Serum lutein is a promising biomarker for type 2 diabetes mellitus and diabetic kidney disease in the elderly.

OBJECTIVE: To investigate the relationship between serum lutein and type 2 diabetes mellitus (T2DM) and diabetic kidney disease (DKD) in elderly individuals. METHODS: A total of 60 T2DM patients over 60 years were subgrouped into a DKD group and a non-DKD group according to their urinary microalbumin-to-creatinine ratio (UACR), while 30 age-matched non-T2DM patients were recruited in the control group. Baseline characteristics, laboratory examination results, and serum lutein levels were compared, and their correlations were analyzed. Receiver operating characteristic (ROC) curves were plotted to identify the diagnostic potential of lutein in T2DM and DKD. RESULTS: The lutein level in the T2DM group was significantly lower than that in the control group and was also significantly lower in the DKD group than in the non-DKD group (p < 0.001). Lutein levels were negatively correlated with body mass index, glycosylated hemoglobin, fasting blood glucose, triglyceride, and UACR and positively correlated with high-density lipoprotein cholesterol (p < 0.05). T2DM patients were divided into four groups according to the quartile of their lutein level. The proportion of T2DM and DKD gradually decreased with increasing lutein levels (p < 0.001). The area under the ROC curve of serum lutein in diagnosing T2DM and DKD was 0.880 and 0.779, respectively, with corresponding cut-off values of 0.433 µmol/L and 0.197 µmol/L (p < 0.001). CONCLUSION: The serum level of lutein is negatively correlated with the incidence of T2DM and DKD in the elderly and can serve as a diagnostic marker for T2DM and DKD.

Peptidome analysis reveals the involvement of endogenous peptides in mouse pancreatic dysfunction with aging.

Type 2 diabetes (T2D) is a glucose regulation disorder that has significantly enhanced mortality and the global disease burden. The prevalence of T2D has increased worldwide and is higher in the elderly. The function of pancreatic islets decreases with age, which is one important reason for the occurrence of diabetes in the elderly. Recently, peptidome analysis has attracted attention. However, the role of age-related peptides in pancreatic dysfunction has not been investigated extensively. Here, we conducted a comparison of endogenous peptides between pancreas from adult and aging mice by liquid chromatography tandem mass spectrometry (LC-MS/MS). A total of 2,089 peptides originating from 1,280 protein precursors were identified, of which 232 were upregulated and 183 were downregulated in the aging mice (fold change ≥ 2 and p < 0.05), suggesting that the expression of pancreatic peptides in mice varied with age. The molecular weight of most peptides was <3.0 kDa, and the isoelectric point distribution had a bimodal characteristic. Further analysis of cleavage site patterns indicated that proteases cleaved pancreatic proteins according to their rules. Moreover, Gene Ontology and pathway analyses showed that the differentially expressed peptides potentially had specific effects on pancreatic dysfunction. Some differential peptides were located within the domains of precursor proteins that were closely associated with the development of diabetes. We believe that our research may advance the current understanding of pancreas-derived peptides and that certain peptides may be involved in the etiology of diabetes.

Expression characteristics of long noncoding RNA uc.322 and its effects on pancreatic islet function.

Increasing evidence indicates that long noncoding RNAs (lncRNAs) perform special biological functions by regulating gene expression through multiple pathways and molecular mechanisms. The aim of this study was to explore the expression characteristics of lncRNA uc.322 in pancreatic islet cells and its effects on the secretion function of islet cells. Bioinformatics analysis was used to detect the lncRNA uc.322 sequence, location, and structural features. Expression of lncRNA uc.322 in different tissues was detected by quantitative polymerase chain reaction analyses. Quantitative polymerase chain reaction, Western blot analysis, adenosine triphosphate determination, glucose-stimulated insulin secretion, and enzyme-linked immunosorbent assay were used to evaluate the effects of lncRNA uc.322 on insulin secretion. The results showed that the full-length of lncRNA uc.322 is 224 bp and that it is highly conserved in various species. Bioinformatics analysis revealed that lncRNA uc.322 is located on chr7:122893196-122893419 (GRCH37/hg19) within the SRY-related HMG-box 6 gene exon region. Compared with other tissues, lncRNA uc.322 is highly expressed in pancreatic tissue. Upregulation of lncRNA uc.322 expression increases the insulin transcription factors pancreatic and duodenal homeobox 1 and Forkhead box O1 expression, promotes insulin secretion in the extracellular fluid of Min6 cells, and increases the adenosine triphosphate concentration. On the other hand, knockdown of lncRNA uc.322 has opposite effects on Min6 cells. Overall, this study showed that upregulation of lncRNA uc.322 in islet ß-cells can increase the expression of insulin transcription factors and promote insulin secretion, and it may be a new therapeutic target for diabetes.

GLP-1RA promotes brown adipogenesis of C3H10T1/2 mesenchymal stem cells via the PI3K-AKT-mTOR signaling pathway.

