Characterization of the effects of cannabinoid receptor deletion on energy metabolism in female C57BL mice.

Background: Despite the evidence that energy balance is regulated differently in females and that the endocannabinoid system is sexually dimorphic, previous studies on the endocannabinoid system and energy balance predominantly used male models. Here, we characterize the effects of cannabinoid receptor deletion on body weight gain and glucose metabolism in female C57BL mice. Methods: Female mice lacking the cannabinoid-1 receptor (CB1R-/-), cannabinoid-2 receptor (CB2R-/-), or both receptors (CB1R-/-/CB2R-/-) and wild-type (WT) mice were fed with a low (LFD; 10% of calories from fat) or high-fat diet (HFD; 45% of calories from fat) for six weeks. Results: Female WT mice fed with HFD gained significantly more weight than WT mice fed with LFD (p < 0.001). Similar pattern was observed for CB2/- mice fed with HFD compared to CB2R-/- mice fed with LFD (p < 0.001), but not for CB1R-/- fed with HFD vs. LFD (p = 0.22) or CB1R-/-/CB2R-/- fed with HFD vs. LFD (p = 0.96). Comparing the 4 groups on LFD, weight gain of CB1R-/- mice was greater than all other genotypes (p < 0.05). When fed with HFD, the deletion of CB1R alone in females did not attenuate weight gain compared to WT mice (p = 0.72). Female CB1R-/-/CB2R-/- mice gained less weight than WT mice when fed with HFD (p = 0.007) despite similar food intake and locomotor activity, potentially owing to enhanced thermogenesis in the white adipose tissue. No significant difference in weight gain was observed for female CB2R-/- and WT mice on LFD or HFD. Fasting glucose, however, was higher in CB2R-/- mice fed with LFD than all other groups (p < 0.05). Conclusion: The effects of cannabinoid receptor deletion on glucose metabolism in female mice were similar to previously published findings on male mice, yet the effects on body weight gain and thermogenesis were attenuated in CB1R-/- mice.

Theory, methods, and operational results of the Young Women's Health History Study: a study of young-onset breast cancer incidence in Black and White women.

PURPOSE: The etiology of young-onset breast cancer (BC) is poorly understood, despite its greater likelihood of being hormone receptor-negative with a worse prognosis and persistent racial and socioeconomic inequities. We conducted a population-based case-control study of BC among young Black and White women and here discuss the theory that informed our study, exposures collected, study methods, and operational results. METHODS: Cases were non-Hispanic Black (NHB) and White (NHW) women age 20-49 years with invasive BC in metropolitan Detroit and Los Angeles County SEER registries 2010-2015. Controls were identified through area-based sampling from the U.S. census and frequency matched to cases on study site, race, and age. An eco-social theory of health informed life-course exposures collected from in-person interviews, including socioeconomic, reproductive, and energy balance factors. Measured anthropometry, blood (or saliva), and among cases SEER tumor characteristics and tumor tissue (from a subset of cases) were also collected. RESULTS: Of 5,309 identified potentially eligible cases, 2,720 sampled participants were screened and 1,812 completed interviews (682 NHB, 1140 NHW; response rate (RR): 60%). Of 24,612 sampled control households 18,612 were rostered, 2,716 participants were sampled and screened, and 1,381 completed interviews (665 NHB, 716 NHW; RR: 53%). Ninety-nine% of participants completed the main interview, 82% provided blood or saliva (75% blood only), and SEER tumor characteristics (including ER, PR and HER2 status) were obtained from 96% of cases. CONCLUSIONS: Results from the successfully established YWHHS should expand our understanding of young-onset BC etiology overall and by tumor type and identify sources of racial and socioeconomic inequities in BC.

Regulation of lipid metabolism in pancreatic beta cells by interferon gamma: A link to anti-viral function.

