Demonstrated brain insulin resistance in Alzheimer's disease patients is associated with IGF-1 resistance, IRS-1 dysregulation, and cognitive decline

Abstract

While a potential causal factor in Alzheimer's disease (AD), brain insulin resistance has not been demonstrated directly in that disorder. We provide such a demonstration here by showing that the hippocampal formation (HF) and, to a lesser degree, the cerebellar cortex in AD cases without diabetes exhibit markedly reduced responses to insulin signaling in the IR→IRS-1→PI3K signaling pathway with greatly reduced responses to IGF-1 in the IGF-1R→IRS-2→PI3K signaling pathway. Reduced insulin responses were maximal at the level of IRS-1 and were consistently associated with basal elevations in IRS-1 phosphorylated at serine 616 (IRS-1 pS⁶¹⁶) and IRS-1 pS⁶³⁶/⁶³⁹. In the HF, these candidate biomarkers of brain insulin resistance increased commonly and progressively from normal cases to mild cognitively impaired cases to AD cases regardless of diabetes or APOE ε4 status. Levels of IRS-1 pS⁶¹⁶ and IRS-1 pS⁶³⁶/⁶³⁹ and their activated kinases correlated positively with those of oligomeric Aβ plaques and were negatively associated with episodic and working memory, even after adjusting for Aβ plaques, neurofibrillary tangles, and APOE ε4. Brain insulin resistance thus appears to be an early and common feature of AD, a phenomenon accompanied by IGF-1 resistance and closely associated with IRS-1 dysfunction potentially triggered by Aβ oligomers and yet promoting cognitive decline independent of classic AD pathology.

Figures

Figure 1. Ex vivo stimulation is a valid method for studying insulin signaling in postmortem HF.

(A) Representative dose-response tests in 1 of 5 N adult humans at 0–100 nM insulin in immunoblots of phosphorylated or bound antigens from immunoprecipitates of the indicated antigens. (B) Sample blots on the rat HF, showing that PMIs up to 16 hours (n = 4 per PMI) had no substantial effect on basal levels of IRβ or signaling evoked by 1 or 10 nM insulin. Effects of insulin on IGF-1 signaling were also tested on the same samples (Supplemental Figure 2). (C) PMI effects on IRβ pY and IGF-1Rβ pY relative to total receptor levels (mean ± SEM). 1 nM insulin activated IRβ (P = 0.0015), but not IGF-1Rβ (see blots). 10 nM insulin induced greater activation of IRβ (P = 0.0063) as well as IGF-1Rβ (P = 0.0009). (D) PMI effects on IRS-1 bound to IRβ and IGF-1Rβ relative to total receptor levels (mean ± SEM). 1 nM insulin induced IRβ binding (P = 0.0002), but not IGF-1Rβ binding (see blots), of IRS-1. 10 nM insulin induced greater IRβ binding (P = 0.0009) as well as IGF-1Rβ binding (P = 0.0007) of IRS-1. kDa values correspond to the molecular weight marker closest to the bands shown. *P < 0.005.

Figure 2. At near-physiological doses (1 nM), insulin and IGF-1 activate different IRS signaling pathways.

This was demonstrated with ex vivo stimulation of HF and cerebellar cortex samples from 8 N humans with low PMIs. Data from the HF are shown. The effect of 0, 1, and 10 nM insulin and IGF-1 is shown on IRβ and IGF-1Rβ activation (A–D), IRS-1 and IRS-2 binding of IRβ and IGF-1Rβ (E–H), IRS-1 and IRS-2 activation (I–L), and PI3K p85α binding to IRS-1 and IRS-2 (M–P). 1 nM insulin activated IRβ, but not IGF-1Rβ, and bound IRS-1, but not IRS-2, to its receptor. In contrast, 1 nM IGF-1 activated IGF-1Rβ, but not IRβ, and bound IRS-2, but not IRS-1, to its receptor. Values (mean ± SEM) are ratios of phosphorylated or bound molecules to total levels of those molecules or of the molecules to which they were bound. #P < 0.01, *P < 0.001 vs. baseline (0 nM). Sample Western blots on which these graphs were based are shown in Figures 3 and 4.

