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Hepatic adaptations to maintain metabolic homeostasis in response to fasting and refeeding in mice

View Article: PubMed Central - PubMed

ABSTRACT

Background: The increased incidence of obesity and associated metabolic diseases has driven research focused on genetically or pharmacologically alleviating metabolic dysfunction. These studies employ a range of fasting-refeeding models including 4–24 h fasts, “overnight” fasts, or meal feeding. Still, we lack literature that describes the physiologically relevant adaptations that accompany changes in the duration of fasting and re-feeding. Since the liver is central to whole body metabolic homeostasis, we investigated the timing of the fast-induced shift toward glycogenolysis, gluconeogenesis, and ketogenesis and the meal-induced switch toward glycogenesis and away from ketogenesis.

Methods: Twelve to fourteen week old male C57BL/6J mice were fasted for 0, 4, 8, 12, or 16 h and sacrificed 4 h after lights on. In a second study, designed to understand the response to a meal, we gave fasted mice access to feed for 1 or 2 h before sacrifice. We analyzed the data using mixed model analysis of variance.

Results: Fasting initiated robust metabolic shifts, evidenced by changes in serum glucose, non-esterified fatty acids (NEFAs), triacylglycerol, and β-OH butyrate, as well as, liver triacylglycerol, non-esterified fatty acid, and glycogen content. Glycogenolysis is the primary source to maintain serum glucose during the first 8 h of fasting, while de novo gluconeogenesis is the primary source thereafter. The increase in serum β-OH butyrate results from increased enzymatic capacity for fatty acid flux through β-oxidation and shunting of acetyl-CoA toward ketone body synthesis (increased CPT1 (Carnitine Palmitoyltransferase 1) and HMGCS2 (3-Hydroxy-3-Methylglutaryl-CoA Synthase 2) expression, respectively). In opposition to the relatively slow metabolic adaptation to fasting, feeding of a meal results in rapid metabolic changes including full depression of serum β-OH butyrate and NEFAs within an hour.

Conclusions: Herein, we provide a detailed description of timing of the metabolic adaptations in response to fasting and re-feeding to inform study design in experiments of metabolic homeostasis. Since fasting and obesity are both characterized by elevated adipose tissue lipolysis, hepatic lipid accumulation, ketogenesis, and gluconeogenesis, understanding the drivers behind the metabolic shift from the fasted to the fed state may provide targets to limit aberrant gluconeogenesis and ketogenesis in obesity.

No MeSH data available.


Mechanisms that regenerate NAD+ to allow for continued metabolic flux through β-oxidation and the tricarboxylic acid cycle. First, we present a β-OH butyrate dehydrogenase 1 (BDH 1) activity and b BDH1 mRNA expression to understand the potential regeneration of NAD+ as acetoacetate is converted to β-OH butyrate by β-OH butyrate dehydrogenase 1. c BDH2 converts β-OH butyrate to acetoacetate and in turn reduces NAD+ to NADH. d By assessing the relative ratio of BDH1:BDH2 we can see that as fasting duration is extended so is the flux from acetoacetate to β-OH butyrate which will increase the regeneration of NAD+. Finally, we shown that uncoupling protein 2 expression increases with fasting duration (e), leading to decreased synthesis of ATP and decreased hepatic ATP content (f). a,bBars that do not share a common letter differ significantly (P < 0.05; n = 6)
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Fig4: Mechanisms that regenerate NAD+ to allow for continued metabolic flux through β-oxidation and the tricarboxylic acid cycle. First, we present a β-OH butyrate dehydrogenase 1 (BDH 1) activity and b BDH1 mRNA expression to understand the potential regeneration of NAD+ as acetoacetate is converted to β-OH butyrate by β-OH butyrate dehydrogenase 1. c BDH2 converts β-OH butyrate to acetoacetate and in turn reduces NAD+ to NADH. d By assessing the relative ratio of BDH1:BDH2 we can see that as fasting duration is extended so is the flux from acetoacetate to β-OH butyrate which will increase the regeneration of NAD+. Finally, we shown that uncoupling protein 2 expression increases with fasting duration (e), leading to decreased synthesis of ATP and decreased hepatic ATP content (f). a,bBars that do not share a common letter differ significantly (P < 0.05; n = 6)

