Therapeutic extracellular vesicle production is substantially increased by inhibition of cellular cholesterol biosynthesis.

Extracellular vesicles (EVs) are a new therapeutic modality with the promise to treat many diseases through their ability to deliver diverse molecular cargo. As with other emerging modalities transitioning into the industrialization phase, all aspects of the manufacturing process are rich with opportunities to enhance the ability to deliver these medicines to patients. With the goal of improving cell culture EV productivity, we have utilized high throughput siRNA screens to identify the underlying genetic pathways that regulate EV productivity to inform rational host cell line engineering and media development approaches. The screens identified multiple metabolic pathways of potential interest; one of which was validated and shown to be a ready implementable, cost-effective strategy to increase EV titers. We show that both EV volumetric and specific productivity from HEK293 and CHO-S were increased in a dose and cell line-dependent manner up to ninefold when cholesterol synthesis was inhibited by the inclusion of statins in the cell culture media. In addition, we show in response to statin treatment, elevation of EV markers in mesenchymal stem cell (MSC) cell culture media suggesting this approach can also be applicable to MSC EVs. Furthermore, we show that the EVs produced from statin-treated HEK293 cultures are effectively loaded by both endogenous and exogenous loading methods and have equivalent in vitro or in vivo potency relative to EVs from untreated cultures.

Exosome-mediated genetic reprogramming of tumor-associated macrophages by exoASO-STAT6 leads to potent monotherapy antitumor activity.

Effectiveness of checkpoint immunotherapy in cancer can be undermined by immunosuppressive tumor-associated macrophages (TAMs) with an M2 phenotype. Reprogramming TAMs toward a proinflammatory M1 phenotype is a novel approach to induce antitumor immunity. The M2 phenotype is controlled by key transcription factors such as signal transducer and activator of transcription 6 (STAT6), which have been "undruggable" selectively in TAMs. We describe an engineered exosome therapeutic candidate delivering an antisense oligonucleotide (ASO) targeting STAT6 (exoASO-STAT6), which selectively silences STAT6 expression in TAMs. In syngeneic models of colorectal cancer and hepatocellular carcinoma, exoASO-STAT6 monotherapy results in >90% tumor growth inhibition and 50 to 80% complete remissions. Administration of exoASO-STAT6 leads to induction of nitric oxide synthase 2 (NOS2), an M1 macrophage marker, resulting in remodeling of the tumor microenvironment and generation of a CD8 T cell-mediated adaptive immune response. Collectively, exoASO-STAT6 represents the first platform targeting transcription factors in TAMs in a highly selective manner.

ExoSTING, an extracellular vesicle loaded with STING agonists, promotes tumor immune surveillance.

Cyclic dinucleotide (CDN) agonists of the STimulator of InterferoN Genes (STING) pathway have shown immune activation and tumor clearance in pre-clinical models. However, CDNs administered intratumorally also promote STING activation leading to direct cytotoxicity of many cell types in the tumor microenvironment (TME), systemic inflammation due to rapid tumor extravasation of the CDN, and immune ablation in the TME. These result in a failure to establish immunological memory. ExoSTING, an engineered extracellular vesicle (EV) exogenously loaded with CDN, enhances the potency of CDN and preferentially activates antigen presenting cells in the TME. Following intratumoral injection, exoSTING was retained within the tumor, enhanced local Th1 responses and recruitment of CD8+ T cells, and generated systemic anti-tumor immunity to the tumor. ExoSTING at therapeutically active doses did not induce systemic inflammatory cytokines, resulting in an enhanced therapeutic window. ExoSTING is a novel, differentiated therapeutic candidate that leverages the natural biology of EVs to enhance the activity of CDNs.

A versatile platform for generating engineered extracellular vesicles with defined therapeutic properties.

Extracellular vesicles (EVs) are an important intercellular communication system facilitating the transfer of macromolecules between cells. Delivery of exogenous cargo tethered to the EV surface or packaged inside the lumen are key strategies for generating therapeutic EVs. We identified two "scaffold" proteins, PTGFRN and BASP1, that are preferentially sorted into EVs and enable high-density surface display and luminal loading of a wide range of molecules, including cytokines, antibody fragments, RNA binding proteins, vaccine antigens, Cas9, and members of the TNF superfamily. Molecules were loaded into EVs at high density and exhibited potent in vitro activity when fused to full-length or truncated forms of PTGFRN or BASP1. Furthermore, these engineered EVs retained pharmacodynamic activity in a variety of animal models. This engineering platform provides a simple approach to functionalize EVs with topologically diverse macromolecules and represents a significant advance toward unlocking the therapeutic potential of EVs.

