Regulation of beige adipocyte thermogenesis by the cold-repressed ER protein NNAT

Abstract

Objective: Cold stimuli trigger the conversion of white adipose tissue into beige adipose tissue, which is capable of non-shivering thermogenesis. However, what process drives this activation of thermogenesis in beige fat is not well understood. Here, we examine the ER protein NNAT as a regulator of thermogenesis in adipose tissue.

Methods: We investigated the regulation of adipose tissue NNAT expression in response to changes in ambient temperature. We also evaluated the functional role of NNAT in thermogenic regulation using Nnat null mice and primary adipocytes that lack or overexpress NNAT.

Results: Cold exposure or treatment with a β3-adrenergic agonist reduces the expression of adipose tissue NNAT in mice. Genetic disruption of Nnat in mice enhances inguinal adipose tissue thermogenesis. Nnat null mice exhibit improved cold tolerance both in the presence and absence of UCP1. Gain-of-function studies indicate that ectopic expression of Nnat abolishes adrenergic receptor-mediated respiration in beige adipocytes. NNAT physically interacts with the ER Ca²⁺-ATPase (SERCA) in adipocytes and inhibits its activity, impairing Ca²⁺ transport and heat dissipation. We further demonstrate that NHLRC1, an E3 ubiquitin protein ligase implicated in proteasomal degradation of NNAT, is induced by cold exposure or β3-adrenergic stimulation, thus providing regulatory control at the protein level. This serves to link cold stimuli to NNAT degradation in adipose tissue, which in turn leads to enhanced SERCA activity.

Conclusions: Our study implicates NNAT in the regulation of adipocyte thermogenesis.

Keywords: Beige fat; NHLRC1; NNAT; SERCA2; Thermogenesis.

Figure 1

NNAT expression is highly enriched in white fat and repressed during cold exposure (A) Volcano plot of RNA-seq data. Nine-week-old male mice were maintained at thermoneutrality (30 °C) or acclimated to cold (7 °C) for 3 weeks (n = 3 for both groups). The log₂ fold-changes at 7 °C compared to at 30 °C are indicated on the x-axis. The y-axis represents the log₁₀ p-values. The dotted lines on the x- and y-axes show a 2-fold change and p-value of 0.05, respectively. (B) Relative mRNA levels of Nnat in the indicated tissues. (C) NNAT protein levels in various tissues. (D) The mRNA levels of Nnat in iWAT exposed to chronic cold or housed at 30 °C. (E) Expression of NNAT in iWAT determined by immunoblotting under 30 °C, 22 °C, or 7 °C conditions for 3 weeks. (F and G) The expression of Nnat in iWAT after acute cold exposure determined by qPCR (F) and immunoblotting (G). (H and I) NNAT protein levels in iWAT of mice administered to saline or CL316,243 for 3 days (H) and 7 days (I). (J) NNAT protein levels in iWAT after acclimation from 22 °C to 30 °C for 0–6 days. P-values are determined by two-tailed Student's t-test.

Figure 2

Loss of NNAT enhances thermogenic function in vivo (A) NNAT protein level in indicated tissues of Nnat KO mice and WT-littermate. (B to D) Metabolic cage studies of Nnat KO (n = 9) and WT-littermate (n = 8). Raw energy expenditure (EE) data were plotted relative to lean mass (LM) and fitted by linear regression (B, C, D). ANCOVA analysis with lean mass as a covariate was utilized to calculate P-values. (E and F) Effect of acute cold exposure from 30 °C to 22 °C (G) and that from 22 °C to 7 °C (H) on core rectal temperature of Nnat KO and WT-littermate (n = 5 for both groups). (G) Changes in the iWAT temperature following CL316,243 treatment. WT-littermate, n = 3; Nnat KO, n = 4. (H) FDG-PET standardized uptake value (SUV) measures for iWAT from Nnat KO (n = 5) and WT-littermate (n = 5) groups. The Mann–Whitney test reveals a significant increase in FDG uptake for KO compared to WT (p = 0.016). (I) Coronal FDG-PET images of the chest and abdomen for representative animals from the two groups in (E). From each group, the individual with the SUV value closest to the group mean is shown. Due to differences in animal positioning, the cross section images for the two animals do not necessarily align exactly. The iWAT uptake (arrowheads) is seen as hot spots bilateral to the bladder and is stronger for KO. The bladder uptake (bottom midline) is due to concentration and excretion of FDG and is expected. P-value is determined by two-tailed Student's t-test unless otherwise indicated.

Figure 3

NNAT controls the thermogenic capacity of beige adipocytes in a UCP1-independent manner (A) NNAT protein level during differentiation of primary iWAT cells to beige adipocytes. (B) NNAT protein level after the treatment with 10 μM CL316,243 for 48 h in primary iWAT cells. (C) OCR in the iWAT cells derived from Nnat KO or WT-littermate in the absence (DMSO only) or presence of 10 μM CL316,243. (D) OCR in iWAT cells derived from WT mice and ectopically expressing LacZ or Nnat OE with or without treatment with 10 μM of CL316,243. An immunoblot image of NNAT expression is shown. (E and F) Effect of acute cold exposure from 30 °C to 22 °C (E) and that from 22 °C to 7 °C (F) on core rectal temperature of Ucp1 KO and the Ucp1/Nnat double KO mice. n = 5 for both groups. P-value is determined by two-tailed Student's t-test.

