Reduced absorption of saturated fatty acids and resistance to diet-induced obesity and diabetes by ezetimibe-treated and Npc1l1-/- mice

Abstract

The impact of NPC1L1 and ezetimibe on cholesterol absorption are well documented. However, their potential consequences relative to absorption and metabolism of other nutrients have been only minimally investigated. Thus studies were undertaken to investigate the possible effects of this protein and drug on fat absorption, weight gain, and glucose metabolism by using Npc1l1(-/-) and ezetimibe-treated mice fed control and high-fat, high-sucrose diets. Results show that lack of NPC1L1 or treatment with ezetimibe reduces weight gain when animals are fed a diabetogenic diet. This resistance to diet-induced obesity results, at least in part, from significantly reduced absorption of dietary saturated fatty acids, particularly stearate and palmitate, since food intake did not differ between groups. Expression analysis showed less fatty acid transport protein 4 (FATP4) in intestinal scrapings of Npc1l1(-/-) and ezetimibe-treated mice, suggesting an important role for FATP4 in intestinal absorption of long-chain fatty acids. Concomitant with resistance to weight gain, lack of NPC1L1 or treatment with ezetimibe also conferred protection against diet-induced hyperglycemia and insulin resistance. These unexpected beneficial results may be clinically important, given the focus on NPC1L1 as a target for the treatment of hypercholesterolemia.

Fig. 1.

Ezetimibe treatment or Niemann-Pick type C-1 like protein 1 (NPC1L1) gene inactivation prevents diet-induced weight gain. A: body weight of control mice fed diabetogenic diet with or without added ezetimibe and of Npc1l1^−/− mice fed diabetogenic diet was measured for several weeks (n = 4–6 per group). B: data expressed as change from starting weight over time. Differences between control mice fed diabetogenic diet and all other groups are significant at 3 wk and all times later.

Fig. 2.

Dietary fat and cholesterol absorption are decreased in ezetimibe-treated and Npc1l1^−/− mice. Cholesterol absorption (left, n = 3–4) was measured by the dual-isotope method and fat absorption (right, n = 4–5) was measured by the sucrose polybehenate method as described in materials and methods. Mice were fed basal chow. *P < 0.001 compared with controls; **P = 0.033, ***P = 0.025 compared with controls.

Fig. 3.

Western blot analysis of proteins potentially important for fatty acid absorption. The protein from mucosal scrapings of the small intestine from control, ezetimibe-fed, and Npc1l1^−/− mice were subjected to Western blot for analysis of expression of fatty acid transport protein 4 (FATP4), liver fatty acid binding protein (L-FABP), CD36, and NPC1L1. L-FABP was assayed in cytosolic fractions; all others were assayed in total cell membrane fractions. Mice were fasted 12 h before sample collection. Each blot is representative of at least 3 independent sample collections.

Fig. 4.

Ezetimibe-treated and Npc1l1^−/− mice are protected from diet-induced glucose intolerance. Mice were subjected to a glucose tolerance test while fed basal chow (dotted lines, open symbols) and again after 10 wk of diabetogenic diet (solid lines, solid symbols). After baseline glucose was measured, tests were initiated by intraperitoneal injection of 2 g glucose/kg body wt. Blood glucose was monitored from the tail vein for 2 h thereafter (n = 6–8 per group). *P < 0.05 control basal vs. control diabetogenic, **P < 0.05 control diabetogenic vs. ezetimibe diabetogenic and Npc1l1^−/− diabetogenic.

Fig. 5.

Npc1l1^−/− and ezetimibe-treated mice are protected from diet-induced loss of insulin sensitivity. A: control and Npc1l1^−/− mice were subjected to an insulin tolerance test after 6 wk of Western diet. After baseline glucose was measured, animals were given insulin (0.75 U/kg) by intraperitoneal injection and glucose was monitored for 2 h thereafter (n = 8–10 per group). Groups were significantly different, *P < 0.01, at all times after time 0. B: insulin sensitivity was similarly measured in different groups of mice fed Western diet with or without ezetimibe for 3 wk (n = 8 per group). Groups were significantly different, *P < 0.02, at 15 and 30 min.