Exosome Surface Display of IL12 Results in Tumor-Retained Pharmacology with Superior Potency and Limited Systemic Exposure Compared with Recombinant IL12.

The promise of IL12 as a cancer treatment has yet to be fulfilled with multiple tested approaches being limited by unwanted systemic exposure and unpredictable pharmacology. To address these limitations, we generated exoIL12, a novel, engineered exosome therapeutic that displays functional IL12 on the surface of an exosome. IL12 exosomal surface expression was achieved via fusion to the abundant exosomal surface protein PTGFRN resulting in equivalent potency in vitro to recombinant IL12 (rIL12) as demonstrated by IFNγ production. Following intratumoral injection, exoIL12 exhibited prolonged tumor retention and greater antitumor activity than rIL12. Moreover, exoIL12 was significantly more potent than rIL12 in tumor growth inhibition. In the MC38 model, complete responses were observed in 63% of mice treated with exoIL12; in contrast, rIL12 resulted in 0% complete responses at an equivalent IL12 dose. This correlated with dose-dependent increases in tumor antigen-specific CD8+ T cells. Rechallenge studies of exoIL12 complete responder mice showed no tumor regrowth, and depletion of CD8+ T cells completely abrogated antitumor activity of exoIL12. Following intratumoral administration, exoIL12 exhibited 10-fold higher intratumoral exposure than rIL12 and prolonged IFNγ production up to 48 hours. Retained local pharmacology of exoIL12 was further confirmed using subcutaneous injections in nonhuman primates. This work demonstrates that tumor-restricted pharmacology of exoIL12 results in superior in vivo efficacy and immune memory without systemic IL12 exposure and related toxicity. ExoIL12 is a novel cancer therapeutic candidate that overcomes key limitations of rIL12 and thereby creates a therapeutic window for this potent cytokine.

Pancreatic ß-cell dysfunction in polycystic ovary syndrome: role of hyperglycemia-induced nuclear factor-κB activation and systemic inflammation.

In polycystic ovary syndrome (PCOS), oxidative stress is implicated in the development of ß-cell dysfunction. However, the role of mononuclear cell (MNC)-derived inflammation in this process is unclear. We determined the relationship between ß-cell function and MNC-derived nuclear factor-κB (NF-κB) activation and tumor necrosis factor-α (TNF-α) secretion in response to a 2-h 75-g oral glucose tolerance test (OGTT) in normoglycemic women with PCOS (15 lean, 15 obese) and controls (16 lean, 14 obese). First- and second-phase ß-cell function was calculated as glucose-stimulated insulin secretion (insulin/glucose area under the curve for 0-30 and 60-120 min, respectively) × insulin sensitivity (Matsuda Index derived from the OGTT). Glucose-stimulated NF-κB activation and TNF-α secretion from MNC, and fasting plasma thiobarbituric acid-reactive substances (TBARS) and high-sensitivity C-reactive protein (hs-CRP) were also assessed. In obese women with PCOS, first- and second-phase ß-cell function was lower compared with lean and obese controls. Compared with lean controls, women with PCOS had greater change from baseline in NF-κB activation and TNF-α secretion, and higher plasma TBARS. ß-Cell function was inversely related to NF-κB activation (1st and 2nd) and TNF-α secretion (1st), and plasma TBARS and hs-CRP (1st and 2nd). First- and second-phase ß-cell function also remained independently linked to NF-κB activation after adjustment for body fat percentage and TBARS. In conclusion, ß-cell dysfunction in PCOS is linked to hyperglycemia-induced NF-κB activation from MNC and systemic inflammation. These data suggest that in PCOS, inflammation may play a role in impairing insulin secretion before the development of overt hyperglycemia.

The altered mononuclear cell-derived cytokine response to glucose ingestion is not regulated by excess adiposity in polycystic ovary syndrome.

