitors, compound C and nicotinamide, respectively. These findings implicate that Leptin normally acts as a signal for energy homeostasis in neurons. Perhaps Leptin deficiency in AD contributes to a neuronal imbalance in handling energy requirements, leading to higher Aβ and phospho-tau, which can be restored by replenishing low Leptin levels. This may also be a legitimate strategy for therapy.

Highlights

► Leptin treatment of neuronal cells reduces Aβ and phospho-tau levels via AMPK and SIRT1. ► Metabolic disturbances in neurons could precede pathological changes in AD. ► AMPK or SIRT1 could be drug targets for AD.

Introduction

Several pieces of evidence suggest that brain metabolic disturbances may precede the pathological cascades characteristic of AD. For example, functional neuroimaging studies, including 2-deoxy-2[(18)F]fluoro-d-glucose (FDG) positron emission topography (PET), have illustrated regional hypometabolism in the early AD brain [1], [2], [3], and that the pattern correlates with typical brain atrophy in AD [4]. Interestingly, pyramidal neurons of the hippocampus have particularly demanding energy needs [5], rendering the hippocampus a region more sensitive to states of metabolic distress.

Both genetic and environmental factors are likely contributors in this interconnection between brain metabolic state and disease. For example, carriers of one copy of the APOE4 gene, involved in lipid metabolism, are three to fourfold more likely to develop AD than APOE3 carriers [6]. Further, carriers of the very long isotype of the TOMM40 gene polyT region among APOE3 carriers develop AD, on average, 7 years earlier than carriers of the short isotype [7].

Rodents fed high fat/caloric diets with limited exercise demonstrate impaired learning and memory performance compared to similar animals on lower energy diets [8]. In humans, a large longitudinal analysis showed a significant correlation between central obesity in midlife and an increased risk of dementia independent of diabetes and cardiovascular co-morbidities later in life [9].

Once AD pathological cascades are initiated because of these metabolic disturbances, these could further and cyclically exacerbate hypometabolic states regionally. Aβ oligomers induce oxidative stress [10], while activation of GSK-3β (promoted by Aβ and inhibited by Leptin), one of the many kinases that can phosphorylate tau, leads to decreased mitochondrial membrane potential and ATP production [11]. Studies utilizing animal models of aging and AD have shown that achieving optimal energy balance (i.e. through feeding and exercise) can improve cognitive function and prevent an age-related decline in learning [12], [13].

The main focus of our research efforts is to identify and characterize metabolic factors, the levels of which are altered during normal brain aging or during the conversion of a healthy brain to that with dementia and AD. Leptin, primarily secreted from adipocytes, can function as a modulator of energy metabolism [14]. Within the arcuate nucleus of the hypothalamus, Leptin acts on neuropeptide Y/agouti-related peptide (NPY/AgRP) and pro-opiomelanocortin (POMC) neurons to regulate food intake, energy expenditure and hepatic glucose production [15]. However, other larger regions in the brain known to express high levels of the Ob-Rb, the long isoform of Leptin receptor known to transduce signaling, include the cortex and the hippocampus [16], [17]. Peripherally, Leptin acts directly on fat and skeletal muscle to stimulate fatty acid oxidation, increase glucose uptake and inhibit lipogenesis [18]. In both settings, Leptin production is stimulated by a positive energy balance, and acts to restore energy homeostasis through suppressing anabolic and boosting catabolic pathways.

To date a number of reports have shown that there is a positive correlation between reduced levels of circulating Leptin and AD risk [19], [20], severity of dementia [21] and cognitive decline [22], [23]. Most notably, a study involving 785 cognitively-normal elderly followed for a median of 8.3 years showed that those with plasma Leptin levels in the lowest quartile at baseline were at four times greater risk for developing AD than those in the highest quartile [24]. At the physiological level, it is known that there is a high concentration of Leptin receptors in the hippocampus [16], which are functional, and direct injection of Leptin in that region can improve memory processing and modulate long term potentiation and synaptic plasticity [25]. Further, Leptin administration improves memory in SAMP-8 mice, an accelerated senescence rodent model that develops amyloid plaques [26]. Moreover, the diabetic/obese db/db mice, which lack a functional Leptin receptor exhibit cognitive impairment and impaired synaptic function and neurogenesis [27]. Interestingly, it has been suggested that one of Leptin’s roles could involve the prevention of excess accumulation of lipids in non-adipocytes, including neurons, which could be poisoning [28].

We have previously reported that Leptin reduces tau phosphorylation and Aβ production in neuronal cells and transgenic mice models of AD [29], [30], [31]. Leptin’s effects in vitro were dependent on activation of the cellular energy sensor, AMP-activated protein kinase (AMPK) [32]. AMPK is ubiquitously expressed throughout the body and is activated in states of low cellular energy by an elevated AMP/ATP ratio [33]. Besides ATP the only other small molecule in cells that indicates energy status is NAD+, which is necessary for activation of a family of evolutionarily conserved energy sensors, the sirtuins (SIRT) [34]. The sirtuins are histone deacetylases that play important roles in a number of physiological processes, including stress resistance [35], replicative senescence [36], aging and differentiation [37]. Notably SIRT1 has been associated with the anti-aging effects of caloric restriction and, most recently, inhibition of amyloidogenic pathways in laboratory models of AD [38], [39], [40]. Additionally, caloric restriction has been shown to indirectly activate SIRT1 through a linear pathway involving AMPK [41]. To this end, we investigated the extent to which activation of cellular energy sensors, involving AMPK and the sirtuins, is involved in Leptin’s beneficial effects on AD-related biochemical pathways.

Section snippets

Reagents and antibodies

Minimum essential medium (MEM) was purchased from ATCC (Manassas, VA). Fetal bovine serum (FBS), all-trans retinoic acid, nicotinamide and recombinant human Leptin were purchased from Sigma–Aldrich (St. Louis, MO). Compound C was purchased from EMD Biosciences (San Diego, CA). Rabbit anti-tau (pThr181) was purchased from Abcam (Cambridge, MA). Tau (tau46) mAb was purchased from Cell Signaling.

Culture and stable transfection of cell lines

The human neuroblastoma cell line, SH-SY5Y, was purchased from ATCC. Cell culture was performed

Leptin activates AMPK and SIRT in neuronal cells

The initial set of studies investigated whether treatment of neuronal cells with Leptin can boost cellular metabolism by directly increasing AMPK kinase activity and total sirtuin (SIRT) deacetylase activity (Fig. 1). We have previously shown that Leptin induces phosphorylation of AMPK in neurons [32], but have yet to determine its effects on AMPK kinase activity. SY5Y neuroblastoma cells were differentiated with retinoic acid (RA-SY5Y) and then treated with Leptin for 6 h in the presence or

Discussion

The link between dysfunctional brain energy metabolism and AD has become progressively clearer with the advent of sophisticated technologies that enable functional neuroimaging. Studies using FDG-PET have illustrated that areas of the brain sequentially presenting with the AD pathology are preceded by a matching distribution of regional hypometabolism. The etiologic basis for such cascade of events in AD is not entirely clear. It is possible that metabolic diseases, such as obesity and diabetes

Acknowledgment

This work was supported by the National Institute on Aging (SBIR – 1R43AG029670).

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