Anti-obesity effects of microalgae

Int. J. Mol. Sci. 2020, 21, 41; doi:10.3390/ijms21010041 www.mdpi.com/journal/ijms Review Anti-Obesity Effects of Microalgae Saioa Gómez-Zorita 1,2,†, Jenifer Trepiana 1,†, Maitane González-Arceo 1, Leixuri Aguirre 1,2, Iñaki Milton-Laskibar 1,2, Marcela González 3, Itziar Eseberri 1,2,*, Alfredo Fernández-Quintela 1,2,* and María P. Portillo 1,2 1 Nutrition and Obesity Group, Department of Nutrition and Food Science, University of the Basque Country (UPV/EHU) and Lucio Lascaray Research Institute, 01006 Vitoria, Spain; [email protected] (S.G.-Z.); [email protected] (J.T.); [email protected] (M.G.-A.); [email protected] (L.A.); [email protected] (I.M.-L.); [email protected] (M.P.P.) 2 CIBEROBN Physiopathology of Obesity and Nutrition, Institute of Health Carlos III, 01006 Vitoria, Spain 3 Nutrition and Food Science Department, Faculty of Biochemistry and Biological Sciences, National University of Litoral and National Scientific and Technical Research Council (CONICET), Santa Fe 3000, Argentina; [email protected] (M.G.) * Correspondence: itziar.eseb[email protected]s (I.E.); alfredo.fernan[email protected] (A.F.-Q.); Tel.: +34-945014363 (I.E.); Tel.: +34-945013066 (A.F.-Q.) † These authors contributed equally to this work. Received: 14 October 2019; Accepted: 17 December 2019; Published: 19 December 2019 Abstract: In recent years, microalgae have attracted great interest for their potential applications in nutraceutical and pharmaceutical industry as an interesting source of bioactive medicinal products and food ingredients with anti-oxidant, anti-inflammatory, anti-cancer, and anti-microbial properties. One potential application for bioactive microalgae compounds is obesity treatment. This review gathers together in vitro and in vivo studies which address the anti-obesity effects of microalgae extracts. The scientific literature supplies evidence supporting an anti-obesity effect of several microalgae: Euglena gracilis, Phaeodactylum tricornutum, Spirulina maxima, Spirulina platensis, or Nitzschia laevis. Regarding the mechanisms of action, microalgae can inhibit pre-adipocyte differentiation and reduce de novo lipogenesis and triglyceride (TG) assembly, thus limiting TG accumulation. Increased lipolysis and fatty acid oxidation can also be observed. Finally, microalgae can induce increased energy expenditure via thermogenesis activation in brown adipose tissue, and browning in white adipose tissue. Along with the reduction in body fat accumulation, other hallmarks of individuals with obesity, such as enhanced plasma lipid levels, insulin resistance, diabetes, or systemic low-grade inflammation are also improved by microalgae treatment. Not only the anti-obesity effect of microalgae but also the improvement of several comorbidities, previously observed in preclinical studies, has been confirmed in clinical trials. Keywords: microalgae; obesity; triglyceride; adipose tissue; adipocyte; mice 1. Introduction Microalgae are prokaryotic or eukaryotic microscopic single-cell organisms, found in fresh water and marine systems. They produce approximately half of the atmospheric oxygen and use the greenhouse gas carbon dioxide to grow photo-autotrophically. Together with bacteria, microalgae provide energy for all the trophic levels above them. Although microalgae show a great biodiversity, the ones most studied are Chlorella, Spirulina, Haematococus, Dunaniella y Scenedesmus. Microalgae produce a great variety of compounds, such as photosynthetic pigments (carotenoids and chlorophylls), sterols, polyunsaturated fatty acids, vitamins, minerals, fiber, polysaccaharides, enzymes, peptides, and toxins. It is important to emphasize that the chemical Int. J. Mol. Sci. 2020, 21, 41 2 of 21 composition of microalgae depends on the species and the cultivation conditions, such as temperature, illumination, pH, CO2 supply, salt, and nutrients [1,2]. They have attracted great interest in recent years due to their potential applications in nutraceutical and pharmaceutical industries, and are a major source of bioactive medicinal