Acute dietary fat intake initiates alterations in energy metabolism and insulin resistance

Abstract

Background: Dietary intake of saturated fat is a likely contributor to nonalcoholic fatty liver disease (NAFLD) and insulin resistance, but the mechanisms that initiate these abnormalities in humans remain unclear. We examined the effects of a single oral saturated fat load on insulin sensitivity, hepatic glucose metabolism, and lipid metabolism in humans. Similarly, initiating mechanisms were examined after an equivalent challenge in mice.

Methods: Fourteen lean, healthy individuals randomly received either palm oil (PO) or vehicle (VCL). Hepatic metabolism was analyzed using in vivo 13C/31P/1H and ex vivo 2H magnetic resonance spectroscopy before and during hyperinsulinemic-euglycemic clamps with isotope dilution. Mice underwent identical clamp procedures and hepatic transcriptome analyses.

Results: PO administration decreased whole-body, hepatic, and adipose tissue insulin sensitivity by 25%, 15%, and 34%, respectively. Hepatic triglyceride and ATP content rose by 35% and 16%, respectively. Hepatic gluconeogenesis increased by 70%, and net glycogenolysis declined by 20%. Mouse transcriptomics revealed that PO differentially regulates predicted upstream regulators and pathways, including LPS, members of the TLR and PPAR families, NF-κB, and TNF-related weak inducer of apoptosis (TWEAK).

Conclusion: Saturated fat ingestion rapidly increases hepatic lipid storage, energy metabolism, and insulin resistance. This is accompanied by regulation of hepatic gene expression and signaling that may contribute to development of NAFLD.REGISTRATION. ClinicalTrials.gov NCT01736202.

Funding: Germany: Ministry of Innovation, Science, and Research North Rhine-Westfalia, German Federal Ministry of Health, Federal Ministry of Education and Research, German Center for Diabetes Research, German Research Foundation, and German Diabetes Association. Portugal: Portuguese Foundation for Science and Technology, FEDER - European Regional Development Fund, Portuguese Foundation for Science and Technology, and Rede Nacional de Ressonância Magnética Nuclear.

Conflict of interest statement

The authors have declared that no conflict of interest exists.

Figures

Figure 1. CONSORT flow diagram.

Forty-four patients underwent screening, which included a medical history, BMI analyses, and bioimpedance and an oral glucose tolerance tests. Of the 44 participants, 19 did not meet the inclusion criteria, 2 declined to participate, and 2 were excluded for other reasons. Twenty-one participants were allocated to receive the intervention, two of whom did not receive the allocated intervention. An additional 5 volunteers were excluded due to changes in inclusion and exclusion criteria (n = 2), as well as changes in the experimental procedure, which yielded data that could not be compared with subsequently acquired data (n = 3). Ultimately, data from 14 individuals were analyzed, except for hepatic lipid and energy analyses (n = 12) and some hepatic glucose flux measurements (n = 9).

Figure 2. Time courses of circulating metabolites and hormones in humans.

VCL (gray triangles) or PO (black circles) was administered at 0 minutes to lean, healthy men, and the hyperinsulinemic-euglycemic clamp was started at 360 minutes. TG circulating in plasma (solid line) and in chylomicrons (dashed line). The AUC was 59% and 156% higher after PO ingestion, respectively (A). Circulating FFA (B). Time courses for insulin (C), C-peptide (AUC 28% higher after PO) (D), blood glucose (E), and glucagon (AUC 41% higher after PO) (F). Values represent the mean ± SEM. n = 14; chylomicron TG n = 6. ***P < 0.001, **P < 0.005, and *P < 0.05, by paired, 2-tailed t test. Large asterisks refer to AUC differences; small asterisks refer to differences per time point.

Figure 3. Parameters of insulin resistance in human volunteers after VCL or PO during clamp experiments.

VCL, white bars; PO, black bars. (A) WBIS, reflected by the M value. (B) Rd and its components GOX and nonoxidative glucose disposal (NOXGD). (C) EGP denoting hepatic insulin sensitivity at baseline (–180 min), after PO or VCL ingestion (240 min), and under insulin-stimulated conditions during the clamp (480 min). (D) Insulin-induced EGP suppression as an indicator of hepatic insulin sensitivity. (E) Insulin-induced FFA suppression reflecting adipose tissue insulin sensitivity and (F) the percentage of insulin-induced TG suppression. Data shown represent the mean ± SEM. n = 14. ****P = 0.0005, **P < 0.01, and *P < 0.05; #P < 0.05, for GOX PO versus VCL. P values were determined by 2-tailed t test and ANOVA.

