Inflammation and ER stress regulate branched-chain amino acid uptake and metabolism in adipocytes

Abstract

Inflammation plays a critical role in the pathology of obesity-linked insulin resistance and is mechanistically linked to the effects of macrophage-derived cytokines on adipocyte energy metabolism, particularly that of the mitochondrial branched-chain amino acid (BCAA) and tricarboxylic acid (TCA) pathways. To address the role of inflammation on energy metabolism in adipocytes, we used high fat-fed C57BL/6J mice and lean controls and measured the down-regulation of genes linked to BCAA and TCA cycle metabolism selectively in visceral but not in subcutaneous adipose tissue, brown fat, liver, or muscle. Using 3T3-L1 cells, TNFα, and other proinflammatory cytokine treatments reduced the expression of the genes linked to BCAA transport and oxidation. Consistent with this, [(14)C]-leucine uptake and conversion to triglycerides was markedly attenuated in TNFα-treated adipocytes, whereas the conversion to protein was relatively unaffected. Because inflammatory cytokines lead to the induction of endoplasmic reticulum stress, we evaluated the effects of tunicamycin or thapsigargin treatment of 3T3-L1 cells and measured a similar down-regulation in the BCAA/TCA cycle pathway. Moreover, transgenic mice overexpressing X-box binding protein 1 in adipocytes similarly down-regulated genes of BCAA and TCA metabolism in vivo. These results indicate that inflammation and endoplasmic reticulum stress attenuate lipogenesis in visceral adipose depots by down-regulating the BCAA/TCA metabolism pathway and are consistent with a model whereby the accumulation of serum BCAA in the obese insulin-resistant state is linked to adipose inflammation.

Figure 1.

Obesity down-regulates the expression of genes of BCAA metabolism. Schematic diagram of cellular BCAA metabolism (A) and expression of BCAA metabolism genes in epididymal (B) or inguinal white adipose tissue (C) in C57BL/6J mice maintained on a low-fat (filled bars) or high-fat (open bars) diet. D, Expression of BCAA metabolism genes in brown adipose tissue, liver, and gastrocnemius skeletal muscle tissue. Error bars represent SEM. *, P < .05 (n equals 6–12 per group).

Figure 2.

Inflammatory factors down-regulate the expression of genes of BCAA metabolism. A, Expression of BCAA metabolism genes in 3T3-L1 adipocytes treated on day 8 after differentiation for 24 hours with 1 nM TNFα. B, Expression of BCAA metabolism genes in 3T3-L1 adipocytes treated on day 8 after differentiation for 24 hours with varying concentrations of TNFα. C, Expression of BCAA metabolism genes in 3T3-L1 adipocytes treated on day 8 after differentiation for 24 hours with 1 nM of the indicated cytokine. D, Expression of Bcat2, Sdha, Bckdha, Atp5a, and β-actin in 3T3-L1 adipocytes treated for 24 hours on day 8 after differentiation with 1 nM TNFα. E, Expression of BCAA metabolism genes in 3T3-L1s treated on day 8 after differentiation for 24 hours with 1 nM TNFα and 10 μM Bay11–7085. $, P < .05 compared with TNFα treatment. ND, not detectable. Error bars represent SEM. *, P < .05 compared with control (n = 6 per treatment group).

Figure 3.

Obesity and inflammation down-regulate TCA cycle and anaplerotic reaction enzyme gene expression. A, Expression of TCA cycle metabolism genes in 3T3-L1 adipocytes treated with or without 1 nM TNFα for 24 hours. Expression of TCA cycle genes in inguinal white adipose tissue (B) and epididymal white adipose tissue (C) from low-fat or high-fat fed C57BL/6J mice. Error bars represent SEM. *, P < .05 (n = 6–12 per group).

Figure 4.

Leucine transport and metabolism in 3T3-L1 adipocytes. A, Expression of leucine transport proteins in 3T3-L1 adipocytes treated for 24 hours with 1 nM TNFα. B, Expression of leucine transport proteins in inguinal and epididymal white adipose tissue from mice maintained on chow (black bars) or high-fat diet (white bars) for 12 weeks. C, Total [³H]-leucine uptake in 3T3-L1 adipocytes treated with 1 nM TNFα. [¹⁴C]-leucine fractioned into lipid (D) or protein (E) after 24 hours TNFα treatment. Error bars represent SEM. *, P < .05 (n = 6 per group).

Figure 5.

Metabolomics analysis of 3T3-L1 cells. 3T3-L1 adipocytes were treated for 24 hours with 1 nM TNFα and the intracellular levels of branched chain amino acids (A), amino acids (B), and organic acids (C) analyzed. Error bars represent SEM. *, P < .05 (n = 6 per group).

Figure 6.

ER stress down-regulates BCAA metabolism genes. A, 3T3-L1 adipocytes were treated for 24 hours with 5 μM tunicamycin, 1 μM thapsigargin, or dimethylsulfoxide (DMSO), and the expression of BCAA/TCA genes were analyzed. B, Expression of spliced XBP1 in epididymal and subcutaneous fat of wild-type and FIXs mice. Expression of BCAA/TCA metabolism genes in wild-type and transgenic mice overexpressing spliced XBP1 maintained on a chow diet in the inguinal (C) or epididymal (D) depot are shown. Error bars represent SEM. *, P < .05 (n equals 4–8 per group).