On a chow diet, adult Gpihbp1−/−
mice have plasma triglyceride and cholesterol levels of ~3000–5000 mg/dl and ~300–500 mg/dl, respectively.7
mice were switched from the chow diet to a “western” diet containing 20% milk fat and 0.15% cholesterol, the hyperlipidemia worsened. After one week on the western diet, the plasma triglycerides were 20,000–25,000 mg/dl, more than fourfold higher than baseline levels on a chow diet (). Also, the plasma cholesterol levels increased to >1,700 mg/dl (). At that time point, the whole blood of Gpihbp1−/−
mice was pink; the plasma was cream-colored; and the blood vessels on the surface of the heart were white (). After consuming the western diet for 4–8 weeks, the plasma triglyceride levels in Gpihbp1−/−
mice fell to ~10,000–12,000 mg/dl ().
Higher plasma triglyceride levels and gene perturbations in Gpihbp1−/− mice on a western diet containing ezetimibe
Despite the severity of the hyperlipidemia, >90% of Gpihbp1−/− mice on the western diet appeared healthy, although they gained less weight than wild-type littermates (Gpihbp1+/+) () and exhibited reduced adiposity (). These phenotypes were not due to ill health. Food intake was actually higher in Gpihbp1−/− mice (), and activity levels were also increased (). A small percentage of Gpihbp1−/− mice stopped consuming food and water during the first 14 days on the western diet and were euthanized; those mice invariably had typhlitis ().
Effects of an LPL–V5 adenovirus on plasma triglyceride levels in Gpihbp1−/− and Gpihbp1+/+ mice
The cholesterol content of the diet modulates plasma triglyceride levels in Gpihbp1−/− mice
To test the impact of cholesterol intake on plasma triglyceride levels and Lpl
expression in the liver, we fed 8-week-old Gpihbp1−/−
mice a western diet containing 0.005% ezetimibe (a dose that inhibits intestinal cholesterol absorption in mice19
). We predicted that reduced cholesterol absorption would lower Lpl
expression in the liver and lead to higher plasma triglyceride levels. Indeed, the plasma triglyceride levels were significantly higher in ezetimibe-treated Gpihbp1−/−
= 0.0034, as judged by a repeated measures paired t
test) (). The plasma cholesterol levels were significantly lower in ezetimibe-treated Gpihbp1−/−
mice at the 1-week time point (P
< 0.001) and tended to be lower at the subsequent time points (). At each time point, the ratio of triglycerides to cholesterol in the plasma was significantly higher (P
= 0.0046) in ezetimibe-treated mice than in untreated mice (). The plasma triglycerides in wild-type (Gpihbp1+/+
) mice consuming the western diet for 4 weeks were very low (~15 mg/dl); as expected, ezetimibe treatment had no effect on triglyceride levels ().
Gene-expression perturbations in Gpihbp1−/− and Gpihbp1+/+ mice on a western diet containing either high (1.3%) or low (0.05%) levels of cholesterol
The higher triglyceride levels in ezetimibe-treated Gpihbp1−/−
mice were accompanied by significantly lower levels of Lpl
expression in the liver (measured after 13 weeks of diet) (); however, ezetimibe had no effect on Lpl
transcript levels in heart, skeletal muscle, and adipose tissue (). As expected from earlier studies,20
ezetimibe treatment increased HMG-CoA reductase (Hmgcr
) expression in the liver (). In addition to reducing Lpl
expression in the liver, ezetimibe reduced expression of three additional LXR-regulated genes in the liver—Abca1, Abcg5,
Our presumption was that LPL expression in the liver would lower plasma triglyceride levels, even in the absence of GPIHBP1, because the fenestrations in the sinusoidal capillaries would allow access of LPL to lipoproteins in the bloodstream. To test this idea, we injected either an adenovirus encoding either V5-tagged LPL (LPL–V5) or V5-tagged β-galactosidase (LacZ–V5) into Gpihbp1−/− and control mice. Within 2 days of injection, the plasma triglyceride levels in Gpihbp1−/− mice injected with the LPL–V5 adenovirus fell by 90% (P < 0.0001 compared to mice given the LacZ–V5 adenovirus) (). This difference was also evident 4 h after delivering 75 μl of soybean oil by gavage (P < 0.0001). The plasma triglyceride levels in wild-type mice given the LPL–V5 adenovirus tended to be lower, but the changes were not statistically significant (). In control experiments, we found that both adenoviruses yielded high levels of protein expression (). Neither adenovirus increased transaminase levels in the plasma.
