VLCAD−/− mice are resistant to high-fat diet induced obesity due to decreased energy intake and decreased respiratory quotient
Preliminary hyperinsulinemic-euglycemic clamp study of the VLCAD−/−
mice fed standard diet showed no difference in insulin sensitivity compared to WT mice (Supplemental Data, Fig.1
). To investigate why a major fatty acid oxidation enzyme deficiency did not lead to insulin resistance, we subjected the VLCAD−/−
mice to a high-fat diet. Interestingly, two weeks of ad lib
feeding of the high fat diet caused a 40% increase in body weight in the WT mice, while only causing a 10% increase in body weight in the VLCAD−/−
mice within the same period (, Day 14). Another 3 months of high-fat feeding led to further body weight gains in both groups with the WT gaining slightly more than the VLCAD−/−
mice (, P<0.05 from day 9 to day 100). Food intake measurements showed that VLCAD−/−
mice had significantly less food intake than WT mice for the first two weeks of the feeding period ().
Figure 1 (A) VLCAD−/− mice have significantly lower body weight (P<0.05) than WT mice when fed a high-fat diet ad lib for 100 days. (B) VLCAD−/− mice have significantly lower food intake (P<0.05) as compared to WT (more ...)
We next analyzed whole body energy homeostasis in these mice fed the same high fat diet using indirect calorimetry. Interestingly, despite the absence of a key enzyme in mitochondrial β-oxidation, VLCAD−/− mice had relatively higher percentage of fatty acid oxidation compared to WT controls reflected by a 20% decrease in RQ (calculated from area under the curves of , P<0.01). Overall energy expenditure was 15% lower (calculated from area under the curves of , P<0.05) in the VLCAD−/− mice. Measurements of intestinal fat absorption in VLCAD−/− and WT mice demonstrated no differences in fat absorption between the two groups when fed the same high fat diet ().
Metabolic profile in plasma of the WT and VLCAD−/− mice pair-fed high-fat diet for 3 weeks
Taken together these data suggest that the VLCAD−/−
mice are resistant to high-fat diet induced obesity due to decreased food intake. Since previous studies with carnitine palmitoyltransferase-1 inhibition in the hypothalamus have implicated increased hypothalamic long-chain acyl-CoA concentrations in reducing food intake (Obici et al., 2003
) we examined hypothalamic long-chain acyl-CoA concentrations in fat fed VLCAD−/−
and WT mice but found no differences in long-chain acyl-CoA concentrations ().
Since there was a marked difference in body weight between the two groups of mice after ad lib high-fat feeding, which is known to impact insulin stimulated glucose metabolism we subsequently studied VLCAD−/− and WT mice after 3 weeks of pair-feeding the high-fat diet. As expected, VLCAD−/− mice and WT mice pair-fed the high-fat diet had a similar body weight but VLCAD−/− mice had a 36% decrease in whole body fat content (P<0.01) as compared to WT control mice (). Basal plasma concentrations of glucose, insulin, triglyceride, β-hydroxybutyrate and cholesterol were similar in the two groups. However, plasma fatty acid levels were 30% higher in the VLCAD−/− mice consistent with their inborn error of metabolism as compared to the WT control mice. There were no differences in plasma concentrations of IL-6, TNFα or resistin between the two groups of mice (). However, adiponectin and leptin concentrations were both 40% lower in the VLCAD−/− mice compared to the WT controls.
VLCAD−/− mice were protected from high-fat diet induced insulin resistance
To investigate the effect of VLCAD−/− deficiency on glucose metabolism, we performed hyperinsulinemic-euglycemic clamp studies on WT and VLCAD−/− mice pair-fed the high-fat diet for 3 weeks. Reduced food intake in the WT mice (~450 kcal/kg/d) due to pair-feeding, as compared to their ad lib intake, did not protect the mice from severe insulin resistance caused by high-fat feeding (). In stark contrast, VLCAD−/− mice required a significantly higher glucose infusion rate () in order to maintain euglycemia during the clamp reflecting increased whole body insulin sensitivity (). This increased whole body insulin responsiveness in the VLCAD−/− mice could be attributed to both increased peripheral glucose uptake () and increased suppression of hepatic glucose production during the clamp (). Consistent with these data VLCAD−/− mice also had significantly higher insulin stimulated 2-deoxy glucose uptake in skeletal muscle () during the clamp compared to the WT mice.
Figure 2 VLCAD−/− mice are resistant to high-fat induced insulin resistance as demonstrated by (A) a significantly higher glucose infusion rate, (B) significantly higher whole body glucose uptake (Rd), (C) significantly higher insulin-mediated (more ...)
Consistent with the increased insulin suppression of hepatic glucose production in liver and insulin stimulated glucose uptake in muscle, insulin stimulated Akt2 activities in the liver and muscle of the VLCAD−/− mice were increased by 50% and 120%, respectively, compared to the WT controls (). These data suggest that the VLCAD−/− mice are protected from high-fat diet induced insulin resistance both in liver and in skeletal muscle. This was further supported by glucose tolerance testing, with a significantly lower plasma glucose levels () and a trend of lower plasma insulin levels measured during the test ().
