Alzheimer's Disease: A Review of Pathology, Current Treatments, and the Potential Therapeutic Effect of Decreasing Oxidative Stress by Combined Vitamin D and l-Cysteine Supplementation.

Significance: Excess oxidative stress and neuroinflammation are risk factors in the onset and progression of Alzheimer's disease (AD) and its association with amyloid-ß plaque accumulation. Oxidative stress impairs acetylcholine (ACH) and N-methyl-d-aspartate receptor signaling in brain areas that function in memory and learning. Glutathione (GSH) antioxidant depletion positively correlates with the cognitive decline in AD subjects. Treatments that upregulate GSH and ACH levels, which simultaneously decrease oxidative stress and inflammation, may be beneficial for AD. Recent Advances: Some clinical trials have shown a benefit of monotherapy with vitamin D (VD), whose deficiency is linked to AD or with l-cysteine (LC), a precursor of GSH biosynthesis, in reducing mild cognitive impairment. Animal studies have shown a simultaneous decrease in ACH esterase (AChE) and increase in GSH; combined supplementation with VD and LC results in a greater decrease in oxidative stress and inflammation, and increase in GSH levels compared with monotherapy with VD or LC. Therefore, cosupplementation with VD and LC has the potential of increasing GSH, downregulation of oxidative stress, and decreased inflammation and AChE levels. Future Directions: Clinical trials are needed to determine whether safe low-cost dietary supplements, using combined VD+LC, have the potential to alleviate elevated AChE, oxidative stress, and inflammation levels, thereby halting the onset of AD. Goal of Review: The goal of this review is to highlight the pathological hallmarks and current Food and Drug Administration-approved treatments for AD, and discuss the potential therapeutic effect that cosupplementation with VD+LC could manifest by increasing GSH levels in patients. Antioxid. Redox Signal. 40, 663-678.

Positive association of acetylcholinesterase (AChE) with the neutrophil-to-lymphocyte ratio and HbA1c, and a negative association with hydrogen sulfide (H2S) levels among healthy African Americans, and H2S-inhibition and high-glucose-upregulation of AChE in cultured THP-1 human monocytes.

The incidence of Alzheimer's disease (AD) is higher in people over the age of 65 and in African Americans (AA). Elevated acetylcholinesterase (AChE) activity has been considered a major player in the onset of AD symptoms. As a result, many FDA-approved AD drugs target AChE inhibition to treat AD patients. Hydrogen sulfide (H2S) is a signaling molecule known to downregulate oxidative stress and inflammation. The neutrophil-to-lymphocyte ratio (NLR) in the blood is widely used as a biomarker to monitor inflammation and immunity. This study examined the hypothesis that plasma AChE levels have a negative association with H2S levels and that a positive association exists between levels of NLR, HbA1c, and ROS with the AChE in the peripheral blood. The fasting blood sample was taken from 114 African Americans who had provided written informed consent approved by the IRB. The effect of H2S and high-glucose treatment on AChE activity levels was also investigated in THP-1 human monocytes. There was a significant negative relationship between AChE and the levels of H2S (r = -0.41, p = 0.001); a positive association between the levels of AChE with age (r = 0.26, p = 0.03), NLR (r = 0.23, p = 0.04), ROS (r = 0.23, p = 0.04) and HbA1c levels (r = 0.24, p = 0.04), in AA subjects. No correlation was seen between blood levels of AChE and acetylcholine (ACh). Blood creatinine had a negative correlation (r = -0.23, p = 0.04) with ACh levels. There was a significant effect of H2S on AChE inhibition and of high glucose in upregulating AChE activity in cultured monocytes. This study suggests hyperglycemia and lower H2S status can contribute to an increase in the AChE activity levels. Future clinical studies are needed to examine the potential benefits of supplementation with hydrogen sulfide pro-drugs/compounds in reducing the AChE and the cognitive dysfunctions associated with AD.

Overview of gene expression techniques with an emphasis on vitamin D related studies.