Interferons (IFN) have been shown to alter lipid metabolism in immune and some non-hematopoietic cells and this affects host cell response to pathogens. In type 1 diabetes, IFNγ acts as a proinflammatory cytokine that, along with other cytokines, is released during pancreatic beta cell autoinflammation and contributes to immune response and beta cell dysfunction. The hypothesis tested herein is that IFN modifies beta cell lipid metabolism and this is associated with enhanced anti-viral response and beta cell stress. Treatment of INS-1 cells with IFNγ for 6 to 24 h led to a dynamic change in TAG and lipid droplet (LD) levels, with a decrease at 6 h and an increase at 24 h. The later accumulation of TAG was associated with increased de novo lipogenesis (DNL), and impaired mitochondrial fatty acid oxidation (FAO). Gene expression results suggested that IFNγ regulates lipolytic, lipogenic, LD and FAO genes in a temporal manner. The changes in lipid gene expression are dependent on the classical Janus kinase (JAK) pathway. Pretreatment with IFNγ robustly enhanced anti-viral gene expression induced by the viral mimetic polyinosinic: polycytidylic acid (PIC), and this potentiating effect of IFNγ was markedly attenuated by inhibitors of DNL. The IFNγ-induced accumulation of lipid, however, was insufficient to cause endoplasmic reticulum (ER) stress. These studies demonstrated a non-canonical effect of IFNγ in regulation of pancreatic beta cell lipid metabolism that is intimately linked with host cell defense and might alter cellular function early in the progression to type 1 diabetes.

miR-216a-targeting theranostic nanoparticles promote proliferation of insulin-secreting cells in type 1 diabetes animal model.

Aberrant expression of miRNAs in pancreatic islets is closely related to the development of type 1 diabetes (T1D). The aim of this study was to identify key miRNAs dysregulated in pancreatic islets during T1D progression and to develop a theranostic approach to modify their expression using an MRI-based nanodrug consisting of iron oxide nanoparticles conjugated to miRNA-targeting oligonucleotides in a mouse model of T1D. Isolated pancreatic islets were derived from NOD mice of three distinct age groups (3, 8 and 18-week-old). Total RNA collected from cultured islets was purified and global miRNA profiling was performed with 3D-Gene global miRNA microarray mouse chips encompassing all mouse miRNAs available on the Sanger miRBase V16. Of the miRNAs that were found to be differentially expressed across three age groups, we identified one candidate (miR-216a) implicated in beta cell proliferation for subsequent validation by RT-PCR. Alterations in miR-216a expression within pancreatic beta cells were also examined using in situ hybridization on the frozen pancreatic sections. For in vitro studies, miR-216a mimics/inhibitors were conjugated to iron oxide nanoparticles and incubated with beta cell line, ßTC-6. Cell proliferation marker Ki67 was evaluated. Expression of the phosphatase and tensin homolog (PTEN), which is one of the direct targets of miR-216a, was analyzed using western blot. For in vivo study, the miR-216a mimics/inhibitors conjugated to the nanoparticles were injected into 12-week-old female diabetic Balb/c mice via pancreatic duct. The delivery of the nanodrug was monitored by in vivo MRI. Blood glucose of the treated mice was monitored post injection. Ex vivo histological analysis of the pancreatic sections included staining for insulin, PTEN and Ki67. miRNA microarray demonstrated that the expression of miR-216a in the islets from NOD mice significantly changed during T1D progression. In vitro studies showed that treatment with a miR-216a inhibitor nanodrug suppressed proliferation of beta cells and increased the expression of PTEN, a miR-216a target. In contrast, introduction of a mimic nanodrug decreased PTEN expression and increased beta cell proliferation. Animals treated in vivo with a mimic nanodrug had higher insulin-producing functionality compared to controls. These observations were in line with downregulation of PTEN and increase in beta cell proliferation in that group. Our studies demonstrated that miR-216a could serve as a potential therapeutic target for the treatment of diabetes. miR-216a-targeting theranostic nanodrugs served as exploratory tools to define functionality of this miRNA in conjunction with in vivo MR imaging.

Diet-Induced Obesity in Cannabinoid-2 Receptor Knockout Mice and Cannabinoid Receptor 1/2 Double-Knockout Mice.