products and food ingredients with anti-oxidant, antiinflammatory, anti-cancer, and anti-microbial properties [2,3]. One of the potential application fields for the microalgae bioactive compounds is obesity, which has become a serious health problem due to its high prevalence, and because it is a major risk factor for a wide range of chronic diseases, including diabetes, cardiovascular diseases, and cancer [4–6]. Nowadays, approved new-generation anti-obesity medications offer a safe and tolerable adjunct to lifestyle interventions for the majority of individuals with obesity. Nevertheless, depending on patient tolerability to side effects, poor adherence or discontinuation can be treatment limitations. In fact, this situation reduces treatment benefits [7]. In this context, the present review gathers in vitro and in vivo studies addressed to analyze the anti-obesity effects of microalgae extracts, but not those where isolated microalgae compounds have been used. 2. In Vitro Studies To date, several in vitro studies have been conducted to analyze the effects of microalgae extracts on adipogenesis and metabolic processes involved in triglyceride (TG) accumulation (Table 1; Figure 1). Int. J. Mol. Sci. 2020, 21, 41 3 of 21 Table 1. Effects of microalgae in pre-adipocytes and mature adipocytes. Reference Numbers Cell Line Microalgae and Doses Experimental Design Effects Mechanisms [8] Human adipose-derived stem cells Euglena gracilis Z extract (5%,10%, or 20%) Cells were treated during differentiation (7 days) and adipocyte maturation (7 additional days) At 10% and 20%: ↓ lipid content 44% and 74% respectively At 20%: ↓ Adipogenesis At 20%: ↓ C/EBPα and PPARγ gene and protein expressions ↓ Creb, Srebp1c, C/ebpβ and C/ebpδ gene expression ↓ Fabp4 and Lpl gene expression [9] 3T3-L1 pre-adipocytes Spirulina maxima extract (50 and 100 µg/mL) Cells were treated on day 0, 2, 4, and 6 of differentiation (cell harvesting on day 8) At 100 µg/mL: ↓ Adipogenesis (dose dependent) At 100 µg/mL: ↓ FAS and C/EBPα protein expression At 50 and 100 µg/mL: ↓ PPARγ and aP2 protein expression ↓ SREBP1c, ACC, LPAATβ, lipin1, and DGAT-1 protein expression C3H10T1/2 mesenchymal stem cells Spirulina maxima extract (100 µg/mL) Cells were treated on day 0, 2, 4, and 6 of differentiation (cell harvesting on day 8) ↓ Adipogenesis ↓ C/EBPα, PPARγ, and aP2 protein expression ↓ SREBP1c, FAS, ACC, LPAATβ, lipin1 and DGAT-1 protein expression [10] 3T3-L1 pre-adipocytes Phaeodactylum tricornutum extract (250 and 400 µg/mL) Cells were treated during differentiation (6 days) At 250 µg/mL ↓ Lipid accumulation At 400 µg/mL ↓ Adipogenesis At 400 µg/mL ↓ PPARγ and ↑ UCP1 protein expression [11] 3T3-L1 pre-adipocytes Phaeodactylum tricornutum extract (100 mg/L) Cells were treated on day 7 of differentiation for 24 h (cell harvesting on day 8) No differences in lipid content or cytotoxicity ↑ Cd36 and Cpt1 gene expression Int. J. Mol. Sci. 2020, 21, 41 4 of 21 Special note: Doted lines incorporated for separation between different studies. ACC: Acetyl-CoA carboxylase, AP2: fatty acid binding protein, C/EBP: CCAATenhancer-binding protein, CD36: cluster of differentiation 36, CPT1: carnitine palmitoyltransferase 1, CREB: cAMP regulatory element-binding protein; DGAT-1: diacylglycerol O-acyltransferase, FABP4: fatty acid-binding protein 4, FAS: fatty acid synthase, LPAATβ: lysophosphatidic acid acyltransferase β, LPL: lipoprotein lipase, PPARγ: peroxisome proliferator activated receptor γ, SREBP1c: sterol regulatory element-binding protein 1c, UCP: uncoupling protein. ↑ significant increase, ↓: significant decrease. Int. J. Mol. Sci. 2020, 21, 41 5 of 21 Figure 1. Anti-obesity mechanisms of action described in in vitro studies (* ex vivo). ACC: acetyl-CoA carboxylase, AP2: fatty acid binding protein, C/EBP: CCAAT-enhancer-binding protein, CPT1: carnitine palmitoyltransferase 1, CREB: cAMP regulatory element-binding protein; DGAT-1: diacylglycerol O-acyltransferase, FABP4: fatty acid-binding protein 4, FAS: fatty acid synthase, LPAATβ: lysophosphatidic acid acyltransferase β, PGC-1α: