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. 2015 Apr 8;35(14):5851-9.
doi: 10.1523/JNEUROSCI.5180-14.2015.

Neuronal LRP1 regulates glucose metabolism and insulin signaling in the brain

Chia-Chen Liu  1 , Jin Hu  1 , Chih-Wei Tsai  2 , Mei Yue  2 , Heather L Melrose  2 , Takahisa Kanekiyo  2 , Guojun Bu  3
Affiliations

Neuronal LRP1 regulates glucose metabolism and insulin signaling in the brain

Chia-Chen Liu et al. J Neurosci. .

Abstract

Alzheimer's disease (AD) is a neurological disorder characterized by profound memory loss and progressive dementia. Accumulating evidence suggests that Type 2 diabetes mellitus, a metabolic disorder characterized by insulin resistance and glucose intolerance, significantly increases the risk for developing AD. Whereas amyloid-β (Aβ) deposition and neurofibrillary tangles are major histological hallmarks of AD, impairment of cerebral glucose metabolism precedes these pathological changes during the early stage of AD and likely triggers or exacerbates AD pathology. However, the mechanisms linking disturbed insulin signaling/glucose metabolism and AD pathogenesis remain unclear. The low-density lipoprotein receptor-related protein 1 (LRP1), a major apolipoprotein E receptor, plays critical roles in lipoprotein metabolism, synaptic maintenance, and clearance of Aβ in the brain. Here, we demonstrate that LRP1 interacts with the insulin receptor β in the brain and regulates insulin signaling and glucose uptake. LRP1 deficiency in neurons leads to impaired insulin signaling as well as reduced levels of glucose transporters GLUT3 and GLUT4. Consequently, glucose uptake is reduced. By using an in vivo microdialysis technique sampling brain glucose concentration in freely moving mice, we further show that LRP1 deficiency in conditional knock-out mice resulted in glucose intolerance in the brain. We also found that hyperglycemia suppresses LRP1 expression, which further exacerbates insulin resistance, glucose intolerance, and AD pathology. As loss of LRP1 expression is seen in AD brains, our study provides novel insights into insulin resistance in AD. Our work also establishes new targets that can be explored for AD prevention or therapy.

Keywords: Alzheimer's disease; LRP1; apolipoprotein E; glucose metabolism; insulin signaling.