OBJECTIVE: Evidence suggests that cannabinoid-1 receptor (CB1R) activation is associated with increased food intake and body weight gain. Human epidemiological studies, however, show decreased prevalence of obesity in cannabis users. Given the overlapping and complementary functions of the cannabinoid receptors (CB1R and CB2R), mice lacking CB2R and mice lacking both CB1R and CB2R were studied. METHODS: A high-fat diet was used to study metabolic changes in male mice lacking CB2R (CB2-/- ) or lacking both CB1R and CB2R (double-knockout [CB-DKO]) compared with wild-type mice. RESULTS: When CB2-/- mice were maintained on a high-fat diet, their weight gain was not different from wild-type mice (gaining 19 and 21 g, respectively), whereas CB-DKO mice gained only 5 g. There were no significant differences in food intake or locomotor activity between the three groups. Respiratory exchange rate and heat production were elevated in CB-DKO mice, with upregulation of adipose tissue thermogenic genes. Glucose tolerance test and insulin levels indicated increased insulin sensitivity in CB-DKO mice, whereas CB2-/- displayed signs of impaired glucose clearance. CONCLUSIONS: These results indicate that lacking both CB1R and CB2R protected mice from diet-induced obesity, possibly through the prominent role of CB1R in obesity or through an interactive effect of both receptors.

Impact of alogliptin and pioglitazone on lipid metabolism in islets of prediabetic and diabetic Zucker Diabetic Fatty rats.

Prolonged exposure of pancreatic beta (ß) cells to elevated glucose and free fatty acids (FFA) as occurs in type 2 diabetes results in loss of ß cell function and survival. In Zucker Diabetic Fatty (ZDF) rats, ß cell failure is associated with increased triacylglyceride (TAG) synthesis and disruption of the glycerolipid/FFA (GL/FFA) cycle, a critical arm of glucose-stimulated insulin secretion (GSIS). The aim of this study was to determine the impact of activation of PPARγ and increased incretin action via dipeptidyl-peptidase inhibition using pioglitazone and/or alogliptin, respectively, on islet lipid metabolism in prediabetic and diabetic ZDF rats. Transition of control prediabetic ZDF rats to diabetes was associated with reduced plasma insulin levels, reduced islet insulin content and GSIS, reduced stearoyl-CoA desaturase 2 (SCD 2) expression, and increased islet TAG, diacylglyceride (DAG) and ceramides species containing saturated FA. Treatment of prediabetic ZDF rats with a combination of pioglitazone and alogliptin, but not individually, prevented the transition to diabetes and was associated with marked lowering of islet TAG and DAG levels. Pioglitazone and alogliptin, however, did not restore SCD2 expression, the degree of FA saturation in TAG, DAG or ceramides, islet insulin content, or lower ceramide levels. These findings are consistent with activation of PPARγ and increased incretin action working in concert to restore GL/FFA cycle in ß cells of ZDF rats. Restoration of the GL/FFA cycle without correcting islet FA desaturation, production of islet ceramides, and/or insulin sensitivity, however, may place these islets at risk for ß cell failure.

A role for aberrant protein palmitoylation in FFA-induced ER stress and ß-cell death.

Exposure of insulin-producing cells to elevated levels of the free fatty acid (FFA) palmitate results in the loss of ß-cell function and induction of apoptosis. The induction of endoplasmic reticulum (ER) stress is one mechanism proposed to be responsible for the loss of ß-cell viability in response to palmitate treatment; however, the pathways responsible for the induction of ER stress by palmitate have yet to be determined. Protein palmitoylation is a major posttranslational modification that regulates protein localization, stability, and activity. Defects in, or dysregulation of, protein palmitoylation could be one mechanism by which palmitate may induce ER stress in ß-cells. The purpose of this study was to evaluate the hypothesis that palmitate-induced ER stress and ß-cell toxicity are mediated by excess or aberrant protein palmitoylation. In a concentration-dependent fashion, palmitate treatment of RINm5F cells results in a loss of viability. Similar to palmitate, stearate also induces a concentration-related loss of RINm5F cell viability, while the monounsaturated fatty acids, such as palmoleate and oleate, are not toxic to RINm5F cells. 2-Bromopalmitate (2BrP), a classical inhibitor of protein palmitoylation that has been extensively used as an inhibitor of G protein-coupled receptor signaling, attenuates palmitate-induced RINm5F cell death in a concentration-dependent manner. The protective effects of 2BrP are associated with the inhibition of [(3)H]palmitate incorporation into RINm5F cell protein. Furthermore, 2BrP does not inhibit, but appears to enhance, the oxidation of palmitate. The induction of ER stress in response to palmitate treatment and the activation of caspase activity are attenuated by 2BrP. Consistent with protective effects on insulinoma cells, 2BrP also attenuates the inhibitory actions of prolonged palmitate treatment on insulin secretion by isolated rat islets. These studies support a role for aberrant protein palmitoylation as a mechanism by which palmitate enhances ER stress activation and causes the loss of insulinoma cell viability.

