Introduction
Dimethyl sulfoxide (DMSO) is an organic, amphiphilic molecule with a wide range of applications in cell biology and biomedicine. While it's recognized for properties like vasodilation, anti-inflammatory action, and its use as a solvent for hydrophobic drugs, the effects of DMSO on weight loss and related metabolic processes are complex and warrant careful examination. This article aims to provide a comprehensive overview of existing research on DMSO, focusing on its potential impact on weight management, lipid metabolism, and cellular function.
What is DMSO?
Dimethyl sulfoxide (DMSO, (CH3)2SO) is an organic amphiphilic molecule that is widely used in cell biology for various applications. It is an effective solvent and cytoprotectant agent that can induce diverse actions in experimental settings, ranging from metabolic stress to cytotoxic effects depending on the concentration used.
DMSO's Properties and Uses
DMSO exhibits a number of capabilities, including vasodilatory, diuretic, anti-inflammatory and bacteriostatic properties. In vitro, DMSO is routinely used for cryopreservation of cells, and for many researchers, DMSO has been the preferred solvent for the dissolution of small hydrophobic drug molecules. DMSO is known to interact strongly with phospholipids, which makes it efficient at facilitating movement of molecules, especially drugs across biological membranes. In addition, DMSO is also a free radical scavenger demonstrating antioxidant activities at low concentrations but becoming pro-oxidant at higher concentrations. Despite the potential for physiological interference and cytotoxicity, DMSO remains a solvent of choice in biomedical research.
DMSO and Adipocytes
An in vitro model of 3T3-L1 adipocytes represents an accomplished system to investigate adipocytic lipid metabolism and adiposity. During the progression of obesity, adipose tissue metabolism is dysregulated, accompanied by reduced metabolic activity, generation of oxidative stress, and subsequent cell damage. Consequently, there has been a drastic increase in the use of 3T3-L1 adipocytes as a model to screen pharmacological and natural compounds, including those dissolved in DMSO, for their therapeutic potential against obesity and its associated complications.
While it has been reported that DMSO can inhibit the differentiation of 3T3 T mesenchymal stem cells or promote glucose transporter 4 translocation in insulin-stimulated 3T3-L1 adipocytes, less is known about the dose-dependent effect of DMSO on these cells.
Read also: Uncover the benefits of DMSO for weight loss in our overview.
Concentration-Dependent Effects of DMSO on Adipocytes
A study investigated the concentration-dependent (0.01-100%) effect of DMSO on lipid content and cell viability in 3T3-L1 adipocytes. Furthermore, as contradicting evidence has emerged on the role of DMSO in the modulation of oxidative stress in different cell types, this study investigated the effect of DMSO on the oxidative stress response in 3T3-L1 adipocytes.
Impact on Lipid Content
ORO staining confirmed that 3T3-L1 adipocytes presented with enhanced lipid content in the model. Thereafter, various doses of DMSO were tested for their effect on lipid content in 3T3-L1 adipocytes after one hour of exposure. In comparison to an experimental control (DPBS exposed only cells), DMSO doses of 0.01-1% did not have any effect on lipid content, while doses of 10 and 100% significantly reduced lipid content by 14% (p < 0.05) and 47% (p < 0.001), respectively.
Impact on Metabolic Activity
Exposing adipocytes to 0.1-100% DMSO resulted in a concentration-dependent suppression of metabolic activity when compared to the experimental control. In comparison to an experimental control (DPBS exposed only cells), a DMSO dose of 0.01% did not have any effect on metabolic activity, while doses of 0.1, 1, 10 and 100% significantly reduced metabolic activity by 11% (p < 0.001), 14% (p < 0.001), 40% (p < 0.001) and 90% (p < 0.001), respectively.
Impact on Mitochondrial Membrane Potential
Although no significant effect was observed with 0.01% DMSO when compared to the experimental control, doses of 0.1, 1, 10 and 100% markedly increased mitochondrial membrane potential by 0.3 RFI units (p < 0.5), 0.5 RFI units (p < 0.01), 0.6 RFI units (p < 0.01), 1.7 RFI units (p < 0.01), respectively. The increase in mitochondrial membrane potential was evident by enhanced green fluorescent staining of depolarized cells compared to the experimental control.
