High-fat diet induces hepatic insulin resistance and impairment of synaptic plasticity

Abstract

High-fat diet (HFD)-induced obesity is associated with insulin resistance, which may affect brain synaptic plasticity through impairment of insulin-sensitive processes underlying neuronal survival, learning, and memory. The experimental model consisted of 3 month-old C57BL/6J mice fed either a normal chow diet (control group) or a HFD (60% of calorie from fat; HFD group) for 12 weeks. This model was characterized as a function of time in terms of body weight, fasting blood glucose and insulin levels, HOMA-IR values, and plasma triglycerides. IRS-1/Akt pathway was assessed in primary hepatocytes and brain homogenates. The effect of HFD in brain was assessed by electrophysiology, input/output responses and long-term potentiation. HFD-fed mice exhibited a significant increase in body weight, higher fasting glucose- and insulin levels in plasma, lower glucose tolerance, and higher HOMA-IR values. In liver, HFD elicited (a) a significant decrease of insulin receptor substrate (IRS-1) phosphorylation on Tyr608 and increase of Ser307 phosphorylation, indicative of IRS-1 inactivation; (b) these changes were accompanied by inflammatory responses in terms of increases in the expression of NFκB and iNOS and activation of the MAP kinases p38 and JNK; (c) primary hepatocytes from mice fed a HFD showed decreased cellular oxygen consumption rates (indicative of mitochondrial functional impairment); this can be ascribed partly to a decreased expression of PGC1α and mitochondrial biogenesis. In brain, HFD feeding elicited (a) an inactivation of the IRS-1 and, consequentially, (b) a decreased expression and plasma membrane localization of the insulin-sensitive neuronal glucose transporters GLUT3/GLUT4; (c) a suppression of the ERK/CREB pathway, and (d) a substantial decrease in long-term potentiation in the CA1 region of hippocampus (indicative of impaired synaptic plasticity). It may be surmised that 12 weeks fed with HFD induce a systemic insulin resistance that impacts profoundly on brain activity, i.e., synaptic plasticity.

Fig 1. Characterization of the HFD-induced mouse model of obesity.

Mice were fed with 12-week HFD or normal diet and different parameters were monitored weekly or monthly. (A) Body weight; (B) Energy intake; (C) Fasting blood glucose concentration; (D) Fasting insulin concentration; (E) Insulin resistance index, HOMA-IR; (F) Fasting triglyceride levels; (G) Glucose tolerance test at 12 week; (H) Area under curve (AUC) analyses for glucose tolerance tests. Data presented as mean ± SD, n ≥ 3 mice/group, *p < 0.05, **p < 0.01 versus control group.

Fig 2. HFD led to impairment of IRS-1/Akt pathway and activation of inflammatory responses in primary hepatocytes.

Primary hepatocytes were isolated from mice fed a normal diet or HFD for 12 weeks (as described in the Materials and Methods section). (A) Representative western blots of IRS-1/Akt pathway proteins and the respective densitometry measurements; (B) Representative western blots of inflammatory proteins and the corresponding densitometry measurements. Data presented as mean ± SD, n ≥3 mice/group, *p < 0.05, ** p < 0.01 versus control group.

Fig 3. HFD-induced mitochondria dysfunction in primary hepatocytes.

Primary hepatocytes were isolated from mice fed a normal diet or HFD for 12 weeks as described in the Materials and Methods section. (A) Time course of Oxygen Consumption Rate (OCR), determined using XF-24 Extraflux Analyzer as described in the Materials and Methods section. (B) Quantification of OCR (bioenergetic) parameters in primary hepatocytes (from data in Fig 3A). (C) Effect of HFD on PGC1α expression (western blots and the corresponding densitometry analysis). (D) Effect of HFD on mitochondrial biogenesis (expressed as the ratio of COX2 (mitochondrial DNA) to globin (nuclear DNA)), measured by real-time PCR with specific primers. Data presented as mean ± SD, n ≥ 3 mice/group, *p < 0.05, **p < 0.01 versus control group.

Fig 4. HFD led to a decrease in the levels of membrane-associated glucose transporters.

Representative western blots of expression of neuronal glucose transporter 3 (GLUT3) and neuronal glucose transporter 4 (GLUT4). Quantification against a control for total membrane (Na⁺/K⁺ ATPase). Data presented as mean ± SD, n ≥ 3 mice/group, *p < 0.05 versus control group.

Fig 5. Effect of HFD on insulin signaling: IRS and ERK/CREB pathways.

(A) Western blots of the expression of phosphorylated IRS-1, total IRS-1, phosphorylated Akt, and total Akt. Densitometry analyses on the right of panel A. (B) Western blots pERK and ERK expression. Densitometry analysis on the right of panel B. (C) Western blots of pCREB and CREB and densitometry analysis on the right. Whole brain homogenates were used for (A) and (B) and nuclei extraction fractions for (C). Data presented as mean ± SD, n ≥ 3 mice/group, *p < 0.05, ** p < 0.01 versus control group.

Fig 6. HFD induced compromised hippocampal synaptic plasticity.

Input/Output (I/O) and LTP changes were measured in mice fed with normal chow or HFD. (A) I/O relationships after applying increasing stimulation to the stratum radiatum of the CA1 region in the hippocampus for control group mice and HFD group mice and recording the output (electrophysiology as described in the Materials and Methods section). fEPSP slope (mV/ms) plotted against the corresponding stimulation intensity of HFD group (open circles) and control group (close circles). Bar graphs showing the maximum (B) and minimum (C) fEPSP slope values at 400 μA, n ≥ 6 slices/group and at least 3–4 animals/group. (D) LTP was induced at baseline intensity using theta burst stimulation (TBS) consisting of ten trains of five 100 Hz stimulation repeated at 5 Hz. Slope of EPSPs was measured and results normalized to the average value measured during the 10 min baseline period. Recording continued for at least 30 min following TBS and the last 5 min was used to calculate the LTP. It shows the first 10 min of baseline followed by the percentage of the baseline response elicited after TBS for 30 min for control and HFD mice. (E) Bar graphs showing the measured LTP using % EPSP for the last 5 min of the response to TBS stimulation for control and HFD mice. Total n = 20 slices, n ≥ 9 slices/group and at least 3–4 animals/group. Data presented as mean ± SD, *p < 0.05, **p < 0.01 versus control group.