CONTEXT: Excess adipose tissue is a source of inflammation. Polycystic ovary syndrome (PCOS) is a proinflammatory state and is often associated with excess abdominal adiposity (AA) alone and/or frank obesity. OBJECTIVE: To determine the effect of glucose ingestion on cytokine release from mononuclear cells (MNC) in women with PCOS with and without excess AA and/or obesity. DESIGN: A cross-sectional study. SETTING: Academic medical center. PATIENTS: Twenty-three women with PCOS (seven normal weight with normal AA, eight normal weight with excess AA, eight obese) and 24 ovulatory controls (eight normal weight with normal AA, eight normal weight with excess AA, eight obese). INTERVENTION: Three-hour 75-g oral glucose tolerance test (OGTT). MAIN OUTCOME MEASURES: Body composition was measured by dual energy x-ray absorptiometry. Insulin sensitivity was derived from the OGTT (ISOGTT). TNFα, IL-6, and IL-1ß release was measured in supernatants of cultured MNC isolated from blood samples drawn while fasting and 2 hours after glucose ingestion. RESULTS: Insulin sensitivity was lower in obese subjects regardless of PCOS status and in normal-weight women with PCOS compared with normal-weight controls regardless of body composition status. In response to glucose ingestion, MNC-derived TNFα, IL-6, and IL-1ß release decreased in both normal-weight control groups but failed to suppress in either normal-weight PCOS group and in obese women regardless of PCOS status. For the combined groups, the cytokine responses were negatively correlated with insulin sensitivity and positively correlated with abdominal fat and androgens. CONCLUSIONS: Women with PCOS fail to suppress MNC-derived cytokine release in response to glucose ingestion, and this response is independent of excess adiposity. Nevertheless, a similar response is also a feature of obesity per se. Circulating MNC and excess adipose tissue are separate and distinct sources of inflammation in this population.

Hyperandrogenism induces a proinflammatory TNFα response to glucose ingestion in a receptor-dependent fashion.

CONTEXT: Hyperandrogenism and inflammation are related in polycystic ovary syndrome (PCOS). Hyperandrogenemia can induce inflammation in reproductive-age women, but the mechanism for this phenomenon is unclear. OBJECTIVE: We examined the in vivo and in vitro effects of hyperandrogenism on mononuclear cell (MNC)-derived androgen receptor (AR) status and TNFα release. DESIGN: This study combined a randomized, controlled, double-blind protocol with laboratory-based cell culture experiments. SETTING: This work was performed in an academic medical center. PARTICIPANTS: Lean, healthy, reproductive-age women were treated with 130 mg of dehydroepiandrosterone (DHEA) or placebo (n = 8 subjects each) for 5 days and also provided untreated fasting blood samples (n = 12 subjects) for cell culture experiments. MAIN OUTCOME MEASURES: AR mRNA content and TNFα release were measured before and after DHEA administration in the fasting state and 2 hours after glucose ingestion. TNFα release in the fasting state was also measured in cultured MNCs exposed to androgens with or without flutamide preincubation. RESULTS: At baseline, subjects receiving DHEA or placebo exhibited no significant difference in androgens and TNFα release from MNCs before and after glucose ingestion. Compared with placebo, DHEA administration raised levels of T, androstenedione, and DHEA sulfate, and increased MNC-derived AR mRNA content and TNFα release in the fasting state and in response to glucose ingestion. Compared with MNC exposure to baseline concentrations of DHEA (175 ng/dL) or T (50 ng/dL), the absolute change in TNFα release increased after exposure to T concentrations of 125 and 250 ng/dL and a DHEA concentration of 1750 ng/dL. Preincubation with flutamide reduced the TNFα response by ≥ 60% across all T concentrations. CONCLUSION: Androgen excess in vivo and in vitro comparable to what is present in PCOS increases TNFα release from MNCs of lean healthy reproductive-age women in a receptor-dependent fashion. Hyperandrogenemia activates and sensitizes MNCs to glucose in this population.

Glucose-stimulated oxidative stress in mononuclear cells is related to pancreatic ß-cell dysfunction in polycystic ovary syndrome.

CONTEXT: Oxidative stress induced by reactive oxygen species (ROS) is involved in the development of pancreatic ß-cell dysfunction. OBJECTIVE: We determined the relationship between mononuclear cell (MNC)-derived ROS generation and p47phox protein content in response to glucose ingestion and ß-cell function in women with polycystic ovary syndrome (PCOS). DESIGN: This was a cross-sectional study. SETTING: This study was conducted at an academic medical center. PARTICIPANTS: Twenty-nine normoglycemic women with PCOS (13 lean, 16 obese) and 25 ovulatory controls (16 lean, 9 obese) underwent a 3-h 75-g oral glucose tolerance test (OGTT). MAIN OUTCOME VARIABLES: Pancreatic ß-cell function was calculated as glucose-stimulated insulin secretion (insulin/glucose area under the curve0-30 min; GSIS)×Matsuda index-derived insulin sensitivity (ISOGTT). ROS generation was measured by chemiluminescence, and p47phox protein was quantified by Western blotting in MNC isolated from blood samples obtained at 0 and 2 hours of the OGTT. RESULTS: Compared with controls, women with PCOS exhibited a higher percent change from baseline in ROS generation and p47phox protein in conjunction with greater GSIS and a tendency toward lower ß-cell function. Lean women with PCOS exhibited a greater percent change from baseline in ROS generation and p47phox protein yet had similar GSIS responses compared with lean controls despite having lower ISOGTT. For the combined groups, ß-cell function was inversely related to ROS generation and p47phox protein. GSIS was directly related to body mass index, central obesity, and circulating androgens. CONCLUSION: In normoglycemic women, obesity plays a role in exaggerating GSIS. However, MNC-derived oxidative stress is independent of obesity and may contribute to the decline in ß-cell function in women with PCOS.