Effects of a western diet containing either high (1.3%) or low (0.05%) levels of cholesterol on the livers of Gpihbp1−/− mice
To gain further insights into the relationship between cholesterol intake and plasma triglyceride levels in Gpihbp1−/− mice, we examined plasma lipid levels in 8-week-old Gpihbp1−/− mice consuming western diets containing either low (0.05%) or high (1.3%) levels of cholesterol. The plasma triglyceride levels in mice on the low-cholesterol diet were 20,000–25,000 mg/dl, higher than on the high-cholesterol diet (P = 0.0015). On the high-cholesterol diet, the triglyceride levels initially increased to ~15,000 mg/dl but then fell to ~6000–7000 mg/dl (). At the 1-week time point, the plasma cholesterol levels in mice on the high-cholesterol diet were higher than in mice on the low-cholesterol diet (). However, at the 2-, 3-, and 4-week time points, the cholesterol levels were lower in mice on the high-cholesterol diet (P = 0.0129, as judged by a repeated measures paired t test) (). In wild-type mice, the high-cholesterol diet had little or no effect on plasma triglyceride levels () but resulted in slightly higher plasma cholesterol levels (P = 0.0086) ().
The lower plasma triglyceride levels in Gpihbp1−/−
mice on the high-cholesterol diet were accompanied by significantly higher Lpl
expression levels in the liver after 5 weeks on the diet (). Dietary cholesterol did not affect Lpl
expression in heart, skeletal muscle, or adipose tissue (). The high-cholesterol diet also increased expression of Abca1
8 in the liver (). The expression of Cyp7a1
, an LXR-regulated gene21
that controls bile acid synthesis, was also increased on the high-cholesterol diet, but two FXR target genes, Bsep
, were unaffected (). The expression of Apoe
was slightly but significantly increased in the livers of Gpihbp1−/−
mice fed the high-cholesterol western diet ().
Hepatic steatosis in Gpihbp1−/− mice on a western diet containing high levels of cholesterol, and protection from steatosis with ezetimibe treatment
We suspected that changing mice from a chow to a western diet would gradually increase Lpl expression levels in the liver [explaining the fall in plasma lipids that occurs in Gpihbp1−/− mice after one week on the western diet with 0.15% cholesterol () and the western diet with 1.3% cholesterol ()]. Indeed, after three days on the western diet, the hepatic Lpl expression levels are higher than baseline levels on the chow diet (). The hepatic Lpl expression levels were even higher after 21 days on the western diet ().
We predicted that higher levels of Lpl expression in livers of Gpihbp1−/− mice on the high-cholesterol western diet might lead to increased lipid stores within the liver. Indeed, after only 5 weeks on diet, liver mass was significantly higher (P < 0.001) in Gpihbp1−/− mice on the high-cholesterol diet than in Gpihbp1−/− mice fed the low-cholesterol diet (). Furthermore, as judged by nuclear magnetic resonance, the lipid content of the liver in Gpihbp1−/− mice on the high-cholesterol diet was significantly higher than in mice fed the low-cholesterol diet (). In line with those findings, the livers of Gpihbp1−/− mice on the high-cholesterol diet contained higher levels of triglycerides () and cholesterol (). The high-cholesterol diet was not accompanied by significant increases in the expression of acyl-CoA carboxylase (Acc), fatty acid synthase (Fas), and sterol regulatory element binding protein 1C (Srebp-1c), either in Gpihbp1−/− or wild-type mice ().
Also, livers of Gpihbp1−/− mice maintained on the 1.3% cholesterol western diet for 5 weeks exhibited steatosis, as judged by hematoxylin and eosin staining (). In contrast, mice fed the same western diet with 0.05% cholesterol had no evidence of steatosis (). Gpihbp1−/− mice maintained on the 0.15% cholesterol western diet for three months also exhibited hepatic steatosis (), but mice maintained on the same diet with 0.005% ezetimibe had no steatosis ().