peroxisome proliferator-activated receptor gamma co-activator 1α, PRDM16: PR domain-containing 16, PPARγ: peroxisome proliferator activated receptor γ, SREBP1c: sterol regulatory element-binding protein 1c. ↑ significant increase, ↓: significant decrease. Sugimoto et al. [8] used an aqueous extract of Euglena gracilis Z (Euglena), unicellular photosynthesizing green algae. Euglena contains vitamins, minerals, unsaturated fatty acids, and accumulates crystalline β-1,3-glucan, a polysaccharide also known as paramylon, which is considered a functional dietary fiber. The authors obtained human adipose-derived stem cells (hASCs) from a non-diabetic female donor with a body mass index (BMI) of 26 kg/m2, and differentiated these cells into adipocytes for 7 days (0–7 days). Cells were cultured for an additional 7-day maturing period (8–14 days). Cytotoxicity was not observed in cells treated with any of the checked Euglena extract dilutions used (1.25%, 2.5%, 5%, 10%, 20%, or 40%). When the lipid content of cells incubated with the extract at doses of 5%, 10%, or 20% was analyzed, the authors observed that Euglena extract reduced cellular TG content 17%, 44%, and 74%, in line with the increased concentration of the extract in the medium. In order to explore the mechanism underlying this effect, the authors studied the adipogenic pathway. Adipogenesis is a tightly regulated cellular differentiation process, which allows adipose tissue expansion. In this process, mesenchymal stem cells become pre-adipocytes and pre-adipocytes differentiate into mature adipocytes, the cells that are able to accumulate triglycerides into lipid droplets [12]. For this purpose, they measured gene and protein expressions of peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT-enhancer-binding protein α (C/EBPα), the master regulators of adipocyte-differentiation. While the gene expression of Pparγ and C/ebpα were increased during adipocyte-differentiation in the control cells, these were repressed by 23% when 20% of Euglena extract was added to the medium. Protein amounts of PPARγ and C/EBPα were also significantly reduced, which is consistent with this result. The authors also observed an inhibition induced by the Euglena extract in gene expression of adipogenic markers expressed downstream in the adipocyte differentiation process, and regulated by PPARγ and C/EBPα, such as fatty acid binding protein (Ap2) (also known as fatty acid bonding protein 4, Fabp4) and lipoprotein lipase (Lpl). These results show that Euglena extract inhibits adipocyte-differentiation through suppression of master regulators involved in that metabolic pathway. Furthermore, since Pparγ expression is enhanced at the early phase of adipocyte-differentiation by two members of the C/EBP protein family, C/EBPβ and C/EBPδ, as well as by sterol regulatory element-binding transcription factor 1c (SREBP1c) and cAMP regulatory element-binding protein (CREB), their gene expression was also determined in hASCs. For this purpose, cells were cultured with or without Euglena extract (20%) during the first three days in the differentiation process. All these genes were downregulated when Euglena extract was present in the medium, showing that its inhibitory effect on adipocyte-differentiation was caused by repressing the early stage of adipocyte- Int. J. Mol. Sci. 2020, 21, 41 6 of 21 differentiation. These observations were confirmed when adipogenesis was evaluated by determining Oil Red O from cells treated with 20% of extract during adipocyte-differentiation (days 0–7) and from those cells treated during adipocyte maturation (days 8–14). Constant supplementation (day 0–14) with Euglena extract inhibited lipid accumulation by approximately 50% as compared to the control cells. When supplementation with the alga extract took place only during the adipocyte differentiation period (days 0–7) lipid accumulation was inhibited by 60%, but approximately 96% of the accumulated lipids remained in the cells treated with the extract on days 7–14. Therefore, the authors concluded that Euglena extract suppresses