Each cell controls when and how its genes must be expressed for proper function. Every function in a cell is driven by signaling molecules through various regulatory cascades. Different cells in a multicellular organism may express very different sets of genes, even though they contain the same DNA. The set of genes expressed in a cell determines the set of proteins and functional RNAs it contains, giving it its unique properties. Malfunction in gene expression harms the cell and can lead to the development of various disease conditions. The use of rapid high-throughput gene expression profiling unravels the complexity of human disease at various levels. Peripheral blood mononuclear cells (PBMC) have been used frequently to understand gene expression homeostasis in various disease conditions. However, more studies are required to validate whether PBMC gene expression patterns accurately reflect the expression of other cells or tissues. Vitamin D, which is responsible for a multitude of health consequences, is also an immune modulatory hormone with major biological activities in the innate and adaptive immune systems. Vitamin D exerts its diverse biological effects in target tissues by regulating gene expression and its deficiency, is recognized as a public health problem worldwide. Understanding the genetic factors that affect vitamin D has the potential benefit that it will make it easier to identify individuals who require supplementation. Different technological advances in gene expression can be used to identify and assess the severity of disease and aid in the development of novel therapeutic interventions. This review focuses on different gene expression approaches and various clinical studies of vitamin D to investigate the role of gene expression in identifying the molecular signature of the disease.

Gene optimizations are essential in maintaining biological functions. Gene dysregulation results in disease progression. Advanced analytical techniques determine the link between impaired genes and disease conditions. This knowledge can be applied to design clinical trials to aid novel therapeutic interventions and disease prevention.

l-Cysteine Stimulates the Effect of Vitamin D on Inhibition of Oxidative Stress, IL-8, and MCP-1 Secretion in High Glucose Treated Monocytes.

Objective: Vitamin D deficiency is common in the general population and diabetic patients, and supplementation with vitamin D is widely used to help lower oxidative stress and inflammation. The cytokine storm in SARS-CoV2 infection has been linked with both diabetes and Vitamin D deficiency. This study examined the hypothesis that supplementation with vitamin D, in combination with l-cysteine (LC), is better at reducing oxidative stress and thereby, more effective, at inhibiting the secretion of the pro-inflammatory cytokines, Interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) in U937 monocytes exposed to high glucose concentrations. Methods: U937 monocytes were pretreated with 1,25 (OH)2 vitamin D (VD, 10 nM) or LC (250 µM) or VD + LC for 24 h and then exposed to control or high glucose (HG, 25 mM) for another 24 h. Results: There were significantly greater reactive oxygen species (ROS) levels in monocytes treated with HG than those in controls. Combined supplementation with VD and LC showed a more significant reduction in ROS (46%) in comparison with treatment with LC (19%) or VD (26%) alone in monocytes exposed to HG. Similarly, VD supplementation, together with LC, caused a more significant inhibition in the secretion of IL-8 (36% versus 16%) and MCP-1 (46% versus 26%) in comparison with that of VD (10 nM) alone in high-glucose treated monocytes. Conclusions: These results suggest that combined supplementation with vitamin D and LC has the potential to be more effective than either VD or LC alone in lowering the risk of oxidative stress and inflammation associated with type 2 diabetes or COVID-19 infection. Further, this combined vitamin D with LC/N-acetylcysteine may be a potent alternative therapy for SARS-CoV2 infected subjects. This approach can prevent cellular damage due to cytokine storm in comorbid systemic inflammatory conditions, such as diabetes, obesity, and hypertension.

l-Cysteine and Vitamin D Co-Supplementation Alleviates Markers of Musculoskeletal Disorders in Vitamin D-Deficient High-Fat Diet-Fed Mice.

Vitamin D (VD) deficiency is associated with musculoskeletal disorders. This study examines whether co-supplementation of l-cysteine (LC) and VD is better than monotherapy with LC or VD at alleviating musculoskeletal dyshomeostasis in the skeletal muscle of VD-deficient high-fat diet (HFD-VD-) fed mice. Mice were fed a healthy diet or an HFD; for VD-deficient animals, the mice were maintained on a HFD-VD-diet (16 weeks); after the first 8 weeks, the HFD-VD-diet-fed mice were supplemented for another 8 weeks with LC, VD-alone, or the same doses of LC + VD by oral gavage. Saline and olive oil served as controls. Myotubes were exposed with high-glucose, palmitate, Monocyte Chemoattractant Protein 1 (MCP-1), and Tumor Necrosis Factor (TNF), to mimic the in vivo microenvironment. In vitro deficiencies of glutathione and hydrogen sulfide were induced by knockdown of GCLC and CSE genes. Relative gene expression of biomarkers (myogenic: MyoD, Mef2c, Csrp3; muscle dystrophy: Atrogin1, Murf1, and Myostatin; bone modeling and remodeling: RANK, RANKL, OPG) were analyzed using qRT-PCR. Co-supplementatoin with LC + VD showed beneficial effects on gene expression of myogenic markers and OPG but reduced markers of dystrophy, RANK/RANKL in comparison to LC or VD alone-supplementation. In vitro myotubes treated with glutathione (GSH) precursors also showed a positive effect on OPG and the myogenesis genes, and inhibited RANK/RANKL and muscle-dystrophy markers. This study reveals that the co-supplementation of LC with VD significantly alleviates the markers of musculoskeletal disorders in the skeletal muscle better than monotherapy with LC or VD in HFD-VD-fed mice.

