Susceptibility to diet-induced obesity and glucose intolerance in the APP SWE/PSEN1 A246E mouse model of Alzheimer’s disease is associated with increased brain levels of protein tyrosine phosphatase 1B (PTP1B) and retinol-binding protein 4 (RBP4), and basal phosphorylation of S6 ribosomal protein
Obesity is a major risk factor for development of insulin resistance, a proximal cause of type 2 diabetes and is also associated with an increased relative risk of Alzheimer’s disease. We therefore investigated the susceptibility of transgenic mice carrying human mutated transgenes for amyloid precursor protein (APP SWE) and presenilin 1 (PSEN1 A246E) (APP/PSEN1), or PSEN1 A246E alone, which are well-characterised animal models of Alzheimer’s disease, to develop obesity, glucose intolerance and insulin resistance, and whether this was age- and/or diet-dependent.
We analysed the effects of age and/or diet on body weight of wild-type, PSEN1 and APP/PSEN1 mice. We also analysed the effects of diet on glucose homeostasis and insulin signalling in these mice.
While there were no body weight differences between 16–17- and 20–21-month-old PSEN1 mice, APP/PSEN1 mice and their wild-type controls on standard, low-fat, chow diet, the APP/PSEN1 mice still exhibited impaired glucose homeostasis, as investigated by glucose tolerance tests. This was associated with increased brain protein tyrosine phosphatase 1B protein levels in APP/PSEN1 mice. Interestingly, short-term high-fat diet (HFD) feeding of wild-type, PSEN1 and APP/PSEN1 mice for a period of 8 weeks led to higher body weight gain in APP/PSEN1 than in PSEN1 mice and wild-type controls. In addition, HFD-feeding caused fasting hyperglycaemia and worsening of glucose maintenance in PSEN1 mice, the former being further exacerbated in APP/PSEN1 mice. The mechanism(s) behind this glucose intolerance in PSEN1 and APP/PSEN1 mice appeared to involve increased levels of brain retinol-binding protein 4 and basal phosphorylation of S6 ribosomal protein, and decreased insulin-stimulated phosphorylation of Akt/protein kinase B and extracellular signal-regulated kinase 1/2 in the brain.
Our results indicate that Alzheimer’s disease increases susceptibility to body weight gain induced by HFD, and to the associated glucose intolerance and insulin resistance.
KeywordsDiet-induced obesity Insulin resistance Glucose intolerance Type 2 diabetes Alzheimer’s disease Human mutations Transgenes
Amyloid precursor protein
Mice carrying human mutated transgenes for amyloid precursor protein (APP SWE) and presenilin 1 (PSEN1 A246E)
Extracellular signal-regulated kinase
Glucose tolerance test
Akt/protein kinase B
Protein tyrosine phosphatase 1B
Retinol-binding protein 4
Alzheimer’s disease, the most common type of progressive dementia in the elderly , is characterised by neurofibrillary tangles and deposits of β-amyloid. Three genes have been identified in familial early-onset Alzheimer’s disease: (1) amyloid-β protein precursor (AβPP, also known as APP); (2) presenilin 1 (PSEN1); and (3) PSEN2 . The generation of transgenic mice with β-amyloid plaques has improved our understanding of pathophysiological processes associated with Alzheimer’s disease [3, 4]. Thus, transgenic mice carrying human mutated transgenes for APP, PSEN1 or PSEN2/APP+PSEN1 mutated transgenes are a well-characterised animal model of Alzheimer’s disease.
Increasing evidence suggests that insulin resistance, i.e. the inability of tissues to respond to the circulating effects of insulin, is a risk factor associated with pathogenesis of Alzheimer’s disease . Therefore, insulin-sensitising drugs such as the peroxisome proliferator-activated receptor γ agonist, rosiglitazone, have been found to improve cognitive function in transgenic APP mouse models of Alzheimer’s disease . Studies using analogues for the gut hormone glucagon-like peptide-1 (GLP-1) such as liraglutide, which facilitate insulin release, have shown that even short-term treatment can improve learning and memory, in addition to improving glucose homeostasis in mice with diet-induced obesity . Moreover, an increase of GLP-1 signalling in the brain has also been suggested as a promising strategy to ameliorate the degenerative processes observed in Alzheimer’s disease , while studies crossing APP mice to mouse models with alterations in brain insulin-signalling have revealed an important role for insulin signalling and glucose homeostasis in cognitive behaviour in these mice [9, 10, 11].
