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. 2024 Feb 13;19(1):20240900.
doi: 10.1515/med-2024-0900. eCollection 2024.

Metformin plus L-carnitine enhances brown/beige adipose tissue activity via Nrf2/HO-1 signaling to reduce lipid accumulation and inflammation in murine obesity

Guojin Liang  1 , Jie Fang  2 , Pingping Zhang  2 , Shuxia Ding  2 , Yudan Zhao  2 , Yueying Feng  3
Affiliations

Metformin plus L-carnitine enhances brown/beige adipose tissue activity via Nrf2/HO-1 signaling to reduce lipid accumulation and inflammation in murine obesity

Guojin Liang et al. Open Med (Wars). .

Abstract

This study investigated how Metformin (Met) combined with L-carnitine (L-car) modulates brown adipose tissue (BAT) to affect obesity. High-fat-induced obese rats received daily oral gavage with Met and/or L-car, followed by serum biochemical analysis, histopathological observation on adipose tissues, and immunochemistry test for the abdominal expression of BAT-specific uncoupling protein 1 (UCP1). Mouse-embryonic-fibroblast cells were induced into adipocytes, during which Met plus L-car was added with/without saturated fatty acid (SFA). The role of nuclear factor erythroid 2-related factor 2 (Nrf2) in adipocyte browning was investigated by gene silencing. Mitochondria biogenesis in adipocytes was inspected by Mitotracker staining. Nrf2/heme oxygenase-1 (HO-1)/BAT-related genes/proinflammatory marker expressions in adipose tissues and/or adipocytes were analyzed by Western blot, qRT-PCR, and/or immunofluorescence test. Met or L-car improved metabolic disorders, reduced adipocyte vacuolization and swelling, upregulated levels of BAT-related genes including UCP1 and downregulated proinflammatory marker expressions, and activated the Nrf2/HO-1 pathway in adipose tissues of obese rats. Met and L-car functioned more strongly than alone. In adipocytes, Met plus L-car upregulated BAT-related gene levels and protected against SFA-caused inflammation promotion and mitochondria degeneration, which yet was attenuated by Nrf2 silencing. Met plus L-car enhances BAT activity and white adipose tissue browning via the Nrf2/HO-1 pathway to reduce lipid accumulation and inflammation in obese rats.

Keywords: Nrf2/HO-1 pathway; brown adipose tissues; inflammation; obesity; white adipose tissue browning.

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Conflict of interest statement

Conflict of interest: The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Met combined with L-car improved metabolic disorders, reduced adipocyte vacuolization and swelling and upregulated BAT-specific UCP1 level in abdominal subcutaneous adipose tissues of obese rats. SD rats were fed with normal/high-fat food, and those fed with high-fat food received daily oral gavage of Met and/or L-car. (a) The level of fasting blood glucose was measured by a glucose oxidase method-based assay kit. (b) Fasting insulin concentration was determined via radioimmunoassay. (c) HOMA-IR was calculated. (d) Histological abnormalities in adipose tissue under rat abdominal skin were observed via hematoxylin-eosin staining (magnification, 100×; scale bar, 50 µm). (e) Fat vacuoles of adipose tissue. (f and g) The expression of BAT-specific UCP1 in adipose tissue under rat abdominal skin was detected by the immunochemistry test (magnification, 100×; scale bar, 50 µm). + P < 0.05; && P < 0.01; *** P or ### P or +++ P or &&& P < 0.001; * vs NF; #vs HF; + vs HF + Met; & vs HF + L-car (NF, normal fat food; HF, high fat food; Met, Metformin; L-car, L-carnitine; HOMA-IR, homeostasis model assessment of insulin resistance; BAT, brown adipose tissues; UCP1, uncoupling protein 1).
Figure 2
Figure 2
Met combined with L-car upregulated BAT-related gene levels, suppressed inflammation, and activated the Nrf2/HO-1 pathway in abdominal subcutaneous adipose tissues of obese rats. SD rats were fed with normal/high-fat food, and those fed with high-fat food received daily oral gavage with Met and/or L-car. (a–e) The expressions of UCP1, PRDM16, PGC1α, IL-6, and TNF-α in adipose tissue under rat abdominal skin were analyzed by qRT-PCR, with GAPDH used as the normalizer. (f–h). The expressions of Nrf2 and HO-1 in adipose tissue under rat abdominal skin were measured by Western blot, with GAPDH used as the normalizer. # P or + P or & P < 0.05; ## P or ++ P or && P < 0.01; *** P or ### P or +++ P or &&& P < 0.001; * vs NF; # vs HF; + vs HF + Met; & vs HF + L-car (NF, normal fat food; HF, high fat food; Met, Metformin; L-car, L-carnitine; BAT, brown adipose tissues; UCP1, uncoupling protein; PRDM16, 1 PR domain containing 16; PGC1α, peroxisome proliferator-activated receptor-gamma co-activator 1-alpha; IL-6, interleukin-6; TNF-α, tumor necrosis factor alpha; Nrf2, nuclear factor (erythroid-derived 2)-like 2; HO-1, heme oxygenase 1; qRT-PCR, quantitative reverse transcription polymerase chain reaction).
Figure 3
Figure 3
Nrf2 silencing counteracted the combined effect of Met and L-car on upregulation of BAT-related genes in mouse adipocytes. The expression of Nrf2 in SiNrf2/SiNC-transfected 3T3-L1 cells (a) and the expressions of UCP1, PRDM16 and PGC1α (b–d) in SiNrf2/SiNC-transfected 3T3-L1 cells that underwent adipogenic differentiation and treatment with 0.5 mM Met for 24 h and 5 mM L-car for 48 h were analyzed by qRT-PCR, with GAPDH used as the normalizer. ** P or ## P < 0.01; &&& P or *** P or ### P < 0.001; & vs SiNC; * vs Con; # vs Met + L-car + SiNrf2 (Met, Metformin; L-car, L-carnitine; BAT, brown adipose tissues; UCP1, uncoupling protein; PRDM16, 1 PR domain containing 16; PGC1α, peroxisome proliferator-activated receptor-gamma co-activator 1-alpha; Nrf2, nuclear factor (erythroid-derived 2)-like 2; SiNrf2, small interfering RNA targeting Nrf2; SiNC, small interfering RNA targeting negative control; qRT-PCR, quantitative reverse transcription polymerase chain reaction).
Figure 4
Figure 4
Nrf2 silencing reversed the combined effect of Met and L-car on SFA-caused inflammation promotion and mitochondria degeneration in mouse adipocytes. SiNrf2/SiNC-transfected 3T3-L1 cells underwent adipogenic differentiation and treatment with 5 mM L-car for 48 h in the presence or absence of 2 mM SFA (48 h), accompanied by treatment with 0.5 mM Met for 24 h before cell harvest. (a and b) The expressions of IL-6 and TNF-α in adipocytes were analyzed by qRT-PCR, with GAPDH used as the normalizer. (c) The expression of UCP1 and the activity of mitochondria in adipocytes were detected by immunofluorescence test combined with Mitotracker staining (magnification, × 200; scale bar, 50 µm). *** P or ### P or &&& P < 0.001; * vs Con; # vs SFA; & vs SFA + Met + L-car + SiNC (Metformin; L-car, L-carnitine; SFA, saturated fatty acid; UCP1, uncoupling protein; IL-6, interleukin-6; TNF-α, tumor necrosis factor alpha; Nrf2, nuclear factor (erythroid-derived 2)-like 2; SiNrf2, small interfering RNA targeting Nrf2; SiNC, small interfering RNA targeting negative control; qRT-PCR, quantitative reverse transcription polymerase chain reaction).
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