Modulation of palmitate-induced endoplasmic reticulum stress and apoptosis in pancreatic ß-cells by stearoyl-CoA desaturase and Elovl6.

Induction of endoplasmic reticulum (ER) stress and apoptosis by elevated exogenous saturated fatty acids (FAs) plays a role in the pathogenesis of ß-cell dysfunction and loss of islet mass in type 2 diabetes. Regulation of monounsaturated FA (MUFA) synthesis through FA desaturases and elongases may alter the susceptibility of ß-cells to saturated FA-induced ER stress and apoptosis. Herein, stearoyl-CoA desaturase (SCD)1 and SCD2 mRNA expression were shown to be induced in islets from prediabetic hyperinsulinemic Zucker diabetic fatty (ZDF) rats, whereas SCD1, SCD2, and fatty acid elongase 6 (Elovl6) mRNA levels were markedly reduced in diabetic ZDF rat islets. Knockdown of SCD in INS-1 ß-cells decreased desaturation of palmitate to MUFA, lowered FA partitioning into complex neutral lipids, and increased palmitate-induced ER stress and apoptosis. Overexpression of SCD2 increased desaturation of palmitate to MUFA and attenuated palmitate-induced ER stress and apoptosis. Knockdown of Elovl6 limited palmitate elongation to stearate, increasing palmitoleate production and attenuating palmitate-induced ER stress and apoptosis, whereas overexpression of Elovl6 increased palmitate elongation to stearate and palmitate-induced ER stress and apoptosis. Overall, these data support the hypothesis that enhanced MUFA synthesis via upregulation of SCD2 activity can protect ß-cells from elevated saturated FAs, as occurs in prediabetic states. Overt type 2 diabetes is associated with diminished islet expression of SCD and Elovl6, and this can disrupt desaturation of saturated FAs to MUFAs, rendering ß-cells more susceptible to saturated FA-induced ER stress and apoptosis.

Soraphen A, an inhibitor of acetyl CoA carboxylase activity, interferes with fatty acid elongation.

Acetyl CoA carboxylase (ACC1 and ACC2) generates malonyl CoA, a substrate for de novo lipogenesis (DNL) and an inhibitor of mitochondrial fatty acid ß-oxidation (FAO). Malonyl CoA is also a substrate for microsomal fatty acid elongation, an important pathway for saturated (SFA), mono- (MUFA) and polyunsaturated fatty acid (PUFA) synthesis. Despite the interest in ACC as a target for obesity and cancer therapy, little attention has been given to the role ACC plays in long chain fatty acid synthesis. This report examines the effect of pharmacological inhibition of ACC on DNL and palmitate (16:0) and linoleate (18:2, n-6) metabolism in HepG2 and LnCap cells. The ACC inhibitor, soraphen A, lowers cellular malonyl CoA, attenuates DNL and the formation of fatty acid elongation products derived from exogenous fatty acids, i.e., 16:0 and 18:2, n-6; IC(50)∼5nM. Elevated expression of fatty acid elongases (Elovl5, Elovl6) or desaturases (FADS1, FADS2) failed to override the soraphen A effect on SFA, MUFA or PUFA synthesis. Inhibition of fatty acid elongation leads to the accumulation of 16- and 18-carbon unsaturated fatty acids derived from 16:0 and 18:2, n-6, respectively. Pharmacological inhibition of ACC activity will not only attenuate DNL and induce FAO, but will also attenuate the synthesis of very long chain saturated, mono- and polyunsaturated fatty acids.

Role of fatty acid elongases in determination of de novo synthesized monounsaturated fatty acid species.