Mentions: The flux of fatty acids through β-oxidation and acetyl-CoA through the tricarboxylic acid cycle increases hepatic mitochondrial NADH production. Without regeneration of NAD+, there would be limited flux of fatty acids through β-oxidation and decreased production of acetyl-CoA, which would limit ketogenesis. The liver has adapted 2 methods to regenerate NAD+ during a fast. First, it can increase the ratio of β-OH butyrate to acetoacetate production by altering the expression of BDH1 and BDH2. BDH1 primarily catalyzes the conversion of acetoacetate to β-OH butyrate and simultaneously NADH to NAD+, while BDH2 catalyzes the reverse reaction. Hepatic BDH1 activity increased within 4 h of fasting (P = 0.02; Fig. 4a). This preceded a significant increase in BDH1 mRNA, which was significantly elevated by fasting at 8 and 12 h (P < 0.05; Fig. 4b). Fasting decreased BDH2 mRNA expression significantly by 16 h (P < 0.05; Fig. 4c). By increasing BDH1 and decreasing BDH2, fasting increased the BDH1:BDH2 ratio to favor synthesis of β-OH butyrate and NAD+ (Fig. 4d). Alternatively, the liver can regenerate NAD+ by uncoupling electron transport and oxidative phosphorylation through upregulation of uncoupling protein 2, a PPARα responsive gene. We observe a robust fasting induced increase in UCP2 expression (P < 0.0001; Fig. 4e). This increase in hepatic UCP2 expression is expected to decrease hepatic ATP synthesis, explaining the reduction in hepatic ATP content following an overnight fast [30, 31]. Accordingly, liver ATP content decreased as the fasting duration went from 4 and 8 to 16 h (P = 0.02; Fig. 4f).Fig. 4


Hepatic adaptations to maintain metabolic homeostasis in response to fasting and refeeding in mice
Mechanisms that regenerate NAD+ to allow for continued metabolic flux through β-oxidation and the tricarboxylic acid cycle. First, we present a β-OH butyrate dehydrogenase 1 (BDH 1) activity and b BDH1 mRNA expression to understand the potential regeneration of NAD+ as acetoacetate is converted to β-OH butyrate by β-OH butyrate dehydrogenase 1. c BDH2 converts β-OH butyrate to acetoacetate and in turn reduces NAD+ to NADH. d By assessing the relative ratio of BDH1:BDH2 we can see that as fasting duration is extended so is the flux from acetoacetate to β-OH butyrate which will increase the regeneration of NAD+. Finally, we shown that uncoupling protein 2 expression increases with fasting duration (e), leading to decreased synthesis of ATP and decreased hepatic ATP content (f). a,bBars that do not share a common letter differ significantly (P < 0.05; n = 6)
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Fig4: Mechanisms that regenerate NAD+ to allow for continued metabolic flux through β-oxidation and the tricarboxylic acid cycle. First, we present a β-OH butyrate dehydrogenase 1 (BDH 1) activity and b BDH1 mRNA expression to understand the potential regeneration of NAD+ as acetoacetate is converted to β-OH butyrate by β-OH butyrate dehydrogenase 1. c BDH2 converts β-OH butyrate to acetoacetate and in turn reduces NAD+ to NADH. d By assessing the relative ratio of BDH1:BDH2 we can see that as fasting duration is extended so is the flux from acetoacetate to β-OH butyrate which will increase the regeneration of NAD+. Finally, we shown that uncoupling protein 2 expression increases with fasting duration (e), leading to decreased synthesis of ATP and decreased hepatic ATP content (f). a,bBars that do not share a common letter differ significantly (P < 0.05; n = 6)
Mentions: The flux of fatty acids through β-oxidation and acetyl-CoA through the tricarboxylic acid cycle increases hepatic mitochondrial NADH production. Without regeneration of NAD+, there would be limited flux of fatty acids through β-oxidation and decreased production of acetyl-CoA, which would limit ketogenesis. The liver has adapted 2 methods to regenerate NAD+ during a fast. First, it can increase the ratio of β-OH butyrate to acetoacetate production by altering the expression of BDH1 and BDH2. BDH1 primarily catalyzes the conversion of acetoacetate to β-OH butyrate and simultaneously NADH to NAD+, while BDH2 catalyzes the reverse reaction. Hepatic BDH1 activity increased within 4 h of fasting (P = 0.02; Fig. 4a). This preceded a significant increase in BDH1 mRNA, which was significantly elevated by fasting at 8 and 12 h (P < 0.05; Fig. 4b). Fasting decreased BDH2 mRNA expression significantly by 16 h (P < 0.05; Fig. 4c). By increasing BDH1 and decreasing BDH2, fasting increased the BDH1:BDH2 ratio to favor synthesis of β-OH butyrate and NAD+ (Fig. 4d). Alternatively, the liver can regenerate NAD+ by uncoupling electron transport and oxidative phosphorylation through upregulation of uncoupling protein 2, a PPARα responsive gene. We observe a robust fasting induced increase in UCP2 expression (P < 0.0001; Fig. 4e). This increase in hepatic UCP2 expression is expected to decrease hepatic ATP synthesis, explaining the reduction in hepatic ATP content following an overnight fast [30, 31]. Accordingly, liver ATP content decreased as the fasting duration went from 4 and 8 to 16 h (P = 0.02; Fig. 4f).Fig. 4

