Background
Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease in the Western world [
1] and is closely associated with dyslipidemia [
2,
3]. Affecting more than 30 % of the general adult population and with the potential to progress from simple steatosis to irreversible and life-threatening non-alcoholic steatohepatitis (NASH), it is an important public health concern [
1,
4]. Disease progression is instigated by a series of parallel hits such as inflammation and oxidative stress, causing hepatocyte damage (e.g. metabolic dysfunction, DNA injury and apoptosis) and irreversible fibrosis, ultimately leading to cirrhosis and liver failure [
5]. Although the etiology of NAFLD is not yet fully elucidated, changes in food composition are believed to play an essential role in disease progression [
4]. Diets rich in saturated fat, cholesterol and non-complex carbohydrates (e.g. the disaccharide: sucrose) have been shown to induce dyslipidemia and hepatic lipid accumulation and are suggested to play a key role in the development of NASH in human patients [
6‐
9]. However, potential interactions of different dietary components and whether certain components are more likely to cause NAFLD has not been determined [
10]. Fat and cholesterol promotes oxidative stress, hepatocellular apoptosis, steatosis and NASH [
5]. Furthermore, previous results from our group have indicated that sucrose may affect the development of NAFLD in guinea pigs [
11]. Guinea pigs are one of the few species carrying the majority of their cholesterol in low density lipoprotein (LDL) particles; hence exhibiting a lipoprotein profile similar to that of humans [
12‐
14]. Furthermore, we and others have reported that guinea pigs subjected to long-term feeding with diets high in fat, cholesterol and sucrose develop hepatic steatosis in accordance with NAFLD [
11,
15,
16].
In the present study, we investigated the specific effects of dietary sucrose and fat/cholesterol, alone and in combination, on the development of dyslipidemia and NAFLD or NASH.
Discussion
The present study shows that dietary fat and cholesterol, but not sucrose, are the main factors driving the progression of dyslipidemia and NAFLD to NASH in a guinea pig model. Additionally, adding sucrose to a high-fat diet does not exacerbate the metabolic or hepatic consequences of a high-fat diet per se.
Expectedly, addition of cholesterol to the dietary regime increased circulating levels of TC, similar to findings of other studies utilizing cholesterol-rich [
25,
26] or high-fat diet [
27]. Though Plasma TC and TG concentrations were not affected by addition of sucrose consistent with results from mice and rats fed sucrose at levels of 32–35 % of total caloric intake [
28,
29], circulating levels of TC and TG upon sucrose feeding have also been reported [
30‐
33] It is possible that sucrose induced dyslipidemia differs mechanistically from the high fat induced [
31], and moreover very high levels (≥60 %) of sucrose are applied to induce dyslipidemia [
31‐
33]. Thus, it is possible that higher dietary sucrose concentrations may have been necessary to promote dyslipidemia in the current study; indeed calories originating from sucrose and fat were not equal potentially confounding the effect of sucrose. However, the translational relevance of models utilizing extremely high levels of sucrose have been questioned [
34]. Consequently, the levels of dietary sucrose in this study may have more relevance to human consumption, albeit still being high.
Decreased levels of hepatic LDL-receptors and concomitant increased levels of circulating TC and LDL-C has been reported in guinea pigs subjected to a high-fat diet [
35]. Accordingly, we found that LDL-C and VLDL-C increased upon high-fat feeding, regardless of dietary content of sucrose. This contradicts previous findings of a sucrose imposed elevation of TC and LDL-C when added to a high-fat diet in male guinea pigs [
13,
36]. The observed dissimilarity may be due to differences in dietary composition as the latter high-fat diets did not contain excess cholesterol [
13,
36] and/or a gender associated effect, as female rats—in contrast to males—proved resistant to sucrose-induced hypertriglyceridemia [
37]. In agreement with our findings, circulating levels of lipids did not increase in humans placed on eucaloric diets, consuming 20 % of calories as sucrose for 10 weeks [
38].
BW increased in all groups over time, but high-fat fed animals do not become obese and compared to the control and vHS groups, this is in accordance with previous results by us and others [
11,
13,
16,
35]. Lack of comparable weight gain despite similar caloric intake might partly be due to hepatic lipid accumulation, rendering lipids unavailable for other tissues. After 16 weeks, HFHS and HFvHS increased energy-intake compared to control and consequently weight differences were eliminated between HFHS, HFvHS and control. Compared to control, HF also tended to increase energy-intake, but not enough to completely eliminate the weight difference. The apparent increase in BW observed for vHS after week 16 was due to the randomization procedure: animals were randomized, but not block-randomized based on weight at euthanasia and by chance, most of the animals with the highest BW were randomly chosen to continue on the diet. Regardless, vHS did not develop dyslipidemia or NAFLD. Thus, weight differences are not thought to influence the results of this study, and animals exposed to either vHS or high-fat diet were not obese compared to controls.