Enhanced production of monounsaturated fatty acids (FA) derived from carbohydrate-enriched diets has been implicated in the development of obesity and insulin resistance. The FA elongases Elovl-5 and Elovl-6 are regulated by nutrient and hormone status, and have been shown using intact yeast and mammalian microsome fractions to be involved in the synthesis of monounsaturated FAs (MUFA). Herein, targeted knockdown and overexpression of Elovl-5 or Elovl-6 was used to determine their roles in de novo synthesis of specific MUFA species in mammalian cells. Treatment of rat insulinoma (INS)-1 cells with elevated glucose increased de novo FA synthesis and the ratio of MUFAs to saturated FAs. Elovl-5 knockdown decreased elongation of 16:1,n-7. Elovl-5 overexpression increased synthesis of 18:1,n-7; however, this increase was dependent on stearoyl-CoA desaturase-driven 16:1,n-7 availability. Knockdown of Elovl-6 decreased elongation of 16:0 and 16:1,n-7, resulting in accumulation of 16:1,n-7. Elovl-6 overexpression preferentially drove synthesis of 16:0 elongation products 18:0 and 18:1,n-9 but not 18:1,n-7. These findings demonstrate that coordinated induction of FA elongase and desaturase activity is required for balanced synthesis of specific n-7 versus n-9 MUFA species. Given the relative abundance of 16:0 to 16:1,n-7 and the specificity of Elovl-6 for 16:0, Elovl-6 is a major elongase for 18:1,n-9 production.

Activation of autophagy through modulation of 5'-AMP-activated protein kinase protects pancreatic beta-cells from high glucose.

Chronic hyperglycaemia is detrimental to pancreatic beta-cells by causing impaired insulin secretion and diminished beta-cell function through glucotoxicity. Understanding the mechanisms underlying beta-cell survival is crucial for the prevention of beta-cell failure associated with glucotoxicity. Autophagy is a dynamic lysosomal degradation process that protects organisms against metabolic stress. To date, little is known about the physiological function of autophagy in the pathogenesis of diabetes. In the present study, we explored the roles of autophagy in the survival of pancreatic beta-cells exposed to high glucose using pharmacological and genetic manipulation of autophagy. We demonstrated that chronic high glucose increases autophagy in rat INS-1 (832/13) cells and pancreatic islets, and that this increase is enhanced by inhibition of 5'-AMP-activated protein kinase. Our results also indicate that stimulation of autophagy rescues pancreatic beta-cells from high-glucose-induced cell death and inhibition of autophagy augments caspase-3 activation, suggesting that autophagy plays a protective role in the survival of pancreatic beta-cells. Greater knowledge of the molecular mechanisms linking autophagy and beta-cell survival may unveil novel therapeutic targets needed to preserve beta-cell function.

Elevated insulin secretion from liver X receptor-activated pancreatic beta-cells involves increased de novo lipid synthesis and triacylglyceride turnover.

Increased basal and loss of glucose-stimulated insulin secretion (GSIS) are hallmarks of beta-cell dysfunction associated with type 2 diabetes. It has been proposed that elevated glucose promotes insulin secretory defects by activating sterol regulatory element binding protein (SREBP)-1c, lipogenic gene expression, and neutral lipid storage. Activation of liver X receptors (LXRs) also activates SREBP-1c and increases lipogenic gene expression and neutral lipid storage but increases basal and GSIS. This study was designed to characterize the changes in de novo fatty acid and triacylglyceride (TAG) synthesis in LXR-activated beta-cells and determine how these changes contribute to elevated basal and GSIS. Treatment of INS-1 beta-cells with LXR agonist T0901317 and elevated glucose led to markedly increased nuclear localization of SREBP-1, lipogenic gene expression, de novo synthesis of monounsaturated fatty acids and TAG, and basal and GSIS. LXR-activated cells had increased fatty acid oxidation and expression of genes involved in mitochondrial beta-oxidation, particularly carnitine palmitoyltransferase-1. Increased basal insulin release from LXR-activated cells coincided with rapid turnover of newly synthesized TAG and required acyl-coenzyme A synthesis and mitochondrial beta-oxidation. GSIS from LXR-activated INS-1 cells required influx of extracellular calcium and lipolysis, suggesting production of lipid-signaling molecules from TAG. Inhibition of diacylglyceride (DAG)-binding proteins, but not classic isoforms of protein kinase C, attenuated GSIS from LXR-activated INS-1 cells. In conclusion, LXR activation in beta-cells exposed to elevated glucose concentrations increases de novo TAG synthesis; subsequent lipolysis produces free fatty acids and DAG, which are oxidized to increase basal insulin release and activate DAG-binding proteins to enhance GSIS, respectively.

