Main Research Findings

1) Co-administration of vitamin E and Atorvastatin improves insulin sensitivity, as well as the expression of peroxisome proliferator–activated receptor-gamma, in patients with type 2 diabetes mellitus.

2) Dietary administration of vitamin E improves total antioxidant status and decreases levels of oxidized low-density lipoprotein.

 

Selected Data

1) The research team of Tabaei et al carried out a randomized, double-blind clinical trial to evaluate the effects of co-administering vitamin E with atorvastatin in female patients with type 2 diabetes mellitus. The sample size was calculated based on a previous study that assessed the impact of atorvastatin on low-density lipoprotein cholesterol. Inclusion criteria were women aged 18–65 years with a body mass index of 25–35 kg/m², an HbA1c level between 7% and 9%, and a daily intake of 20 mg atorvastatin for low-density lipoprotein cholesterol control. Participants were required to be on similar glucose-lowering medications. Exclusion criteria included recent use of thiazolidinediones, vitamin E or other supplements, recent significant weight loss, thyroid dysfunction, pregnancy or breastfeeding, smoking, use of weight-loss drugs, and any chronic illness [1].

A total of 43 female type 2 diabetes mellitus patients were screened, and 30 eligible patients were randomly assigned to two equal groups: one receiving atorvastatin with a placebo and the other receiving atorvastatin with vitamin E. Randomization was performed using block randomization with a block size of six to ensure balanced allocation. To reduce selection bias, treatment packages were placed in indistinguishable boxes labeled with a random sequence, keeping both the care providers and participants blinded to the group assignments.

The atorvastatin with a placebo group received 20 mg atorvastatin daily along with 25 μg/d of lactose as placebo, while the atorvastatin with vitamin E group received 20 mg atorvastatin and 400 IU of vitamin E for 12 weeks. Identical packaging was used to ensure blinding, and medication adherence was monitored weekly by checking unused portions of the medications. Participants were instructed to maintain their usual diet throughout the study period. A dietitian supervised dietary adherence and assessed three-day dietary recalls at the end of each month. Supplement intake was monitored through weekly phone calls and verified using a food frequency questionnaire. Physical activity levels were assessed at the beginning of the study using the International Physical Activity Questionnaire [1].

The study aimed to evaluate the effects of combining vitamin E with atorvastatin on anthropometric parameters, fasting blood sugar, insulin levels and sensitivity, lipid profile, and peroxisome proliferator-activated receptor gamma (PPAR-γ) mRNA expression. Anthropometric measurements included body weight and height for BMI calculation and waist-to-hip ratio to assess fat distribution. Waist circumference was measured midway between the lowest rib and the iliac crest, while hip circumference was taken at the widest point of the buttocks. These measurements were taken at baseline and after 12 weeks, except for height [1].

Biochemical parameters were assessed using fasting blood samples collected at baseline and after the intervention. Blood was drawn from the antecubital vein into both EDTA-coated and regular tubes. To avoid hormonal influences on lipid levels, samples were not collected during the menstrual phase. Serum lipid profile, fasting blood sugar, two-hour plasma glucose, HbA1c, and insulin levels were measured using commercial immunoassay kits and analyzed with an automated Hitachi analyzer. Serum insulin was measured separately using an ELISA kit, and insulin resistance was calculated using the Homeostatic Model Assessment for Insulin Resistance formula.

To analyze gene expression, peripheral blood mononuclear cells were isolated from the EDTA blood samples through density-gradient separation. The isolated peripheral blood mononuclear cells were then processed for RNA extraction using a commercial kit. The concentration and purity of the extracted RNA were assessed using a NanoDrop spectrophotometer and agarose gel electrophoresis. Complementary DNA was synthesized for real-time PCR analysis. The expression of PPAR-γ was quantified using a SYBR Green chemistry PCR system, with GAPDH serving as the housekeeping gene. Primer sequences were designed using Gene Runner software. The PCR amplification involved an initial denaturation step followed by 40 cycles of denaturation and annealing/extension [1].

Serum vitamin E levels, specifically alpha-tocopherol, were measured using high-performance liquid chromatography. Internal standards were obtained from Sigma-Aldrich, and alpha-tocopherol was separated using a C18 column. Methanol served as the mobile phase, with the system operated at a flow rate of 0.8 mL/min and temperature of 30°C. The peak areas for tocopherols were measured at 280 nm. Special precautions, including nitrogen-filled, foil-wrapped tubes, were taken to prevent vitamin E oxidation during sample preparation and storage. The extraction involved mixing serum with alcohol, followed by hexane extraction and centrifugation, with the final solution injected into the HPLC system. The column was thoroughly washed between runs to maintain performance consistency [1].