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Figures

Figure 1.
Figure 1.
Deficiency of LRP1 in primary neurons and mouse brain leads to decreased insulin receptor levels and signaling. A, B, GT1–7 neuronal cells infected with control or LRP1 shRNA were incubated with serum-free medium for 4 h and then stimulated with insulin (Ins; 100 nm) for 30 min. Lysates from GT1–7 cells were blotted for LRP1, IRβ, total Akt, p-Akt, and β-actin. C, D, Primary neurons infected with control or LRP1 shRNA were incubated with serum-free medium for 4 h and then treated with insulin (100 nm) for 30 min. Lysates were blotted for LRP1, IRβ, Akt, p-Akt, and β-actin. E, Levels of LRP1, IRβ, Akt, p-Akt, and β-actin were analyzed by Western blot in the cortex of nLrp1−/− mice (Lrp1flox/flox, CaMKII-Cre+/−) or Ctrl (Lrp1flox/flox, CaMKII-Cre−/−) mice at the age of 18 months (n = 7/group). Data are mean ± SEM. *p < 0.05. **p < 0.01. N.S., Not significant. F, Lysates from wild-type mouse brain were immunoprecipitated with either an LRP1 antibody or control rabbit IgG. Immunoprecipitates were then subjected to Western blot analysis with an anti-IRβ antibody. LRP1 antibody, but not control IgG, immunoprecipitated IRβ.
Figure 2.
Figure 2.
Downregulation of LRP1 leads to a reduction of glucose uptake. A, MEF1 (wild-type) and MEF2 (LRP1-deficient) cells were incubated with indicated concentrations of fluorescent 2-NBDG for 20 min at 37°C. The intracellular fluorescence of 2-NBDG was detected by flow cytometry. B, MEF1 (wild-type) and MEF2 (LRP1-deficient) cells were incubated with 2-NBDG (300 μm) for indicated amounts of time at 37°C. The intracellular fluorescence of 2-NBDG was detected by flow cytometry. C, MEF1 (wild-type) and MEF2 (LRP1-deficient) cells were incubated with 2-NBDG (300 μm) for 20 min at 37°C. The intracellular fluorescence of 2-NBDG was examined by confocal microscopy. D, GT1–7 neuronal cells infected with control or LRP1 shRNA were incubated with serum-free medium for 4 h and stimulated with insulin (100 nm) for 20 min. Cells were then treated with 2-NBDG (300 μm) in the presence or absence of insulin for 20 min. The intracellular fluorescence was detected by flow cytometry. E, Levels of LRP1, IRβ, GLUT3, GLUT4, and β-actin in primary neurons expressing control or LRP1 shRNA were analyzed by Western blot and quantified. Data are mean ± SEM. *p < 0.05. **p < 0.01. F, Levels of GLUT3, GLUT4, and β-actin were analyzed by Western blot in the cortex of nLrp1−/− mice (Lrp1flox/flox, CaMKII-Cre+/−) or Ctrl (Lrp1flox/flox, CaMKII-Cre−/−) mice at the age of 18 months (n = 7–9/group). Data are mean ± SEM. *p < 0.05.
Figure 3.
Figure 3.
Neuronal deletion of LRP1 results in glucose intolerance in mouse brain. A, B, Brain glucose tolerance test was performed on nLrp1−/− mice (Lrp1flox/flox, CaMKII-Cre+/−) or Ctrl (Lrp1flox/flox, CaMKII-Cre−/−) mice at 20–22 months of age. Mice were fasted overnight, and d-glucose at 2 g/kg of body weight was injected intraperitoneally as indicated by the arrow after baseline ISF collection. In vivo microdialysis was used to assess the ISF glucose concentration in the hippocampus of free moving mice. The baseline ISF glucose level is higher in nLrp1−/− mice compared with control mice (A). B, The brain glucose level in each fraction measure by glucose assay was plotted against time. nLrp1−/− mice had significantly decreased tolerance in the brain in response to exogenous glucose. Data are mean ± SEM. n = 7/each group. *p < 0.05. C, Evans blue dye (30 mg/kg) was injected intravenously in mice without surgery, 30 min or 24 h after surgery. After 12 h, mice were perfused with saline and brains were collected. Representative images of PBS-perfused brains were shown. The Evans blue was extracted using N,N-dimethyl formamide and quantified using a fluorometer (620 nm excitation; 695 nm emission). Data are mean ± SEM. n = 3/each group. **p < 0.01. N.S., Not significant.
Figure 4.
Figure 4.
Insulin induces cell surface presentation of LRP1 in neuronal cells. A–C, SH-SY5Y human neuronal cells were treated with or without insulin (100 nm) for 20 min, and the surface proteins were examined by cell surface biotinylation assay. The levels of total and cell surface LRP1, GLUT4, IRβ, and β-tubulin in SH-SY5Y cells treated with or without insulin were examined by Western blot analysis (A) and quantified (B, C). Induction of p-Akt expression confirmed an increase in insulin signaling upon insulin treatment. Data are mean ± SEM. N.S., Not significant. **p < 0.01.
Figure 5.
Figure 5.
Hyperglycemia and insulin deficiency suppress LRP1 expression in the brain. A, SH-SY5Y cells were treated with low glucose DMEM supplemented with various concentrations of glucose for 48 h. The levels of LRP1 and β-actin in SH-SY5Y cells were examined by Western blot analysis. The LRP1 level in SH-SH5Y cells treated with regular growth medium DMEM/glucose (25 mm) was set as 1.0. Data are mean ± SEM. *p < 0.05. B, Neuronal GT1–7 cells cultured in serum-free DMEM growth medium were treated with (+Ins, 100 nm) or without insulin (−Ins) for 24 h. The levels of LRP1 and β-actin were examined by Western blot analysis. C, D, Brain glucose tolerance test was performed on STZ-injected diabetic mice and control mice at 3 months of age. Mice were fasted overnight, and d-glucose at 2 g/kg of body weight was injected intraperitoneally as indicated by the arrow after baseline ISF collection. In vivo microdialysis was used to assess ISF glucose levels in the hippocampus of freely moving mice. The brain glucose level in each fraction measure by glucose assay was plotted against time (C). The rate of glucose clearance was shown as a function of the slope of the natural log of glucose levels against time (D). Data are mean ± SEM. n = 4/each group. **p < 0.01. N.S., Not significant. E, Blood glucose in STZ-injected diabetic mice and control mice was measured by glucose monitor. F, The levels of LRP1 in the cortex and hippocampus of STZ-injected diabetic mice and control mice were examined by Western blot analysis and quantified. Data are mean ± SEM. n = 4/each group. *p < 0.05.
Figure 6.
Figure 6.
Model of neuronal LRP1 in the regulation of insulin signaling and glucose metabolism. LRP1 interacts with IRβ through which it regulates insulin signaling. LRP1 also affects glucose metabolism in neurons through modulation of glucose transporter expression and function. Hyperglycemia suppresses the levels of LRP1, which might further impair insulin signaling and glucose homeostasis in the brain.
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