Can Vitamin D and L-Cysteine Co-Supplementation Reduce 25(OH)-Vitamin D Deficiency and the Mortality Associated with COVID-19 in African Americans?

Early reports indicate an association between the severity of the COVID-19 infection and the widespread 25-hydroxy vitamin D deficiency known to exist in populations around the world. Vitamin D deficiency is extremely common among African American (AA) communities, where the COVID-19 infection rate is three-fold higher, and the mortality rate nearly six-fold higher, compared with rates in predominantly white communities. COVID-19 infection primarily affects the lungs and airways. Previous reports have linked 25-hydroxy vitamin D deficiency with subclinical interstitial lung disease. AA are at risk for lower cellular glutathione (GSH) levels, and GSH deficiency epigenetically impairs VD biosynthesis pathway genes. Compared with vitamin D alone, co-supplementation of vitamin D and L-cysteine (a GSH precursor) showed a better efficacy in improving levels of GSH and VD-regulatory genes at the cellular/tissue level, increasing 25(OH) vitamin D levels, and reducing inflammation biomarkers in the blood in mice studies. We propose that randomized clinical trials are needed to examine the potential of co-supplementation with anti-inflammatory antioxidants, vitamin D and L-cysteine in correcting the 25(OH)VD deficiency and preventing the 'cytokine storm,' one of the most severe consequences of infection with COVID-19, thereby preventing the adverse clinical effects of COVID-19 infection in the vulnerable AA population.

Hyperglycemia (high-glucose) decreases L-cysteine and glutathione levels in cultured monocytes and blood of Zucker diabetic rats.

L-Cysteine (LC) is an essential precursor of GSH biosynthesis. GSH is a major physiological antioxidant, and its depletion increases oxidative stress. Diabetes is associated with lower blood levels of LC and GSH. The mechanisms leading to a decrease in LC in diabetes are not entirely known. This study reports a significant decrease in LC in human monocytes exposed to high glucose (HG) concentrations as well as in the blood of type 2 diabetic rats. Thus, a significant decrease in the level of LC in response to exposure to HG supports the assertion that uncontrolled hyperglycemia contributes to a reduction of blood levels of LC and GSH seen in diabetic patients. Increased requirement of LC to replace GSH needed to scavenge excess ROS generated by hyperglycemia can result in lower levels of LC and GSH. Animal and human studies report that LC supplementation improves GSH biosynthesis and is beneficial in lowering oxidative stress and insulin resistance. This suggests that hyperglycemia has a direct role in the impairment of LC and GSH homeostasis in diabetes.

Glutathione deficiency alters the vitamin D-metabolizing enzymes CYP27B1 and CYP24A1 in human renal proximal tubule epithelial cells and kidney of HFD-fed mice.

Chronic kidney disease (CKD) is a worldwide public health problem with an estimated prevalence of 8.2%. This study reports glutathione deficiency, excess oxidative stress, and altered vitamin D metabolism in the kidney of mice fed a high-fat diet (HFD). The levels of GCLC and GCLM gene expression were significantly downregulated and the protein carbonylation level, a hallmark of oxidative damage, was significantly increased in the kidney of HFD-fed mice. While the levels of VD-regulatory genes 1-alpha-hydroxylase (CYP27B1), VDR, and RXRα were significantly downregulated in the kidney of mice fed a HFD, those of 24-hydroxylase (CYP24A1) were significantly elevated. In vitro, GSH deficiency per se causes excess oxidative damage (protein carbonylation), and significantly decreases the levels of VD-regulatory genes (CYP27B1, VDR, and RXRα), but increases levels of CYP24A1 in human renal proximal tubule epithelial cells (RPTEC), similar to findings in the kidney of HFD-fed diabetic mice. L-cysteine supplementation restores GSH and prevents oxidative damage in RPTEC. These studies suggest a potential role of GSH precursor in reducing excess oxidative stress and renal injury that commonly accompanies obesity/diabetes.

Glucose-6-phosphate dehydrogenase deficiency increases cell adhesion molecules and activates human monocyte-endothelial cell adhesion: Protective role of l-cysteine.