Impact on Glutathione Content and Reactive Oxygen Species
The lower doses of DMSO (0.01 and 0.1%) increased GSH content of cells by 12% (p < 0.001) and 23% (p < 0.001), respectively, when compared to the experimental control. While doses of 1, 10 and 100% significantly reduced GSH levels in cells by 17% (p < 0.001), 40% (p < 0.001) and 61% (p < 0.001), respectively. On the other hand, ROS levels as measured by DCFH-DA stain were dose-dependently increased (p < 0.001) when compared to the experimental control. Doses of 10 and 100% DMSO further showed significant difference in comparison to the lowest DMSO dose tested (0.01%) for both GSH content and ROS production.
Read also: Weight Loss Guide Andalusia, AL
Impact on Apoptosis and Necrosis
Except for the lowest dose of DMSO tested (0.01%), all doses induced a dose-dependent increase in apoptosis and necrosis when compared to the experimental control. Furthermore, doses of 0.1-100% for apoptosis, and doses of 1-100% for necrosis showed a marked increase when compared to the lowest dose of DMSO tested (0.01%).
DMSO and Autophagy
Induction of autophagy is known not only to regulate cellular homeostasis but also to decrease triglyceride accumulation in hepatocytes. In HepG2 cells, treatment with 0.5 mM palmitate for six hours significantly increased lipid and triglyceride (TG) accumulation, assessed by Oil-red O staining and TG quantification assay. Treatment with 0.01% DMSO for 16 h statistically reduced palmitate-induced TG contents. Pretreatment of 10 mM 3-methyladenine (3MA) for 2 h restored hepatocellular lipid contents, which were attenuated by treatment with DMSO. DMSO increased LC3-II conversion and decreased SQSTM1/p62 expression in a time and dose-dependent manner.
In addition, the number of autophagosomes and autolysosomes, as seen under an electron microscopy, as well as the percentage of RFP-LAMP1 colocalized with GFP-LC3 dots in cells transfected with both GFP-LC3 and RFP-LAMP1, as seen under a fluorescent microscopy, also increased in DMSO-treated HepG2 cells. DMSO also suppressed p-eIF2α/p-EIF2S1, ATF4, p-AKT1, p-MTOR and p-p70s6k/p-RPS6KB2 expression as assessed by western blotting. Knockdown of ATF4 expression using siRNA suppressed ATF4 expression and phosphorylation of AKT1, MTOR and RPS6KB2, but increased LC3-II conversion. DMSO reduced not only soluble but also insoluble mtHTT (mutant huntingtin aggregates) expressions, which were masked in the presence of an autophagy inhibitor.
DMSO Reduces Lipid Accumulation in Hepatocytes
HepG2 cells exposed to 0.5 mM palmitate for 24 h showed significantly decreased cell proliferation (68.52 ± 3.03, p < 0.001), and pre-treatment with 0.01, 0.05, 0.1, 0.5 and 1.0% DMSO for 16 h partially prevented palmitate-induced HepG2 cell toxicity with significance.
Intracellular lipid accumulation was analyzed by microscopy after staining cells with Oil red O (ORO), and quantification using a spectrophotometer at 520 nm. Since triglyceride (TG) is the major component of the lipid droplets, the TG content of the cells in this experiment was also checked. Thereby, visible lipid droplets were observed in cells incubated with palmitate. Compared with the untreated controls, HepG2 cells treated with 0.01% DMSO for 16 h showed minimally decreased assessable lipid content (0.23 ± 0.01 vs. 0.21 ± 0.003) without statistical significance as assessed by fat quantization with ORO. HepG2 cells exposed to 0.5 mM palmitate for 6 h showed significantly increased lipid accumulation (0.23 ± 0.01 vs. 0.40 ± 0.02. p < 0.001) and TG determination (34.98 ± 5.76 vs. 66.01 ± 5.40 p < 0.001). Pretreatment with 0.01% DMSO before exposure to 0.5 mM palmitate for 6 h also partially inhibited free fatty acid-induced lipid contents (0.40 ± 0.02 vs. 0.35 ± 0.02, p < 0.001) and TG accumulation.