adipocyte-differentiation at the early stage, thus contributing to its anti-obesity effect. Another microalga studied by several authors is Spirulina maxima. It contains pigment proteins such as chlorophyll a and C-phycocyanin, which have been reported as possessing anti-oxidant, antiinflammatory (both pigments), and anti-diabetic actions (C-phycocyanin). Seo et al. [9] performed an in vitro study to explore whether an ethanolic extract of this microalga also showed anti-obesity and adipocyte browning properties. For this purpose, 3T3-L1 pre-adipocytes and C3H10T1/2 cells, a cellular line functionally similar to mesenchymal stem cells, were treated during the differentiation period (0–8 days) with 50 or 100 µg/mL of the microalga extract. Previously no cytotoxicity had been confirmed at these concentrations. In 3T3-L1 pre-adipocytes, the addition of the extract to the differentiation medium decreased TG accumulation in a dose dependent manner. This effect was due to lower protein expression of the adipogenic regulators C/EBPα, PPARγ, and aP2, meaning that adipogenesis was inhibited. The same results were observed when the authors treated C3H10T1/2 cells with the extract at a concentration of 100 µg/mL. In addition, the authors explored lipogenesis, the metabolic process through which fatty acids are esterified with glycerol for their storage as triglycerides, and that allows adipocytes to increase their size. More specifically, the authors measured enzymes involved in de novo lipogenesis, the biosynthetic pathway by which acetyl-CoA is converted to fatty acids before they are esterified with glycerol to synthesize triglycerides. For that purpose, authors measured proteins such as acyl-CoA carboxylase (ACC) and fatty acid synthase (FAS), as well as markers involved in triglyceride assembly, such as lysophosphatidic acid acyltransferase β (LPAATβ), lipin-1 and diacylglycerol acyltransferase-1 (DGAT1). In this regard, they observed that treating 3T3-L1 pre-adipocytes during differentiation with the ethanolic extract led to reductions in the protein expressions of SREBP1, ACC, and FAS, as well as of LPAATβ, lipin-1, and DGAT1. As far as C3H10T1/2 cells are concerned, incubating cells with the extract obtained from Spirulina maxima during differentiation reduced protein expressions of the three latter lipogenic markers (LPAATβ, lipin-1, and DGAT1). The authors concluded that this extract significantly suppressed lipogenesis either in 3T3-L1 adipocytes or C3H10T1/2 cells. Finally, the authors reported browning effects ex vivo, in cells obtained from the stromal vascular fraction of mice fed a high-fat diet (HFD) supplemented with an ethanolic extract of the alga (150 or 450 mg/kg/day). Cells were differentiated into white adipocytes, and higher protein expression of PR domain containing 16 (PRDM16) and uncoupling protein 1 (UCP1) was detected in the adipose primary cells. The expression of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) was upregulated only by the higher dose. Using Phaeodactylum tricornutum, a diatom microalga rich in eicosapentanoic acid (EPA) and the carotenoid fucoxanthin, Koo et al. [10] aimed to evaluate the anti-obesity effect of a commercially available extract, containing 3.5–6% fucoxanthin (w/w), on lipid accumulation in 3T3-L1 adipocytes. The cells were cultured during the differentiation period for six days with the Phaeodactylum tricornutum extract (100, 125, 200, 250, and 400 µg/mL), fucoxanthin (active principle; 10, 20, and 40 µg/mL) or curcumin as control (20 µg/mL). The microalga extract reduced adipogenesis in 3T3-L1 preadipocytes at a concentration of 250 µg/mL, and consequently reduced cellular lipid accumulation was observed. When looking at the mechanisms underlying this effect, the authors reported that although no changes were observed in the protein expression of the adipogenic factor C/EBPα, Phaeodactylum tricornutum extract decreased PPARγ protein expression and increased that of UCP1, mainly at the highest dose (400 µg/mL). Therefore, the authors concluded that Phaeodactylum Int. J. Mol. Sci. 2020, 21, 