Currently, several potential mechanisms have been suggested that connect insulin resistance to Alzheimer’s disease. One is that insulin crosses the blood–brain barrier from the periphery to the central nervous system and competes with β-amyloid for insulin degrading enzyme in the brain . Peripheral hyperinsulinaemia may also inhibit brain insulin production, which, in turn, results in impaired amyloid clearance and a higher risk of Alzheimer’s disease [13, 14]. Other mechanisms have also been suggested, such as advanced glycation end-products, which can be identified immunohistochemically in senile plaques and neurofibrillary tangles, the pathological hallmarks of Alzheimer’s disease . In addition, associations between adipokine levels and Alzheimer’s disease have also been made . Thus, obesity and the associated hyperinsulinaemia and hyperleptinaemia have been found to play an important role in early-onset learning deficits in APP mouse model of Alzheimer’s disease, as crossing these mice to genetically obese ob/ob mice results in profound learning and memory deficits in mice as young as 8 weeks of age .
In our studies, we asked whether the presence of Alzheimer’s disease genes was a risk factor for development of diet-induced obesity and glucose intolerance. Thus, we used the well-characterised double mutant mice carrying human mutated transgenes for amyloid precursor protein (APP SWE) and presenilin 1 (PSEN1 A246E) (APP/PSEN1) mice, as these mice develop numerous amyloid deposits much earlier (9 months onwards) than age-matched mice expressing APP SWE (positive at 18 months), PSEN A246E or wild-type mice . In addition, behavioural and physiological deficits in APP/PSEN1 mice are obvious, starting at around 6 months of age, prior to the presence of amyloid deposits, [3, 17]. We set out to investigate if the presence of Alzheimer’s disease genes increases the risk of age- and diet-associated obesity and associated glucose intolerance and insulin resistance, by comparing aged APP/PSEN1, PSEN1 and wild-type mice on standard rodent chow or when challenged with a high-fat diet (HFD).
All animal studies were performed under a project licence approved by the Home Office under the Animals (Scientific Procedures) Act 1986. Mice were maintained on a 12 h light/dark cycle in a temperature-controlled barrier facility, with free access to water and food. Transgenic mice were purchased (Jackson Laboratories, Bar Harbor, ME, USA) and breeding colonies established at the University of Aberdeen, UK, as described previously .
For body weight analysis studies on chow diet, we used one cohort of male mice, which we weighed longitudinally (APP/PSEN1 n = 11, wild-type n = 8 and PSEN1 n = 3). For the effects of chow vs HFD feeding on body weight gain and glucose tolerance, we used another cohort of male mice, which we examined pre- (i.e. while on chow) and post-HFD feeding (APP/PSEN n = 18, PSEN1 n = 9 and wild-type n = 8).
Tail blood glucose was determined using a Accu-Check glucometer (Roche Diagnotics, Mannheim, Germany). Glucose tolerance tests (GTT) were performed as described previously [18, 19, 20, 21]. Briefly, mice were fasted overnight (16 h) and experiments started at 09:00 hours the following day. Mice were separated into individual cages, and their body weight and fasting blood glucose determined, followed by i.p. injection of a bolus of glucose (2 g/kg body weight). Blood glucose levels were determined immediately before and at 15, 30, 60 and 120 min post-injection. For HFD studies, mice were placed on diet containing 55% kcal from fat, primarily hydrogenated vegetable shortening, TD93075 (Harlan Teklad, Madison, WI, USA) for a period of 8 weeks.