Glucose-6-phosphate dehydrogenase is a major enzyme that supplies the reducing agent nicotinamide adenine dinucleotide phosphate hydrogen (NADPH), which is required to recycle oxidized/glutathione disulfide (GSSH) to reduced glutathione (GSH). G6PD-deficient cells are susceptible to oxidative stress and a deficiency of GSH. Endothelial dysfunction is characterized by the loss of nitric oxide (NO) bioavailability, which regulates leukocyte adhesion to endothelium. G6PD-deficient endothelial cells (EC) demonstrate reduced expression of endothelial nitric oxide synthase (eNOS) and NO levels along with reduced GSH. Whether G6PD deficiency plays any role in EC dysfunction is unknown. The chronic inflammation commonly seen in those with metabolic syndrome, characterized by elevated levels of tumor necrosis factor (TNF) and monocyte chemoattractant protein 1 (MCP-1), provided an incentive for investigation of these cytokines as well. A GSH/G6PD-deficient model was created using human umbilical vein endothelial cells (HUVEC) treated with either buthionine sulfoximine (BSO), a pharmacological inhibitor of the rate-limiting enzyme of GSH biosynthesis (γ-glutamylcysteine synthetase), or with 6-aminonicotinamide (6-AN), an inhibitor of G6PD or G6PD siRNA. Normal and G6PD-deficient cells were also treated with pro-atherosclerotic stimuli such as high glucose, TNF, and MCP-1. After inhibiting or knocking down G6PD/GSH, the capacity of endothelial cells for monocyte recruitment was assessed by determining the expression of the adhesion molecules intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1), which was upregulated by G6PD deficiency and accompanied by the presence of the oxidative stress markers NADPH oxidase 4 (NOX4), inducible nitric oxide synthase (iNOS), and reactive oxygen species (ROS). Treatment with the inhibitors BSO and 6-AN caused increased levels of adhesion molecule mRNA and monocyte-EC adhesion. Following treatment with high glucose, G6PD-deficient cells showed an increase in levels of ICAM-1 and VCAM-1 mRNA, as well as monocyte-EC adherence, compared with results seen in control cells. Treatment with l-cysteine (a precursor of GSH) protected endothelial cells by increasing GSH and attenuating ROS, ICAM-1, VCAM-1, and monocyte-EC adhesion. These results suggest that G6PD/GSH deficiency plays a role in endothelial dysfunction and that supplementation with l-cysteine can restore GSH levels and reduce the EC activation markers in G6PD-deficient conditions.

Glutathione Stimulates Vitamin D Regulatory and Glucose-Metabolism Genes, Lowers Oxidative Stress and Inflammation, and Increases 25-Hydroxy-Vitamin D Levels in Blood: A Novel Approach to Treat 25-Hydroxyvitamin D Deficiency.

AIMS: 25-Hydroxyvitamin D [25(OH)VD] deficiency/inadequacy is a major public health issue affecting more than 1 billion people worldwide. A convincing association exists between low levels of circulating 25(OH)VD and the poor health outcomes associated with chronic diseases. However, high supraphysiological doses of VD are needed to achieve the required 25(OH)VD levels in the blood, because many subjects respond poorly to supplementation. RESULTS: This study reports a link between 25(OH)VD deficiency and a reduction in glutathione (GSH) in obese adolescents. The improvement in GSH status that results from cosupplementation with VD and l-cysteine (LC; a GSH precursor) significantly reduced oxidative stress in a mouse model of 25(OH)VD deficiency. It also positively upregulated VD regulatory genes (VDBP/VD-25-hydroxylase/VDR) in the liver and glucose metabolism genes (PGC-1α/VDR/GLUT-4) in muscle, boosted 25(OH)VD, and reduced inflammation and insulin resistance (IR) levels in the blood compared with supplementation with VD alone. In vitro GSH deficiency caused increased oxidative stress and downregulation of VDBP/VD-25-hydroxylase/VDR and upregulation of CYP24a1 in hepatocytes and downregulation of PGC-1α/VDR/GLUT-4 in myotubes. This study demonstrates that improvement in the GSH status exerts beneficial effects on the blood levels of 25(OH)VD, as well as on the inflammation and IR in a VD-deficient mouse model. Thus, the VD supplements widely consumed by the public are unlikely to be successful unless the GSH status is also corrected. INNOVATION: These studies demonstrate a previously undiscovered mechanism by which GSH status positively upregulates the bioavailability of 25(OH)VD. CONCLUSION: Supplementation with a combination of VD and LC or GSH precursor, rather than supplementation with VD alone, is beneficial and helps achieve more successful VD supplementation. Antioxid. Redox Signal. 00, 000-000.