Liver protein kinase Cε and muscle protein kinase Cθ activity are decreased in VLCAD−/− mice along with reduced intracellular diacylglycerol content
To determine if decreased activation of protein kinase Cε in liver and protein kinase Cθ in skeletal muscle played a role in protecting VLCAD−/− from fat induced insulin resistance we next measured protein kinase Cε and protein kinase Cθ activity in these tissues as reflected by the membrane to cytosol ratio of these proteins. Consistent with this hypothesis we found that protein kinase Cε and protein kinase Cθ activity were reduced by 35% and 50% respectively in the VLCAD−/− mice compared to the WT mice (). Diacylglycerol (DAG) is a known activator of the novel PKCs and VLCAD−/− mice had less intracellular diacylglycerol (DAG) and triglyceride (TG) content compared to the WT mice (). Acyl-CoA concentrations were significantly higher (P<0.01 [liver] and P< 0.05 [muscle]) in the VLCAD−/− mice as compared to the WT mice (); however, ceramide, another putative mediator of fat induced insulin resistance, was not different in either liver or skeletal muscle in the two groups ().
Figure 3 (A) Protein kinase Cε or θ membrane translocation (activation) in liver and muscle of VLCAD−/− mice was significantly decreased as compared to WT controls; (B) diacylglycerol (DAG) and (C) triglyceride (TG) concentrations (more ...)
AMPK was activated in VLCAD−/− mice fed a high-fat diet
We found a 220% increase in liver and a 100% increase in muscle in AMPK activity in the VLCAD−/−
mice compared to WT controls (). We hypothesized that AMPK activation due to excess long-chain fatty acids in these tissues (Clark et al., 2004
; Za’tara, et al., 2008
) might explain the increased AMPK activity and serve as a compensatory mechanism to increase fat oxidation with a reduction in substrate available for DAG synthesis. The AMPK downstream target, acetyl-CoA carboxylase (ACC), was found to be phosphorylated 3 and 4 fold higher in the liver and muscle of the VLCAD−/−
mice (). Consistent with increased ACC phosphorylation, which leads to decreased ACC activity, liver malonyl-CoA content was also found to be lower in the VLCAD−/−
mice (). In contrast, there were no differences in liver or muscle AMPK activity in the LCAD deficient mice (). In vitro measurements with freshly isolated liver, muscle and brown adipose tissue showed significantly higher rates of fatty acid oxidation in the VLCAD−/−
mice compared to WT control mice (). These data suggest that increased long-chain fatty acyl-CoAs () in the VLCAD−/− mice were able to activate AMPK and compensate partially for the loss of function in fatty acid oxidation, while significantly increasing insulin sensitivity in these mice. In contrast, however, the deficiency of fatty acid oxidation in the LCAD−/−
mice did not lead to activation of AMPK () in liver or muscle, thus there was no compensation in fatty acid oxidation resulting in exacerbated hepatic insulin resistance in these mice (Zhang. et al., 2007
). The predominant substrates that build up in LCAD deficiency are the medium-chain length C14:1
acyl-CoA and C14:1
acylcarnitine (Kurtz, et al, 1998; Zhang, et al., 2007
Figure 4 AMPK activity and phospho-acetyl-CoA carboxylase. AMPK activity is significantly elevated in (A) VLCAD−/− liver and skeletal muscle; but not in (B) LCAD−/− liver and skeletal muscle; VLCAD−/− mice have significantly (more ...)
We then measured acylcarnitines in plasma, muscle and liver of these mice since acylcarnitine measurement is an important clinical indicator for disorders in fatty acid oxidation. Plasma long chain acylcarnitines (C14 to C18) were slightly increased, however, liver and muscle long chain acylcarnitines were not statistically different (Supplemental Fig. 2
In order to further understand the metabolic phenotype in these mice, we performed quantitative RT-PCR to measure liver, muscle and brown adipose tissue expression of genes involved in fatty acid metabolism in the WT and VLCAD−/− mice pair-fed high-fat diet. We found a dramatic compensatory increase in muscle and brown adipose tissue gene expression of PPARα and LCAD in the VLCAD−/− mouse group (). There was no difference in liver gene expression of PPARα and LCAD, probably because these genes in WT mice were also up regulated in liver due to high fat feeding. In addition, there was no difference in gene expression of PGC1α, PPARδ and AOX in liver, muscle or brown adipose tissue. Interestingly, muscle PGC1β and SREBP1 gene expression was down regulated significantly, indicating a strong transcriptional mechanism that reduces lipogenesis in skeletal muscle in the VLCAD−/− mice. In contrast, PGC1β and SREBP1 gene expression in liver and brown adipose tissue were not different. In addition, there was a 23% increase in UCP1 gene expression in brown adipose tissue of the VLCAD−/− mice.
Gene expression relative to 18S in the liver, muscle, and brown adipose tissue of high-fat fed WT and VLCAD−/− mice (×108)