Excess dietary carbohydrate affects mitochondrial integrity as observed in brown adipose tissue

Abstract

Hyperglycemia affects over 400 million individuals worldwide. The detrimental health effects are well studied at the tissue level, but the in vivo effects at the organelle level are poorly understood. To establish such an in vivo model, we used mice lacking TXNIP, a negative regulator of glucose uptake. Examining mitochondrial function in brown adipose tissue, we find that TXNIP KO mice have a lower content of polyunsaturated fatty acids (PUFAs) in their membrane lipids, which affects mitochondrial integrity and electron transport chain efficiency and ultimately results in lower mitochondrial heat output. This phenotype can be rescued by a ketogenic diet, confirming the usefulness of this model and highlighting one facet of early cellular damage caused by excess glucose influx.

Keywords: BAT; PUFA; TXNIP; cold stress; glucose; ketogenic diet; lipid; mitochondria.

Figure 1.. TXNIP KO BAT has defects in thermogenesis

(A) BAT morphology, H&E stain at RT. (B) BAT and eWAT gene expression at RT by qPCR (n = 7). (C) BAT morphology and H&E stain after 4-h cold stress. (D) Quantitation of lipid droplets from BAT H&E slides. (E) Quantitation of TAG content in BAT. (F) BAT gene expression by qPCR (n = 7). (G) BAT western blot. (H)Intrascapular and lumbar temperature during cold stress (n = 10). For gene expression and lipid quantification, error bars show the mean ± SD; *p < 0.05; ^**p < 0.01; ^***p < 0.001; ^****p < 0.0001 (Student’s t test, unpaired, two-tailed).

Figure 2.. TXNIP KO BAT mitochondria functional defects rescued by a ketogenic diet

(A) BAT TEM, before and after 4 h of cold stress. (B) BAT TEM, after a 4 h cold stress followed by 4 h recovery at RT. (C) Representative Seahorse OCR of isolated mitochondria from BAT after 4 h of cold stress. (D) Quantification of TAG in BAT on ketogenic diet. (E) Ucp1, Pgc1a, and Dio2 expression levels in BAT on different diets at RT by qPCR. (F) Western blot of BAT on different diets at RT. (G) BAT TEM on different diets after 4 h of cold stress. (H) Seahorse OCR measured as in (C) from animals on ketogenic diet.

Figure 3.. TXNIP KO BAT has lower expression of genes in PUFA transport and elongation

(A) Volcano plot showing genome-wide transcriptional changes between TXNIP WT and KO BAT after 4 h of cold stress for the control diet (left) and a ketogenic diet (right). Red dots represent genes with significant differential expression (FDR <0.05) between WT and KO on the control diet but not on the ketogenic diet (FDR ≥ 0.05). All other genes are represented by gray dots. The horizontal dashed line indicates the p value 0.05, while the vertical demarcates twice up- or downregulated genes. (B) GO-based gene set enrichment for genes having significant differential expression between TXNIP WT and KO on the control but not the ketogenic diet after a 4-h cold stress. Gene ratio (horizontal axis) is M/N where M = number of genes in our gene list in a given pathway, N = number of predefined unique genes in the same pathway. (C) The same as (A) for RT. (D) The same as (B) for RT. (E) Gene expression in fatty acid uptake, elongation, desaturation, and cardiolipin synthesis and remodeling pathways from mRNA-seq data on control diet. (F) qPCR verification of selected gene expression from the heatmap (n = 7). (G) qPCR verification of gene expression on the ketogenic diet at RT (n = 7). For gene expression, error bars show mean ± SD; *p < 0.05; ^**p < 0.01; ^***p < 0.001 (Student’s t test).

Figure 4.. Lipidomics analysis of BAT showing less PUFA in KO (n = 8, males)

(A–E) Total fatty acid acyl-chain species (A), acyl-chain composition of TAG (B), PC (C), and PE (D), and total acyl-chain (E) of isolated mitochondria from BAT of TXNIP WT and KO animals at RT on control diet. Only the most abundant species are shown for clarity. (F) Membrane fluidity measured as Laurdan general polarization in isolated mitochondria from WT and KO on the control and ketogenic diet. (G–J) Total acyl-chain species (G), acyl-chain composition of TAG (H), PC (I), and PE (J) of WT and KO BAT on a ketogenic diet. (K–O) Total acyl-chain (K), acyl-chain composition of TAG (L), PC (M), PE (N), and cardiolipin (O) of WT and KO heart on the control diet. *p = 0.01 < p < 0.05; ^**p = 0.001 < p ≤ 0.01; ^***p ≤ 0.001