Figure 4. Hepatic glucose fluxes in humans.

The rates of GNG, GP flux, glycogen cycling (cycling), and GLYnet were analyzed using in vivo 13C/31P/1H and ex vivo 2H MRS combined with 2H2O ingestion, after either VCL (white bars) or PO (black bars) treatment, in lean, insulin-sensitive, male volunteers. Data represent the mean ± SEM. n = 14; GP and cycling n = 9. *P < 0.05 and #P = 0.085, by 2-tailed t test.

Figure 5. Time course of parameters of energy metabolism in lean, healthy volunteers after VCL and PO.

VCL, white bars; PO, black bars. Parameters were obtained by performing an indirect calorimetry. The time points indicated are basal (–5 min), 300 minutes after intervention, and 420 minutes under insulin-stimulated conditions. Effects on RQ (A), REE (B), LOX (C), and GOX (D). Data represent the mean ± SEM. n = 14. ***P < 0.001, **P < 0.005, and *P < 0.05, by ANOVA.

Figure 6. Circulating metabolites and hormones in mice.

VCL (white bars) or PO (black bars) was administered via gavage to identical mouse cohorts at minute 0, after a 6-hour fast. Hyperinsulinemic-euglycemic clamp experiments were performed from 120 to 240 minutes. (A) The TG AUC tended to be higher after PO than after VCL. (B) The FFA AUC was increased after PO administration. Blood glucose (C) and insulin (D) levels were not different between groups. Data represent the mean ± SEM. n = 6–10. **P < 0.005 and #P = 0.08, by 2-tailed t test.

Figure 7. Parameters of insulin resistance in mice.

VCL (white bars) or PO (black bars) was administered via gavage to identical mouse cohorts at minute 0, after a 6-hour fast. Hyperinsulinemic-euglycemic clamp experiments were performed from 120 to 240 minutes. The M value trended toward a reduction after PO (A), while the Rd was unchanged (B). EGP at basal (0 min) and at clamp steady state (210–240 min) (C). EGP suppression was impaired after PO (D). Insulin-induced FFA suppression (E) and TG suppression (F) are also shown. Data represent the mean ± SEM. n = 9–10. **P < 0.005, *P < 0.05, and #P = 0.07, by 2-tailed t test.

Figure 8. Transcriptome analysis after PO in murine hepatic tissue.

Tissue was harvested from clamped and nonclamped murine cohorts. (A) Relevant canonical pathways predicted by Ingenuity software in hepatic samples from mice after PO under insulin- (black bars) and noninsulin-stimulated (gray bars) conditions. (B) Predicted canonical pathways with a P value of less than 0.05 during insulin-stimulated (turquoise) and noninsulin-stimulated conditions (blue). (C) Noteworthy predicted upstream regulators and (D) predicted upstream regulators with a P value of less than 0.01 (asterisk denotes an activation Z score above 1.9). (E) Genes that were upregulated in both cohorts (green), downregulated in both cohorts (yellow), and downregulated under insulin-stimulated, but upregulated under noninsulin-stimulated, conditions (green and yellow, respectively) after PO treatment, with a P value of less than 0.05, a fold change greater than 1.3, and an average expression in at least 1 group of greater than 4 (n = 9–10, 273, and 327 probe sets, respectively).

Figure 9. Human study design.

Lean, healthy male adults randomly received either PO or VCL on 2 occasions. Hepatic metabolism was measured using in vivo 13C, 31P, 1H and ex vivo 2H MRS combined with 2H2O and acetaminophen ingestion before and during hyperinsulinemic-euglycemic clamps with D-[6,6-2H2]glucose–labeled 20% glucose infusion.

Figure 10. Mouse study design.

Lean, adult male C57BL/6NTac mice were matched for BM and littermates and then divided into 2 cohorts. One cohort underwent hyperinsulinemic-euglycemic clamps after receiving either PO or vehicle via gavage (A), whereas another identical cohort underwent analysis of tissue and blood samples (B).