Figure 3. Ex vivo stimulation revealed IRS-1–associated insulin resistance and IRS-2–associated IGF-1 resistance in the cerebellar cortex of AD cases.

(A–C) Western blots from a representative matched pair of N and AD cases showed decreased signaling responses in the AD case to 1 and 10 nM insulin without affecting IRS-2 (specifically, reductions in IRβ activation; IRS-1 binding of IRβ, IRS-1 activation [pY] and suppression [pS]; and PI3K p85α binding of IRS-1). (D–F) Western blots from a representative matched pair of N and AD cases showed decreased signaling responses in the AD case to 1 and 10 nM IGF-1 without affecting IRS-1 (specifically, reductions in IGF-1Rβ activation; IRS-2 binding of IGF-1Rβ, IRS-2 activation and suppression; and PI3K p85α binding of IRS-2). See Figure 5 and Tables 1–4 for quantification.

Figure 4. Ex vivo stimulation revealed IRS-1–associated insulin resistance and IRS-2–associated IGF-1 resistance in HF of AD cases.

(A–C) Western blots from a representative matched pair of N and AD cases showed decreased signaling responses in the AD case to 1 and 10 nM insulin without affecting IRS-2 (specifically, reductions in IRβ activation; IRS-1 binding of IRβ, IRS-1 activation [pY] and suppression [pS]; and PI3K p85α binding of IRS-1). (D–F) Western blots from a representative matched pair of N and AD cases showed decreased signaling responses in the AD case to 1 and 10 nM IGF-1 without affecting IRS-1 (specifically, reductions in IGF-1Rβ activation; IRS-2 binding of IGF-1Rβ, IRS-2 activation and suppression; and PI3K p85α binding of IRS-2). See Figure 5 and Tables 1–4 for quantification.

Figure 5. Direct demonstration of insulin and IGF-1 resistance in the cerebellar cortex (A–L) and HF (M–X) of AD cases without diabetes.

Both structures showed reduced responsiveness to near-physiological doses (1 nM) of insulin and IGF-1, as seen in receptor activation (A, B, G, H, M, N, S, and T); IRS-1 bound to IRβ and IRS-2 bound to IGF-1Rβ (C, I, O, and U); IRS-1 or IRS-2 activation (pY) or suppression (pS) (D, E, J, K, P, Q, V, and W); and PI3K p85α bound to IRS-1 or IRS-2 (F, L, R, and X). Values (mean ± SEM) for N and AD cases denote percent increase in signaling responses above baseline (0 nM) in the same diagnostic group. Percentages were calculated from response strengths expressed as ratios of the phosphorylated or bound molecule to the total level of the same molecule or of the molecule to which it was bound (Supplemental Tables 2–5). Unlike the HF, insulin resistance to 1 nM insulin in the cerebellar cortex was overcome at most levels of the signaling pathway by 10 nM insulin. Unlike insulin resistance, IGF-1 resistance was profound even at the receptor level. †P < 0.05, #P < 0.01, *P < 0.001 vs. N. See Tables 1–4 for quantification.

Figure 6. CA1 pyramidal cells in AD display marked elevation in cytosolic levels of IRS-1 pS species and their activated kinases.

The heat map summarizes relative basal levels of select insulin signaling molecules (A), activation states of those and related molecules (B), activation states of IRS-1 serine kinases (C), protein phosphatases that regulate insulin signaling (D), and neuropathological parameters (E). Data are shown for the 24 N and 24 matched AD cases in the UPenn cohort. Each row displays mean qIHC data on the respective analyte; each cell shows mean cellular levels of an analyte in a given case relative to all 48 cases studied. See Table 5 for measures used to quantify each analyte. P values denote differences between N and AD cases. Note that AD cases typically showed high levels of IRS-1 pS species and of activated IRS-1 pS kinases (GSK-3, IKK, JNK, mTOR, and PKCζ/λ). All amino acid sequence numbers are for the human proteins. tAβ, total Aβ; oAβ, oligomeric Aβ.