Read also: Beef jerky: A high-protein option for shedding pounds?
DMSO Induces Autophagy
To elucidate the possible mechanism of macrolipophagy-mediated clearance of lipid droplets, steps of autophagy that can be monitored were performed. First, LC3 (microtubule-associated protein 1 light chain 3)-II conversion of HepG2 cells treated with 0.01 to 1.0% DMSO for 16 h was increased in a dose-dependent manner, monitored by western blotting. HepG2 cells exposed to 0.05 and 0.1% DMSO for 8 to 16 h were shown to increase LC3-II conversion and decrease SQSTM1/p62 (hereafter SQSTM1) in a dose- and time-dependent manner.
Second, it was tested for whether increased DMSO could be due to either the induction of autophagy or the inhibition of lysosomal degradation by using autophagic flux assay. In the presence of 50 µM lysomal inhibitor chloroquine, more decrease in SQSTM1 expression and increased expression of LC3-II conversion were found in a time- and dose-dependent manner than by DMSO alone. To determine the RNA levels of LC3, reverse transcription polymerase chain reaction (RT-PCR) was employed. Time and dose-dependent expression of LC3 transcription was also found in HepG2 cells treated with 0.01% DMSO.
Third, HepG2 cells were infected with a specific autophagic marker GFP-LC3 (comprising green fluorescent protein fused to the N-terminus of LC3), and visualized by fluorescence microscopy. A punctate pattern of GFP-LC3 accumulated upon the challenge with 0.01% DMSO, an excessive number of dots appeared in DMSO-treated cells compared the control groups (6.86 ± 4.37 vs. 23.38 ± 16.32, p < 0.05). In addition, HepG2 cells were also co-transfected with both GFP-LC3 and RFP-LAMP1 (lysosome associated membrane protein 1-tagged with red fluorescent protein) and then treated with 0.01% DMSO for 16 h. Fluorescence microscopy showed RFP-LAMP1 colocalized with GFP-LC3 in the cytoplasm. Finally, one pathognomonic feature of autophagy is the ultrastructural evidence of autophagosomes. DMSO-treated HepG2 cells were examined using an electron microscope to confirm the induction of autophagy.
To verify the hypothesis stating that DMSO could attenuate free fatty acid-induced lipid accumulation via macrolipophagy induction, its autophagic role in the presence or absence of (1) MG132 for ubiquitin-proteasome inhibition, (2) 3-methyladenie (3MA) for autophagy inhibition, and (3) chloroquine for lysosomal inhibition was investigated. Similar to previous results, 0.5 mM palmitate at 6 h significantly increased lipid accumulation; however, 0.01% DMSO partially attenuated palmitate-induced lipid accumulation.
In the treatment of MG132, a potent membrane-permeable proteasome inhibitor, no statistical difference in lipid accumulation was found between cells treated with palmitate + DMSO and those with palmitate + DMSO + MG132 (0.34 ± 0.05 vs. 0.35 ± 0.06, p > 0.05), suggesting no involvement of proteasome on the lipid-reducing effects of DMSO. In the treatment of 3-MA, which inhibits autophagy by blocking autophagosome formation via the inhibition of type III phosphatidylinositol 3-kinases (PtdIns3K) at the sequestration stage, where a double-membrane structure forms around a portion of the cytosol and sequesters it from the rest of cytoplasm to form the autophagosomes, a significant difference in lipid content was found between cells treated with palmitate + DMSO and those with palmitate + DMSO + 3-MA (0.35 ± 0.02 vs. 0.42 ± 0.01, p < 0.001). Chloroquine is known to accumulate in lysosomes due to its weak base properties and inhibit the lysosomal pathway of protein degradation. There was a significant difference in lipid content between cells treated with palmitate + DMSO and those treated with palmitate + DMSO + chloroquine.
