Here, we demonstrate that despite spontaneous isoenergetic intake, rats fed ad libitum a HF diet accumulated substantially more visceral and subcutaneous fat than rats fed standard chow. In fact, within 3 to 4 days on the HF diet, food consumption was adjusted to precisely match the energy intake elicited by control rats. This was observed either when food intake was assessed relative to body weight or in amounts per animal, indicating that alterations in the energy density of the diet were rapidly detected and food intake was self-regulated accordingly throughout the entire duration of the study. It was remarkable that the masses of visceral (epididymal and retroperitoneal) and subcutaneous (inguinal) fat pads were ~1.85- to 2.5-fold higher in HF rats than controls, even though energy intake was similar between both groups of animals and body mass in HF rats was only ~8% higher than controls. In fact, the weight gained was strictly towards fat accumulation, since no differences in LBM between control and HF rats were found after 8 weeks of dietary intervention. This is in line with previous observations that rats fed a HF diet for 10 weeks displayed only a 10% increase in total body weight, but fat pads weighed 30-45% more than those of animals fed a low-fat diet . This is also compatible with the fact that the density of fat is significantly lower than other components of fat-free mass , which reduces the impact of increased adiposity on total body mass. We also found that 24 h O2 consumption was ~16% higher in HF than controls in weeks 1 and 2, and then reduced to ~8% from weeks 3 to 8. Whole-body energy expenditure was also higher in HF rats than controls at weeks 1 and 2, a difference that no longer existed after week 3, indicating that a transient increase in energy expenditure occurred in HF rats. Interestingly, RER was reduced within the first two weeks of the animals being on the HF diet, demonstrating that whole-body substrate metabolism was progressively shifted towards fat oxidation. Fat actually supplied ~25%, 39.6%, and 60.5% of the energy expended at baseline, week 1, and 2 of HF feeding, respectively. From week 3 to week 8, RER remained relatively constant. Therefore, although food intake was adjusted within 3 to 4 days, it took ~2 weeks for whole-body substrate partitioning to be fully adjusted once the animals were placed on a HF diet.
We had previously demonstrated that the dark-cycle ambulatory activity was significantly reduced in rats exposed for 8 weeks to HF diet . However, it was not clear at what point during the course of HF feeding this adaptation occurred. An early reduction in spontaneous physical activity in HF animals could restrain energy expenditure and contribute to obesity development. In fact, we found a progressive reduction in dark-cycle ambulatory activity in HF rats; reaching values ~38% lower than controls at week 8. The onset of this reduction occurred at week 2 of HF diet when adjustments in food intake and whole-body substrate partitioning had already occurred. These findings are consistent with sensing energy availability and triggering alterations in peripheral metabolism that regulate energy expenditure accordingly [14, 23, 24]. However, the progressive reduction in dark-cycle ambulatory activity in rats fed a HF diet seems counterintuitive at first, since energy expenditure would be expected to increase in an attempt to maintain body mass relatively constant over time. Although our data demonstrated that energy expenditure of HF rats was indeed higher than controls in Weeks 1 and 2 of the study, this was not sustained thereafter. Importantly, equalization of energy expenditure between the two groups coincided with the time of reduction in ambulatory activity in HF rats. This indicates that the initially increased thermogenic response in HF rats was counteracted by a reduction in dark cycle spontaneous physical activity, which decreased energy expenditure and facilitated adipose tissue expansion in these animals.
This study assessed several components of the complex system that regulates whole-body energy homeostasis and revealed that besides energy density, the nutrient composition of the diet played a major role in determining whether whole-body energy expenditure was increased or reduced. The mechanisms underlying these adaptive metabolic responses are still unclear. Previous studies have suggested that HF diet-induced obesity is associated with increased calories per meal rather than per day . Time and rhythmicity of feeding have indeed been associated with obesity in rodents and humans . However, it still does not explain the origin of the energy surplus required for HF rats to have markedly higher than control adiposity [9–12], since both groups of animals elicited isoenergetic daily intake. Importantly, in this study and those of others [9–11] energy intake was assessed based on the amount of food consumed without precisely determining nutrient absorption by the gastrointestinal (GI) tract. It is possible that alterations in the nutrient composition of the diet could alter the gut microbiota toward more efficient extraction of energy from the diet and lead to obesity. In fact, a gut microbiome with increased capacity for energy harvest has been associated with obesity in humans . Moreover, recent studies have provided evidence that switching humanized gnotobiotic mice from a low-fat-plant poly-saccharide rich-diet to a high-fat/high-sugar Western diet quickly (within one day) shifted the structure of the gut microbiota and altered microbiome gene expression , indicating that the macronutrient composition of the diet may drastically affect GI tract function. Therefore, further studies are warranted to investigate whether feeding a HF diet alters the gut microbiota in a way that increases nutrient absorbance by the GI tract and facilitates the development of obesity in HF diet-induced obesity.