Insulin infusion suppresses while glucose infusion induces Toll-like receptors and high-mobility group-B1 protein expression in mononuclear cells of type 1 diabetes patients.

The purpose of this study was to determine whether an insulin infusion exerts an anti-inflammatory effect and whether the infusion of small amounts of glucose results in oxidative and inflammatory stress in patients with type 1 diabetes. Ten patients with type 1 diabetes were infused with either 2 U/h of insulin with 100 ml 5% dextrose/h to or just dextrose (100 ml/h) or physiological saline (100 ml/h) for 4 h after an overnight fast on three separate days. Blood samples were collected at 0, 2, 4, and 6 h. Insulin with glucose infusion led to the maintenance of euglycemia and a significant suppression of reactive oxygen species (ROS) generation, p47(phox) expression, Toll-like receptor (TLR)-4, TLR-2, TLR-1, CD14, high-mobility group-B1 (HMGB1), p38 mitogen-activated protein (MAP) kinase, c-Jun NH2-terminal kinase (JNK)-1, and platelet/endothelial cell adhesion molecule expression and a fall in serum concentrations of C-reactive protein, HMGB1, and rapid upon activation T cell expressed and secreted. Glucose infusion led to an increase in plasma glucose concentration from 115 (fasting) to 215 (at 4 and 6 h) mg/dl and to an increase in ROS generation, the expression of TLR-4, TLR-2, TLR-1, HMGB1, p38 MAP kinase, and JNK-1, and plasma concentrations of HMGB1. While insulin reduces indexes of oxidative and inflammatory stress in patients with type 1 diabetes, even small amounts of glucose (20 g over 4 h) induce oxidative and inflammatory stress. These effects are reflected in TLR, p38 MAP kinase, and HMGB1 expression. The induction of significant oxidative and inflammatory stress by small amounts of glucose in patients with type 1 diabetes may have important pathophysiological and therapeutic implications.

Hyperglycemia-induced oxidative stress is independent of excess abdominal adiposity in normal-weight women with polycystic ovary syndrome.

STUDY QUESTION: What is the effect of glucose ingestion on leukocytic reactive oxygen species (ROS) generation in normal-weight women with polycystic ovary syndrome (PCOS) with and without excess abdominal adiposity (AA)? SUMMARY ANSWER: Normal-weight women with PCOS exhibit an increase in leukocytic ROS generation in response to glucose ingestion, and this increase is independent of excess AA. WHAT IS KNOWN ALREADY: Excess adipose tissue is a source of oxidative stress. Normal-weight women with PCOS exhibit oxidative stress and can have excess AA. STUDY DESIGN AND SIZE: This is a cross-sectional study involving 30 reproductive-age women. PARTICIPANTS/MATERIALS, SETTING AND METHODS: Fourteen normal-weight women with PCOS (6 normal AA, 8 excess AA) and 16 body composition-matched controls (8 normal AA, 8 excess AA) underwent body composition assessment by dual-energy absorptiometry and an oral glucose tolerance test (OGTT) at a university medical center. Insulin sensitivity was derived from the OGTT (IS(OGTT)). Blood was drawn while fasting and 2 h after glucose ingestion to measure leukocytic ROS generation and p47(phox) protein content and plasma thiobarbituric acid-reactive substances (TBARS) and C-reactive protein (CRP). MAIN RESULTS AND THE ROLE OF CHANCE: Compared with controls, both PCOS groups exhibited lower IS(OGTT) (43-54%) and greater percentage change (% change) in ROS generation (96-140%), p47(phox) protein (18-28%) and TBARS (17-48%). Compared with women with PCOS with excess AA, those with normal AA exhibited higher testosterone levels (29%) and lower CRP levels (70%). For the combined groups, IS(OGTT) was negatively correlated with the % change in ROS generation and p47(phox) protein. CRP was positively correlated with abdominal fat. The % change in p47(phox) protein was positively correlated with CRP and androgens. LIMITATIONS, REASONS FOR CAUTION: Although this study is adequately powered to assess differences in ROS generation between the women with PCOS and control participants, the modest sample size merits caution when interpreting the corroborative results of the additional measures of oxidative stress and inflammation. WIDER IMPLICATIONS OF THE FINDINGS: This study highlights the unique pro-oxidant contribution of circulating leukocytes in the development of insulin resistance and hyperandrogenism in PCOS. STUDY FUNDING/COMPETING INTEREST(S): Supported by NIH grant HD-048535 to F.G. The authors have nothing to disclose.

