Whole body MondoA deletion protects against diet-induced obesity through uncontrolled multi-organ substrate utilization and futile cycling

Abstract

Objective –: Delineating the nodal control points that maintain whole-body energy homeostasis is critical for understanding potential treatments of obesity and cardiometabolic diseases. The nutrient-sensing transcription factor MondoA is a regulator of skeletal muscle fuel storage, where muscle-specific inhibition improves glucose tolerance and insulin sensitivity. However, the role of MondoA in whole body energy metabolic homeostasis is not understood.

Methods –: Generalized MondoA knockout (gKO) mice were generated and assessed for glucose tolerance and insulin sensitivity, body composition, energy expenditure, cold tolerance, and tissue specific transcriptional changes in response to high fat diet. Complementary studies in cultured human adipocytes assessed the impact of MondoA deficiency on substrate utilization and lipolysis.

Results –: gKO mice are protected from diet-induced obesity and insulin resistance, through increased whole body energy expenditure. gKO mice exhibit reduced brown and inguinal white adipose tissue mass, without evidence of beiging. The gKO mice are hyperlactatemic and isolated MondoA-deficient adipocytes have increased 2-deoxyglucose uptake and glycolytic function. Lastly, gKO mice and KO adipocytes display increased circulating glycerol relative to free fatty acids in response to adrenergic stimulus consistent with elevated re-esterification. However, this phenotype is not recapitulated in adipocyte-specific KO mice.

Conclusions –: MondoA deficiency alters cellular sensing of nutrient availability and storage/utilization mechanisms. In the whole-body setting, this results in increased energy expenditure, potentially related to increased glucose uptake and glycolytic flux driving glycerol synthesis to supply high rates of lipolysis and lipid re-esterification. These results suggest that MondoA functions to maintain fuel storage and when lost, inter-organ futile cycling ensues.

Keywords: Diet induced obesity; adaptive thermogenesis; energy homeostasis; futile cycling.

Fig 1.. Whole-body MondoA deficiency protects against diet-induced obesity with reduced adiposity.

A) Body weight trends for WT and gKO MondoA mice on chow and 60% kcal HFD over sixteen weeks (n=14–25). B) MRI body composition data (n=3–5) and c) gross weights of brown (BAT), inguinal white (iWAT) and epididymal white (eWAT) adipose tissues (n=7–9). CD, chow diet; HFD, high fat diet; KO, knockout; WT, wildtype. P values * < 0.05, *** <0.001, ****<0.0001, ns, not significant displayed on graphs; ‡ < 0.05 and † < 0.001 compared to WT CD. The data represent mean ± SD. All statistical significance determined by 2-way ANOVA with Tukey’s multiple-comparisons post hoc test.

Fig 2.. MondoA gKO mice have improved glucose tolerance, insulin sensitivity, and reduced dyslipidemia.

A) Fasting whole blood glucose in WT and gKO male littermates on chow and HFD for 10 weeks (n=8–12). B) Glucose and C) insulin tolerance tests (left panel) and area under the curve (right panel). D) In the same cohort of mice, fasted plasma measurements of insulin, free fatty acids (FFA), total cholesterol, and triglycerides. CD, chow diet; HFD, high fat diet; KO, knockout; WT, wildtype. Data displayed as mean ± SEM. Two-way analysis of variance (ANOVA) followed by multiple-comparisons test. P values * < 0.05, **< 0.01, *** <0.001, ****<0.0001 displayed on graphs.

Fig 3.. Lean HFD-fed gKO mice have increased energy expenditure despite higher caloric intake.