Whole-cell lysates were prepared by extraction in RIPA buffer (10 mmol/l TRIS-HCl, pH 7.4; 150 mmol/l NaCl; 0.1% [wt/vol.] SDS; 1% [vol./vol.] Triton X-100; 1% [wt/vol.] sodium deoxycholate; 5 mmol/l EDTA; 1 mmol/l NaF; 1 mmol/l sodium orthovanadate; protease inhibitors) at 4°C, followed by clarification at 14,000 g, as described previously . For western blots, proteins were resolved by SDS-PAGE using pre-cast 18-well 10% gels (BioRad Laboratories, Hemel Hempstead, UK) and transferred to nitrocellulose membranes. Immunoblots were performed with rabbit polyclonal antibodies against phospho-AKT, phospho-extracellular signal-regulated kinase (ERK)1/ERK2, phospho-S6, mouse monoclonal ERK2 (all from Cell Signaling/New England Biolabs, Hitchin UK), protein tyrosine phosphatase 1B (PTP1B) (Upstate/Millipore, Watford, UK) and retinol-binding protein 4 (RBP4, A0040; Dako, Ely UK), following the manufacturer’s directions. Proteins were visualised using enhanced chemiluminescence and quantified using a Fusion CCD imaging system and Bio1D software (Peqlab, Sarisbury Green, UK).
Results are expressed as mean ± SEM. Comparisons between groups were made by ANOVA (one- or two-way, with repeated measures, where appropriate), with values of p < 0.05 considered statistically significant.
APP/PSEN1 mice are more susceptible to diet-induced body weight gain
Mouse models of Alzheimer’s disease are susceptible to diet-induced hyperglycaemia, while presence of APP genotype leads to glucose intolerance
Since measuring circulating blood glucose levels is not the most comprehensive way to determine subtle differences in glucose homeostasis, we also performed GTTs in the mouse groups. On chow diet, no differences in 5 h (data not shown) or 16 h fasted circulating blood glucose levels (Fig. 3a) were observed, whereas GTTs revealed that APP/PSEN1 mice were more glucose-intolerant than their wild-type controls, as evidenced by their slower rate of glucose clearance at 30 and 60 min post-GTT, and more inkolerant than PSEN1 mice at 60 min (two-way repeated-measures ANOVA, interaction F = 2.60, p = 0.01) (Fig. 3b). In addition, PSEN1 mice exhibited an intermediate phenotype with worsening of glucose clearance significantly different from that in wild-type controls at 30 min post-GTT (Fig. 3b). Even though HFD-feeding induced glucose intolerance in all groups of mice, APP/PSEN1 mice had much less efficient glucose clearance than their wild-type controls (Fig. 3c), with significance reached for glucose clearance after 30 min (two-way repeated-measures ANOVA). Again, PSEN1 mice exhibited an intermediate phenotype, suggesting that presence of APP genotype is necessary for diet-induced glucose intolerance to be detectable. Analysis of the AUC (Fig. 3d) during a GTT further confirmed that APP/PSEN1 mice are more glucose-intolerant than their wild-type control littermates with short-term HFD-feeding (two-way repeated-measures ANOVA, interaction F = 4.07, p = 0.02) (Fig. 3d).
Increased brain PTP1B and RBP4 protein levels and nutrient-driven basal S6 phosphorylation in mouse models of Alzheimer’s disease are associated with glucose intolerance in these mice
Total protein levels of brain PTP1B, which is the major insulin receptor phosphatase and negative regulator of insulin and leptin signalling, were increased by Alzheimer’s disease genotype on chow diet (Fig. 4a, i), while HFD-feeding increased brain PTP1B levels in all groups of mice (Fig. 4b, i), consistent with previous findings .
Serum RBP4 is an adipokine found to be elevated in insulin-resistant humans and in many mouse models of obesity and insulin resistance, including HFD , although not all studies show this effect . According to many studies, the level of elevation correlates highly with the degree of insulin resistance in mice and in humans, and serum RBP4 levels were highly predictive of metabolic syndrome risk in a large population-based study . We analysed the levels of brain RBP4 in our mice on chow and HFD. Our data suggest that chow-fed APP/PSEN1 mice tend to have increased levels of brain RBP4 in comparison to wild-type and PSEN1 mice (Fig. 4a, j), while HFD-feeding significantly increases brain RBP4 levels in PSEN1 and APP/PSEN1 mice, relative to wild-type control littermates (two-way ANOVA) (Fig. 4b, j).
We also examined nutrient- and insulin-regulated S6 ribosomal protein phosphorylation, finding that in mice on HFD, basal S6 phosphorylation was dramatically higher in brains from PSEN1 and APP/PSEN1 mice (Fig. 4b, f) and was as high as wild-type insulin-stimulated S6 phosphorylation (two-way ANOVA). This was not the case on standard low-fat chow diet (Fig. 4a, e).