Inflammation in response to glucose ingestion is independent of excess abdominal adiposity in normal-weight women with polycystic ovary syndrome.

CONTEXT: Inflammation and excess abdominal adiposity (AA) are often present in normal-weight women with polycystic ovary syndrome (PCOS). OBJECTIVE: We determined the effects of hyperglycemia on nuclear factor-κB (NFκB) activation in mononuclear cells (MNC) of normal-weight women with PCOS with and without excess AA. DESIGN: This was a prospective controlled study. SETTING: The study was conducted at an academic medical center. PATIENTS: Fifteen normal-weight, reproductive-age women with PCOS (seven normal AA, eight excess AA) and 16 body composition-matched controls (eight normal AA, eight excess AA) participated in the study. MAIN OUTCOME MEASURES: Body composition was measured by dual-energy absorptiometry. Insulin sensitivity was derived from an oral glucose tolerance test (IS(OGTT)). Activated NFκB and the protein content of p65 and inhibitory-κB were quantified from MNC, and TNFα and C-reactive protein (CRP) were measured in plasma obtained from blood drawn while fasting and 2 h after glucose ingestion. RESULTS: Compared with controls, both PCOS groups exhibited lower IS(OGTT), increases in activated NFκB and p65 protein, and decreases in inhibitory-κB protein. Compared with women with PCOS with excess AA, those with normal AA exhibited higher testosterone levels and lower TNFα and CRP levels. For the combined groups, the percent change in NFκB activation was negatively correlated with IS(OGTT) and positively correlated with androgens. TNFα and CRP were positively correlated with abdominal fat. CONCLUSION: In normal-weight women with PCOS, the inflammatory response to glucose ingestion is independent of excess AA. Circulating MNC and excess AA are separate and unique sources of inflammation in this population.

Hyperandrogenism exerts an anti-inflammatory effect in obese women with polycystic ovary syndrome.

We determined the effect of chronic androgen suppression on inflammation in women with polycystic ovary syndrome (PCOS) compared to weight-matched controls. We performed a pilot project using samples from previous prospective, controlled studies. Nine women with PCOS (5 obese, 4 lean) and 9 ovulatory controls (5 obese, 4 lean) participated in the study. Androgens, C-reactive protein (CRP), interleukin-6 (IL-6), free fatty acids (FFA) and body weight were measured before and after 3 and 6 months of gonadotropin-releasing hormone (GnRH) agonist administration. GnRH agonist treatment decreased estradiol, testosterone and androstenedione to similar levels in all subjects. CRP and IL-6 increased in obese women with PCOS, was unaltered in lean women with PCOS and obese controls, and decreased in lean controls after 6 months of treatment. FFA decreased and body weight increased in obese women with PCOS, but did not change significantly in lean women with PCOS and in either control group after 6 months of treatment. The testosterone reduction was related to increases in weight and IL-6. The fall in FFA was related to the rise in CRP. The increases in weight and IL-6 were related to the rise in CRP. We propose that hyperandrogenism in PCOS may exert an anti-inflammatory effect when obesity is present, but may not promote inflammation in the disorder; and that circulating androgens have a pleiotropic effect on inflammation depending on the combination of PCOS and weight status in a given individual.

Reduction in inflammation and the expression of amyloid precursor protein and other proteins related to Alzheimer's disease following gastric bypass surgery.