Figure 7. Key insulin signaling molecules seen immuno­histochemically in CA1 neurons of N, MCI, and AD cases of the ROS cohort.

See Table 5 for numeric data on the antigens. IR/IGF-1Rβ pY (A–C) was reduced in MCI. Total neuronal IRS-1 (D–F) was not reduced in MCI or AD. IRS-1 pS was normally confined to cell nuclei (e.g., arrow in G) with few exceptions, but the density of neurons with detectable cytoplasmic IRS-1 pS616 (G–I) or IRS-1 pS636/639 (J–L) increased markedly from N to MCI to AD. What appears to be high background levels of IRS-1 pS616 in I was actually elevated antigen in the neuropil. (M–O) Akt1 pS473 was barely detectable in N cases, but the density of neurons with detectable cytoplasmic levels of the activated molecule rose markedly from N or MCI to AD. See Supplemental Figures 5 and 6 for other insulin signaling molecules studied in the ROS cohort. Scale bar: 70 μm.

Figure 8. Insulin by itself does not affect glucose uptake in HF slices (A and B) and HF synaptosomes (C and D) of N or AD cases.

Data were derived from the same 8 pairs of cases in which insulin resistance was demonstrated in AD (Figure 5 and Table 2). Shown are (A and C) net [3H] glucose uptake in disintegrations per minute (dpm) and (B and D) percent increase in uptake compared with unstimulated tissue. Whereas 1 and 10 nM insulin had no effect on [3H] glucose uptake in N or AD cases in either tissue preparation, 10 μM glutamate plus 1 μM glycine evoked clear increases in [3H] glucose uptake in both tissue slices and synaptosomes, an effect that was significantly reduced in AD cases. Values are mean ± SEM. *P < 0.0001 vs. N.

Figure 9. The density of CA1 neurons displaying cytoplasmic IRS-1 pS616 is inversely associated with episodic memory (A), working memory (B), and global cognition (C).

Linear regression graphs plot data on all the ROS cases (N, MCI, and AD; n = 88) adjusted for age, sex, and years of education; the linear regression line (solid) is shown flanked by 95% confidence intervals (dashed). Episodic memory, working memory, and global cognition scores are composites of the multiple measures used to assess those cognitive abilities. Raw scores on individual tests were converted to z scores (using population estimates of the mean and SD) and averaged to yield the composite scores (see Supplemental Methods).

Figure 10. Evidence for insulin resistance and its likely proximal causes in the HF of AD cases.

The summary is consistent with the more limited data collected on insulin responses in the cerebellar cortex of these cases. (A) Compared with N cases, AD cases responded to 1 nM insulin with lower IR-A pY1150/1151 and IR-A pY960; less IRS-1 bound to IRβ; lower total IRS-1 pY, including IRS-1 pY612; less IRS-1 bound to PI3K p85α; less activation of downstream molecules, reflected in lesser elevations in Akt1 pS473, GSK-3β pY216, mTOR pS2448, and ERK2 pT185/pY187; and less suppression of GSK-3β, reflected in lesser elevation of GSK-3β pS9. GSK-3β pY216 is not a product of Akt1 activity, but of unknown mammalian kinases and autophosphorylation (87). The question mark indicates an expected, but not tested, reduction in insulin-induced binding of the regulatory and catalytic subunits of PI3K (p85α and p110, respectively) in AD. (B) Basal activation states of insulin signaling and regulating molecules in AD vs. N cases. Consistent with decreased insulin signaling, reduced levels of IR/IGF-1Rβ pY, IRβ pY960, and PIP3 were found with qIHC. The question mark indicates uncertainty regarding the phosphorylation state of the IR catalytic domain, since IHC cannot distinguish this domain in IR and IGF-1R. The increases in IRS-1 pS312, IRS-1 pS616, and IRS-1 pS636/639 are probably caused by correlated increases in activated IRS-1 serine kinases (ERK2, IKK, JNK1/2, mTOR, and PKCζ/λ), potentially resulting from elevated oligomeric Aβ. See Discussion for details.