Regulation of hepatic fatty acid elongase and desaturase expression in diabetes and obesity.

Fatty acid elongases and desaturases play an important role in hepatic and whole body lipid composition. We examined the role that key transcription factors played in the control of hepatic elongase and desaturase expression. Studies with peroxisome proliferator-activated receptor alpha (PPARalpha)-deficient mice establish that PPARalpha was required for WY14643-mediated induction of fatty acid elongase-5 (Elovl-5), Elovl-6, and all three desaturases [Delta(5) desaturase (Delta(5)D), Delta(6)D, and Delta(9)D]. Increased nuclear sterol-regulatory element binding protein-1 (SREBP-1) correlated with enhanced expression of Elovl-6, Delta(5)D, Delta(6)D, and Delta(9)D. Only Delta(9)D was also regulated independently by liver X receptor (LXR) agonist. Glucose induction of l-type pyruvate kinase, Delta(9)D, and Elovl-6 expression required the carbohydrate-regulatory element binding protein/MAX-like factor X (ChREBP/MLX) heterodimer. Suppression of Elovl-6 and Delta(9)D expression in livers of streptozotocin-induced diabetic rats and high fat-fed glucose-intolerant mice correlated with low levels of nuclear SREBP-1. In leptin-deficient obese mice (Lep(ob/ob)), increased SREBP-1 and MLX nuclear content correlated with the induction of Elovl-5, Elovl-6, and Delta(9)D expression and the massive accumulation of monounsaturated fatty acids (18:1,n-7 and 18:1,n-9) in neutral lipids. Diabetes- and obesity-induced changes in hepatic lipid composition correlated with changes in elongase and desaturase expression. In conclusion, these studies establish a role for PPARalpha, LXR, SREBP-1, ChREBP, and MLX in the control of hepatic fatty acid elongase and desaturase expression and lipid composition.

MafA expression and insulin promoter activity are induced by nicotinamide and related compounds in INS-1 pancreatic beta-cells.

Nicotinamide has been reported to induce differentiation of precursor/stem cells toward a beta-cell phenotype, increase islet regeneration, and enhance insulin biosynthesis. Exposure of INS-1 beta-cells to elevated glucose leads to reduced insulin gene transcription, and this is associated with diminished binding of pancreatic duodenal homeobox factor 1 (PDX-1) and mammalian homologue of avian MafA/l-Maf (MafA). Nicotinamide and other low-potency poly(ADP-ribose) polymerase (PARP) inhibitors were thus tested for their ability to restore insulin promoter activity. The low-potency PARP inhibitors nicotinamide, 3-aminobenzamide, or PD128763 increased expression of a human insulin reporter gene suppressed by elevated glucose. In contrast, the potent PARP-1 inhibitors PJ34 or INO-1001 had no effect on promoter activity. Antioxidants, including N-acetylcysteine, lipoic acid, or quercetin, only minimally induced the insulin promoter. Site-directed mutations of the human insulin promoter mapped the low-potency PARP inhibitor response to the C1 element, which serves as a MafA binding site. INS-1 cells exposed to elevated glucose had markedly reduced MafA protein and mRNA levels. Low-potency PARP inhibitors restored MafA mRNA and protein levels, but they had no affect on PDX-1 protein levels or binding activity. Increased MafA expression by low-potency PARP inhibitors was independent of increased MafA protein or mRNA stability. These data suggest that low-potency PARP inhibitors increase insulin biosynthesis, in part, through a mechanism involving increased MafA gene transcription.

Amelioration of obesity and glucose intolerance in high-fat-fed C57BL/6 mice by anthocyanins and ursolic acid in Cornelian cherry (Cornus mas).

