Excerpts - A High-Fat Ketogenic Diet Induces A Unique Metabolic State In Mice
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K etogenic diets have been used as an approach to weight loss on the basis of the theoretical advantage of a low-carbohydrate, high-fat diet. To evaluate the physiological and metabolic effects of such diets on weight we studied mice consuming a very-low-carbohydrate, ketogenic diet (KD). This diet had profound effects on energy balance and gene expression.
C57BL/6 mice animals were fed one of four diets: KD; a commonly used obesogenic high-fat, high-sucrose diet (HF); 66% caloric restriction (CR); and control chow (C). Mice on KD ate the same calories as mice on C and HF, but weight dropped and stabilized at 85% initial weight, similar to CR. This was consistent with increased energy expenditure seen in animals fed KD vs. those on C and CR. Microarray analysis of liver showed a unique pattern of gene expression in KD, with increased expression of genes in fatty acid oxidation pathways and reduction in lipid synthesis pathways. Animals made obese on HF and transitioned to KD lost all excess body weight, improved glucose tolerance, and increased energy expenditure.
Analysis of key genes showed similar changes as those seen in lean animals placed directly on KD. Additionally, AMP kinase activity was increased, with a corresponding decrease in ACC activity. These data indicate that KD induces a unique metabolic state congruous with weight loss.
To examine the effects of gross dietary manipulation, we chose four distinct dietary conditions. First, we utilized a rodent ketogenic diet (KD) previously used extensively in studies of seizure susceptibility and known to induce consistent ketosis (6). We compared this diet to a high-fat (HF) diet that was also high in carbohydrate and commonly used in studies of mouse obesity. The effects of these diets were compared with the effects of ad libitum chow feeding as well as to the effects of restriction of calories to 66% of ad libitum chow (caloric restriction).
We found that mice tolerated the KD well, consuming at least as many calories as mice fed a high-fat diet. However, mice eating KD failed to gain weight despite the high caloric density of the diet. Compared with mice fed standard chow, mice fed KD transiently lost weight and then stabilized at a lower weight than chow-fed animals in a pattern that was the same as that seen calorie-restricted mice. KD fed mice had a unique metabolic and physiological profile, exhibiting increased energy expenditure and very low respiratory quotient. Insulin levels were extremely low compared with both animals fed chow and animals fed high-fat diet. Furthermore, despite the consumption of saturated fat, serum lipids did not increase. Analysis of gene expression using Affymetrix chips revealed that consumption of KD led to a pattern of expression in the liver distinct from all other diets. An interesting feature of gene expression was suppression of both transcription factors and enzymes involved in lipid synthesis. These included fatty acid synthase (FAS), stearoyl-CoA desaturase-1 (SCD-1), and sterol regulatory element-binding protein-1c (SREBP-1c).
These data indicate that dietary manipulation is capable of altering energy balance and metabolic state. In these experiments a high-fat, ketogenic diet not only failed to cause obesity but was capable of reversing diet-induced obesity (DIO) in mice.
Mouse weights did not correlate with calories consumed. C animals had a small 2-g increase in weight (27.8–29.8 g) typical of C57BL/6 mice. As expected, animals placed on the CR diet lost weight and weighed 23.8 g at the end of the study, 3.5 g less than their initial weight, and 6.0 g less than animals fed C. Also as expected, C57BL/6 mice fed HF gained weight to a degree that has been previously described by a number of investigators (25, 28, 48). After 2 mo, these mice were 6 g heavier than mice fed C and weighed 35.9 g. In contrast, mice fed KD, although they ate as many calories as mice fed HF, demonstrated a pattern of weight change that paralleled that seen in the CR group, initially losing weight and then stabilizing at the same weight as CR, i.e., 23.8 g, a weight 6.0 g lower than that of C-fed animals (Fig. 1B).
At 5 wk, DEXA scan was performed to assess body composition. Consistent with the similarity in leptin levels, fat mass was the same in animals fed C, KD, and CR and averaged 3.4 g/animal. Fat mass of animals fed HF was increased twofold and averaged 6.7 g/animal. Lean mass ranged between 21 and 23 g in animals fed either C or HF. Lean mass of animals fed both KD and CR was the similar: 17.6 g for KD animals and 17.5 g for CR-fed animals (Fig. 2, A and B). Data represent mean of eight animals in each group.