L-Cysteine in vitro can restore cellular glutathione and inhibits the expression of cell adhesion molecules in G6PD-deficient monocytes.

L-Cysteine is a precursor of glutathione (GSH), a potent physiological antioxidant. Excess glucose-6-phosphate dehydrogenase (G6PD) deficiency in African Americans and low levels of L-cysteine diet in Hispanics can contributes to GSH deficiency and oxidative stress. Oxidative stress and monocyte adhesion was considered to be an initial event in the progression of vascular dysfunction and atherosclerosis. However, no previous study has investigated the contribution of GSH/G6PD deficiency to the expression of monocyte adhesion molecules. Using human U937 monocytes, this study examined the effect of GSH/G6PD deficiency and L-cysteine supplementation on monocyte adhesion molecules. G6PD/GSH deficiency induced by either siRNA or inhibitors (6AN/BSO, respectively) significantly (p < 0.005) increased the levels of cell adhesion molecules (ICAM-1, VCAM-1, SELL, ITGB1 and 2); NADPH oxidase (NOX), reactive oxygen species (ROS) and MCP-1 were upregulated, and decreases in levels of GSH, and nitric oxide were observed. The expression of ICAM-1 and VCAM-1 mRNA levels increased in high glucose, MCP-1 or TNF-α-treated G6PD-deficient compared to G6PD-normal cells. L-Cysteine treatment significantly (p < 0.005) increased G6PD activity and levels of GSH, and decreased NOX, ROS, and adhesion molecules. Thus, GSH/G6PD deficiency increases susceptibility to monocyte adhesion processes, whereas L-cysteine supplementation can restore cellular GSH/G6PD and attenuates NOX activity and expression of cell adhesion molecules.

1,25(OH)2-vitamin D3 upregulates glucose uptake mediated by SIRT1/IRS1/GLUT4 signaling cascade in C2C12 myotubes.

This study examined the hypothesis that 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) upregulates the insulin-independent signaling cascade of glucose metabolism. C2C12 myotubes were treated with high glucose (HG, 25 mM) and 1,25(OH)2D3 (0-50 nM). 1,25(OH)2D3 supplementation upregulated both insulin-independent (SIRT1) and insulin-dependent (p-IRS) signaling molecules, and stimulated the GLUT4 translocation, and glucose uptake in HG-treated myotubes. The effect of 1,25(OH)2D3 on IRS1 phosphorylation, GLUT4 translocation, and glucose uptake was attenuated in SIRT1-knockdown myotubes. Treatment with 1,25(OH)2D3, coupled with insulin, enhanced GLUT4 translocation and glucose uptake compared to treatment with either insulin or 1,25(OH)2D3 alone in HG-treated myotubes, which suggests that insulin-independent signaling molecules can contribute to the higher glucose metabolism observed in 1,25(OH)2D3 and insulin-treated cells. The data, therefore, suggest that 1,25(OH)2D3 increases glucose consumption by inducing SIRT1 activation, which in turn increases IRS1 phosphorylation and GLUT4 translocation in myotubes.

l-Cysteine supplementation increases insulin sensitivity mediated by upregulation of GSH and adiponectin in high glucose treated 3T3-L1 adipocytes.

Diabetic patients have lower blood levels of l-cysteine (LC) and glutathione (GSH). This study examined the hypothesis that LC supplementation positively up regulates the effects of insulin on GSH and glucose metabolism in 3T3-L1 adipocyte model. 3T3L1 adipocytes were treated with LC (250 µM, 2 h) and/or insulin (15 or 30 nM, 2 h), and high glucose (HG, 25 mM, 20 h). Results showed that HG caused significant increase (95%) in ROS and reduction in the protein levels of DsbA-L (43%), adiponectin (64%), GCLC (20%), GCLM (21%), GSH (50%), and GLUT-4 (23%) in adipocytes. Furthermore, HG caused a reduction in total (35%) and HMW adiponectin (30%) secretion. Treatment with insulin alone significantly (p < 0.05) reduced ROS levels as well as increased DsbA-L, adiponectin, GCLC, GCLM, GSH, and GLUT-4 protein levels, glucose utilization, and improved total and HMW adiponectin secretion in HG treated adipocytes compared to HG alone. Interestingly, LC supplementation along with insulin caused greater reduction in ROS levels and significantly (p < 0.05) boosted the DsbA-L (41% vs LC, 29% vs Insulin), adiponectin (92% Vs LC, 84% Vs insulin) protein levels and total (32% Vs LC, 22% Vs insulin) and HMW adiponectin (75% Vs LC, 39% Vs insulin) secretion compared with the either insulin or LC alone in HG-treated cells. In addition, LC supplementation along with insulin increased GCLC (21% Vs LC, 14% insulin), GCLM (28% Vs LC, 16% insulin) and GSH (25% Vs LC and insulin) levels compared with the either insulin or LC alone in HG-treated cells. Furthermore, LC and insulin increases GLUT-4 protein expression (65% Vs LC, 18% Vs Insulin), glucose utilization (57% Vs LC, 27% Vs insulin) compared with the either insulin or LC alone in HG-treated cells. Similarly, LC supplementation increased insulin action significantly in cells maintained in medium contained control glucose. To explore the beneficial effect of LC is mediated by the upregulation of GCLC, we knocked down GCLC using siRNA in adipoctyes. There was a significant decrease in DsbA-L and GLUT-4 mRNA levels and GSH levels in GCLC knockdown adipocytes and LC supplementation up regulates GCLC, DsbA-L and GLUT-4 mRNA expression and GSH levels in GCLC knockdown cells. These results demonstrated that LC along with insulin increases GSH levels thereby improving adiponectin secretion and glucose utilization in adipocytes. This suggests that LC supplementation can increase insulin sensitivity and can be used as an adjuvant therapy for diabetes.