Given that recent data have shown that crossing APP mice to ob/ob mice resulted in complete absence of phosphorylation of Akt/protein kinase B (PKB) at S473 site in muscle and liver, we also investigated phosphorylation status of this site in brains from our mice. While insulin was able to stimulate phosphorylation of Akt/PKB on S473 site in wild-type mice, which was somewhat diminished with short-term HFD feeding (Fig. 4b–d), insulin was not able to cause any phosphorylation of Akt/PKB in APP/PSEN1 or PSEN1 mice on chow diet (Fig. 4a, c), or on HFD (two-way ANOVA) (Fig. 4b, c). Phosphorylation of ERK1/2 was strikingly higher in insulin-stimulated brains from wild-type mice fed chow diet (Fig. 4a, g) and HFD (Fig. 4b, h) than in PSEN1 and APP/PSEN1 mice on the same diets.
Obesity is a major risk factor for the development of several disorders such as type 2 diabetes, metabolic syndrome, cardiovascular disease, dementia and cancer. In addition, due to improvements in medicine, housing, economic factors and general well-being in the developed countries, people are living much longer. Ageing is also associated with the induction of most of these diseases, with associated costs to healthcare systems in the developed world also rapidly raising. Alzheimer’s disease is the most common form of dementia, characterised by loss of recent memory as one of the first symptoms and a progressive decline in all cognitive skills later on in the disease [3, 26]. Considerable evidence suggests that insulin resistance and obesity may directly contribute to the pathogenesis of Alzheimer’s disease [27, 28, 29, 30, 31]. However, not much emphasis has been put on the role of Alzheimer’s disease in obesity and type 2 diabetes development, as characterised by insulin resistance. In this study, we set out to investigate, in a well-established APP/PSEN1 and PSEN1 mouse model, whether genotypes relevant to Alzheimer’s disease make mice more susceptible to obesity development than their wild-type control littermates with increasing age and under different dietary conditions (low-fat standard chow diet vs HFD). We found that aged PSEN1 and APP/PSEN1 mice weighed exactly the same as their wild-type control mice, so ageing on its own did not seem to make Alzheimer’s disease mice more susceptible to increased body weight gain in comparison to their controls. However, even a short-term dietary treatment with HFD, consisting of 55% fat, made APP/PSEN1 mice significantly more susceptible to body weight gain than PSEN1 littermates or wild-type controls. Thus, APP genotype is a risk factor for diet-induced body weight gain, with potential implications with regard to other secondary co-morbidities that may develop with an increase in body weight e.g. type 2 diabetes. Indeed, our HFD study is in agreement with a similar study using sucrose-rich diet (10% sucrose) as a dietary supplement, which also led to an increase in body weight gain in APP/PSEN1 mice ; however, that study only used non-supplemented diet vs supplemented diet in APP/PSEN1 mice. No wild-type or transgene alone mice were included for a comparison. Our aim, however, was to determine whether the APP gene was the risk factor for increase in body weight gain or just diet alone.
Thus, we next set out to investigate whether an Alzheimer’s disease-like phenotype would make mice glucose-intolerant and insulin-resistant, and whether this was controlled by dietary conditions. Interestingly, even though we observed no differences in body weight between APP/PSEN1, PSEN1 and wild-type mice on chow diet, APP/PSEN1 mice were significantly more glucose-intolerant than PSEN1 transgene mice and/or their wild-type controls. However, at this point, APP/PSEN1 mice were still normoglycaemic, suggesting that we detected early changes in glucose maintenance. However, glucose intolerance induced by diet-induced obesity was strikingly evident in APP/PSEN1 mice, as they also developed diet-induced fasting hyperglycaemia after 8 weeks of HFD feeding. This was accompanied by worsening in glucose maintenance as measured by GTTs. In addition, PSEN1 transgene alone mice exhibited an intermediate phenotype, with fasting hyperglycaemia present under HFD feeding conditions. They were, however, significantly more glucose-tolerant than their APP/PSEN1 control littermates. Thus, presence of APP genotype presents a risk factor for development of glucose intolerance under normal dietary conditions as well as under HFD-feeding.