41 7 of 21 tricornutum extract exhibits anti-obesity effects by controlling lipid metabolism through PPARγ and UCP1. Finally, Gille et al. [11] incubated 3T3-L1 cells on day 7 of differentiation with an ethanolic extract of the same microalga, at a dose of 100 mg/L for 24 h. Moreover, they also tested its bioactive compound fucoxanthin at a concentration of 5 µM. According to the authors, each 100 mg/L of the microalga contained 3.6 µM of fucoxanthin. Regarding the microalga effect, the authors did not appreciate significant effects on lipid content or cell toxicity, although cluster of differentiation 36 (Cd36) and carnitine palmitoyltransferase 1a (Cpt1a) mRNA levels were significantly increased. Furthermore, Cpt1a gene expression was similarly induced by fucoxanthin incubation. Consequently, it can be proposed that the effect of the microalga extract on Cpt1a expression were due, at least in part, to its fucoxanthin content. 3. Animal Studies Studies using animal models and different experimental approaches to analyze the potential anti-obesity effect of microalgae have revealed beneficial effects on body weight management and energy metabolism (Table 2; Figure 2). Int. J. Mol. Sci. 2020, 21, 41 8 of 21 Table 2. Effects of microalgae in animal studies. Reference Numbers Animal Model Microalgae and Doses Experimental Design Effects Mechanisms [9] Male ICR mice (4 weeks old) Spirulina maxima extract (SM70EE; 150 and 450 mg/kg BW/day) 6 weeks HFD: high-fat diet (60% of energy from fat) SM150: HFD + SM70EE 150 mg/kg BW/day SM450: HFD + SM70EE 450 mg/kg BW/day SM450 group: ↑ Serum HDL-c SM150 and SM450 groups: ↓ Final body weight ↓ Body weight gain ↓ Subcutaneous and abdominal WAT ↓ Serum TG, TC, LDL-c and glucose levels ↓ Adipogenesis in WAT SM450 group: ↑ pAMPK, PRDM16 and PGC1α protein expression in WAT ↑ UCP1 protein expression in BAT SM150 and SM450 groups: ↓ C/EBPα, PPARγ, and aP2 protein expression in WAT ↑ UCP1 protein expression in WAT ↑ PRDM16 protein expression in BAT [13] Male SpragueDawley rats (5 weeks old) Spirulina maxima (62.5, 125, and 250 mg/kg BW/day) 4 weeks LFD: low-fat diet (10% of energy from fat) HFD: high-fat diet (60% of energy from fat) SM 62.5: HFD + 62.5 mg/kg BW/day SM 125: HFD + 125 mg/kg BW/day SM 250: HFD + 250 mg/kg BW/day of SM 62.5, 125, and 250 SM groups: ↓ Epididymal adipocyte size ↑ Brown adipose tissue index ↑ Serum adiponectin ↓ Serum TNF-α ↓ HOMA-IR ↑ Serum HDL-c/TC ↓ Serum ALT SM 125 and SM 250 groups: ↓ Body weight gain ↑ Brown adipose tissue ↓ Serum leptin ↓ Serum insulin ↓ Serum TC SM 250 group: ↓ Epididymal adipose tissue index ↓ Serum glucose SM 125 and SM 250 groups: ↓ FAS protein expression (epididymal AT) ↓ Fasn gene expression (epididymal AT) ↑ AdipoR gene expression (epididymal AT) ↑ pAMPK protein expression (epididymal AT and skeletal muscle) ↑ AdipoR1, Cpt1 and Ucp2 gene expression (skeletal muscle) ↓ Srebf1 and Nfκβ gene expression (epididymal AT) SM 250 group: ↑ Atgl and Cpt1 gene expression (epididymal AT) The dose is not specified: Int. J. Mol. Sci. 2020, 21, 41 9 of 21 ↑ pACC, AdipoR1, NAMPT, and SIRT1 protein expression (epididymal AT) ↓ SREBP1 and FAS protein expression (epididymal AT) ↑ AdipoR1, NAMPT and SIRT1 protein expression (skeletal muscle) [11] Male C75BL/6J mice (6–8 weeks old) Phaeodactylum tricornutum (PE) extract (100 and 300 mg/kg BW/day) 26 days HFD: high-fat diet 45% of energy from fat) PE100: HFD + PE 100 mg/kg BW/day PE300: HFD + PE 300 mg/kg BW/day PE300 group: ↓ Final body weight ↓ Total body fat mass ↓ Epididymal and inguinal tissue ↓ Adiposity index ↑ Energy expenditure PE100 and PE300 group: ↑ % of small adipocytes in inguinal WAT PE100 group: ↑Cd36 and Ppargc1a gene expression in BAT PE300 group: ↓Lipe, Plin1, and Lpl gene expression in epididymal WAT ↑Ucp1 and Cpt gene expression in inguinal WAT ↑BAT activation PE100 and PE300 group: ↓Lipe and Fasn gene expression in BAT ↑UCP1 protein expression in BAT [10] Female C57BL/6J mice (8 weeks old) Phaeodactylum tricornutum (PE) extract (0.81, 1.62, and 3.25 mg/kg BW/day) 6 weeks HFD: High-fat diet PE-L: HFD + PE 