Manganese supplementation increases adiponectin and lowers ICAM-1 and creatinine blood levels in Zucker type 2 diabetic rats, and downregulates ICAM-1 by upregulating adiponectin multimerization protein (DsbA-L) in endothelial cells.

Blood and tissue levels of manganese (Mn) are lower in type 2 diabetic and atherosclerosis patients compared with healthy subjects. Adiponectin has anti-diabetic and anti-atherogenic properties. Impairment in Disulfide bond A-like protein (DsbA-L) is associated with low adiponectin levels and diabetes. This study investigates the hypothesis that the beneficial effects of Mn supplementation are mediated by adiponectin and DsbA-L. At 6 weeks of age, Male Zucker diabetic fatty rats (ZDF) were randomly divided into two groups: diabetic controls and Mn-supplemented diabetic rats. Each rat was supplemented with Mn (D+Mn, 16 mg/kg BW) or water (placebo, D+P) daily for 7 weeks by oral gavage. For cell culture studies, Human Umbilical Vein Endothelial Cells (HUVEC) or 3T3L1 adipocytes were pretreated with Mn (0-10 µM MnCl2) for 24 h, followed by high glucose (HG, 25 mM) or normal glucose (5 mM) exposure for another 24 h. Mn supplementation resulted in higher adiponectin (p = 0.01), and lower ICAM-1 (p = 0.04) and lower creatinine (p = 0.04) blood levels compared to those in control ZDF rats. Mn-supplemented rats also caused reduced oxidative stress (ROS) and NADPH oxidase, and higher DsbA-L expression in the liver (p = 0.03) of ZDF rats compared to those in livers of control rats; however, Fe levels in liver were lower but not significant (p = 0.08). Similarly, treatment with high glucose (25 mM) caused a decrease in DsbA-L, which was prevented by Mn supplementation in HUVEC and adipocytes. Mechanistic studies with DsbA-L siRNA showed that the beneficial effects of Mn supplementation on ROS, NOX4, and ICAM-1 expression were abolished in DsbA-L knock-down HUVEC. These studies demonstrate that DsbA-L-linked adiponectin mediates the beneficial effects observed with Mn supplementation and provides evidence for a novel mechanism by which Mn supplementation can increase adiponectin and reduce the biomarkers of endothelial dysfunction in diabetes.

Vitamin D supplementation inhibits oxidative stress and upregulate SIRT1/AMPK/GLUT4 cascade in high glucose-treated 3T3L1 adipocytes and in adipose tissue of high fat diet-fed diabetic mice.