OBJECTIVE: Obesity and type 2 diabetes are associated with an increase in the incidence and prevalence of Alzheimer's disease (AD) and an impaired cognitive function. Because peripheral blood mononuclear cells (MNC) express amyloid precursor protein (APP), the precursor of ß-amyloid, which forms the pathognomonic plaques in the brain, we hypothesized that APP expression diminishes after the marked caloric restriction and weight loss associated with Roux-en-Y gastric bypass (RYGB) surgery. RESEARCH DESIGN AND METHODS: Fifteen type 2 diabetic patients with morbid obesity (body mass index, 52.1 ± 13 kg/m(2)) underwent RYGB, and the expression of inflammatory and AD-related genes was examined before and after 6 months in plasma and in MNC. RESULTS: Body mass index fell to 40.4 ± 11.1 kg/m(2) at 6 months after RYGB. There was a significant fall in plasma concentrations of glucose and insulin and in homeostasis model of assessment for insulin resistance. The expression of APP mRNA fell by 31 ± 9%, and that of protein fell by 36 ± 14%. In addition, there was a reduction in the expression of other AD-related genes including presinilin-2, ADAM-9, GSK-3ß, PICALM, SORL-1, and clusterin (P < 0.05 for all). Additionally, the expression of c-Fos, a subunit of the proinflammatory transcription factor AP-1, was also suppressed after RYGB. These changes occurred in parallel with reductions in other proinflammatory mediators including C-reactive protein and monocyte chemoattractant protein-1. CONCLUSIONS: Thus, the reversal of the proinflammatory state of obesity is associated with a concomitant reduction in the expression of APP and other AD-related genes in MNC. We conclude that obesity and caloric intake modulate the expression of APP in MNC. If indeed, this effect also occurs in the brain, this may have implications for the pathogenesis and the treatment of AD. It is relevant that cognitive function has been shown to improve with weight loss following bariatric surgery.

Exenatide exerts a potent antiinflammatory effect.

OBJECTIVE: Our objective was to determine whether exenatide exerts an antiinflammatory effect. RESEARCH DESIGN AND METHODS: Twenty-four patients were prospectively randomized to be injected sc with either exenatide 10 µg twice daily [n = 12; mean age = 56 ± 3 yr; mean body mass index = 39.8 ± 2 kg/m(2); mean glycosylated hemoglobin (HbA1c) = 8.6 ± 0.4%] or placebo twice daily (n = 12; mean age = 54 ± 4 yr; mean body mass index = 39.1 ± 1.6 kg/m(2); mean HbA1c = 8.5 ± 0.3%) for 12 wk. Fasting blood samples were obtained at 0, 3, 6, and 12 wk. Blood samples were also collected for up to 6 h after a single dose of exenatide (5 µg) or placebo. RESULTS: Fasting blood glucose fell from 139 ± 17 to 110 ± 9 mg/dl, HbA1c from 8.6 ± 0.4 to 7.4 ± 0.5% (P < 0.05), and free fatty acids by 21 ± 5% from baseline (P < 0.05) with exenatide. There was no weight loss. There was a significant reduction in reactive oxygen species generation and nuclear factor-κB binding by 22 ± 9 and 26 ± 7%, respectively, and the mRNA expression of TNFα, IL-1ß, JNK-1, TLR-2, TLR-4, and SOCS-3 in mononuclear cells by 31 ± 12, 22 ± 10, 20 ± 11, 22 ± 9, 16 ± 7, and 31 ± 10%, respectively (P < 0.05 for all) after 12 wk of exenatide. After a single injection of exenatide, there was a reduction by 20 ± 7% in free fatty acids, 19 ± 7% in reactive oxygen species generation, 39 ± 11% in nuclear factor-κB binding, 18 ± 9% in TNFα expression, 26 ± 7% in IL-1ß expression, 18 ± 7% in JNK-1 expression, 24 ± 12% in TLR-4 expression, and 23 ± 11% in SOCS-3 expression (P < 0.05 for all). The plasma concentrations of monocyte chemoattractant protein-1, matrix metalloproteinase-9, serum amyloid A, and IL-6 were suppressed after 12 wk exenatide treatment by 15 ± 7, 20 ± 11, 16 ± 7, and 22 ± 12%, respectively (P < 0.05 for all). CONCLUSIONS: Exenatide exerts a rapid antiinflammatory effect at the cellular and molecular level. This may contribute to a potentially beneficial antiatherogenic effect. This effect was independent of weight loss.

Reduction in endotoxemia, oxidative and inflammatory stress, and insulin resistance after Roux-en-Y gastric bypass surgery in patients with morbid obesity and type 2 diabetes mellitus.