DMSO Reduces Expression of ER Stress Markers and Induces Autophagy
To elucidate the hypothesis stating that downregulation of an unfolded protein response or ER stress markers, such as GRP78/HSPA5 (hereafter HSPA5), p-PERK/p-EIF2AK3 (hereafter EIF2AK3), p-eIF2α/p-EIF2S1 (hereafter p-EIF2S1), ATF4, ATF6, p-IRE1/p-ERN1 (hereafter p-ERN1), sXBP1 and CHOP/DDIT3 (hereafter DDIT3) by DMSO, would induce autophagy, with DMSO acting as a chemical chaperone, a western blotting assay was performed. In the absence of a stressor, DMSO inhibited upstream of unfolded protein response or ER stress pathway, such as phosphorylation of p-EIF2AK3, EIF2S1 and the expression of ATF4, ATF6, p-AKT1 and p-MTOR in a time- and dose-dependent manner.
Similar to previous reports on palmitate-induced increased expression of ER stress markers in various cell lines, HepG2 cells exposed to 0.5 mM palmitate for 6 h significantly activated HSPA5, p-EIF2AK3, p-EIF2S1, ATF4, ATF6, p-ERN1, sXBP1 and DDIT3 with significance. Regarding upregulated LC3II conversion as a self-protecting mechanism in our hypothesis, autophagy induction might be upregulated in response to ER stress, which could be also considered as a stressor marker. As expected, 0.01% DMSO efficiently inhibited palmitate-induced ER stress markers. Of the ER stress markers, EIF2S1 phosphorylation is also known to be a critical part of the PtdIns3K/AKT1/MTOR/ RPS6KB2 pathway, which may regulate either autophagy induction or suppression. The downstream target of phophorylated EIF2S1 is ATF4, and knockdown of ATF4 expression can induce autophagy. As shown, 0.01% DMSO reduced the expression of phophorylated EIF2S1, ATF4, p-AKT1, p-MTOR and p-RPS6KB2, resulting in activated LC3II conversion and increased autophagy flux as confirmed by decreased levels of SQSTM1.
Obesity and Metabolic Phenotypes
Obesity is defined as excessive accumulation or improper distribution of body fat (BF). Other concomitant illnesses include type 2 diabetes mellitus (T2DM), hepatic steatosis, cardiovascular disease, stroke, dyslipidemia, and hypertension, making obesity treatment more essential. Conventional classifications of overweight and obesity have been developed based on the Body Mass Index (BMI) and ethnicity-specific thresholds. Adults with a BMI of 25 to 29.9 kg/m2 were categorized as overweight, those with a BMI of 30 kg/m2 as obese, and those with a BMI of 18.5 to 24.9 kg/m2 as normal (ie, lean) weight. For Asian populations, BMI from 23.0 to 24.9 kg/m2 is considered overweight, BMI ≥25.0 kg/m2 is considered obese, and 18. 5-22.
In the past several decades, the prevalence of obesity has increased nearly three times, reaching epidemic levels. According to the World Health Organization (WHO), more than 650 million adults, or over 13% of the world’s population, had this chronic illness in 2016. According to previous reports, up to 463 million individuals globally and 1 in 11 adults have TD2M. Patients with non-alcoholic fatty liver disease (NAFLD) may have a higher risk of developing diabetes because they often exhibit aberrant glucose metabolism, which is indicative of T2DM and is characterized by elevated blood glucose levels, insulin resistance (IR), and impaired islet cell function. The prevalence rates of NAFLD and non-alcoholic steatohepatitis (NASH) in T2DM were 65.04% and 31.55%, respectively, according to a meta-analysis of 156 studies including 1,832,125 individuals.
Visceral obesity is the principal contributor to insulin resistance, which is the pathophysiological underpinning of Obesity, MAFLD, and T2DM.