0.81 mg/kg BW/day PE-M: HFD + PE 1.62 mg/kg BW/day PE-H: HFD + PE 3.25 mg/kg BW/day PE-H group: ↓ Plasma LDL-c PE-M group: ↓ Plasma TG PE-M and PE-H groups: ↓ Subcutaneous fat volume PE-L, PE-M and PE-H groups: ↓ Body weight gain ↓ inguinal fat depots ↓ Total and abdominal fat volume PE-H group: ↓ C/EBPα protein expression in WAT PE-M and PE-H groups: ↑ UCP1 protein expression in WAT PE-L, PE-M, and PE-H groups: PPARγ protein expression in WAT Int. J. Mol. Sci. 2020, 21, 41 16 of 21 Table 3. Effects of microalgae in clinical studies. Reference Numbers Participants Microalgae and Doses Experimental Design Effects [17] 52 sedentary young men (26 ± 5 years) with BMI ≥ 25 kg/m2 27 subjects with overweight and 25 subjects with obesity Arthrospira (Spirulina) maxima 4.5 g/day Two intervention groups: Sm: S. maxima supplementation C: Control (Placebo) Duration: 6 weeks + 2 weeks of wash-out + 6 weeks (crossover for the supplementation interventions) Intra-group (pretreatment vs. post) comparisons (Sm): ↓TC and TG: only in subjects with dyslipidemia. LDL-c: no changes ↑ HDL-c: only in in subjects with dyslipidemia. Inter-group comparisons (Sm vs. C): ↓TC: only in in subjects with obesity. TG: no changes ↓ LDL-c: in in subjects with overweight, obesity or dyslipidemia. HDL-c: no changes ↓ BMI: in subjects with obesity or dyslipidemia. [18] 50 patients (25 women and 25 men, 25–60 years old) with obesity (BMI ≥ 30 kg/m2) and well-controlled hypertension Arthrospira platensis Four-daily dosage of 500 mg each Two intervention groups: Placebo Spirulina Duration: 12 weeks Effects vs. placebo: ↓ BMI and waist circumference ↓ Serum TC, LDL-c, glucose, and insulin ↑ Total antioxidant state [19] 56 individuals with obesity (20–50 years old) with BMI ≥ 30 kg/m2 Arthrospira platensis Twice-daily dosage of 500 mg each Two intervention groups: Intervention group: supplementation of S. platensis in a dosage of 500 mg twice a day over 12 weeks Control group: two pills of placebo daily over 12 weeks. ↓ BMI (↓ BW) ↓ TC TG: no changes LDL-c: no changes HDL-c: no changes VEGF: no changes ↓ Appetite Int. J. Mol. Sci. 2020, 21, 41 17 of 21 [20] 52 subjects with overweight or obesity with BMI between 25 and 40 kg/m2 Spirulina platensis Four-daily dosage of 500 mg/day Two intervention groups Intervention SP group: supplementation of SP extract (four tablets per day) for 12 weeks with a restricted calorie diet Placebo group: placebo extract (four tablets per day) for 12 weeks with a restricted calorie diet ↓ Body weight ↓ Waist circumference ↑ Body fat reduction ↓ Plasma TG, LDL-c ↓ LDL-c/HDL-c ratio Special note: Doted lines incorporated for separation between different studies. BMI: body mass index; BW: body weight; C: control (Placebo); HDL-c: high density lipoprotein -cholesterol; LDL-c: low density lipoprotein-cholesterol; Sm: Spirulina without exercise; TC: total cholesterol; TG: triglycerides; VEGF: vascular endothelial growth factor. ↑ significant increase, ↓: significant decrease. Int. J. Mol. Sci. 2020, 21, 41 18 of 21 The results showed a significant decrease in TC and TG along with a significant increase in HDLcholesterol in the Sm group after treatment, when compared with the basal levels. These changes were observed only among dyslipidemic subjects. However, LDL-cholesterol showed no change. In addition, the authors compared the variation of each parameter between both experimental groups and observed that plasma TC level decreased significantly in obese subjects in the Sm treatment, and LDL-cholesterol was lower in overweight, obese, and dyslipidemic subjects enrolled in the Sm treatment, when compared to those in the placebo group. By contrast, TG and HDL-cholesterol levels were not modified. As far as BMI is concerned, a significant reduction was only observed in obese and dyslipidemic subjects after treatment. These results suggest that Spirulina maxima supplementation results in a partial improvement of blood lipid profile and BMI in men with excess body weight and dyslipidemia. Using the same microalga, Szulinska et al. [18] carried out a