An alternative possible explanation for our intriguing findings and of others [9–11] is that the high availability of fat disrupted the normal operation of the system that senses and regulates adipose tissue metabolism and whole-body energy expenditure. In fact, in order for the brain to regulate non-exercise activity thermogenesis (NEAT) according to changes in energy balance, the hypothalamus and other brain regions need to integrate external sensory cues of energy availability with internal endocrine and metabolic signals arising from various organs and tissues . In this scenario, the adipose-derived hormone leptin plays a major role communicating to the hypothalamus the amount of energy stored in the organism [23, 27, 28]. As fat mass increases so does the expression and secretion of leptin by adipocytes leading to a central nervous system (CNS)-mediated reduction in food intake and up-regulation of NEAT [23, 27, 28]. In this study, we found that circulating leptin was significantly higher in HF than control rats after just 2 weeks of the dietary intervention. However, we have recently reported that the protein content of the suppressor of cytokine signaling 3 (SOCS3), a marker of leptin resistance, was increased in the hypothalami of HF rats . Also, while hypothalamic AMPK activation is expected to be downregulated by increased leptin , we found that this variable was actually higher in HF than control rats , indicating hypothalamic leptin resistance in our HF rats. Therefore, the reduction in ambulatory activity and the inability to sustain the initial increase in energy expenditure in HF rats could, at least partially, be due to impaired leptin signaling in the CNS of these animals. These centrally-mediated effects must also have increased the ability of the adipose tissue to store fat as demonstrated by the markedly increased adiposity in rats chronically fed a HF diet. Interestingly, major lipogenic organs such as liver and adipose tissue in HF rats markedly reduced their ACC content, which indicates that the de novo lipid synthesis pathway was potently suppressed in these animals. These findings are compatible with the fact that there was no need to generate long-chain fatty acids (LCFAs) in a condition where the diet already supplied large amounts of this substrate. Additionally, suppression of the de novo lipid synthesis pathway must have increased the efficiency of nutrient storage in adipose tissue, since there is a high energy cost associated with building LCFAs from glucose or aminoacids .
In order to assess whether BAT contributed to the shift in whole-body fat oxidation and to the thermogenic response to HF diet, we measured UCP-1 content and palmitate oxidation in this tissue. The increase in UCP-1 content and palmitate oxidation in BAT shows that an adaptive thermogenic response was induced by HF diet. However, it appears that the reduction in NEAT and activation of other tissue-specific metabolic adjustments offset the impact of BAT-induced thermogenesis on whole-body energy expenditure in HF rats. In this context, we found that fat oxidation in the adipocytes isolated from the epididymal fat pad was reduced by 40% in HF rats, which is in line with our previous study showing that fatty acid oxidation was potently reduced in visceral and subcutaneous adipocytes from mice exposed to HF diet for 8 weeks . Although there is no evidence that FA oxidation in adipocytes increases as a means to cope with excess lipid load in obesity, our data suggest that the impairment of FA oxidation in WAT may further contribute to the accumulation of fat mass in both visceral and subcutaneous fat depots under conditions of HF-diet-induced obesity.
In conclusion, ad libitum HF feeding induced time-dependent adaptive responses in food consumption that maintained energy intake at the same level of standard-chow-fed animals. Increased whole-body fat oxidation and UCP-1 content in BAT of HF rats were counteracted by the reduction in spontaneous physical activity during the dark cycle in these animals. The largely expanded adipose tissue of HF rats was very efficient in storing lipids and displayed significantly reduced fat acid oxidation. Also, a marked reduction in the content ACC in fat tissue and liver indicated that the costly process of de novo lipid synthesis was potently suppressed. Interestingly, the increased ability of the adipose tissue to store large amounts of energy under conditions of high dietary fat availability occurred in the absence of overfeeding, indicating that these adaptive responses were mainly driven by the macronutrient composition of the diet. These findings provide novel information regarding time-dependent adaptations to HF diet that may be of relevance for understanding the physiopathology of diet-induced obesity.