Hormonal and metabolic profiles
The different diets were associated with dramatic differences in fed insulin levels (Table 4). As expected, mice fed HF gained weight and became hyperinsulinemic compared with C-fed animals, whereas insulin levels in lean CR animals were lower compared with the C-fed group. In KD animals, insulin levels were dramatically decreased at 1.0 pg/ml. This represents an 84% reduction compared with CR animals of the same weight and a 90% reduction compared with C-fed animals (Fig. 2C). Animals fed KD maintained normal testosterone levels in contrast to CR animals, which showed a substantial decline in testosterone to 30% of initial values (Table 4). Furthermore, in one study of females fed KD, we found they continued to cycle normally through an 8-wk observation period, whereas females that were fed CR stopped cycling within 4 days of CR (data not shown).
Mice fed KD achieved ketosis despite ad libitum feeding. β-hydroxybutyrate levels were fourfold higher than control animals. HF animals had significantly lower ketone levels compared with control animals, whereas CR animals had a slight decrease compared with ad libitum-fed animals (Table 4).
Recently, Lin et al. (32) analyzed livers from HF-fed mice and reported 14 genes to increase acutely in response to a HF diet. We then focused specifically on these 14 genes and found that, in our study, the pattern of gene expression in animals chronically fed high-fat diet was very similar to that previously reported in the acute study. In contrast, animals fed KD had a distinctly different pattern. As is visually apparent from the heat map in Fig. 3B, expression of genes in KD was generally inverse to those seen on HF diet. For example, expression of genes such as FAS and SCD-1, which are essential for fatty acid synthesis, increased in animals fed HF. In contrast, in animals fed KD, expression of both of these genes was substantially suppressed.
Analysis of body composition using DEXA analysis showed the expected increase in fat mass in animals fed HF; this was significantly decreased in the animals that had been transitioned to KD and was not statistically significant from the fat mass of animals fed C (Table 5). Whole body DEXA analysis also showed a decrease in total lean mass in KD-fed animals compared with both HF and C groups. To assess the possibility that the decrease in lean mass was secondary to muscle loss, DEXA analysis of the hind-limbs was performed. This revealed an increase in both fat and lean mass in the limbs of animals fed HF compared with both C-fed and KD-fed animals. There was no difference in either lean mass or fat mass between C-fed and KD animals (Table 5). Analysis of plasma lipids revealed improvement of both plasma triglyceride and plasma NEFA levels compared with HF as well as improved plasma cholesterol compared with HF (Table 5).
In KD animals leptin levels were also lower than those seen in C- and HF-fed animals. HF animals had fivefold higher levels than C-fed animals, whereas KD animals had levels 50% lower than C-fed animals despite similar fat mass (Table 5).
As expected, insulin levels were high in animals fed HF. In animals transitioned to KD, hyperinsulinemia-resolved and fed insulin levels were dramatically lower than animals fed either chow or HF (>95% decreased;Table 5). Fasting glucose in KD animals was significantly lower than both C and HF
Animals fed ketogenic diet ate the same number of calories as animals that were fed either chow or a high-fat diet but nevertheless failed to gain weight. Remarkably, animals eating ketogenic diet lost a small amount of weight and achieved the same weight and body composition as animals that were calorie restricted to 66% of usual daily intake. Fat mass, lean body mass, levels of leptin, and glucose were the same in ketogenic diet-fed and calorie-restricted animals.
Insulin levels were somewhat reduced in calorie-restricted animals compared with the chow-fed group, whereas insulin levels in ketogenic diet-fed animals were dramatically lower to a level that was only 10% of that seen in the calorie-restricted group. The difference in feed efficiency between chow and conventional high-fat diets has been previously reported by us (28) and by others (2); however, this is the first time that the remarkable effect of ketogenic diet on energy expenditure has been documented.
Since feeding ketogenic diet is associated with weight loss and increased energy expenditure, we also examined the possibility that feeding ketogenic diet to animals with diet-induced obesity (from feeding of high-fat chow) would lead to weight loss. Transition to ketogenic diet was associated with rapid loss of excess body weight within 14 days of starting ketogenic diet. Whereas ketogenic diet animals maintained a lower weight through the end of the study, high-fat-fed animals continued to gain weight.
In conclusion, feeding of a ketogenic diet with a high content of fat and very low carbohydrate leads to distinct changes in metabolism and gene expression that appear consistent with the increased metabolism and lean phenotype seen. Through a specific dietary manipulation, weight loss can occur secondary to distinct metabolic changes and without caloric restriction.
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