Prospective observational study and mechanistic evidence showing lipolysis of circulating triglycerides worsens hypertriglyceridemic acute pancreatitis

Abstract

BACKGROUNDWhile most hypertriglyceridemia is asymptomatic, hypertriglyceridemia-associated acute pancreatitis (HTG-AP) can be more severe than AP of other etiologies. The reasons underlying this are unclear. We thus examined whether lipolytic generation of nonesterified fatty acids (NEFAs) from circulating triglycerides (TGs) could worsen clinical outcomes.METHODSAdmission serum TGs, NEFA composition, and concentrations were analyzed prospectively for 269 patients with AP. These parameters, demographics, and clinical outcomes were compared between HTG-AP (TGs >500 mg/dL; American Heart Association [AHA] 2018 guidelines) and AP of other etiologies. Serum NEFAs were correlated with serum TG fatty acids (TGFAs) alone and with the product of TGFA serum lipase (NEFAs - TGFAs × lipase). Studies in mice and rats were conducted to understand the role of HTG lipolysis in organ failure and to interpret the NEFA-TGFA correlations.RESULTSPatients with HTG-AP had higher serum NEFA and TG levels and more severe AP (19% vs. 7%; P < 0.03) than did individuals with AP of other etiologies. Correlations of long-chain unsaturated NEFAs with corresponding TGFAs increased with TG concentrations up to 500 mg/dL and declined thereafter. However, NEFA - TGFA × lipase correlations became stronger with TGs above 500 mg/dL. AP and intravenous lipase infusion in rodents caused lipolysis of circulating TGs to NEFAs. This led to multisystem organ failure, which was prevented by pancreatic TG lipase deletion or lipase inhibition.CONCLUSIONSHTG-AP is made severe by the NEFAs generated from intravascular lipolysis of circulating TGs. Strategies that prevent TG lipolysis may be effective in improving clinical outcomes for patients with HTG-AP.FUNDINGNational Institute of Diabetes and Digestive and Kidney Diseases (NIDDK, NIH) (RO1DK092460 and R01DK119646); Department of Defense (PR191945 under W81XWH-20-1-0400); National Institute on Alcohol Abuse and Alcoholism (NIAAA), NIH (R01AA031257).

Keywords: Gastroenterology; Lipoproteins.

Figure 1. Flowcharts comparing the conventional hypothesis to the new findings and showing the design for patient selection in the current study.

(A) Schematic of conventional hypothesis of severity of HTG pancreatitis compared with new findings. (B) Flowchart of patient selection for the study population of patients with AP and classification as HTG-AP versus non–HTG AP.

Figure 2. Parameters of mice with HTG alone or HTG-AP, those given NEFAs, and in vitro studies using pancreatic acini.

(A–H) Mice were given poloxamer-407 on day –1 to induce HTG (H) alone (blue). HTG-AP (HAP) was induced on day 0 (i.e., 1 day after poloxamer-407) in C57bl/6 WT mice (red) or pancreatic TG lipase–KO mice (PTL KO) (green dots). Dot plots with means are shown for serum lipase (A), serum TGs (B), and serum NEFAs (C) for the days indicated on the x axis. Serum creatinine at necropsy (D), along with carotid pulse distention (E) and rectal temperature (F) recorded before euthanasia are shown. (G) Comparison of the survival curves for the 2 groups with P values based on the log-rank (Mantel-Cox) test. (H and I) Images of H&E-stained pancreatic sections from WT mice (H) and PTL-KO mice (I) with HAP. Scale bars: 200 μm. (J and K) Dot plots showing pancreatic necrosis (J) and pancreatic edema (K) in WT and PTL-KO mice with HAP. (L–N) Dot plots with means comparing the effects of administering linoleic acid (red dots) or palmitic acid (blue dots) on serum BUN levels at necropsy (L), along with carotid pulse distention (M) and rectal temperature (Temp) (N) recorded before euthanasia. P values were determined by 2-way ANOVA was done (A–F), log-rank (Mantel-Cox) test (G), Mann-Whitney U test (J), 2-tailed Student’s t test was done (K), and ordinary 1-way ANOVA (L–N).

Figure 3. Effect on rats of intravenous infusion of TGs versus infusion of TGs with PPL, with or without the lipase inhibitor orlistat.

Bar graphs (with SD and individual values) comparing the effects of infusion of PPL; TG (GTO); PPL plus TG; and PPL plus TG, plus the lipase inhibitor orlistat at baseline (Bas) and after infusion. Parameters are serum lipase (A), NEFAs (B), BUN (C), and ionized calcium (iCa) (D). (E) Representative images of H&E-stained images of pancreatic sections from rats infused with GTO alone or GTO plus PPL. Scale bars: 200 μm. (F–H) Plots of preterminal oxygen saturation (Ox sat) (F), LDH activity in BAL fluid from the lungs (G), and protein concentrations in the BAL (H). (I) Representative lung histologic images after H&E staining from each group mentioned at the top of the image. Scale bars: 100 μm. The rectangle inset is zoomed outside the ×20 image. In the PPL plus GTO group, arrows point to alveolar wall damage, and asterisks show fluid-filled alveoli. All data in graphs were compared by ordinary 1-way ANOVA with multiple comparisons.

Figure 4. Effect of lipase activity on the relationship between individual NEFAs and TGFAs in patients with pancreatitis.

(A) Schematic comparing the Kennedy pathway (left side, green background) by which NEFAs are physiologically incorporated into the TGs for storage (adipose, liver) or transport (intestine) versus the pathological release of NEFAs from intravascular TG lipolysis by pancreatic lipases during HTG-AP (red background on the right side). (B) Correlation of serum lipase activity with individual TGFAs for all patients. Each column shows a unique FA. The upper value shows the correlation coefficient, and the lower number the P value. (C) All patient data were formatted as in B. Middle row (dark gray background) correlates individual serum NEFAs with serum lipase; top row (white background) correlates individual NEFAs and their TGFA concentrations; and bottom row (white background) correlates individual NEFAs and the product of serum lipase × TGFA concentrations. The light gray rows show P values comparing the strength of correlations (COCOR as described in Methods) between the middle row and corresponding top or bottom rows. Those with a P value of less than 0.05 are shown in red. (D) Bar graphs of correlations (R values) arranged by serum TG concentrations (x axis) for individual FAs. Each graph is for a FA (mentioned above) and shows correlations of its NEFAs with corresponding TGFAs (back bars) or NEFAs with the product of the corresponding TGFA concentration × serum lipase (red bars). Asterisks show the bar with significantly stronger correlations versus normal TGs, i.e., TGs below 150 mg/dL. All correlations are Spearman correlations, and P values are 2 tailed. The comparison of COCOR correlations between 2 Spearman coefficients were done as described in Methods, and P values are shown. Asterisks in D indicate a COCOR P value of less than 0.05 versus the normal (<150 mg/dL) TG group.

Figure 5. Schematic showing mechanisms underlying severity of HTG-AP.

Illustration shows elevated TGs (3-limb structures in blue, red shown is within upper blood vessels) being cleaved by pancreatic lipases (green) into FAs during HTG-AP (middle vessel). The images, inset depict saturated FAs as blue lines, and unsaturated FAs as red. The unsaturated FAs cause vascular leakage, resulting in shock along with renal failure and lung injury, resulting in severe AP (lower vessel).