2) An experimental study was conducted by the research team of Rainwater et al, on a population of 251 pedigreed baboons to examine the effects of a high-fat, high-cholesterol diet, with and without vitamin E supplementation, on lipid metabolism, lipoprotein characteristics, and oxidative biomarkers. The research employed a structured dietary intervention, extensive blood sampling, and a range of biochemical and analytical techniques to assess the physiological changes associated with diet and antioxidant supplementation [2].

The study design included a controlled dietary protocol consisting of three phases. Initially, the baboons were fed a basal diet low in fat and cholesterol. This was followed by a seven-week period on a high-fat, high-cholesterol diet containing 40% of calories from lard and 6 mg of cholesterol per gram of diet. Blood samples were collected at the end of this first high-fat, high-cholesterol phase. Afterward, the baboons were returned to the basal diet for a seven-week washout period to allow their lipid profiles to stabilize. Subsequently, the same high-fat, high-cholesterol diet was reintroduced, this time supplemented with 1000 IU/kg of vitamin E acetate, for another seven weeks. A second round of blood samples was collected after this supplementation phase [2].

Previous studies established that maximal low-density and high density lipoprotein (LDL and HDL, respectively) cholesterol responses to the high-fat, high-cholesterol diet occurred within four weeks. A pilot study involving 60 baboons confirmed that LDL and HDL cholesterol levels returned to baseline values after the washout period, indicating that the washout was effective in reestablishing the animals’ pre-high-fat, high-cholesterol diet lipid levels. These findings supported the reliability of the current experimental design.

Blood samples were obtained from the femoral vein of baboons fasted overnight and immobilized using ketamine. Plasma samples were collected using heparinized tubes and treated with the antioxidants diethylenetriaminepentaacetic acid and 3,5-di-tert-butyl-4-hydroxytoluene, in order to stabilize the samples. All samples were processed through low-speed centrifugation, aliquoted, and stored at −80 °C until analysis [2].

Multiple biochemical parameters were assessed in the collected serum and plasma samples. Total serum cholesterol was measured enzymatically using reagents and HDL cholesterol was quantified after selective precipitation of apolipoprotein B-containing lipoproteins using heparin. LDL cholesterol was calculated by subtracting HDL cholesterol from total cholesterol. Serum triacylglycerol concentrations were also measured enzymatically using various reagents..

To evaluate the distribution of cholesterol among various lipoprotein subclasses, the study employed non-denaturing gradient gel electrophoresis stained with Sudan black B. This technique allowed size-based resolution of HDL and LDL particles. Each serum sample was run in duplicate on composite gels, and the researchers calculated a median diameter for HDL and LDL particles, defined as the size at which half of the lipoprotein absorbance signal was above and half below, effectively representing a weighted average particle size [2].

Plasma concentrations of oxidized LDL, a marker of oxidative stress and cardiovascular risk, were measured immunologically using a sandwich enzyme-linked immunosorbent assay that employed two monoclonal antibodies targeting distinct epitopes on oxidized LDL. Total antioxidant status, reflecting the overall capacity of the serum to neutralize free radicals, was assessed using a commercial kit. This assay quantified the ability of serum to prevent the oxidation of a specific chromogenic substrate by metmyoglobin [2].

The study also measured the activity of lipoprotein-associated phospholipase A2 (PLA2), also known as platelet-activating factor acetylhydrolase, using a substrate-based colorimetric assay. The substrate, 2-thio PAF, was hydrolyzed to produce a free thiol, which was quantified using 5,5′-dithiobis-(2-nitrobenzoic acid). This reaction was monitored spectrophotometrically at 405 nm, and the rate of substrate hydrolysis, calculated from the linear phase of the reaction, was expressed in micromoles per minute per liter of plasma.

Overall, this animal study employed a comprehensive experimental protocol to explore the metabolic effects of a high-fat, high-cholesterol diet and the potential protective role of vitamin E supplementation in baboons. Through a rigorous and carefully controlled design involving sequential dietary challenges and washouts, combined with high-fidelity biochemical and lipoprotein analyses, the study generated robust data on lipid metabolism, oxidative stress, and antioxidant capacity. The use of both traditional markers such as cholesterol, triglycerides, apo A-I, and apo B; as well as advanced methodologies such as lipoprotein size profiling, oxidized LDL, PLA2 and paraoxonase activity, TAS, allowed for an in-depth evaluation of diet-induced metabolic changes and the modulating effect of vitamin E [2].

