Previous studies reported that forage: concentrate ratios might affect hepatic lipid metabolism, resulting in altered levels of available energy for weight gain [29, 30]. In the modern intensive livestock systems in China, high concentrated rations (far over 60%) are widely used to provide greater energy. High concentrate diets and lack of exercise are important factors leading to excessive lipid steatosis and fat accumulation in the liver [14]. However, the molecular mechanism of how a high concentrate diet leads to hepatic lipid metabolic disorder is currently unavailable. The current study, using 60% dietary concentrate levels as the control, emphasized lipid metabolism regulatory network (lipid synthesis, decomposition, and transport) status to study the mechanisms of hepatic lipid metabolism affected by an increased concentrate diet in fattening lambs.

Bodyweight is one of the most important indexes that reflect animal growth and health condition. The similar ADG and F/G between two groups in this study were consistent with a previous study that Brand et al. [31] found no significant correlation between dietary energy level and performance (ADG and F/G) under long-term feeding. The unchanged performance with increasing concentrate may have resulted from the fact that the nutritional requirements of the lambs have been met in all the experimental diets. In our study, the daily digestible energy (DE) and crude protein (CP) received by GN60 and GN70 lambs far exceeded the nutritional requirements of 8 MJ DE and 95 g CP recommended in the Feeding Standard of Meat-producing Sheep and Goats of China (2004). Moreover, it has been reported that a slight increase in diet energy level had no significant effect on daily gain [32]. In our study, the diet provided 0.7 MJ/kg DE increase when increasing 10% concentrate from GN60 to GN70, which can be considered as a slight increase in energy.

It is worth noting that Brand et al. [33] found when the energy content of experimental diets exceeds animal requirements, the metabolism and growth of some tissue were affected. Expectedly, biochemical indices served as indicators and were used to monitor the physiological status, providing evidence regarding the metabolism change for animals in this study. The concentration of liver TG is a common biomarker for lipid metabolism. Under normal circumstances, liver stores only small amounts of triglyceride. The triglyceride accumulation within hepatocytes arises from an imbalance between lipid acquisition (i.e., fatty acid uptake and de novo lipogenesis) and removal (export as a component of VLDL particles), which is the hallmark of lipid deposition in liver [34]. In this study, TG which was an essential marker of lipid metabolism was increased in the liver of GN70 group. Within normal physiological ranges, transport rates of TG are consistent with the synthesis rates of TG as a protective response to hepatic metabolic capacity. The increased TG concentration in liver from GN70 lambs with an experimental period of 3 months suggested the synthetic rate of TG was higher than the degradation rate, indicating hepatic lipid metabolic abnormalities of fattening lambs. Interestingly, it has been shown that different animal species had variable metabolic responses in particular on lipid metabolism when the animals underwent excessively high concentrate diets. For instance, a study on dairy cows showed that feeding a high concentrate diet reduced liver TG levels but had a less potent effect on blood TG content [35]. However, Grum et al. [36] have reported that feed high concentrate diets did not affect liver TG of cows. Moreover, Dong et al. [12] reported that blood and hepatic TG levels were elevated in lactating goats fed high concentrate diets. Indeed, it has been reported that there were differences in liver lipid metabolism between species [37, 38]. The variable response to high concentrate diets in different animal species may have resulted from the reason that complex interactions among factors regulate the hepatic lipid metabolism, and the mechanisms underlying need to be further investigated.

Since hepatic lipid metabolism is tightly regulated by multiple interrelated genes, RNA sequencing was used to identify DEGs and to explore the critical pathways in the liver when fed a high concentrate diet using fattening lambs. Clear separation between GN60 and GN70 groups in liver transcriptome PCA showed alteration in hepatic transcription. The majority of enriched KEGG pathways were related to lipid metabolism, including PPAR signaling pathway, fatty acid metabolism, fatty acid degradation, and fatty acid biosynthesis. Enriched GO items of DEGs were positive regulation of triglyceride catabolic process, positive regulation of lipoprotein lipase activity, and positive regulation of lipid catabolic process. Likewise, the critical modules in PPI network were fatty acid degradation and cholesterol metabolism. The result of GO, KEGG pathway, and PPI analysis revealed the mechanism occurring with lipid transport and metabolism related pathway. The up-regulated FASN in GN70 implied more de novo lipogenesis in lambs. A similar result has also been reported by Dong et al. [12], who found an up-regulated expression of FASN in hepatic tissues of goats after being fed a high concentrate diet for 10 weeks. ACSL3 is a member belonging to the long-chain acyl-CoA synthetases (ACSLs) family, which is involved in cellular absorption of fatty acids and plays a crucial role in the synthesis of lipid droplets [39]. Up-regulated ACSL3 indicated increased TG synthesis in the liver of GN70 [40]. Moreover, the process of fatty acid degradation of the GN70 group has changed. Generally, ACSL1 accounts for 90% of the total ACS activity and is located on the outer mitochondrial membrane where it interacts with CPT1 and directs FAs towards mitochondrial, in which CPT2 participates in β-oxidation [41,42,43]. In the present study, down-regulated ACSL1, CPT1A, CPT1B and CPT2 in the hepatic tissue from GN70 group suggested that fatty acid transport and fatty acid oxidation were restrained. This was contrary to the observations of H Dong et al. [5], who observed a higher expression of ACSL1 in lactating goats fed a high concentrate diet, and the expression of CPT1 and CPT2 had no difference compared with goats fed a low concentrate diet. These differences could be explained by the fact that high concentrate diets increased lactating goat milk production, and the milk fat synthesis mobilized liver fat metabolism, thereby enhancing fatty acid oxidation [12]. GK is a critical gene for glycerol utilization, and its overexpression increases the consumption of sugars and triglycerides by cells [44]. MGLL plays an essential role in fatty acid metabolism, converting monoacylglycerides to free fatty acids and glycerol [45]. A similar function of phospholipase activity and triglyceride lipase activity has also been proposed for LIPG [46, 47]. Thus, dramatically descend expressional levels of GK, MGLL and LIPG indicated the attenuation of glycerolipid degradation in the hepatic tissue from GN70.

As expected, the expressions of some lipid transport related genes were changed in GN70. APOA2 and APOA5 are members of the apolipoprotein family closely linked with TG metabolism [48, 49]. In the current study, the expression of APOA2 was up-regulated while the expression of APOA5 was down-regulated. Nevertheless, it has been reported that the expression of APOA2 and APOA5 were considered to have a positive association with TG accumulation in the liver [49, 50]. Moreover, SA van den Berg et al. [51] reported that hepatic TG content was 50% higher in high-fat diet Apoa5−/− mice compared with Wild-type (WT) mice. It was assumed that APOA5 may regulate TG metabolism through different pathways in different environments [51]. In blood, APOA5 was presumed as the only lipoprotein that lowers blood TG, while overexpression of APOA2 was positively associated with TG level [48, 52]. However, in the present study, the blood TG level was not different between GN60 and GN70. It is well known that TG is generally exported as constituents of VLDL into the blood circulation, the progress of which needs apoB package TG to create VLDL particles [53, 54]. No change in APOB gene in GN70 indicated that there were no differences in the corresponding TC and LDL in the blood, resulting in aligned TG secretion rate of GN70 as compared to GN60. Taken together, these results showed enhanced fatty acid synthesis and TG synthesis, inhibited fatty acid transport and oxidation, and unchanged TG secretion which was explained by an improved triglyceride content in the liver.