Introduction
Obesity has become a global health crisis, with rates nearly tripling since 1975. This escalating epidemic is associated with a range of metabolic disorders, including insulin resistance, type 2 diabetes (T2D), non-alcoholic fatty liver disease (NAFLD), and cardiovascular diseases. Research using high-fat diet (HFD) mouse models is crucial for understanding the intricate mechanisms underlying obesity and its related complications. These models allow scientists to investigate the temporal relationships between inflammation in different tissues, the impact of various dietary components, and the effectiveness of potential therapeutic interventions.
Adipose Tissue Inflammation Precedes Hepatic Inflammation in Diet-Induced Obesity
One key area of investigation is the sequence of inflammatory events in adipose tissue (AT) and the liver during diet-induced obesity. A study by van der Heijden et al. (2009) aimed to elucidate this sequence in C57BL/6J male mice fed either a low-fat diet (LFD; 10% kcal fat) or a high-fat diet (HFD; 45% kcal fat) for 24, 40, or 52 weeks. The study revealed that AT inflammation occurred earlier than hepatic inflammation.
After 24 weeks of HFD feeding, AT inflammation was evident, as indicated by an increased presence of crown-like structures (CLS) and the upregulation of pro-inflammatory genes such as Tnf, Il1β, Mcp1, and F4/80. Hepatic inflammation, however, was not detected until 40 weeks of HFD feeding. These findings suggest that AT inflammation is established prior to the development of hepatic inflammation in this model of diet-induced obesity.
Metabolic Inflammation and Insulin Resistance
Obesity and associated metabolic disorders are characterized by a state of inflammation initiated by nutrient overload. Metabolic inflammation in obesity differs from the classical immune response, as it is modest and lacks apparent resolution over time. Both AT and the liver contribute to metabolic inflammation, with the production of pro-inflammatory cytokines by resident macrophages in the liver linked to disruption of hepatic insulin signaling.
Studies have shown that enhanced expression of inflammatory genes in the liver following HFD feeding is associated with reduced insulin sensitivity in mice. The temporal relationship between AT and liver inflammation in the etiology of insulin resistance (IR) has been a subject of debate. While it is generally accepted that AT is an important initiator of the inflammatory response to obesity, engaging in crosstalk with the liver, other studies have suggested that this crosstalk is bidirectional.
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Body Weight, Adiposity, and Insulin Sensitivity
In the study by van der Heijden et al. (2009), mice fed a HFD exhibited a rapid increase in body weight, which was significant from 12 weeks onward. The weights of mesenteric, gonadal/epididymal, and perirenal fat depots were also significantly increased in HFD-fed mice compared to LFD controls. Fasted glucose levels were not affected by HFD feeding, but fasted insulin levels were significantly elevated in mice fed a HFD for 24 and 40 weeks, suggesting a reduction in insulin sensitivity.
Oral glucose tolerance tests (OGTT) revealed that glucose tolerance was negatively affected after 24 weeks of HFD feeding, as evidenced by elevated glucose levels. Insulin levels were also significantly increased in 24-week HFD-fed mice, confirming the existence of IR at this time point. While whole-body glucose tolerance was also significantly impaired in mice fed a HFD for 40 weeks, no signs of apparent glucose intolerance were observed in mice fed a HFD for 52 weeks.
Adipose Tissue Inflammation: Cellular and Molecular Markers
Histological examination of gonadal fat tissues revealed an increased adipocyte size in mice fed the HFD. The number of cells per mm2 was significantly reduced by 24, 40, and 52 weeks of HFD feeding, confirming an increased adipocyte size compared to LFD controls. Crown-like structures (CLS), representing an accumulation of macrophages around dead adipocytes, were apparent in AT samples of mice fed a HFD.
Gene expression analysis confirmed the presence of AT inflammation, with significantly elevated gene expression levels of the pro-inflammatory cytokine Tnf and the genes encoding for proteins involved in macrophage infiltration, Mcp1 and F4/80, in the adipose tissue of HFD mice. The anti-inflammatory gene Il-10 was also significantly elevated in the AT of HFD mice at 24 and 40 weeks.
Hepatic Steatosis and Inflammation
H&E staining of liver slides indicated the presence of HFD-induced hepatic steatosis from 24 weeks onward, which was confirmed by a significant elevation in the amount of neutral lipids in the liver. Histopathological examination of liver sections showed an increase in steatosis grade at 52 weeks compared to 24 weeks of HFD feeding, suggesting a progression in the accumulation of hepatic lipids over time.
