Global IP6K1 deletion enhances temperature modulated energy expenditure which reduces carbohydrate and fat induced weight gain

Abstract

Objective: IP6 kinases (IP6Ks) regulate cell metabolism and survival. Mice with global (IP6K1-KO) or adipocyte-specific (AdKO) deletion of IP6K1 are protected from diet induced obesity (DIO) at ambient (23 °C) temperature. AdKO mice are lean primarily due to increased AMPK mediated thermogenic energy expenditure (EE). Thus, at thermoneutral (30 °C) temperature, high fat diet (HFD)-fed AdKO mice expend energy and gain body weight, similar to control mice. IP6K1 is ubiquitously expressed; thus, it is critical to determine to what extent the lean phenotype of global IP6K1-KO mice depends on environmental temperature. Furthermore, it is not known whether IP6K1 regulates AMPK mediated EE in cells, which do not express UCP1.

Methods: Q-NMR, GTT, food intake, EE, QRT-PCR, histology, mitochondrial oxygen consumption rate (OCR), fatty acid metabolism assays, and immunoblot studies were conducted in IP6K1-KO and WT mice or cells.

Results: Global IP6K1 deletion mediated enhancement in EE is impaired albeit not abolished at 30 °C. As a result, IP6K1-KO mice are protected from DIO, insulin resistance, and fatty liver even at 30 °C. Like AdKO, IP6K1-KO mice display enhanced adipose tissue browning. However, unlike AdKO mice, thermoneutrality only partly abolishes browning in IP6K1-KO mice. Cold (5 °C) exposure enhances carbohydrate expenditure, whereas 23 °C and 30 °C promote fat oxidation in HFD-KO mice. Furthermore, IP6K1 deletion diminishes cellular fat accumulation via activation of the AMPK signaling pathway.

Conclusions: Global deletion of IP6K1 ameliorates obesity and insulin resistance irrespective of the environmental temperature conditions, which strengthens its validity as an anti-obesity target.

Keywords: Diabetes; Energy expenditure; IP6K; Obesity; β-oxidation.

Figure 1

CD-fed IP6K1-KO mice display enhanced carbohydrate oxidation-mediated EE upon fasting/refeeding or following cold exposure. A. At 23 °C, CD-fed (ad libitum) WT and IP6K1-KO mice display similar VO₂ consumption. Fasting reduces VO₂ in CD-WTs to a higher extent, than CD-KOs. Refed CD-KO mice also display higher VO₂ consumption (n = 7–8 mice/group; t-test). B. Fasting lowers carbohydrate-VO₂ in both genotypes; yet, the values are higher in the knockouts. CD-KOs also exhibit higher carbohydrate-VO₂ following refeeding (n = 7–8 mice/group; t-test). C. Fat-VO₂ is similar in fed and fasted WT and IP6K1-KO mice. Refeeding lowers Fat-VO₂ in WT mice to a lower extent; thus, CD-KOs oxidize less fat than WT, at this condition (n = 7–8 mice/group; t-test). D. CD-KO mice display similar VO₂ consumption at 23 °C. Acute cold and cold + fast exposed knockouts consume more oxygen than WT (n = 6 mice/group). E. Average VO₂ consumption is enhanced in the CD-KOs following cold and cold + fast exposures (n = 6 mice/group; t-test). F. Average EE is higher in the CD-KOs following cold and cold + fast exposures (n = 6 mice/group; t-test). G. CD-KO mice display higher carbohydrate-VO₂ than WT at 5 °C (n = 6 mice/group; t-test). H. Fat-VO₂ is largely similar in CD-fed WTs and IP6K1-KOs (n = 6 mice/group; t-test). I. Acute cold + fast exposure decreases body temperature in CD-WT whereas IP6K1-KO mice are protected. Mice were kept at 5 °C for 8 h followed by acute (6 h) fasting at 5 °C (n = 6–8 mice/group; two-way Anova). In all panels, data are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ****P < 0.0001.

Figure 2

Global IP6K1 deletion stimulates adipose tissue browning. A. At 23 °C, CD-KO IWAT appears brownish. B. Immunoblot analyses indicate upregulation of UCP1 and PGC1α proteins CD-KO IWAT and RWAT at 23 °C. C. Immunohistochemistry of UCP1 also reveals browning in CD-KO IWAT and RWAT at 23 °C. D and E. Amplification of the browning and mitochondrial machinery in CD-KO RWAT (n = 5–7 mice/group, t-test) and IWAT (n = 4 mice/group, t-test) at 23 °C. F. RWAT explant isolated from CD-mice at 23 °C displays higher basal and FCCP-induced OCR in the knockouts (n = 4 mice/group, t-test). G. Proton leak is higher in IWAT-beige adipocytes of CD-KO mice (n = 6 mice/preparation, 8 replicates, t-test). H. Carbohydrate induced and maximal oxidative capacity are higher in IWAT-beige adipocytes of CD-KO mice (n = 6 mice/preparation, 8 replicates, t-test). In all panels, data are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 3

