Table 1.

An overview of preclinical studies on the anti-obesity properties of metformin and its capacity to regulate energy expenditure through brown adipose tissue.

Author, Year	Experimental Model and Metformin Dose	Main Findings
Short-term effects of metformin (treatment duration equivalent to 2 weeks or less)
Rouru et al., 1993 [100]	Obese Zucker rats treated with metformin (dissolved in drinking water) at 320 mg/kg/day for 12 days	Reduced body weight and cumulative food intake; however, did not affect thermogenesis, measured using the binding of [3H]GDP to BAT mitochondria and the expression of uncoupling protein mRNA in brown adipose tissue (BAT).
Kumar et al., 2001 [101]	Genetically modified obese (ob/ob) mice treated with metformin (subcutaneously) at 200 mg/kg/day for 10 days	Reduced food intake, but did not affect serum glucose levels. Metformin did not affect the expression of nitric oxide synthase in the BAT of obese mice.
Klein et al., 2004 [102]	SV40T-immortalized brown adipocytes from the FVB strain of mice were treated with metformin (500 µM and 1 mM) for 8 days	Dose dependently reduced leptin secretion without affecting adipocyte differentiation. Metformin also acutely stimulated p44/p42 MAP kinase and inhibited leptin secretion in a dose-dependent manner in BAT.
Hu et al., 2014 [104]	Olanzapine-induced weigh gain in Sprague Dawley rats treated with metformin (oral gavage) at 300 mg/kg for 2 weeks	Prevented weight gain and loss of BAT. Mechanistically, metformin upregulated BAT genes involved in energy expenditure such as AMP-activated protein kinase (AMPK) and uncoupling protein (UCP)3 and those cited in the regulation of lipid metabolism such as resistin, fatty acid synthase, insulin-induced gene 2, CCAAT/enhancer binding protein alpha (C/EBPa), and peroxisome-proliferator-activated receptor gamma (PPARγ)
Yang et al., 2016 [105]	Newborn offspring of C57BL/6 mice fed a high-fat diet (HFD) and injected intraperitoneally with metformin at 250 mg/kg for 15 consecutive days	Rescued obesity-induced suppression of brown adipogenesis and thermogenesis. Metformin also activated AMPKα and upregulated the expression of PR domain containing 16 (Prdm16) in BAT.
Tokubuchi et al., 2017 [32]	Sprague Dawley rats were treated with metformin (dissolved in drinking water) at 2.5 mg/mL for 2 weeks	Increased plasma levels of lactate and pyruvate. Metformin also significantly reduced visceral fat mass, upregulated fat oxidation-related enzyme in the liver, UCP1 in BAT, and UCP3 in the skeletal muscle.
Breining et al., 2018 [31]	Organic cation transporter (Oct)1/2−/− mice on an FVB background received [11C]-metformin (0.2–1.0 GBq) containing 0.1–0.5 µg/mL metformin for 60 min, whereas, brown adipocytes of human origin were treated with metformin (0, 0.1 or 0.5 mM) for 24 h	Metformin was taken up in murine interscapular BAT depots, and this was associated with increased expression of UCP1. Notably, metformin reduced cellular oxygen consumption in human brown adipocyte cells.
Long-term effects of metformin (treatment duration equivalent to >2 weeks)
Savontaus et al., 1998 [106]	Obese Zucker rats treated with metformin (dissolved in drinking water) at 300 mg/kg/day for 3 weeks	Reduced weight gain, as well as food and water intake; however, did not affect mRNA expressions of UCP1, UCP2, or UCP3 in BAT. The observed effect of metformin on the expression of UCPs was when combined with β3-adrenoceptor agonist (BRL 35135) when administered at 0.5 mg/kg/day.
Geerling et al., 2014 [107]	E3L. CETP mice fed a Western-type diet supplemented with 200 mg/kg body weight/day (0.2%, w/w) metformin for 4 weeks	Lowered plasma total cholesterol and triglyceride levels, in addition to reducing BAT mass and lipid droplet. This effect was linked to increases in AMP-activated protein kinase a1 (AMPKa1) expression and activity, including hormone-sensitive lipase and mitochondrial respiratory chain complexes in BAT.
Liang et al., 2016 [33]	Offspring of C57/BL mice fed an HFD and treated with metformin (dissolved in saline) at 200 mg/kg for 21 days	Decreased serotonin concentration and promoted BAT thermogenic activity by upregulating the expression of UCP1.
Kim et al., 2016 [30]	C57BL/6 mice fed an HFD and treated with metformin (oral gavage) at 10 mg/kg or 50 mg/kg for 14 weeks	Improved glucose metabolism and suppressed white adipocyte differentiation via induction of fibroblast growth factor (FGF) 21 in the liver and in white adipocytes.
Mehdi et al., 2018 [112]	C57BL/6 J mice treated with metformin (oral gavage) at 250 mg/kg/day for 45 days	Significantly increased the mRNA expression of perilipin 5 in BAT.
Kim et al., 2018 [113]	Collagen-induced arthritis DBA/1J mice treated with metformin (oral gavage) at 50 mg/kg for 13 weeks	Displayed a small normalizing effect on the metabolic profile of obese mice. In addition, metformin promoted BAT differentiation while increasing the production of pAMPKα and fibroblast growth factor 21 (FGF21).
Karise et al., 2019 [34]	C57Bl/6 mice fed a HFD and treated with metformin (oral gavage) at 250 mg/kg/day for 8 weeks	Increased BAT content and function, as shown by an increase in adipocyte proliferation and differentiation. Metformin further promoted the activation of AMPK and enhanced thermogenic markers (UCP1 and PGC1-α) through adrenergic stimuli and FGF21. Metformin also improved mitochondrial biogenesis in BAT by upregulating nuclear respiratory factor (NRF) 1 and transcription factor A, mitochondrial (TFAM).
Yuan et al., 2019 [29]	C57BL/6J mice fed and treated with metformin (oral gavage) at 200 mg/kg/day for 8 weeks	Improved the body weight and insulin sensitivity, while affecting differential expression of 3486 proteins in BAT that were mainly assigned to the pathways of EIF2 signaling and mitochondrial dysfunction. Furthermore, carnitine palmitoyltransferase (CPT)1b and CPT2 in BAT were downregulated by metformin significantly.
Abdel-Rehim et al., 2019 [118]	Sprague Dawley rats fed an HFD and treated with metformin (oral gavage) at 200 mg/kg/day for 4 weeks	Improved glucose homeostasis and lipid profile parameters. Metformin also significantly reduced the expression of SREBP-1c, which regulates lipid synthesis in BAT.
Stojnic et al., 2021 [119]	C57BL/6J mice fed an HFD and metformin (dissolved in drinking water) at 100 mg/kg/day for 4 weeks	Improved glucose control and insulin sensitivity. Treatment did not affect energy intake, but increased systemic energy expenditure and BAT activation
Pescador et al., 2021 [120]	C75BL/6J mice fed HFD and treated with metformin (oral gavage) at 100 mg/kg/day for 6 weeks	Reduced inflammatory features in BAT such as macrophage infiltration, proinflammatory signaling, and gene expression and restored the response to cold exposure. Furthermore, suppressed a HIF1α-dependent pro-inflammatory program that was likely responsible for a secondary beneficial effect on insulin-mediated glucose uptake and β-adrenergic responses in BAT.