Chow and HFD-fed WT and gKO male littermates were analyzed in metabolic cages (n=3–5). A) Oxygen consumption in light and dark cycles (averaged over 48 hours). B) Respiratory exchange ratio (RER) over time (left panel) and average by light and dark cycles (right panel). C) Total energy expenditure normalized to lean body mass as measured by MRI in Fig. 1B. D) Cumulative distance traveled and E) food consumed over 48 hours. CD, chow diet; HFD, high fat diet; KO, knockout; WT, wildtype. Data displayed as mean ± SEM. Two-way analysis of variance (ANOVA) followed by multiple-comparisons test (within specific light cycle). P values * < 0.05, **< 0.01, *** <0.001, ****<0.0001 and ns, not significant displayed on graphs.

Fig 4.. MondoA gKO mice have increased cold tolerance without evidence of increased UCP-1 expression in BAT and lipolytic signature without beiging in iWAT.

A) Chow-fed WT (n = 16) and MondoA gKO (n = 18) mice age 10–12 weeks were subjected to thermoneutrality for 14 days followed by acute cold exposure (4 °C). Core rectal temperature was monitored over a 5-h period. The change in core temperature ± SEM is shown in the graph as a function of time. B) Quantitative real-time RT-PCR performed from RNA isolated from BAT for targets of classical and nonclassical non-shivering thermogenesis. The values represent mean arbitrary units normalized to a 36B4 transcript (control). QT-PCR targets for C) non-shivering thermogenesis and D) lipid synthesis, storage and utilization from iWAT isolated from chow and HFD-fed WT and MondoA gKO male littermates (n=5–6). Data displayed as mean ± SEM. Two-way analysis of variance (ANOVA) followed by multiple-comparisons test. P values * < 0.05, **< 0.01, *** <0.001, ****<0.0001 displayed on graphs.

Fig. 5.. MondoA deficiency leads to increased energy expenditure which involves increased glycolytic flux.

A) Whole blood lactate levels in HFD-fed WT and gKO male littermates (n=9–11C). B) Measurement of lactate generation from culture media of CRISPR-generated human iPSC-derived control (HPRT1) and MondoA KO adipocytes (hASCs). C) 2-deoxyglucose uptake assay with and without insulin in hASCs. D) Extracellular acidification rate (ECAR) in MondoA, Txnip and Arrdc4 KO hASCs and controls (HPRT1), representative from 3 independent experiments (n=10–11). Mean maximal respiration, glycolytic capacity and reserve ± SEM are quantified (right). CD, chow diet; HFD, high fat diet; KO, knockout; WT, wildtype. Data displayed as mean ± SEM. Student’s two-tailed t-test, 1-way, or 2-way ANOVA with Tukey’s comparison. P values * < 0.05, **<0.01, ****<0.001 displayed on graphs.

Fig. 6.. MondoA deficiency in mouse and human iPSC-derived adipocytes leads to isoproterenol-stimulated FFA release consistent with futile re-esterification.

A) Basal and isoproterenol-stimulated glycerol and free fatty acid (FFA) release. B) Dose response curve for isoproterenol. C) Line graph represents oxygen consumption rates (OCR) in MondoA, Txnip or Arrdc4 KO APCs and controls (HPRT1), representative from 3 independent experiments (n=10–11). D) Bar graphs show the mean spare and maximal respiratory capacity ± SEM. E) In vitro fatty acid oxidation measured from 3H-palmitate in control, MondoA, Txnip and Arrdc4 KO APCs with positive and negative controls of Etomoxir and FCCP (n=9–13). F) Stimulated lipolysis assays for FFA and glycerol release in WT and KO APCs treated with Etomoxir and Triascin C. G) Incorporation of labelled substrate into the total lipid pool. Free fatty acid (FFA) and glycerol serum levels over time in ad lib H) chow-fed and I) HFD –fed WT and gKO male littermates following CL-316,243 stimulated lipolysis. Inset, area under the curve. The data represent mean ± SEM. All statistical significance determined by 1-way ANOVA with Tukey’s multiple-comparisons post hoc test (A, D, E, F) or 2-tailed student’s t-test (G, H,I). P values * < 0.05, ** < 0.01, *** <0.001, ****<0.0001, displayed on graphs.