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The NAFLD activity score (NAS), the number of inflammatory foci, and hepatocellular ballooning were all significantly increased for all HFD groups compared to LFD groups. Tnf, Il1ß, Il-10, Mcp1, and F4/80 expression was significantly upregulated at 40 weeks of HFD feeding but not 24 weeks of HFD feeding compared to mice fed the LFD.
Adipokines and Cytokines
Plasma leptin levels were significantly increased in mice fed a HFD compared to LFD, reaching a maximum around 24 weeks. Serial adiponectin levels did not change in time or as a result of HFD feeding. After 24 weeks of HFD, the circulating pro-inflammatory mediators, TNF, IL-6, and mKC, as well as the anti-inflammatory mediator IL-10 were up-regulated.
The Impact of Mouse Strain on Obesity Development
The choice of mouse strain is a critical factor in obesity research, as different strains exhibit varying tendencies for obesity and the ability to regulate blood glucose and lipid levels. Common strains used in obesity research include C57BL/6, BALB/c, Kunming, and ICR mice.
A study compared the performance of these different mouse strains when used to develop obese models. Mice were fed either a standard diet or a high-fat diet for 10 weeks, and body weight, adipose tissue index, liver weight, glucose tolerance, lipid profile, and inflammatory cytokines were assessed.
Body Weight Gain and Food Intake
The study found that mouse strain exerts a significant impact on body weight gain during HFD treatment. C57BL/6 mice exhibited the most consistent and widely reported increase in body weight. Kunming mice were heavier than control mice from the 2nd week of the experiment, while C57BL/6 mice were heavier than control from the 3rd week. BALB/c mice showed a slower weight gain, becoming heavier than control from the 6th week. ICR mice also showed a significant increase in body weight compared to controls.
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Food intake was also monitored during the experiment, providing insights into the relationship between diet and weight gain in different strains.
Adipose Tissue and Liver Characteristics
Adipose tissue and liver characteristics are important indicators of obesity. The study found that the liver was heavier in HFD-fed mice compared to controls in most strains. Histological examination revealed differences in lipid accumulation and inflammation in the liver cells of different strains.
Kunming mice showed cells with no accumulation of lipid droplets and inflammation cell infiltration in the control group, while the obese group showed lipid droplets alongside the liver cells. C57BL/6 and BALB/c mice exhibited similar patterns.
Glucose Tolerance and Insulin Sensitivity
Glucose tolerance tests (GTT) and insulin tolerance tests (ITT) were performed to assess the impact of HFD on glucose metabolism in different mouse strains. The results showed that HFD-fed mice generally exhibited impaired glucose tolerance and insulin sensitivity compared to controls.
Lipid Profile
The lipid profile of obese mice was also greatly impacted by HFD. Triglyceride (TG) levels were significantly higher in all four strains compared to control groups. Total cholesterol (TC) and LDL-c levels were also elevated in HFD-fed mice.
Inflammatory Cytokines
The study analyzed the expression of pro-inflammatory cytokines (TNF-α, IL-6, IL-1β) and the anti-inflammatory cytokine IL-10 in different mouse strains. The results showed that HFD feeding generally led to an increase in pro-inflammatory cytokines and a decrease in anti-inflammatory cytokines.
Behavioral Deficits in HFD-Fed Mice
Research has also explored the impact of HFD on behavior in mice. A study investigated the occurrence of motor behavior deficits over time in young mice chronically exposed to HFD. The study found that HFD-exposed mice exhibited decreased motor behavior, deficits in sensorimotor integration, and impaired balance/coordination.
Diet Composition and Control Diets
The composition of the high-fat diet and the choice of control diet are important considerations in HFD mice research. Purified high-fat diets used to induce obesity and obesity-related complications typically have 40-60% of energy derived from fat. Control diets can be designed in several ways, depending on what features the researcher wants to modify relative to the high-fat diet. Options include AIN-93M or AIN-93G diets, which have a moderate amount of sucrose and fat from soybean oil, or grain-based diets.
Cafeteria Diet Models
In addition to HFD models, cafeteria diet (CAF) models are used to mimic the diversity of highly palatable, high-salt, high-fat, and low-fiber foods accessible in Western societies. CAF models have been shown to promote voluntary hyperphagia, rapid weight gain, increased fat pad mass, and glucose and insulin intolerance.
Experimental Considerations
Several factors are important to consider when using HFD mouse models, including the genetic background of the mice, gender, and environmental factors. Male mice are generally more affected by diabetes than female mice and are thus used more often in diet-induced obesity studies. Obese mice are sensitive to stress, and their environmental settings can affect experimental results.