Enteric lactoferrin attenuates the development of high-fat and high-cholesterol diet-induced hypercholesterolemia and atherosclerosis in Microminipigs

<div><p>Previously, we found that enteric lactoferrin (eLF) could reduce the visceral fat accumulation known to associate strongly with metabolic syndrome symptoms and consequently with an increased risk of atherosclerosis. In this study, the atherosclerosis-preventive potential of LF was assessed in a high-fat and high-cholesterol diet (HFCD)-induced hypercholesterolemia and atherosclerosis model using Microminipig™. Eight-week orally administered eLF remarkably reduced the HFCD-induced serum total and low-density lipoprotein cholesterol levels but not high-density lipoprotein cholesterol levels. A histological analysis of 15 arteries revealed that eLF systemically inhibited the development of atherosclerotic lesions. Pathway analysis using identified genes that characterized eLF administration in liver revealed significant changes in the steroid biosynthesis pathway (ssc00100) and all affected genes in this pathway were upregulated, suggesting that cholesterol synthesis inhibited by HFCD was recovered by eLF. In summary, eLF could potentially prevent the hypercholesterolemia and atherosclerosis through protecting homeostasis from HFCD-induced dysfunction of cholesterol metabolism.</p></div

Sequential sections subjected to immunohistochemical staining of hyperphosphorylated tau (AT8), 3R-tau, and 4R-tau in the CA1 region of a neonate brain (A), an adult brain without AT8-positive aggregates (B), and an adult brain with AT8-positive aggregates (C). (A) Only the 3R-tau isoform was expressed in the brain of the neonatal leopard cat.

<p>(B, C) In the adult brains, both the 3R-tau and 4R-tau isoforms were expressed regardless of the presence or absence of AT8-positive aggregates. Bar = 100 µm.</p

Immunohistochemical scoring of Aβ42 and AT8.

<p>Aβ42 deposition, −: none, +: diffuse deposition in the cerebral cortex,</p><p>++: diffuse deposition in the cerebral cortex and hippocampus,</p><p>+++: additional speckled deposits. NFT, −: none, +:a few AT8-positive cells were found in the parahippocampal gyrus, ++: AT8-positive cells were found in the parahippocampal gyrus and hippocampal CA1 region,</p><p>+++: in addition to the parahippocampal gyrus and hippocampal CA1 region, AT8-positive cells were also found in the ectosylvian gyrus and hippocampal CA3 region.</p>a<p>Male;</p>b<p>Female;</p>c<p>Age unknown.</p

Senile plaque and neurofibrillary tangle formation in humans, leopard cats, and rodents.

<p>Senile plaque and neurofibrillary tangle formation in humans, leopard cats, and rodents.</p

Double immunofluorescence staining of hyperphosphorylated tau (AT8)/GSK-3β (A), AT8/Phospho-GSK-3β (Ser9) (B), AT8/ubiquitin (C), and hyperphosphorylated tau (AT100)/tau (D).

<p>(A, B) AT8 colocalized with GSK-3β but not with phospho-GSK-3β (Ser9). Bars = 100µm. (C) Granular staining of ubiquitin was observed in the hyperphosphorylated tau-positive neurons. Bar = 50 µm. (D) AT100 colocalized with aggregated tau. Bar = 50 µm.</p

Primary antibodies used in the present study.

<p>PHF-tau, paired helical filament tau; MAP2, microtubule-associated protein; GFAP, glial fibrillary acidic protein; Olig2, oligodendrocyte transcription factor 2; GSK-3β, glycogen synthase kinase 3 beta.</p

Electron micrographs of NFT.

<p>(A) Some neuronal somata and neurites were filled with filamentous bundles (black arrows). Bar = 1 µm. (B, C) The filaments formed paired structures with diameters of 10–20 nm. Straight laminar filaments (white arrowheads) and constrictions (white arrows) suggesting helical structure. Bar = 50 nm.</p

Double immunofluorescence staining of hyperphosphorylated tau (AT8)/MAP2 (A), AT8/GFAP (B), and AT8/Olig2 (C).

<p>Hyperphosphorylated tau was mainly localized in neuronal cells (MAP2+) and a few oligodendrocytes (Olig2+) (A, C), but not in astrocytes (GFAP+) (B). Bars = 100 µm.</p

Sequential sections subjected to immunohistochemical examinations of Aβ and tau protein expression in the cerebral cortex.

<p>Sequential sections subjected to immunohistochemical examinations of Aβ and tau protein expression in the cerebral cortex.</p

Nucleic acid and amino acid sequences of leopard cat and human Aβ region.

<p>In humans, the 7th amino acid residue of the Aβ peptide is aspartic acid (D), while in leopard cats it is glutamic acid (E).</p