Figure 4

NNAT regulates calcium cycling and thermogenic respiration via SERCA2 (A) SERCA2 protein levels during differentiation of primary iWAT cells to beige adipocytes. (B and C) Immunoblot of SERCA2 and NNAT after immunoprecipitation by SERCA2 or control IgG in non-crosslinked iWAT cell lysate (B) or crosslinked iWAT lysate (C). For (C), two same sample sets were loaded in different lanes and one was probed with SERCA2 monoclonal antibody and the other with NNAT monoclonal antibody above 100 kDa. (D) Change in the intracellular calcium levels in WT and Nnat KO iWAT cells after treatment with 2 μM of thapsigargin (TG). WT, n = 8; Nnat KO, n = 9.(E) Change in the intracellular calcium levels in LacZ control and Nnat OE iWAT cells after treatment with 2 μM of TG. LacZ, n = 5; Nnat OE, n = 5. (F) Protein expression of Ryanodine receptor 1 (RYR1), Ryanodine receptor 2 (RYR2), and IP3 receptor isoform 1 (IP3R1) in the stromal vascular fraction (SVF) of iWAT, differentiated iWAT cells, and indicated tissues. (G) Change in the intracellular calcium levels in WT and Nnat KO iWAT cells after treatment with 50 μM of 2-aminoethyl diphenylborinate (2-APB). WT, n = 10; Nnat KO, n = 10. (H) Change in the intracellular calcium levels in LacZ control and Nnat OE iWAT cells after treatment with 50 μM of 2-APB. LacZ, n = 6; Nnat OE, n = 6. (I and J) OCR in WT and Nnat KO iWAT cells treated with 2 μM TG or 50 μM 2-APB for 30 min in the absence (I) or presence of 10 μM CL316,243 (J). At least biological triplicates were used. P-value is determined by two-tailed Student's t-test (I, J) or two-way ANOVA followed by Fisher's LSD test (D, E, G, H).

Figure 5

The E3 ubiquitin ligase NHLRC1 is induced by cold and regulates NNAT degradation (A) NHLRC1 protein level during differentiation of primary iWAT cells to beige adipocytes. (B to E) The mRNA and protein expression of NHLRC1 in iWAT after chronic cold (B, C) and acute cold exposure (D, E). (F and G) NHLRC1 protein level in iWAT of mice injected with saline or CL316,243 for 3 days (F) or 7 days (G). (H) The protein levels of NHLRC1 in differentiated primary iWAT cells treated with varying concentrations of CL316,243 for 24 h. (I) The relative protein levels of NHLRC1 in differentiated primary iWAT cells treated with DMSO, 10 μM of CL316,243, or 10 μM of CL316,243 + 1 μM of CREB-inhibitor (666–15, Sigma). (J) Immunoblot of NHLRC1 and NNAT after immunoprecipitation by NNAT or control IgG in MG132-treated iWAT cell lysate. (K) In the absence or presence of MG132, iWAT cells were treated with cycloheximide (CHX) and harvested at the indicated time. (L) Differentiated iWAT cells were treated with either DMSO or MG132 for 4 h before the addition of CHX and CL316,243. Cells were harvested at 6 h post CHX treatment. (M) NHLRC1 mediates ubiquitination of NNAT. 293FT cells were transfected with plasmids encoding HA-Ubiquitin and NNAT, along with NHLRC1-FLAG or empty-FLAG and incubated for 48 h. After treatment with 10 μM MG132 for 6 h, cell lysate was prepared and used for IP using polyclonal NNAT antibody. Input (9%) and IP samples were immunoblotted with anti-HA antibody. Vinculin was used as a loading control. (N) NHLRC1 and NNAT protein levels in differentiated iWAT cells expressing a scrambled shRNA (control) or the Nhlrc1 shRNA. (O) OCR in differentiated iWAT cells expressing a scrambled RNA or the Nhlrc1 shRNA in the presence and the absence of 10 μM CL316,243. P-value is determined by two-tailed Student's t-test.

Figure 6

Overview of functional and physical interaction between NNAT and SERCA. A schematic model of regulation of noncanonical thermogenesis by NNAT in adipocytes. At thermoneutrality, NNAT interacts with SERCA2 and inhibits calcium cycling. Cold exposure or treatment with a β3-adrenergic agonist increases intracellular cAMP levels, which induces NHLRC1 expression. The E3 ubiquitin ligase NHLRC1 then ubiquitinates and degrades NNAT, leading to activation of SERCA2. The futile calcium cycling mediated by SERCA2 dissipates energy as heat.