This study examined the hypothesis that vitamin-D prevents oxidative stress and upregulates glucose metabolism via activating insulin-independent signaling molecules in 3T3-L1 adipocytes and in high fat diet (HFD)-fed mice. To investigate the mechanism 3T3L1 adipocytes were treated with high glucose (HG, 25 mM) and 1,25(OH)2D3 (1,25-dihydroxyvitamin D3) (0-50 nM). Results showed that 1,25(OH)2D3 supplementation decreased NOX4 expression, ROS production, NF-κB phosphorylation, and increased the expression of Nrf2 and Trx in HG-treated cells. 1,25(OH)2D3 supplementation upregulated SIRT1 expression and AMPK phosphorylation and stimulated the IRS1/PI3K/PIP3/AKT/PKCζ signaling cascade, GLUT4 expression, and glucose uptake in HG-treated adipocytes. The effect of 1,25(OH)2D3 on the phosphorylation of both AMPK and IRS1, GLUT4 expression, and glucose uptake was significantly inhibited in SIRT1-knockdown adipocytes. This suggests the role of insulin-independent signaling molecules (SIRT1, AMPK) in mediating the effect of 1,25(OH)2D3 on the signaling cascade of glucose uptake. In addition, cholecalciferol supplementation significantly upregulated pAMPK, SIRT-1 and GLUT-4 levels in adipose tissue of mice fed with HFD. This study demonstrates a novel molecular mechanism by which vitamin-D can prevent oxidative stress and upregulates glucose uptake via SIRT1/AMPK/IRS1/GLUT4 cascade in HG-treated adipocytes and in adipose tissue of HFD diabetic mice.

1,25(OH)2D3 inhibits oxidative stress and monocyte adhesion by mediating the upregulation of GCLC and GSH in endothelial cells treated with acetoacetate (ketosis).

BACKGROUND: There is a significantly higher incidence of cardiovascular disease (CVD) among type 1 diabetic (T1D) patients than among non-diabetic subjects. T1D is associated with hyperketonemia, a condition with elevated blood levels of ketones, in addition to hyperglycemia. The biochemical mechanism by which vitamin D (VD) may reduce the risk of CVD is not known. This study examines whether VD can be beneficial in reducing hyperketonemia (acetoacetate, AA) induced oxidative stress in endothelial cells. METHODS: HUVEC were pretreated with 1,25(OH)2D3, and later exposed to the ketone body acetoacetate. RESULTS: The increases in ROS production, ICAM-1 expression, MCP-1 secretion, and monocyte adhesion in HUVEC treated with AA were significantly reduced following treatment with 1,25(OH)2D3. Interestingly, an increase in glutathione (GSH) levels was also observed with 1,25(OH)2D3 in ketone treated cells. The effects of 1,25(OH)2D3 on GSH, ROS, and monocyte-endothelial adhesion were prevented in GCLC knockdown HUVEC. This suggests that 1,25(OH)2D3 inhibits ROS, MCP-1, ICAM-1, and adherence of monocytes mediated by the upregulation of GCLC and GSH. CONCLUSION: This study provides evidence for the biochemical mechanism through which VD supplementation may reduce the excess monocyte adhesion to endothelium and inflammation associated with T1D.

L-Cysteine supplementation increases adiponectin synthesis and secretion, and GLUT4 and glucose utilization by upregulating disulfide bond A-like protein expression mediated by MCP-1 inhibition in 3T3-L1 adipocytes exposed to high glucose.

Adiponectin is an anti-diabetic and anti-atherogenic adipokine; its plasma levels are decreased in obesity, insulin resistance, and type 2 diabetes. An adiponectin-interacting protein named disulfide bond A-like protein (DsbA-L) plays an important role in the assembly of adiponectin. This study examined the hypothesis that L-cysteine (LC) regulates glucose homeostasis through the DsbA-L upregulation and synthesis and secretion of adiponectin in diabetes. 3T3L1 adipocytes were treated with LC (250 and 500 µM, 2 h) and high glucose (HG, 25 mM, 20 h). Results showed that LC supplementation significantly (p < 0.05) upregulated the DsbA-L, adiponectin, and GLUT-4 protein expression and glucose utilization in HG-treated adipocytes. LC supplementation significantly (p < 0.05) promoted the secretion of total and HMW adiponectin secretion in HG-treated adipocytes. In addition, LC significantly (p < 0.05) decreased ROS production and MCP-1 secretion in HG-treated cells. We further investigated whether MCP-1 has any role of LC on DsbA-L expression and adiponectin levels in 3T3-L1 cells. Treatment with LC prevented the decrease in DsbA-L, adiponectin, and GLUT-4 expression in 3T3L1 adipocyte cells exposed to MCP-1. Thus, this study demonstrates that DsbA-L and adiponectin upregulation mediates the beneficial effects of LC on glucose utilization by inhibiting MCP-1 secretion in adipocytes and provides a novel mechanism by which LC supplementation can improve insulin sensitivity in diabetes.

L-cysteine supplementation upregulates glutathione (GSH) and vitamin D binding protein (VDBP) in hepatocytes cultured in high glucose and in vivo in liver, and increases blood levels of GSH, VDBP, and 25-hydroxy-vitamin D in Zucker diabetic fatty rats.

