A casein hydrolysate based formulation attenuates obesity and associated non-alcoholic fatty liver disease and atherosclerosis in LDLr-/-.Leiden mice

Abstract

Background: Obesity frequently associates with the development of non-alcoholic fatty liver disease (NAFLD) and atherosclerosis. Chronic inflammation in white adipose tissue (WAT) seems to be an important driver of these manifestations.

Objective: This study investigated a combination of an extensively hydrolyzed casein (eHC), docosahexaenoic acid (DHA), arachidonic acid (ARA), and Lactobacillus Rhamnosus GG (LGG) (together referred to as nutritional ingredients, NI) on the development of obesity, metabolic risk factors, WAT inflammation, NAFLD and atherosclerosis in high-fat diet-fed LDLr-/-.Leiden mice, a model that mimics disease development in humans.

Methods: LDLr-/-.Leiden male mice (n = 15/group) received a high-fat diet (HFD, 45 Kcal%) for 21 weeks with or without the NI (23.7% eHC, 0.083% DHA, 0.166% ARA; all w/w and 1x109 CFU LGG gavage 3 times/week). HFD and HFD+NI diets were isocaloric. A low fat diet (LFD, 10 Kcal%) was used for reference. Body weight, food intake and metabolic risk factors were assessed over time. At week 21, tissues were analyzed for WAT inflammation (crown-like structures), NAFLD and atherosclerosis. Effects of the individual NI components were explored in a follow-up experiment (n = 7/group).

Results: When compared to HFD control, treatment with the NI strongly reduced body weight to levels of the LFD group, and significantly lowered (P<0.01) plasma insulin, cholesterol, triglycerides, leptin and serum amyloid A (P<0.01). NI also reduced WAT mass and inflammation. Strikingly, NI treatment significantly reduced macrovesicular steatosis, lobular inflammation and liver collagen (P<0.05), and attenuated atherosclerosis development (P<0.01). Of the individual components, the effects of eHC were most pronounced but could not explain the entire effects of the NI formulation.

Conclusions: A combination of eHC, ARA, DHA and LGG attenuates obesity and associated cardiometabolic diseases (NAFLD, atherosclerosis) in LDLr-/-.Leiden mice. The observed reduction of inflammation in adipose tissue and in the liver provides a rationale for these comprehensive health effects.

Fig 1. Effects of nutritional ingredients on body composition and food intake in LDLr-/-.Leiden mice.

Changes in Body weight over time (A) and average daily food intake (B). Data are presented as mean ± SEM, n = 15. Significant diet effects are shown by *P<0.05 or **P<0.01 compared to HFD. LFD = Low Fat Diet; HFD = High Fat Diet + control gavage; HFD + nutritional ingredients (including eHC, ARA, DHA, and gavage with LGG).

Fig 2. Effects of nutritional ingredients on cardiovascular risk factors in LDLr-/-.Leiden mice.

Plasma levels at 21 weeks of dietary intervention for cholesterol (A), triglycerides (B), glucose (C), insulin (D). Values are presented as mean ± SEM, n = 15. Significant diet effects are shown by *P<0.05 or **P<0.01 compared to HFD. LFD = Low Fat Diet; HFD = High Fat Diet + control gavage; HFD + nutritional ingredients (including eHC, ARA, DHA, and gavage with LGG).

Fig 3. Effects of nutritional ingredients on markers of systemic inflammation and vascular activation in LDLr-/-.Leiden mice.

Plasma levels of SAA (A) and VCAM-1 (B) at 21 weeks of dietary exposure. Data are presented as mean ± SEM, n = 15 Significant diet effects are shown by **P<0.01 or ^#P = 0.06 compared to HFD. LFD = Low Fat Diet; HFD = High Fat Diet + control gavage; HFD + nutritional ingredients (including eHC, ARA, DHA, and gavage with LGG).

Fig 4. Effects of nutritional ingredients on adipose tissue quantity and quality in LDLr-/-.Leiden mice.

Body composition analysis at 21 weeks of feeding regimes with fat and lean mass (A), with weights of epididymal fat, mesenteric fat and inguinal fat depots (B), representative histology of epidydimal WAT with inflammatory cells forming crown like structures (CLS, black arrows) (C) and quantitative analysis of CLS in different adipose tissue depots (D) (C). Data are presented as mean ± SEM, n = 15. Significant diet effects are shown by **P<0.01 compared to HFD. LFD = Low Fat Diet; HFD = High Fat Diet + control gavage; HFD + nutritional ingredients (including eHC, ARA, DHA, and gavage with LGG).

Fig 5. Liver integrity and non-alcoholic fatty liver disease in LDLr-/-.Leiden mice.

Liver mass at 21 weeks of feeding regimes (A), alanine aminotransferase (ALAT) over time (B), liver macrovascular steatosis (C) and lobular inflammation (D). Representative Sirius Red staining of liver (E) and quantitative biochemical analysis of hepatic total collagen (F) at 21 weeks of dietary interventions. Data are presented as mean ± SEM, n = 15. Significant diet effects are shown by *P<0.05 or **P<0.01. Low Fat Diet; HFD = High Fat Diet + control gavage; HFD + nutritional ingredients (including eHC, ARA, DHA, and gavage with LGG).

Fig 6. Vasculoprotective effects of nutritional ingredients in LDLr-/-.Leiden mice.

Albuminurea at 15 weeks (A) and Atherosclerosis (B) at 21 weeks of dietary interventions. Data are presented as mean ± SEM, n = 15. Significant diet effects are shown by *P<0.05 or **P<0.01 compared to HFD. Low Fat Diet; HFD = High Fat Diet + control gavage; HFD + nutritional ingredients (including eHC, ARA, DHA, and gavage with LGG).