 

Discussion

1) The clinical study performed by the research team of Tabaei et al investigated the effects of co-administering atorvastatin and vitamin E on insulin sensitivity, glycemic control, lipid profiles, and gene expression related to metabolic regulation in patients with type 2 diabetes mellitus. The primary aim was to determine whether combining these two compounds could mitigate some of the adverse metabolic effects typically associated with statin use and improve overall metabolic health in patients with type 2 diabetes mellitus.

The study found that the co-administration of atorvastatin and vitamin E led to improvements in insulin sensitivity, as evidenced by reduced serum insulin levels and lower Homeostatic Model Assessment for Insulin Resistance scores. These markers suggest a beneficial effect on insulin function, indicating that the intervention may reduce insulin resistance. While reductions in other markers such as HbA1c, two-hour plasma glucose, triglycerides, and total cholesterol were observed in the group receiving both atorvastatin and vitamin E, these reductions were not statistically significant when compared to the group receiving atorvastatin and placebo. However, from a clinical standpoint, the observed reductions, particularly the 80 mg/dL decrease in two-hour plasma glucose, may still be meaningful [1].

Previous research has suggested that atorvastatin, despite its beneficial effects on lipid profiles, may negatively impact glycemic control by impairing insulin secretion and exacerbating insulin resistance. This effect is believed to be mediated through the downregulation of glucose transporter type 4 in adipose tissue, which plays a critical role in glucose uptake. Therefore, combining atorvastatin with agents like vitamin E, which possess antioxidant and anti-inflammatory properties, may counteract these negative effects and enhance treatment outcomes for patients with type 2 diabetes mellitus [1].

The rationale for using vitamin E in combination therapy stems from its potential to improve glycemic control and reduce oxidative stress. In the present study, the combination therapy led to a statistically significant reduction in HbA1c levels within the atorvastatin with vitamin E group, whereas the reduction observed in the atorvastatin with a placebo group was neither statistically nor clinically significant. These findings are consistent with several previous studies that reported HbA1c reductions with high-dose vitamin E supplementation, including daily doses ranging from 100 IU to 1600 IU.

A noteworthy finding of the current study was the significant upregulation of PPAR-γ gene expression in peripheral blood mononuclear cells of the atorvastatin with vitamin E group. PPAR-γ plays a central role in regulating insulin sensitivity, inflammation, lipid metabolism, and glucose homeostasis. Its activation leads to enhanced expression of genes involved in antioxidant production, cholesterol efflux, and anti-inflammatory pathways, which collectively improve metabolic function. This molecular effect may help explain the improvements observed in insulin sensitivity following combination therapy [1].

In conclusion, the co-administration of vitamin E and atorvastatin appears to improve insulin sensitivity and modulate PPAR-γ gene expression in patients with type 2 diabetes mellitus. Although some of the observed benefits were not statistically significant, they may hold clinical relevance. The findings support the potential of combination therapy in managing the metabolic complications associated with statin use in diabetic patients and pave the way for future research into personalized and tissue-specific interventions [1].

2) This study completed by researchers Rainwater et al investigated the effects of vitamin E supplementation on cardiovascular disease (CVD) risk factors in baboons fed a high-fat diet. The researchers administered a diet containing high levels of fat and cholesterol, supplemented with vitamin E at a concentration of 1000 IU/kg, and analyzed a range of biochemical and physiological variables associated with CVD. Their findings shed light on the complex and sometimes contradictory role of vitamin E in modulating lipid metabolism and oxidative stress, and may help explain the inconsistent outcomes observed in human clinical trials evaluating the cardiovascular benefits of vitamin E [2].

One of the key observations from the study was a significant increase in plasma α-tocopherol levels, consistent with earlier research indicating a dose-dependent rise in this antioxidant with dietary intake. In the current experiment, vitamin E supplementation resulted in approximately a 2.5-fold elevation in α-tocopherol levels compared to the high-fat diet without supplementation. This increase in antioxidant levels was associated with various changes in CVD-related biochemical markers.

Total antioxidant status, which reflects the capacity of the serum to neutralize oxidative agents, was significantly higher in animals receiving the vitamin E-enriched diet. This result supports the idea that vitamin E improves serum resistance to oxidative damage, confirming findings from numerous earlier studies. The enhancement of total antioxidant status underscores vitamin E’s role as a potent antioxidant in vivo [2].

While the concentration and size characteristics of apo B-containing lipoproteins, such as LDL, did not change significantly with vitamin E supplementation, oxidized LDL levels declined by about 10%. This reduction is noteworthy because oxidized LDL is known to be a key contributor to atherogenesis. Thus, even without altering LDL cholesterol levels, vitamin E improved LDL quality by making it less susceptible to oxidative damage. These results are consistent with similar findings in human studies [2].