Much attention has been focused on food that may be beneficial in preventing diet-induced body fat accumulation and possibly reduce the risk of diabetes and heart disease. Cornelian cherries (Cornus mas) are used in the preparation of beverages in Europe and also to treat diabetes-related disorders in Asia. In this study, the most abundant bioactive compounds in C. mas fruits, the anthocyanins and ursolic acid, were purified, and their ability to ameliorate obesity and insulin resistance in C57BL/6 mice fed a high-fat diet was evaluated. Mice were initially fed a high-fat diet for 4 weeks and then switched to a high-fat diet containing anthocyanins (1 g/kg of high-fat diet) and ursolic acid (500 mg/kg of high-fat diet) for an additional 8 weeks. The high-fat diet induced glucose intolerance, and this was prevented by anthocyanins and ursolic acid. The anthocyanin-treated mice showed a 24% decrease in weight gain. These mice also showed decreased lipid accumulation in the liver, including a significant decrease in liver triacylglycerol concentration. Anthocyanin and ursolic acid treated mice exhibited extremely elevated insulin levels. Both treatments, however, showed preserved islet architecture and insulin staining. Overall, these data suggest that anthocyanins and ursolic acid purified from C. mas fruits have biological activities that improve certain metabolic parameters associated with diets high in saturated fats and obesity.

Insulin secretion by bioactive anthocyanins and anthocyanidins present in fruits.

Anthocyanins are responsible for a variety of bright colors including red, blue, and purple in fruits, vegetables, and flowers and are consumed as dietary polyphenols. Anthocyanin-containing fruits are implicated in a decrease in coronary heart disease and are used in antidiabetic preparations. In the present study, we have determined the ability of anthocyanins, cyanidin-3-glucoside (1), delphinidin-3-glucoside (2), cyanidin-3-galactoside (3), and pelargonidin-3-galactoside (4), and anthocyanidins, cyanidin (5), delphinidin (6), pelargonidin (7), malvidin (8), and petunidin (9), to stimulate insulin secretion from rodent pancreatic beta-cells (INS-1 832/13) in vitro. The compounds were tested in the presence of 4 and 10 mM glucose concentrations. Our results indicated that 1 and 2 were the most effective insulin secretagogues among the anthocyanins and anthocyanidins tested at 4 and 10 mM glucose concentrations. Pelargonidin-3-galactoside is one of the major anthocyanins, and its aglycone, pelargonidin, caused a 1.4-fold increase in insulin secretion at 4 mM glucose concentration. The rest of the anthocyanins and anthocyanidins tested in our assay had only marginal effects on insulin at 4 and 10 mM glucose concentrations.

Elevated glucose attenuates human insulin gene promoter activity in INS-1 pancreatic beta-cells via reduced nuclear factor binding to the A5/core and Z element.

Chronic exposure of pancreatic beta-cells to elevated glucose reduces insulin gene promoter activity, and this is associated with diminished binding of two beta-cell-enriched transcription factors, Pdx-1 and MafA. In this study using INS-1 beta-cells, overexpression of MafA, but not Pdx-1, was able to restore expression of a human insulin reporter gene (-327 to +30 bp) suppressed by elevated glucose. At issue, however, was that MafA also markedly stimulated an insulin reporter gene (-230 to +30 bp) that was only marginally suppressed by glucose, suggesting that glucose-mediated suppression of the insulin promoter involved elements upstream of -230. Using serial truncations and mini-enhancer constructs of the human insulin promoter, the majority of glucose suppression was localized to regulatory elements between -327 and -261. Nuclear extracts from INS-1 cells exposed to elevated glucose had reduced binding activities to the A5/core (-319 to -307), and to a palindrome (-284 to -267) and an E box (-273 to -257, E3) contained within the Z element. The A5/core binding complex was determined to contain MafA, Pdx-1, and an A2-like binding factor. Two mini-enhancer constructs containing the A5/core were suppressed by glucose and strongly activated by MafA. Glucose-mediated suppression of the Z mini-enhancer was not attenuated by overexpression of MafA or Pdx-1. Site-directed mutation of the A5/core, palindrome, and E3 elements attenuated glucose-mediated suppression. These data indicate that glucose suppression of human insulin promoter activity in INS-1 cells involves reduced binding of MafA to the A5/core. Changes in nuclear factor binding to the Z element, which functions as a strong activator element in primary islets and a negative regulatory element in simian virus 40 or T antigen transformed beta-cell lines, also participate in glucose suppression of insulin promoter activity.

Oct4 expression in adult human stem cells: evidence in support of the stem cell theory of carcinogenesis.