BACKGROUND: Roux-en-Y gastric bypass (RYGB) results in profound weight loss and resolution of type 2 diabetes mellitus (T2DM). The mechanism of this remarkable transition remains poorly defined. It has been proposed that endotoxin (lipopolysaccharide [LPS]) sets inflammatory tone, triggers weight gain, and initiates T2DM. Because RYGB may diminish LPS from endogenous and exogenous sources, we hypothesized that LPS and the associated cascade of oxidative and inflammatory stress would diminish after RYGB. METHODS: Fifteen adults with morbid obesity and T2DM undergoing RYGB were studied. After an overnight fast, a baseline blood sample was collected the morning of surgery and at 180 days to assess changes in glycemia, insulin resistance, LPS, mononuclear cell nuclear factor (NF)-κB binding and mRNA expression of CD14, TLR-2, TLR-4, and markers of inflammatory stress. RESULTS: At 180 days after RYGB, subjects had a significant decrease in body mass index (52.1 ± 13.0 to 40.4 ± 11.1), plasma glucose (148 ± 8 to 101 ± 4 mg/dL), insulin (18.5 ± 2.2 mµU/mL to 8.6 ± 1.0 mµU/mL) and HOMA-IR (7.1 ± 1.1 to 2.1 ± 0.3). Plasma LPS significantly reduced by 20 ± 5% (0.567 ± 0.033 U/mL to 0.443 ± 0.022 E U/mL). NF-κB DNA binding decreased significantly by 21 ± 8%, whereas TLR-4, TLR-2, and CD-14 expression decreased significantly by 25 ± 9%, 42 ± 8%, and 27 ± 10%, respectively. Inflammatory mediators CRP, MMP-9, and MCP-1 decreased significantly by 47 ± 7% (10.7 ± 1.6 mg/L to 5.8 ± 1.0 mg/L), 15 ± 6% (492 ± 42 ng/mL to 356 ± 26 ng/mL) and 11 ± 4% (522 ± 35 ng/mL to 466 ± 35 ng/mL), respectively. CONCLUSION: LPS, NF-κB DNA binding, TLR-4, TLR-2, and CD14 expression, CRP, MMP-9, and MCP-1 decreased significantly after RYGB. The mechanism underlying resolution of insulin resistance and T2DM after RYGB may be attributable, at least in part, to the reduction of endotoxemia and associated proinflammatory mediators.

Insulin suppresses the expression of amyloid precursor protein, presenilins, and glycogen synthase kinase-3beta in peripheral blood mononuclear cells.

OBJECTIVE: Our objective was to determine whether peripheral blood mononuclear cells express amyloid precursor protein (APP) and other mediators involved in the pathogenesis of Alzheimer's disease and whether their expression is suppressed by insulin. RESEARCH DESIGN AND METHODS: Ten obese type 2 diabetic patients were infused with insulin (2 U/h with 100 ml 5% dextrose/h) for 4 h. Patients were also infused with 5% dextrose/h or normal physiological saline for 4 h, respectively, on two other days as controls. Blood samples were obtained at 0, 2, 4, and 6 h. RESULTS: Insulin infusion significantly suppressed the expression of APP, presenilin-1, presenilin-2, and glycogen synthase kinase-3ß in peripheral blood mononuclear cells. Dextrose and saline infusions did not alter these indices. Insulin infusion also caused significant parallel reductions in nuclear factor-κB binding activity and plasma concentrations of serum amyloid A and intercellular adhesion molecule-1. CONCLUSIONS: A low dose infusion of insulin suppresses APP, presenilin-1, presenilin-2, and glycogen synthase kinase-3ß, key proteins involved in the pathogenesis of Alzheimer's disease, in parallel with exerting its other antiinflammatory effects.

A resveratrol and polyphenol preparation suppresses oxidative and inflammatory stress response to a high-fat, high-carbohydrate meal.