randomized, double-blind, placebocontrolled trial addressed on 25–60 year old individuals with obesity (BMI ≥ 30 kg/m2), with wellcontrolled hypertension and without other comorbidities. Participants were divided into two experimental groups: placebo group (four capsules per day of microcrystalline cellulose over 3 months) and spirulina group (four capsules per day of Hawaiian Spirulina over 3 months). Each spirulina capsule contained 0.5 g of Spirulina maxima. At the end of the experimental period, spirulina group showed lower BMI, waist circumference, serum TC, LDL-cholesterol, glucose and insulin and total antioxidant state than the placebo group. No differences in serum HDL-cholesterol and TG were observed between groups. In another randomized doubled-blind, placebo-controlled trial conducted by Zeinalian et al. [19], the effect of Spirulina platensis supplementation on BMI, serum lipids, appetite, and serum vascular endothelial growth factor (VEGF) was studied. Individuals with obesity were divided into two groups, the placebo group and the group that received Spirulina platensis twice daily (500 mg each dose). After 12 weeks of intervention, a decrease in body weight, and thus in BMI, was observed, along with a reduction appetite in the group treated with Spirulina platensis. With regard to serum lipids, the only change was a significant reduction in TC, while LDL-cholesterol and TG remained unchanged after the intervention. Despite a significant increase in HDL-cholesterol in both treated and placebo groups at the end of the experimental period, there was no change in the mean differences between the two groups. VEGF is an important angiogenic factor implicated in normal and pathological vessel formation that can be an important biomarker of obesity and obesity-related cancer progression. In this study, VEGF remained unchanged after treatment with Spirulina platensis. The authors concluded that a dose of 1 g/day of Spirulina platensis for 12 weeks had beneficial effects modulating body weight and appetite, while it only modified the serum lipid profile partially. Spirulina platensis was also used in a randomized, double-blinded, placebo-controlled clinical trial reported by Yousefi et al. [20]. Obese or overweight subjects (BMI: 25–40 kg/m2) were distributed into two groups, a placebo and a Spirulina platensis-treated group, who followed a restricted calorie diet for 12 weeks. The microalga was administered in four tablets of 500 mg/capsule daily. At the end of the intervention, body weight and waist circumference were reduced in the microalgasupplemented group compared to the control group. Moreover, in this group body fat reduction was higher than that observed in the placebo group. Regarding plasma parameters, TG, LDL-cholesterol, and the LDL/HDL ratio were reduced at the end of the treatment period compared with the baseline in the microalga-treated group. Based on these results, the authors suggested that Spirulina platensis could be a useful as a complementary therapy to reduce weight and TG levels. 5. Concluding Remarks Data reported in the literature, and gathered in the present review, show that there is scientific evidence supporting the anti-obesity effect of several microalgae: Euglena gracilis, Phaeodactylum tricornutum, Spirulina maxima, Spirulina platensis, and Nitzschia laevis. With the exception of one study, the published works carried out in animal models have addressed the effects of microalgae in animals submitted to an obesogenic feeding pattern. Consequently, the results have shown the ability of microalgae to total or partially prevent obesity development associated to this dietary pattern. Int. J. Mol. Sci. 2020, 21, 41 19 of 21 Preclinical studies have revealed some of the mechanisms of action underlying this effect. Depending on the species and concentration, microalgae can inhibit pre-adipocyte differentiation, thus reducing the number of mature adipocytes ready to accumulate TG. Moreover, they reduce de novo lipogenesis and TG assembly, thus limiting the amount of TG to be stored. An increase in lipolysis and fatty acid