However, the study also revealed a decrease in the activity of two enzymes associated with lipoproteins including: paraoxonase 1 (PON1) and phospholipase A2 (PLA2), both of which play roles in reducing lipoprotein oxidation. The decline in these enzymes’ activity, particularly PON1, that is associated with HDL particles and protects against lipid oxidation, could potentially be interpreted as pro-atherogenic. This is in contrast to previous studies in other species that reported increased PON1 activity in response to vitamin E. The discrepancy might stem from species-specific differences in HDL composition, or it might suggest that reduced oxidative stress due to vitamin E lessens the need for these enzymes, triggering a feedback reduction in their expression [2].

Vitamin E supplementation also led to two contrasting effects on HDL, a class of lipoproteins associated with lower CVD risk. On one hand, HDL particles became smaller, with a reduced median diameter and a decreased proportion of cholesterol in the HDL2 subfraction. This aligns with previous studies that showed reductions in HDL2 following antioxidant vitamin intake. Since smaller HDL particles are generally linked to higher CVD risk, this change could be considered adverse. On the other hand, apo A-I concentrations increased significantly. Apo A-I is a key structural protein of HDL and is associated with a decreased risk of CVD. The accumulation of small, protein-rich HDL particles without changes in HDL cholesterol concentration suggests that these altered HDLs may have enhanced capacity for reverse cholesterol transport, a protective mechanism against atherosclerosis.

The increase in apo A-I concentrations was positively correlated with plasma α-tocopherol levels, suggesting that vitamin E may promote apo A-I production or stability. Further analysis revealed that the increase was primarily in the pro-form of apo A-I. Although differences between mature and pro-apo A-I forms exist, both are functionally capable of facilitating reverse cholesterol transport. Hence, the rise in pro-apo A-I may still confer cardiovascular protection [2].

The study also examined which CVD-related traits were most strongly associated with the change in total antioxidant status. Of all measured variables, only apo A-I showed a significant independent correlation with total antioxidant status, suggesting that apo A-I might play a role in the body’s antioxidant defenses. However, given the modest increase in apo A-I relative to the changes in total antioxidant status and α-tocopherol, it is unlikely that apo A-I alone explains the improvement in antioxidant capacity.

Genetic analyses revealed that certain responses to vitamin E were heritable. Notably, the proportion of cholesterol in HDL2 and the activity of PON1 showed significant genetic influence. Approximately 37% of the variance in HDL2 cholesterol and 22% of the variance in PON1 activity could be attributed to genetic factors. This is the first report indicating that genes may regulate HDL response to dietary vitamin E in any species. These findings suggest that individual genetic differences can influence how vitamin E affects lipid metabolism and antioxidant defenses. The mechanisms may involve direct regulation of gene transcription or indirect effects mediated through changes in the redox environment [2].

The major conclusion of this research is that vitamin E supplementation has a marked impact on HDL metabolism in baboons. The supplementation results in both a reduction in HDL size, particularly in the HDL2 subfraction, as it is a change that is under genetic control, as well as an increase in apo A-I concentrations that is correlated with antioxidant status but is not genetically regulated. These two changes have opposing implications for CVD risk. While smaller HDL size is typically associated with higher risk, elevated apo A-I levels are protective. Therefore, whether vitamin E is ultimately pro- or anti-atherogenic may depend on which of these effects predominates in a given individua [2]l.

 

Disclaimer

**LAB USE ONLY**
*This information is for educational purposes only and does not constitute medical advice. THE PRODUCTS DESCRIBED HEREIN ARE FOR RESEARCH USE ONLY. All clinical research must be conducted with oversight from the appropriate Institutional Review Board (IRB). All preclinical research must be conducted with oversight from the appropriate Institutional Animal Care and Use Committee (IACUC) following the guidelines of the Animal Welfare Act (AWA).

 

Citations

[1] Tabaei BS, Mousavi SN, Rahimian A, Rostamkhani H, Mellati AA, Jameshorani M. Co-Administration of Vitamin E and Atorvastatin Improves Insulin Sensitivity and Peroxisome Proliferator-Activated Receptor-γ Expression in Type 2 Diabetic Patients: A Randomized Double-Blind Clinical Trial. Iran J Med Sci. 2022;47(2):114-122. doi:10.30476/ijms.2021.89102.1981

[2] Rainwater DL, Mahaney MC, VandeBerg JL, Wang XL. Vitamin E dietary supplementation significantly affects multiple risk factors for cardiovascular disease in baboons. Am J Clin Nutr. 2007;86(3):597-603. doi:10.1093/ajcn/86.3.597

 

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