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

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.

Keywords: Fasting; Gluconeogenesis; Hepatic lipid accumulation; Ketogenesis; Lipolysis.

Fig. 1

Changes in serum metabolites in response to fasting duration. Serum concentration of a glucose, b non-esterified fatty acid (NEFA), c triacylglycerol (TAG), and d β-OH Butyrate in mice that were fasted for 0, 4, 8, 12, and 16 h. ^a,b,c,dBars that do not share a common letter differ significantly (P < 0.05; n = 6)

Fig. 2

Hepatic glucoregulatory responses to fasting duration. Liver a glycogen (mg/g tissue) content, b Glucose 6 phosphatase (G6Pase) mRNA expression, c Phosphoenolpyruvate carboxykinase (PEPCK) mRNA expression, d PEPCK activity, and e cAMP concentration (pM/g tissue). ^a,b,c Bars that do not share a common letter differ significantly (P < 0.05; n = 6)

Fig. 3

Hepatic lipid storage and metabolism responses to increasing fasting duration. Liver a Triacylglycerol (TAG) content, b Non-Esterified Fatty Acid, c Peroxisome proliferator-activated receptor alpha (PPARα) mRNA expression, d Carnitine palmitoyl transferase I (CPT1) mRNA expression, and e Hydroxymethylglutaryl Coenzyme A Synthase 2 (HMGCS2) mRNAexpression. ^a,b,c,dBars that do not share a common letter differ significantly (P < 0.05; n = 6)

Fig. 4

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)

Fig. 5

Serum metabolites in response to re-feeding. Serum a glucose, b non-esterified fatty acids (NEFA), c triacylglycerol (TAG), and d β-OH butyrate concentrations in mice fasted for 0, 8, or 16 h then allowed to re-feed for 0 (white bars), 1 (grey bars), or 2 (black bars) hours. ^*Denotes a significant difference from 0 h fasting within re-feeding duration (P < 0.05). ^a,bBars that do not share a common letter differ significantly within fasting duration (P < 0.05; n = 3–6). NS, no significant differences within a fasting duration (P > 0.05)

Fig. 6

Hepatic glucoregulatory responses to re-feeding after a fast. Liver a glycogen (mg/g tissue) content, b Glucose 6 phosphatase (G6Pase) mRNA expression, c Phosphoenolpyruvate carboxykinase (PEPCK) mRNA expression, d PEPCK activity, and e cAMP concentration (pM/g tissue). ^*Denotes a significant difference from 0 h fasting within re-feeding duration (P < 0.05). ^a,bBars that do not share a common letter differ significantly within fasting duration (P < 0.05; n = 3–6). NS, no significant differences within a fasting duration (P > 0.05)

Fig. 7

Hepatic lipid storage and metabolism responses to re-feeding after a fast. Liver a Triacylglycerol (TAG) content, b Non-Esterified Fatty Acid (NEFA) content, c Peroxisome proliferator-activated receptor alpha (PPARα) mRNA expression, d Carnitine palmitoyl transferase I (CPT1) mRNA expression, and e Hydroxymethylglutaryl Coenzyme A Synthase 2 (HMGCS2) mRNA expression. ^*Denotes a significant difference from 0 h fasting within re-feeding duration (P < 0.05). ^a,b,cBars that do not share a common letter differ significantly within fasting duration (P < 0.05; n = 3–6). NS, no significant differences within a fasting duration (P > 0.05)

Fig. 8

Re-feeding induced changes in hepatic a β-OH butyrate dehydrogenase I (BDH1), b β-OH butyrate dehydrogenase II (BDH2), c BDH1:BDH2 mRNA expression ratio, and d uncoupling protein 2 (UCP2) mRNA expression. ^*Denotes a significant difference from 0 h fasting within re-feeding duration (P < 0.05). ^a,b Bars that do not share a common letter differ significantly within fasting duration (P < 0.05; n = 3–6). NS, no significant differences within a fasting duration (P > 0.05)