oxidation can also be observed. Finally, microalgae can induce an increase in energy expenditure via thermogenesis activation in brown adipose tissue, as well as by inducing browning in white adipose tissue. It could be thought that a potential toxic effect of some constituent common in microalgae could be responsible, at least in part, for the reduced lipid retention and weight reduction. However, this possibility can be discarded because in vitro studies have shown no cytotoxicity of microalgae extracts in a wide range of doses. In parallel with the reduction in body fat accumulation, other features which are typical of individuals with obesity, such as enhanced plasma lipid levels, insulin resistance or diabetes, and low-grade inflammation, are also improved by microalgae treatment. The anti-obesity effect of microalgae, as well as the improvement of several comorbidities observed in preclinical studies, has been confirmed in clinical trials. In this case, due to the experimental design characteristics, the role of microalgae in obesity treatment, rather than in obesity prevention, has been evidenced. Concerning the limitations of the reported studies, it should be pointed out that more research is needed to determine which bioactive compounds, present in microalgae, are responsible for their anti-obesity effects, as well as to look for potential synergies among them. In addition, although several mechanisms have been proposed to explain the anti-obesity effects of microalgae, further studies are needed in order to gain more insight concerning this issue. For instance, in several studies increased expression of genes related to thermogenesis has been found, suggesting the activation of this process, but additional studies are needed to confirm that in fact thermogenesis, and consequently energy expenditure, are increased. Author contributions: Conceptualization, S.G.-Z. and J.T.; Writing—Original Draft Preparation, S.G.-Z., J.T., M.G.-A., L.A., I.M.-L., M.G., I.E. and A.F.-Q.; Writing—Review and Editing, M.P.P., S.G.-Z., I.E. and A.F.-Q.; Project Administration, M.P.P.; Funding Acquisition, M.P.P. and A.F.-Q. All authors have read and agreed to the published version of the manuscript. Funding: This study was supported by grants from the Government of the Basque Country (ELKARTEK) under grant KK-2019/00031, Instituto de Salud Carlos III (CIBERobn) under Grant CB12/03/30007, and University of the Basque Country under Grant GIU18-173. Conflicts of Interest: The authors declare no conflict of interest. Abbreviations ACC acyl-CoA carboxylase AdipoR1 adiponectin receptor ALT alanine aminotransferase Ap2 fatty acid binding protein ATGL adipose triglyceride lipase AUC area under the curve BAT brown adipose tissue BMI body mass index CLS crown-like structures C/EBPα CCAAT-enhancer-binding protein α CD36 cluster of differentiation 36 CPT1a carnitine palmitoyltransferase 1a CREB cAMP regulatory element-binding protein DGAT1 diacylglycerol acyltransferase-1 EPA eicosapentanoic acid FABP4 fatty acid binding protein 4 FAS fatty acid synthase Int. J. Mol. Sci. 2020, 21, 41 20 of 21 G6PDH glucose-6-phosphate dehydrogenase hASCs human adipose-derived stem cells HDL high-density lipoprotein HFD high-fat diet HOMA-IR Homeostatic Model Assessment for Insulin Resistance HSL hormone sensitive lipase LDL low-density lipoprotein LPAATβ lysophosphatidic acid acyltransferase β LPL lipoprotein lipase MAC-2 macrophage marker galectin-3 ME malic enzyme MEST mesoderm-specific transcript homolog protein NAMPT nicotinamide phosphoribosyltransferase ND normal diet NFκΒ nuclear factor κΒ pAMPK 5’ AMP-activated protein kinase Plin1 perilipin 1 PGC1α peroxisome proliferator-activated receptor gamma coactivator 1-alpha PPAR γ peroxisome proliferator-activated receptor γ PRDM16 PR domain containing 16 SIRT1 sirtuin 1 SREBP1c sterol regulatory element-binding transcription factor 1c TC total cholesterol TG triglyceride UCP1 uncoupling protein 1 VEGF vascular endothelial growth factor WAT white adipose tissue References 1. 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