Obesity is a significant risk factor for metabolic syndrome (MS), type 2 diabetes mellitus (T2DM), non-alcoholic fatty liver disease (NAFLD), and non-alcoholic steatohepatitis (NASH). These conditions are characterized by hepatic steatosis, inflammation, fibrosis, and cell damage. While obesity is a major contributor, metabolic health, specifically adipose tissue expansion mechanisms, is also crucial. The adipose tissue expandability hypothesis suggests that the capacity to expand fat mass to store lipids is more important than the absolute amount of fat. Some individuals maintain metabolic health despite obesity (metabolically healthy obese, MHO). Minipigs have been used as obesity models, but they often exhibit limited hepatic steatosis compared to humans. This study aimed to elucidate the molecular mechanisms underlying the limited hepatic steatosis in Göttingen Minipigs fed an FFC diet, using high-throughput qPCR to characterize transcriptional changes in liver, SAT, and VAT after 13 months of dietary intervention. The study focuses on understanding why these pigs, despite being morbidly obese and dyslipidemic, do not exhibit the severe hepatic abnormalities often observed in human obesity.

Several studies have explored the use of minipigs (Bama, Ossabaw, Göttingen) as models for MS and obesity, with varying success in replicating human liver disease. While some studies have shown potential for liver fibrosis, inflammation, insulin resistance, and steatohepatitis using westernized or atherogenic diets, the observed hepatic steatosis is often limited, primarily microvesicular rather than the macrovesicular type seen in humans. This discrepancy suggests that factors other than steatosis drive inflammation and fibrosis in these porcine models. Previous research has indicated a genetic predisposition for an MHO-like phenotype in Göttingen Minipigs. The expandability hypothesis, which emphasizes the capacity of an individual to expand fat mass to store lipids rather than the absolute amount of fat, is central to understanding metabolically healthy obesity.

Castrated male Göttingen Minipigs (n=29) aged 6-7 months were divided into three groups: standard diet (SD), high-fat, fructose, and cholesterol diet (FFC), and FFC diet with streptozotocin-induced diabetes (FFCDIA). The FFC diet contained 2% cholesterol for the first five months, reduced to 1% for the remaining eight months. The FFCDIA group received 1% cholesterol throughout. Pigs were fed for 13 months. RNA was isolated from liver, SAT, and VAT samples using the Tri Reagent protocol. RNA integrity was assessed, and samples with adequate RQI were included. cDNA was synthesized, and high-throughput qPCR was performed using the Biomark HD system and/or Mx3005P platform. Expression levels of 96 (liver) and 98 (SAT and VAT) genes were measured. Four reference genes were used for normalization. Data were processed using Genex 6 Pro software, and statistical analysis included t-tests, ANOVA, Kruskal–Wallis test, and Pearson correlation. Histopathological examination of SAT and VAT sections was also conducted to assess macrophage infiltration.

The FFC and FFCDIA groups developed morbid obesity and dyslipidemia. In the liver, 12 genes showed significant differential expression (FFC vs. SD), with four upregulated (LPL, CD68, PPARG, CD36) and eight downregulated. The FFCDIA group showed even more pronounced changes, with additional genes significantly altered. Interestingly, insulin-signaling pathways remained largely unaffected in both FFC groups. In adipose tissues, the fold changes were significantly lower than in the liver. Histopathological examination revealed no increase in macrophage infiltration in the adipose tissues. Analysis of PPARG and its target genes (FABP4, LPL, CD36) showed that these were expressed at much higher levels in the liver of the FFC and FFCDIA groups than in the adipose tissues. Body fat and visceral fat expansion were highly correlated with PPARG, LPL, and CD36 expression in the liver but not with hepatic triglyceride content. The lack of increase in macrophage infiltration in the adipose tissue is confirmed by the low number of upregulated pro-inflammatory cytokines found, with only IL6 showing a significant increase in SAT.

This study demonstrates that despite significant upregulation of lipogenic genes (PPARG, FABP4, CD36, LPL) in the liver of Göttingen Minipigs, hepatic steatosis remained limited. The remarkable ability of these minipigs to expand their adipose tissue compartments appears to be a key factor in preventing the development of severe hepatic abnormalities associated with obesity. The data support the concept that adipose tissue expandability is more important than absolute fat mass in determining metabolic health. The observed upregulation of lipogenic genes in the liver may be a compensatory mechanism, redirecting lipid storage away from the liver and toward adipose tissues. The limited inflammation in adipose tissue, despite significant fat expansion, contrasts with findings in humans and suggests a distinct metabolic response in these animals.

Severely obese Göttingen Minipigs exhibit a remarkable capacity for adipose tissue expansion, protecting them from many metabolic and hepatic abnormalities associated with obesity. The upregulation of lipogenic genes in the liver, rather than causing steatosis, may contribute to the redirection of lipid storage to adipose tissue. This highlights the importance of adipose tissue expandability in metabolic health and offers a valuable model for studying obesity-related metabolic complications. Future studies could investigate the genetic and epigenetic factors responsible for this unique adaptation.

The study used a relatively small number of animals. Further research is needed to validate the findings in larger cohorts and to explore the long-term effects of the FFC diet. The study focused on gene expression, and additional analyses (e.g., proteomics, metabolomics) could provide a more complete picture of the metabolic processes involved. The use of a specific pig breed and dietary model might limit the generalizability of the findings to other populations and dietary interventions.