Weight Loss and MAFLD Treatment
As a result, treating metabolic fatty liver includes treating both IR and obesity, in addition to the liver itself, and weight loss continues to be the cornerstone of MAFLD treatment, as is prompt and adequate treatment. Not only do ideal therapies result in considerable weight loss, hepatic steatosis remission, and fibrosis regression, but they also reduce IR and prevent the onset of T2DM.
The diversity of obesity, which includes a wide range of potential causes, is highlighted by the occurrence of several “phenotypes of obesity” with varying metabolic and cardiovascular disease (CVD) risks.
Obesity Phenotypes
De Lorenzo et al identified three distinct obesity phenotypes: MHO, NWO, and MUO. Individuals with normal weight who have hereditary obesity and are in the early stages of low-grade inflammation are considered to have Normal Weight Obesity (NWO) syndrome. There is a considerable loss of lean mass, equivalent to at least 1.5 kg (FFM kg), especially in the lower limb muscle mass, when the percentage of body fat (PBF) approaches 30%. They exhibit elevated levels of TNF-α, IL-1, and IL-8 as well as oxidative stress caused by metabolic anomalies. An alteration in a set of genes linked to aging and inflammation was revealed by NWO. Metabolically Obese Normal Weight (MONW), metabolically abnormal with no obesity (MANO), and metabolically unhealthy normal weight phenotype (MUHNW) are other names for this condition. The phenotype has an elevated Visceral Adipose Tissue (VAT) and abdominal Subcutaneous Adipose Tissue (SAT), a normal BMI (18.5-25 kg/m2), and decreased lean mass. Adiposity and ectopic fat distribution also increased. Diet quality may be an independent determinant of metabolic health. Individuals with MetS have a higher incidence of clinical features that are frequently identified because they tend to overlook clinical therapy or prevention.
At present, there is no international standard for the identification of MHO, and more than 30 distinct criteria have been used to operationalize symptoms. The MHO has been proposed to have a combination of obesity and absence of components of metabolic syndrome (in some definitions with the exception of waist circumference), with the exception of normal lipid and blood pressure profiles, good insulin sensitivity, obesity with BMI over 30 kg/m2, and no metabolic anomalies. Reduced visceral adiposity in relation to high total fat levels may contribute to increased insulin sensitivity and decreased inflammation. MHO are often young, physically active, and have good eating habits. According to various classification criteria, the MHO group comprises 6-75% of the obese population. Several underlying reasons, including reduced VAT and ectopic fat deposition (including less hepatic steatosis) compared to the more expandable subcutaneous fat depots, have been hypothesized to explain the better profile in those with MHO.
Compared to healthy individuals of normal weight, patients with MHO have a higher risk of developing metabolic syndrome. The fatty acid composition of myristic, palmitic, stearic, oleic, and linoleic acids may help explain why MHO has a lower inflammatory state. All MHO patients have a healthy Homeostasis Model Assessment of Insulin Resistance (HOMA-IR), Quantitative Insulin Sensitivity Check Index (QUICKI), insulin sensitivity index (ISI) (Mffm/l), hsCRP, and IL-6. Other biomarkers, such as leptin, have increased in MHO patients. Pro-inflammatory proteins such as histamine releasing peptide (HRP), hsCRP, Complement Component 4a (C4A), and inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4) are downregulated, while anti-inflammatory molecules such as Alpha-2 Heremans Schmid Glycoprotein (AHSG), Heregulin (HRG), and retinol-binding protein -4 (RBP4) are overexpressed in MHO.
MHO still has the risk of developing into an unhealthy phenotype and is linked to a number of serious chronic illnesses, such as CVD, hypertension, T2DM, chronic kidney disease, and several types of cancer. Obesity-related inflammation and metabolic problems are exacerbated by macrophage infiltration into adipose tissue, which is a significant pathogenic component. Alanine aminotransferase (ALT) is a metabolic syndrome-related biomarker that can be significantly increased.
