High Fat Diet Impairs Insulin Receptor-Mediated Insulin Uptake by the Brain Endothelium

Background: Insulin acts on the brain to regulate satiety, body weight, glucose and lipid metabolism, and may have a role in treating neurodegenerative disorders. Despite its importance, little is known of how insulin reaches brain interstitial fluid to act on target neurons. Our previous work demonstrated insulin gains rapid access to brain tissue despite its slow entry into cerebrospinal fluid, suggesting insulin is transported across the blood-brain barrier (BBB). The mechanisms that regulate this transport and how they may be altered by insulin resistance remain unknown. We hypothesized that the BBB endothelial cells (ECs) have an insulin receptor (IR)-mediated vesicular transport system and that high-fat diet (HFD)-induced insulin resistance could interfere with this process.  Methods: Male Sprague-Dawley rats were given ad libitum access to normal chow (ND) or HFD (60% fat) for 4 weeks before brain ECs were isolated and cultured for 5-7 days. ECs were serum starved and treated with ¹²⁵I-insulin (¹²⁵I-ins, 200 pM) for 15 min before ECs were lysed and radioactivity was quantified. Insulin-stimulated signaling was determined by treating cells with 10 nM insulin and quantifying phosphorylation of key proteins via western blot. Expression of the IR was determined via RT-PCR and western blot. To examine the pathway of insulin uptake, we used purchased rat brain microvascular ECs (RBMVECs, Cell Applications) and measured the time course of insulin uptake and sensitivity to agents interfering with insulin signaling or lipid raft formation. Results: In RBMVECs, ¹²⁵I-ins uptake reached a plateau after 15 min and was completely blocked by the IR-specific antagonist S-961 (p<0.001), but not by an IGF-I receptor neutralizing antibody (Ab-3). Inhibiting insulin signaling pathways with a PI3-kinase inhibitor (wortmannin) or MEK inhibitor (PD90859) did not significantly decrease ¹²⁵I-ins uptake. Depleting cholesterol from lipid rafts with methyl-β-cyclodextran or filipin decreased EC ¹²⁵I-ins uptake (p<0.05, each). ECs isolated from rats fed HFD had decreased ¹²⁵I-ins uptake (p<0.01) compared to chow-fed rats, despite having similar insulin-stimulated phosphorylation events. IR mRNA expression did not differ, but IR-β protein tended to be greater in ECs from HFD rats (p=0.057). Conclusions: HFD feeding compromises insulin transport in brain ECs despite increasing IR-β content. This transport is mediated by the IR and lipid rafts and does not involve PI3-kinase or MEK. Future work will elucidate the mechanisms by which HFD impairs insulin uptake in brain ECs.