BACKGROUND: High-fat, high-carbohydrate (HFHC) meals are known to induce oxidative and inflammatory stress, an increase in plasma endotoxin concentrations, and an increase in the expression of suppressor of cytokine signaling-3 (SOCS-3). HYPOTHESIS: The intake of a nutritional supplement containing resveratrol and muscadine grape polyphenols reduces HFHC meal-induced oxidative and inflammatory stress and stimulates the activity of the antioxidant transcription factor, NF-E2-related factor-2 (Nrf-2), and its downstream targets. METHODS: Ten normal, healthy subjects were given a 930-kcal HFHC meal either with placebo or with the supplement. Indices of oxidative stress, inflammation, Nrf-2 binding activity, the concentrations of endotoxin (lipopolysaccharide) and lipoprotein binding protein (LBP), and the expression of toll-like receptor 4 (TLR-4), CD14, IL-1ß, TNFα, SOCS-3, Keap-1, NAD(P)H:quinone oxidoreductase-1 (NQO-1), and GST-P1 were measured. RESULTS: The intake of the supplement suppressed the meal-induced elevations of plasma endotoxin and LBP concentrations, the expression of p47(phox), TLR-4, CD14, SOCS-3, IL-1ß, and Keap-1, while enhancing Nrf-2 binding activity and the expression of NQO-1 and GST-P1 genes. CONCLUSION: A supplement containing resveratrol and muscadine polyphenols suppresses the increase in oxidative stress, lipopolysaccharide and LBP concentrations, and expression of TLR-4, CD14, IL-1ß and SOCS-3 in mononuclear cells after an HFHC meal. It also stimulates specific Nrf-2 activity and induces the expression of the related antioxidant genes, NQO-1 and GST-P1. These results demonstrate the acute antioxidant and antiinflammatory effects of resveratrol and polyphenolic compounds in humans in the postprandial state.

An antiinflammatory and reactive oxygen species suppressive effects of an extract of Polygonum cuspidatum containing resveratrol.

BACKGROUND: Resveratrol have been shown to exert an antiinflammatory and antiaging effects in vitro and in animal models. OBJECTIVE: The objective of the study was to investigate the effect of a Polygonum cuspidatum extract (PCE) containing resveratrol on oxidative and inflammatory stress in normal subjects. RESEARCH DESIGN AND METHODS: Two groups (10 each) of normal-weight healthy subjects were randomized to placebo or PCE containing 40 mg resveratrol daily for 6 wk. Fasting blood samples were obtained prior to and after treatment at 1, 3, and 6 wk. Mononuclear cells were prepared for reactive oxygen species generation, RNA isolation, nuclear extract, and total cell homogenate preparation. Indices of oxidative and inflammatory stress, suppressor of cytokine signaling-3, phosphotyrosine phosphatase-1B, jun-N-terminal kinase-1, and inhibitor of kappaB-kinase-beta were measured by RT-PCR and Western blotting. RESULTS: The extract induced a significant reduction in reactive oxygen species generation, the expression of p47(phox), intranuclear nuclear factor-kappaB binding, and the expression of jun-N-terminal kinase-1, inhibitor of kappaB-kinase-beta, phosphotyrosine phosphatase-1B, and suppressor of cytokine signaling-3 in mononuclear cells when compared with the baseline and the placebo. PCE intake also suppressed plasma concentrations of TNF-alpha, IL-6, and C-reactive protein. There was no change in these indices in the control group given placebo. CONCLUSIONS: The PCE-containing resveratrol has a comprehensive suppressive effect on oxidative and inflammatory stress.

Orange juice neutralizes the proinflammatory effect of a high-fat, high-carbohydrate meal and prevents endotoxin increase and Toll-like receptor expression.

BACKGROUND: The intake of glucose or a high-fat, high-carbohydrate (HFHC) meal, but not orange juice, induces an increase in inflammation and oxidative stress in circulating mononuclear cells (MNCs) of normal-weight subjects. OBJECTIVE: We investigated the effect of orange juice on HFHC meal-induced inflammation and oxidative stress and the expression of plasma endotoxin and Toll-like receptors (TLRs). DESIGN: Three groups (10 subjects in each group) of normal, healthy subjects were asked to drink water or 300 kcal glucose or orange juice in combination with a 900-kcal HFHC meal. Blood samples were obtained before and 1, 3, and 5 h after the drinks and meal combinations were consumed. RESULTS: Protein expression of the NADPH oxidase subunit p47(phox), phosphorylated and total p38 mitogen-activated protein kinase, and suppressor of cytokine signaling-3; TLR2 and TLR4 messenger RNA (mRNA) and protein expression; mRNA expression of matrix metalloproteinase (MMP)-9 in MNCs; and plasma concentrations of endotoxin and MMP-9 increased significantly after glucose or water were consumed with the meal but not when orange juice was consumed with the meal. The generation of reactive oxygen species by polymorphonuclear cells was significantly lower when orange juice was added to the meal than when water or glucose was added to the meal. CONCLUSIONS: The combination of glucose or water and the HFHC meal induced oxidative and inflammatory stress and an increase in TLR expression and plasma endotoxin concentrations. In contrast, orange juice intake with the HFHC meal prevented meal-induced oxidative and inflammatory stress, including the increase in endotoxin and TLR expression. These observations may help explain the mechanisms underlying postprandial oxidative stress and inflammation, pathogenesis of insulin resistance, and atherosclerosis.