View Article: PubMed Central - PubMed

ABSTRACT

Background: The increased incidence of obesity and associated metabolic diseases has driven research focused on genetically or pharmacologically alleviating metabolic dysfunction. These studies employ a range of fasting-refeeding models including 4&ndash;24&nbsp;h fasts, &ldquo;overnight&rdquo; fasts, or meal feeding. Still, we lack literature that describes the physiologically relevant adaptations that accompany changes in the duration of fasting and re-feeding. Since the liver is central to whole body metabolic homeostasis, we investigated the timing of the fast-induced shift toward glycogenolysis, gluconeogenesis, and ketogenesis and the meal-induced switch toward glycogenesis and away from ketogenesis.

Methods: Twelve to fourteen week old male C57BL/6J mice were fasted for 0, 4, 8, 12, or 16&nbsp;h and sacrificed 4&nbsp;h after lights on. In a second study, designed to understand the response to a meal, we gave fasted mice access to feed for 1 or 2&nbsp;h before sacrifice. We analyzed the data using mixed model analysis of variance.

Results: Fasting initiated robust metabolic shifts, evidenced by changes in serum glucose, non-esterified fatty acids (NEFAs), triacylglycerol, and &beta;-OH butyrate, as well as, liver triacylglycerol, non-esterified fatty acid, and glycogen content. Glycogenolysis is the primary source to maintain serum glucose during the first 8&nbsp;h of fasting, while de novo gluconeogenesis is the primary source thereafter. The increase in serum &beta;-OH butyrate results from increased enzymatic capacity for fatty acid flux through &beta;-oxidation and shunting of acetyl-CoA toward ketone body synthesis (increased CPT1 (Carnitine Palmitoyltransferase 1) and HMGCS2 (3-Hydroxy-3-Methylglutaryl-CoA Synthase 2) expression, respectively). In opposition to the relatively slow metabolic adaptation to fasting, feeding of a meal results in rapid metabolic changes including full depression of serum &beta;-OH butyrate and NEFAs within an hour.

Conclusions: Herein, we provide a detailed description of timing of the metabolic adaptations in response to fasting and re-feeding to inform study design in experiments of metabolic homeostasis. Since fasting and obesity are both characterized by elevated adipose tissue lipolysis, hepatic lipid accumulation, ketogenesis, and gluconeogenesis, understanding the drivers behind the metabolic shift from the fasted to the fed state may provide targets to limit aberrant gluconeogenesis and ketogenesis in obesity.

No MeSH data available.