This hypothesis was directly investigated by first examining postmortem cases of advanced AD and determining if the neurodegeneration was associated with significant abnormalities in the expression of genes encoding insulin, IGF-1, and IGF-2 peptides, their receptors, and downstream signaling mechanisms.5
In that study, we demonstrated advanced AD to be associated with strikingly reduced levels of insulin and IGF-1 polypeptide and receptor genes in the brain (
). In addition, all the signaling pathways that mediate insulin and IGF-1-stimulated neuronal survival, tau expression, energy metabolism, and mitochondrial function were perturbed in AD. This study carries additional significance because it established that, like all other pancreatic and intestinal polypeptide genes, the insulin gene was also expressed in the adult human brain. Moreover, the results taught us that endogenous brain deficiencies in insulin, IGF-1, IGF-2, and their corresponding receptors, in the absence of T2DM or obesity, could be linked to the most common form of dementia-associated neurodegeneration in the Western hemisphere. Since the abnormalities identified in the brain were quite similar to the effects of T1DM or T2DM (though none of the patients had either of these diseases), including abnormalities in IGFs,81–83
which are important for islet cell function,84,85
we proposed the concept that AD may represent a brain-specific form of diabetes mellitus and coined the term “type 3 diabetes.”
Figure 1. Impaired insulin and IGF (A, C) receptor and (B, D) polypeptide gene expression in late/end-stage AD (A, B) temporal cortex and (C, D) hippocampus.5 Gene expression was measured by qRT-PCR using RNA isolated from the temporal cortex or hippocampus from (more ...)
Even before the initial study had been published, it was realized that if brain insulin/IGF resistance and insulin/IGF deficiency were causal in the pathogenesis of AD, the related abnormalities should be detectable in the early stages of disease and possibly worsen as disease progresses. The investigations were extended to examine the brains of patients with different degrees, i.e., Braak stages,86,87
In that study, we measured the expression of genes encoding insulin, IGF-1, IGF-2 polypeptides, and their corresponding receptors as well as tau and amyloid precursor protein (APP). In addition, we used competitive equilibrium and saturation binding assays to further characterize the degree to which growth factor-transmitted signaling was impaired in the brains with different severities of AD. Finally, the study included the measurement of steady-state levels of adenosine triphosphate and genes regulating acetylcholine homeostasis and energy metabolism.
Using the previously mentioned approaches, we demonstrated progressive AD Braak stage-dependent reductions in insulin, IGF-1, and IGF-2 receptor expression, with more pronounced deterioration in insulin and IGF-1 compared with IGF-2 receptors, and the lowest levels of gene expression in brains with AD Braak Stage 6 (). Therefore, loss of insulin and IGF-1 receptor-bearing neurons begins early and progresses with disease such that, in the advanced stages, the deficits are severe and global. These results provided further evidence that the abnormalities in AD are not restricted to insulin signaling pathways, as they also involve IGF-1 and IGF-2 stimulated mechanisms. Analysis of growth-factor polypeptide genes also revealed AD Braak stage-dependent impairments in insulin, IGF-1, and IGF-2 polypeptide expression, corresponding with progressive trophic factor withdrawal (). Again, the results support the hypothesis that abnormalities in insulin and IGF signaling mechanisms begin early in the course of AD and are therefore likely have an important role in its pathogenesis.
Figure 2. Brain insulin and IGF deficiency and resistance increase with progression of AD.10 Postmortem histopathological studies categorized the brains as having normal aging (Braak 0–1), or mild (Braak 2–3), moderate to severe (Braak 4–5), (more ...)
The eventual paucity of local growth-factor gene expression could substantially impair growth-factor signaling and produce a state of growth-factor withdrawal, which is a well-established mechanism of neuronal death. Therefore, to complement the molecular data, we performed competitive equilibrium and saturation binding assays to determine if reduced levels of growth factor receptor expression were associated with and perhaps mediated by impaired ligand-receptor binding as occurs with insulin/IGF resistance. Those investigations demonstrated progressive declines in equilibrium () and top-level binding (Bmax) to the insulin, IGF-1, and IGF-2 receptors but either unchanged or increased binding affinity, suggesting that impaired insulin/IGF actions in AD brains were mediated by decreased polypeptide and receptor gene expression due to cell loss.
Through a series of in vitro
and in vivo
experiments performed by several groups, including our own, we have been able to draw the conclusion that neuronal and oligodendroglial cell survival and function are integrally related to the integrity of insulin and IGF signaling mechanisms in the brain.10,28,29,31,33,88,89
Similarly, impairments in insulin/IGF signaling lead to deficits in energy metabolism with attendant increased oxidative stress, mitochondrial dysfunction, proinflammatory cytokine activation, and APP expression.4,10,28,89
Correspondingly, the reduced expression of neuronal and oligodendroglial specific genes and the increased expression of astrocytic and microglial inflammatory genes in AD were attributed to progressive brain insulin/IGF deficiency and resistance. Although this point requires the generation of experimental models to demonstrate proof of principle, the finding that microglial, astrocytic, and APP mRNA levels are all increased in the early stages of neurodegeneration supports the inflammatory hypothesis of AD.6
Previous studies demonstrated that microglial activation promotes APP-Aβ accumulation90–92
and that APP gene expression and cleavage increase with oxidative stress.93
Therefore, the mechanism we propose is that impaired insulin/ IGF signaling leads to increased oxidative stress and mitochondrial dysfunction,32,94,95
which induces APP gene expression and cleavage.93
The attendant APP-Aβ accumulations cause local neurotoxicity96–98
and further increase in oxidative stress-induced APP expression and APP-Aβ deposition.
A critical goal in these investigations was to draw connections between brain insulin/IGF deficiency and resistance and the major dementia-associated structural and biochemical abnormalities in AD. In this regard, the postmortem studies demonstrated that the Braak stage-associated declines in tau mRNA paralleled the progressive reductions in insulin and IGF-1 receptor expression in AD. In addition, the studies demonstrated AD Braak stage-associated declines in choline acetyltransferase (ChAT) expression with reduced colocalization of ChAT with insulin or IGF-1 receptor immunoreactivity in cortical neurons. These results correspond with experimental data demonstrating that neuronal tau and ChAT gene expression are regulated by IGF-1 and insulin stimulation.88
Therefore, brain insulin and IGF deficiency and resistance could account for the cytoskeletal collapse, neurite retraction, synaptic disconnection, loss of neuronal plasticity, and deficiencies in acetylcholine production, all of which correlate with cognitive decline and dementia in AD. Altogether, the studies utilizing postmortem human brain tissue provide solid evidence that AD is associated with fundamental abnormalities in insulin/IGF signaling mechanisms that are highly correlated with development and progression of structural, molecular, and biochemical lesions that correlate with dementia. Although the abnormalities noted in AD share features in common with T1MD and T2MD, they are nonetheless distinguished by the dual presence of trophic factor deficiencies and trophic factor receptor resistance, ergo the term “type 3 diabetes.”