HFD-fed IP6K1-KO mice exhibit higher fat oxidation at 23°C, albeit switch to carbohydrate oxidation at 5°C. A. HFD-KOs consume more oxygen than WT, both at 23 °C and 5 °C (n = 6–9 mice/group). B and C. Average VO₂ and EE are enhanced in HFD-KOs under both temperature conditions (n = 6–9 mice/group; t-test). D and E. At 23 °C, HFD-KO mice oxidize more fat. However, cold exposed HFD-KOs switch to carbohydrate oxidation (n = 6–9 mice/group; t-test). F. Cold exposure decreases body temperature in HFD-WT whereas the knockouts are protected (n = 4–5 mice/group; two-way Anova). G. Immunohistochemistry reveals higher UCP1 protein levels in RWAT and IWAT of HFD-KOs. In all panels, data are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 4

Thermoneutrality delays, yet does not abolish the lean phenotype of HFD-fed IP6K1-KO mice. A. After 15-weeks at 30 °C, HFD-fed IP6K1-KO mice display a reduction in total fat, lean, and fluid masses (n = 6–10 mice/group, t-test). B. At this condition, percent (over total body weight) fat is reduced in HFD-KO, whereas percent lean mass is slightly increased. Percent fluid mass is similar in both genotypes (n = 6–10 mice/group; t-test).C. After 15-weeks at 30 °C, HFD-KOs display substantially less weight of various adipose tissue depots and liver but not heart. (n = 6–10 mice/group; t-test). D. HFD-KO mice accumulate less fat in adipocytes of diverse adipose tissue depots. The knockouts are also protected from fatty liver. E–H. Serum cholesterol, TAG, HDL, and LDL levels are lower in HFD-KOs after 15-weeks at 30 °C (n = 6–10 mice/group; t-test). I. Glucose tolerance test (GTT) indicates that 14-weeks HFD-KOs dispose of glucose more efficiently than WT (n = 6–10 mice/group; two-way Anova). J. Area under curve (AUC) values indicate that glucose disposal is significantly improved in HFD-KOs after 14-weeks of HFD at 30 °C. K. Akt stimulatory phosphorylation (S473) is higher in the RWAT and liver of HFD-KOs after 15-weeks at 30 °C. L and M. Serum levels of HMW and LMW AdipoQ are higher in HFD-KOs under the same condition (n = 6 mice/group; t-test). In all panels, data are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 5

HFD-fed IP6K1-KO mice display greater fat oxidation mediated EE at 30°C. A. After 15-weeks at 30 °C, UCP1 mRNA expression is downregulated in the IWAT of HFD-KOs. However, HFD-KO RWAT still display higher UCP1 expression (n = 6–10 mice/group; t-test). B. UCP1 protein level is higher in HFD-KO RWAT at 30 °C. UCP1 is undetectable in the IWAT under this condition. C and D. ImageJ analyses reveal that DIO2 protein level is substantially higher in the RWAT depot of IP6K1-KO mice, whereas its level in the KO-IWAT was also high but to a lesser extent (n = 5–7 mice/group; t-test). E and F. Mitochondrial EE markers are upregulated in HFD-KO IWAT and RWAT at 30 °C (n = 6–10 mice/group; t-test). G. After 15-weeks at 30 °C, HFD-KOs display increased VO₂ consumption (n = 6 mice/group). H. EE is also higher in thermoneutrally placed HFD-KOs (n = 6 mice/group; t-test). I. At 30 °C, HFD-KOs expend more energy by fat oxidation (n = 6 mice/group; t-test). J. Carbohydrate oxidation is similar in HFD-WT and IP6K1-KOs at 30 °C (n = 6 mice/group; t-test). In all panels, data are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 6

IP6K1 enhances cellular fat accumulation by diminishing AMPK mediated energy metabolism. A. IP6K1-KO MEFs accumulate less fat following oleate (OA, 24 h) treatment. B. β (OA)-oxidation is increased in IP6K1-KO MEFs following OA (24 h) treatment. However, β (PA)-oxidation is increased under basal and PA (24 h) treated conditions (n = 3; t-test). C. AICAR treated IP6K1-KO MEFs display enhanced β-oxidation (n = 3; two-way Anova). D. Complementation of IP6K1-KO MEFs with active but not inactive Myc-IP6K1 restores OA-induced fat storage. E. Catalytically active but not inactive Myc-IP6K1 complementation reduces AICAR induced β-oxidation in IP6K1-KO MEFs (n = 3; one-way Anova). F. PGC1α mRNA expression is higher in IP6K1-KO MEFs, which is further enhanced by AICAR treatment (n = 3; two-way Anova). G. PGC1α protein level is higher in OA-treated IP6K1-KO MEFs. AMPK activity on PGC1α is also enhanced whereas PGC1α acetylation is reduced. H. AMPK stimulatory phosphorylation (T172) and its activity on ACC (S79) are enhanced in OA-treated IP6K1-KO MEFs. I. TNP enhances AMPK phosphorylation and activity in glucose starved 3T3L1 preadipocytes. Glucose induction reduces AMPK phosphorylation and activity in control but not in TNP treated cells. J and K. TNP enhances OCR in starved and glucose induced 3T3L1 preadipocytes (8 replicates; t-test). L. TNP enhances AMPK phosphorylation in 3T3L1 adipocytes, under basal conditions. AICAR was used as a positive control. M. TNP, at increasing concentrations, reduces fatty acid biosynthesis in 3T3L1 adipocytes. The ACC inhibitor TOFA was used as a positive control (n = 3; one-way Anova). N. CD-KOs, at 23 °C, display higher AMPK phosphorylation and activity in the RWAT depot. O. CD-KOs, at 23 °C, are sensitive to AICAR induced reduction in blood glucose level (5–6 mice/group; two-way Anova). In all panels, data are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.