The Oct3/4 gene, a POU family transcription factor, has been noted as being specifically expressed in embryonic stem cells and in tumor cells but not in cells of differentiated tissues. With the ability to isolate adult human stem cells it became possible to test for the expression of Oct3/4 gene in adult stem cells and to test the stem cell theory of carcinogenesis. Using antibodies and PCR primers we tested human breast, liver, pancreas, kidney, mesenchyme and gastric stem cells, the cancer cell lines HeLa and MCF-7 and human, dog and rat tumors for Oct4 expression. The results indicate that adult human stem cells, immortalized non-tumorigenic cells and tumor cells and cell lines, but not differentiated cells, express Oct4. Oct4 is expressed in a few cells found in the basal layer of human skin epidermis. The data demonstrate that adult stem cells maintain expression of Oct4, consistent with the stem cell hypothesis of carcinogenesis.

Redox-mediated enrichment of self-renewing adult human pancreatic cells that possess endocrine differentiation potential.

OBJECTIVES: The limited availability of transplantable human islets has stimulated the development of methods needed to isolate adult pancreatic stem/progenitor cells capable of self-renewal and endocrine differentiation. The objective of this study was to determine whether modulation of intracellular redox state with N-acetyl-L-cysteine (NAC) would allow for the propagation of pancreatic stem/progenitor cells from adult human pancreatic tissue. METHODS: Cells were propagated from human pancreatic tissue using a serum-free, low-calcium medium supplemented with NAC and tested for their ability to differentiate when cultured under different growth conditions. RESULTS: Human pancreatic cell (HPC) cultures coexpressed alpha-amylase, albumin, vimentin, and nestin. The HPC cultures, however, did not express other genes associated with differentiated pancreatic exocrine, duct, or endocrine cells. A number of transcription factors involved in endocrine cell development including Beta 2, Islet-1, Nkx6.1, Pax4, and Pax6 were expressed at variable levels in HPC cultures. In contrast, pancreatic duodenal homeobox factor 1 (Pdx-1) expression was extremely low and at times undetectable. Overexpression of Pdx-1 in HPC cultures stimulated somatostatin, glucagon, and carbonic anhydrase expression but had no effect on insulin gene expression. HPC cultures could form 3-dimensional islet-like cell aggregates, and this was associated with expression of somatostatin and glucagon but not insulin. Cultivation of HPCs in a differentiation medium supplemented with nicotinamide, exendin-4, and/or LY294002, an inhibitor of phosphatidylinositol-3 kinase, stimulated expression of insulin mRNA and protein. CONCLUSION: These data support the use of intracellular redox modulation for the enrichment of pancreatic stem/progenitor cells capable of self-renewal and endocrine differentiation.

Differential alterations in sympathetic neurotransmission in mesenteric arteries and veins in DOCA-salt hypertensive rats.

Sympathetic control of arteries and veins may be altered in hypertension. To test this hypothesis, constrictions of mesenteric arteries and veins caused by nerve stimulation and by norepinephrine (NE) and ATP were studied in vitro in tissues from deoxycorticosterone acetate (DOCA)-salt hypertensive and sham normotensive rats. In DOCA-salt arteries, the maximum neurogenic response was greater than that in sham arteries. The P2 receptor antagonist, pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS, 10 microM), greatly reduced neurogenic responses in sham but not DOCA-salt arteries. The alpha1-adrenergic receptor antagonist, prazosin (0.1 microM), inhibited responses in DOCA-salt but not sham arteries. Concentration-response curves for norepinephrine and ATP were similar in sham and DOCA-salt arteries, indicating that reactivity to sympathetic vasoconstrictor transmitters was not changed in DOCA-salt arteries. Neurogenic constrictions in sham and DOCA-salt veins were similar in amplitude, and they were completely blocked by prazosin. However, concentration-response curves for norepinephrine in DOCA-salt veins were right-shifted compared to those in sham veins. Cocaine (10 microM) and corticosterone (10 microM) caused a leftward shift in norepinephrine concentration-response curves in DOCA-salt but not sham veins. Norepinephrine content was decreased in DOCA-salt arteries and veins, and there was an increased norepinephrine transporter (NET) level in DOCA-salt veins. These data indicate that, in DOCA-salt hypertension, there is an increased norepinephrine release from sympathetic nerves associated with mesenteric arteries and veins. In arteries, this results in an increase in the amplitude of neurogenic constrictions. In veins, increased norepinephrine release maintains neurogenic constrictions in the presence of increased NET levels.