To investigate the potential mechanism(s) associated with this glucose intolerance, we examined the levels of PTP1B, a well-known negative regulator of insulin and leptin receptor signalling  whose levels are elevated in obesity in humans [33, 34] and rodents, and under inflammatory conditions [22, 35, 36]. Mice lacking PTP1B globally or specifically in the brain [35, 37, 38] are lean, while those lacking muscle- or liver-PTP1B are protected against HFD-induced insulin resistance and glucose intolerance [18, 19]. Interestingly, we found brain PTP1B protein levels to be increased in APP/PSEN1 mice on chow diet in comparison to wild-type controls, with PSEN1 mice having an intermediate phenotype. As expected, HFD feeding increased PTP1B levels in the brain in all groups of mice, with no statistical differences between groups. However, brain RBP4 protein levels tended to be higher in APP/PSEN1 mice on chow diet and significantly higher in PSEN1 and APP/PSEN1 mice on HFD, suggesting that increased RBP4 levels may be another mechanism by which Alzheimer’s disease genotype predisposes to diet-induced glucose intolerance. RBP4 is an adipokine that was recently found to be elevated in insulin-resistant states as well as in obesity in mice [23, 39, 40] and humans [41, 42, 43, 44], although not all studies have reported this [24, 45]. These data suggest that there are early markers of insulin resistance already present with the APP genotype, making these mice more susceptible to the effects of HFD feeding and associated insulin resistance. They also raise an interesting therapeutic possibility that reduction PTP1B and/or RBP4 levels by pharmacological means  may in turn protect against body weight gain and the associated glucose intolerance in Alzheimer’s disease mouse models. This could be an interesting avenue for further studies.
In addition, we investigated brain insulin-signalling events in all groups of mice. Mice were fasted overnight and injected with a bolus of saline or insulin. Basal and insulin-stimulated phosphorylation events were then examined under chow and HFD feeding conditions. Since HFD is known to promote insulin resistance through nutrient overload-associated increase in S6K1 activity, we used phosphorylation of S6 as a read-out of S6K1 activity . We found that under HFD feeding conditions, basal S6 phosphorylation was significantly higher in brains from PSEN1 and APP/PSEN1 mice and was as high as the insulin-stimulated S6 phosphorylation observed in wild-type mice, consistent with the insulin resistance observed in Alzheimer’s disease mice. Since a decrease in insulin-stimulated Akt/PKB phosphorylation was observed in other mouse models of Alzheimer’s disease in specific regions of brain or peripheral tissues [16, 28], we examined Akt/PKB phosphorylation from whole-brain lysates, finding, as expected from their impaired glucose tolerance, that insulin-stimulated Akt/PKB phosphorylation was decreased in PSEN1 and APP/PSEN1 mice under chow as well as HFD feeding conditions. In addition, insulin-stimulated phosphorylation of ERK1 and -2 was downregulated in PSEN1 and APP/PSEN1 mice in comparison to wild-type controls, both on chow and HFD. Thus it appears that in our Alzheimer’s disease mice, insulin-stimulated Akt/PKB phosphorylation was inhibited, while nutrient-driven S6K signalling became augmented.
In conclusion, we report here that transgenic Alzheimer’s disease mice are susceptible to diet-induced body weight gain, and the associated glucose intolerance and fasting hyperglycaemia, which are associated with increased brain PTP1B and RBP4 levels, and nutrient-driven basal S6 phosphorylation, as well as with decreased insulin-stimulated Akt/PKB and ERK1/2 phosphorylation. Our data suggest that Alzheimer’s disease is a risk factor for diet-induced body weight gain and associated glucose intolerance, and that Alzheimer’s disease patients should be advised to avoid foods high in fat, in order to prevent development of obesity-associated co-morbidities.
This work was funded by the College of Life Sciences and Medicine, University of Aberdeen, start-up funds to M. Delibegovic. M. Delibegovic is also funded by an RCUK Fellowship, Diabetes UK, British Heart Foundation, and the Royal Society. N. Mody is funded by an intermediate basic research fellowship from the British Heart Foundation. G. D. Mcilroy is funded by a BBSRC postdoctoral training studentship.
Duality of interest
The authors declare that there is no duality of interest associated with this manuscript.
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