SCOPE: Vitamin D binding protein (VDBP) status has an effect on and can potentially improve the status of 25(OH) vitamin D and increase the metabolic actions of 25(OH) vitamin D under physiological and pathological conditions. Diabetes is associated with lower levels of glutathione (GSH) and 25(OH) vitamin D. This study examined the hypothesis that upregulation of GSH will also upregulate blood levels of VDBP and 25(OH) vitamin D in type 2 diabetic rats. METHODS AND RESULTS: L-cysteine (LC) supplementation was used to upregulate GSH status in a FL83B hepatocyte cell culture model and in vivo using Zucker diabetic fatty (ZDF) rats. Results show that LC supplementation upregulates both protein and mRNA expression of VDBP and vitamin D receptor (VDR) and GSH status in hepatocytes exposed to high glucose, and that GSH deficiency, induced by glutamate cysteine ligase knockdown, resulted in the downregulation of GSH, VDBP, and VDR and an increase in oxidative stress levels in hepatocytes. In vivo, LC supplementation increased GSH and protein and mRNA expression of VDBP and vitamin D 25-hydroxylase (CYP2R1) in the liver, and simultaneously resulted in elevated blood levels of LC and GSH, as well as increases in VDBP and 25(OH) vitamin D levels, and decreased inflammatory biomarkers in ZDF rats compared with those in placebo-supplemented ZDF rats consuming a similar diet. CONCLUSION: LC supplementation may provide a novel approach by which to raise blood levels of VDBP and 25(OH) vitamin D in type 2 diabetes.

Can L-Cysteine and Vitamin D Rescue Vitamin D and Vitamin D Binding Protein Levels in Blood Plasma of African American Type 2 Diabetic Patients?

AIMS: Vitamin D (VD) deficiency has become a worldwide epidemic, particularly affecting African Americans (AA). VD deficiency has been implicated in the excessive rate of complications associated with diabetes in AA. Blood levels of VD binding protein (VDBP) and glutathione (GSH) are lower in AA compared with those in Caucasians. This study tested the hypothesis that lower GSH levels are linked to VDBP and VD deficiency in AA-type 2 diabetic (AA-T2D) patients. Blood was analyzed from T2D and nondiabetic subjects (N). Experiments examining GSH deficiency and l-cysteine (LC) supplementation were performed using THP-1 monocytes. RESULTS: Plasma levels of LC, GSH, VDBP, and VD were significantly lower in AA-T2D compared with age-matched AA-N or Caucasian-T2D. Lower levels of LC and GSH showed a significant positive correlation with lower VDBP and VD levels in AA-T2D. GSH deficiency investigated using an antisense approach depleted VDBP/vitamin D receptor (VDR); LC supplementation caused significant upregulation of GSH and of VDBP/VDR, while supplementation with VD+LC caused a significantly greater GSH and VDBP/VDR upregulation compared with that of VD alone in monocytes. INNOVATION AND CONCLUSION: The reported observations suggest that VD deficiency may be linked to GSH and LC status and lead to a novel hypothesis that supplementation with LC in combination with VD will be effective in increasing VD levels and reducing health disparities in AA.

Effect of PIP3 on adhesion molecules and adhesion of THP-1 monocytes to HUVEC treated with high glucose.

BACKGROUND: Phosphatidylinositol-3,4,5-triphosphate (PIP3), a well-known lipid second messenger, plays a key role in insulin signaling and glucose homeostasis. Using human umbilical vein endothelial cells (HUVEC) and THP-1 monocytes, we tested the hypothesis that PIP3 can downregulate adhesion molecules and monocyte adhesion to endothelial cells. METHODS: HUVEC and monocytes were exposed to high glucose (HG, 25 mM, 20 h) with or without PIP3 (0-20 nM), or PIT-1 (25 µM), an inhibitor of PIP3. RESULTS: Both HG and PIT-1 caused a decrease in cellular PIP3 in monocytes and HUVEC compared to controls. Treatment with PIT-1 and HG also increased the ICAM-1 (intercellular adhesion molecule 1) total protein expression as well as its surface expression in HUVEC, CD11a (a subunit of lymphocyte function-associated antigen 1, LFA-1) total protein expression as well as its surface expression in monocytes, and adhesion of monocytes to HUVEC. Exogenous PIP3 supplementation restored the intracellular PIP3 concentrations, downregulated the expression of adhesion molecules, and reduced the adhesion of monocytes to HUVEC treated with HG. CONCLUSION: This study reports that a decrease in cellular PIP3 is associated with increased expression of adhesion molecules and monocyte-endothelial cell adhesion, and may play a role in the endothelial dysfunction associated with diabetes.