The pro-inflammatory cytokines IL-6, IL-8, Monocyte chemoattractant protein-1 (MCP-1), regulated upon activation normal T cell expressed and presumably secreted (RANTES), Mps1 interacting protein-1 (MIP1), and plasminogen activator inhibitor-1 (PAI-1) are more incidents of heterogeneous expression seen in VAT, whereas leptin and interferon-gamma (IFN-gamma)-induced protein 10 (IP-10) are mostly expressed in SAT. Leucine rich repeat-containing receptor family NACHT, LRR, and PYD domain-containing protein-3 (NLRP3) gene and IL-1b are increased in VAT, which is infiltrated by pro-inflammatory macrophages in the MUO/MAO subgroup. VAT is associated with metabolic problems and its activation and expression are upregulated. Increased levels of hsCRP and TNF-α were linked to higher waist circumference (WC) in males and BMI in women, according to Marques-Vidal et al.
Sarcopenic obesity (SO), which is characterized by a loss of lean mass and an increase in the percentage of fat mass, is associated with risk factors such as advanced age, a decline in physical activity, atherosclerosis, and pulmonary illness. Individuals of various ages, not just older adults, can develop SO. According to Kim et al’s research, the prevalence of sarcopenic non-obese, sarcopenic, and non-sarcopenic obesity in patients was 10%, 15%, and 20%, respectively. Perna et al stated that according to dual-energy X-ray absorptiometry (DXA) or bioelectrical impedance analysis (BIA), SO refers to those who have extra body fat that is higher than the median or >27% for men and 38% for women, as well as loss of muscle mass and strength. Perna et al reported that sarcopenic visceral obesity is a phenotype that seems to be linked to inflammation, higher risk of fractures, and worse metabolic pattern. SO is associated with increased levels of serum hs-CRP among males, and an increase in MCP-1 levels in the serum indicates a pro-inflammatory state.
The dynamic and ever-changing characteristics of metabolic weight phenotypes make it difficult to predict outcomes. An individual’s health state can transition from metabolically healthy to metabolically unhealthy, for example, from NWO to MONW or MHO to MAO. MHO, NWO, MUO, and MONW may transform into SO as people age.
MAFLD and Obesity
Given the worldwide epidemic incidence of MAFLD and obesity, it is necessary to clarify the pathophysiological relationship between these two conditions.
Increased BMI and waist measurements are linked not only to MAFLD, but also to a higher risk of liver disease progression, especially in elderly individuals. This may be due in part to the fact that visceral fat has a stronger correlation with MAFLD than subcutaneous fat does. Visceral adipose tissue differs from subcutaneous fat in that it releases more pro-inflammatory and pro-fibrogenic mediators, has greater lipolytic rates, and is associated with increased insulin resistance. In addition to aggravating liver/systemic insulin resistance and predisposing to dyslipidemia, MAFLD releases several pro-inflammatory and vasoactive mediators that may facilitate the emergence of cardiometabolic problems linked to obesity.
Elevated serum uric acid (SUA) levels are a novel risk factor for MAFLD. There is evidence that some genetic variants associated with MAFLD interact with obesity.
DMSO: Considerations and Potential Risks
Some DMSO on the market may actually be industrial grade. Industrial grade DMSO may contain a number of impurities that can easily be absorbed into the skin with potentially serious health effects. The most frequent side effects from using DMSO on the skin include:
- Stomach upset
- Skin irritation
- Strong odor of garlic
More serious side effects include:
- Severe allergic reactions
- Headaches
- Itching and burning when applied to the skin
DMSO can also cause a deadly reaction when used in high concentrations.
Using DMSO by mouth can cause:
- Dizziness
- Drowsiness
- Nausea
- Vomiting
- Diarrhea
- Constipation
- Decreased appetite
DMSO can increase the effect of some medicines, which can lead to serious health issues. The biggest concern of DMSO as a solvent is that when it gets on the skin it will cause anything on the skin to be absorbed. So be sure to wash your hands and skin well before using.
Pregnant women and women who are breastfeeding should not use DMSO, since little is known about its possible effects on the fetus or infant.