FFA release increases with increasing fat mass in humans [
39] and the lack of increased FFA concentrations is likely to reflect the absence of obesity as reported in a non-obese rabbit-model of NASH [
40]. Furthermore, plasma TG was not increased in response to the high-fat diet. This could be due to increased TG clearance from the blood and/or decreased hepatic TG production. Accordingly, guinea pigs on high-fat diet (25.1 % fat) displayed reduced plasma TG compared to their low-fat fed counterparts [
13]. Lipoprotein lipase activity was increased by high-fat feeding, most likely contributing to the decreased circulating levels of TG [
13]. Similarly, plasma TG was also lower in rats fed a high-fat, high-cholesterol diet compared to chow or a high-fat diet without cholesterol [
41]. In these rats, hepatic microsomal triglyceride transfer protein mRNA expression was suppressed, potentially limiting hepatic VLDL-TG production [
41]. Indeed, hepatic TG production may be compromised as NAFLD progresses from simple steatosis towards steatohepatitis. In humans, NASH is associated with impaired VLDL synthesis and secretion and reduced apoB100 synthesis [
42,
43]. Hence, hepatic retention of TG, limiting TG availability for storage in adipose tissue, may constitute a causal mechanism in the progression of NASH in the non-obese phenotype of the dyslipidemic guinea pig model.
Guinea pig SAA - a systemic marker of inflammation [
20] - was not induced by high-fat feeding at any of the two time points, similar to results from high fat fed (15 % fat, 1.35 % cholesterol) mice [
44]. Our findings may suggest that systemic inflammation is not prominent in this model, at least when assessed by systemic SAA level. Alternatively, it could be speculated that low SSA levels was due to reduced liver function, supported by histopathology and increased ALT and AST levels, rendering the liver unable to produce and/or secrete SAA.
Our study revealed hepatocyte ballooning after 16 weeks of high-fat feeding, signifying the presence of NASH and distinguishing it from simple steatosis [
45,
46]. This is further supported by the recorded inflammatory foci and progression of fibrosis in the HFD groups. While hepatic fibrosis is not necessary for the diagnosis of NASH, it represents a critical step in the progression of the disease, setting the stage for further liver damage such as cirrhosis and hepatocellular carcinoma [
41]. However, high-fat diet models often induce mild hepatic fibrosis while rarely leading to severe progressive fibrosis [
47,
48], except in guinea pigs [
11]. After 16 weeks, guinea pigs in the HF and HFvHS group exhibited mild fibrosis, while the HFHS group already displayed bridging fibrosis (grade 3). After 25 weeks, all three HFD groups had formed bridging fibrosis. Consistent with our findings of circulating levels of lipids, sucrose feeding alone did not induce NAFLD, nor did it affect hepatic outcomes when added to a high-fat diet. Contrary to our results, 60–70 % sucrose promoted development of hepatic steatosis in both rats [
49‐
51] and mice [
52,
53]. However, while levels of hepatic inflammatory cytokines were increased [
49], hepatic triglycerides were not significantly elevated [
49,
51]. Thus, sucrose is seemingly not able to induce NASH.
Diseases characterized by chronic tissue regeneration, such as cirrhosis ensuing from progressive NASH, ultimately results in telomere shortening [
54]. This promotes genomic instability paralleled by DNA strand damage which may constitute an underlying disease aspect playing an important role in NAFLD, especially with regards to fibrosis progression [
54]. However, DNA strand breaks and telomere length was not different between groups. Consequently, these do not seem to be underlying mechanisms of NAFLD and NASH in this particular animal model. The null results on hepatic DNA damage is in keeping with earlier results in rats showing no altered levels of DNA strand breaks after feeding with saturated fats [
55] and sucrose [
56‐
59]. Alternatively, livers may have to become cirrhotic before notable telomere shortening can be detected, which may also explain the absence of telomere shortenings.
Abbreviations
ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; BW, body weight; FFA, free fatty acids; H&E, Mayer’s haematoxylin and eosin; HDL-C, high density lipoprotein cholesterol; HF, high-fat; HFD, high-fat diet; HFHS, high-fat high-sucrose; HFvHS, high-fat very high-sucrose; LDL-C, low density lipoprotein cholesterol; NAFLD, non-alcoholic fatty liver disease; NASH, non-alcoholic steatohepatitis; OGTT, oral glucose tolerance test; SD, standard deviation; T/S ratio, ratio of telomere repeat copy number (T) to single copy gene (S) copy number; TC, total cholesterol; TG, triglycerides; vHS, very high-sucrose; VLDL-C, very low density lipoprotein cholesterol
Acknowledgements
We would like to thank Annie Bjergby Kristensen, Joan E. Frandsen, Belinda Bringtoft, Lene Winther Takla and Annie Jensen for excellent technical assistance.