OBJECTIVE: In this study, we investigated whether the GLP-1RA, liraglutide, affected differentiation of C3H10T1/2 mesenchymal stem cells (MSCs) to mature brown adipocytes and involvement of PI3K/AKT/mTOR signaling pathway in this process. METHODS: C3H10T1/2 MSCs were induced to differentiate into brown adipocytes and treated with liraglutide (10 nM and 100 nM) for 0, 2, 4, 6 and 8 days with or without PI3K inhibitor LY294002. Oil red O staining was used for lipid droplet staining and cell proliferation was determined by cell counts. Quantitative realtime PCR was employed to determine the expression of adipogenic and mitochondrial genes, mitochondrial DNA (mtDNA). Western blot analyses were used for quantification of protein levels in PI3K/AKT/mTOR signaling pathway. RESULTS: Liraglutide increased proliferation of C3H10T1/2 MSCs and formation of multilocular lipid droplets during differentiation. Adipogenic and mitochondrial genes, mtDNA were promoted by liraglutide. Moreover, liraglutide treatment increased the levels of phosphorylated AKT and mTOR. LY294002 not only attenuated differentiation of C3H10T1/2 MSCs into brown adipocytes, but also reduced phosphorylated AKT and mTOR levels. However, co-treatment with liraglutide and LY294002 decreased the expression of adipogenic and mitochondrial genes, mtDNA, and phosphorylated AKT and mTOR levels compared to C3H10T1/2 MSCs treated with liraglutide 100 nM. CONCLUSION: GLP-1RA promotes brown adipogenesis of C3H10T1/2 mesenchymal stem cells, and PI3K/AKT/mTOR signaling pathway is involved in GLP-1RA-mediated promotion of differentiation.

Age-related change in thyroid-stimulating hormone: a cross-sectional study in healthy euthyroid population.

This cross-sectional study aimed to examine changes in thyroid-stimulating hormone (TSH) concentration over age in China and investigate relationship between TSH and risk factors for cardiovascular disease (CVD) among euthyroid subjects. TSH, free triiodothyronine (FT3), free thyroxine (FT4), blood lipid, and glucose were measured. 7,693 individuals were subdivided into different age groups. Associations between TSH and CVD risk factors [age, body mass index (BMI), systolic and diastolic blood pressure, total cholesterol (TC), triglycerides, low density lipoprotein-cholesterol (LDL-C) and high density lipoprotein-cholesterol (HDL-C) and fasting plasma glucose (FPG)] were evaluated with Pearson correlation analysis. Results showed that 2.5th percentile for TSH was consistent across age groups, whereas 97.5th percentile increased in subjects older than 40 years with upper limit being 6.83 mIU/L in subjects aged 60-69 years and 8.07 mIU/L in those older than 70 years. The age-specific upper limits reclassification rate was higher in all age bands as compared to the common cut-off value. TSH was positively associated with age, SBP, DBP, TC and LDL-C and negatively with FT3 and FT4. Serum TSH within new reference range had a linear correlation with SBP, TC and LDL-C in subjects aged <60 years. There were no significant differences in BMI, blood pressure, lipid profile or FPG among subjects 60-69 and older than or equal to 70 years. Elevated TSH within new reference range is associated with risk factors for CVD in subjects aged <60 years. Thus, there might be age-related difference in the relationship between CVD risk factors and elevated serum TSH.

A Risk Assessment Tool for Identifying Osteoporosis in Older Women with Type 2 Diabetes Mellitus.

PURPOSE: To develop a simple and clinically useful assessment tool for osteoporosis in older women with type 2 diabetes mellitus (T2DM). METHODS: A total of 601 women over 60 years of age with T2DM were enrolled in this study. The levels of serum sex hormones and bone metabolism markers were compared between the osteoporosis and non-osteoporosis groups. The least absolute shrinkage and selection operator regularization (LASSO) model was applied to generate a risk assessment tool. The risk score formula was evaluated using receiver operating characteristic analysis and the relationship between the risk score and the bone mineral density (BMD) and T-value were investigated. RESULTS: Serum sex hormone-binding globulin (SHBG), cross-linked C-telopeptide of type 1 collagen (CTX), and osteocalcin (OC) were significantly higher in the osteoporosis group. After adjustment for age and body mass index (BMI), SHBG was found to be correlated with the T-value or BMD. Then, a risk score was specifically generated with age, BMI, SHBG, and CTX using the LASSO model. The risk score was significantly negatively correlated with the T-value and BMD of the lumbar spine, femoral neck, and total hip (all P<0.05). CONCLUSION: A risk score using age, BMI, SHBG, and CTX performs well for identifying osteoporosis in older women with T2DM.

TNF-alpha upregulates expression of BMP-2 and BMP-3 genes in the rat dental follicle--implications for tooth eruption.

The dental follicle appears to regulate both the alveolar bone resorption and bone formation needed for tooth eruption. Tumor necrosis factor-alpha (TNF-alpha) gene expression is maximally upregulated at postnatal day 9 in the rat dental follicle of the first mandibular molar, a time that correlates with rapid bone growth at the base of the tooth crypt, as well as a minor burst of osteoclastogenesis. TNF-alpha expression is correlated with the expression of bone morphogenetic protein-2 (BMP-2), a molecule expressed in the dental follicle that can promote bone formation. Because BMP-2 signaling may be augmented by bone morphogenetic protein-3 (BMP-3), our objective in this study was to determine if the dental follicle expresses BMP-3 and if TNF-alpha stimulates the dental follicle cells to express BMP-2 and BMP-3. Dental follicles were collected from different postnatal ages of rat pups. Dental follicle cells were incubated with TNF-alpha to study its dosage and time-course effects on gene expression of BMP-2 and BMP-3, as determined by real-time RT-PCR. Next, immunostaining was conducted to confirm if the protein was synthesized and ELISA of the conditioned medium was conducted to determine if BMP-2 was secreted. We found that BMP-3 expression is correlated with the expression of TNF-alpha in the dental follicle and TNF-alpha significantly increased BMP-2 and BMP-3 expression in vitro. Immunostaining and ELISA showed that BMP-2 and BMP-3 were synthesized and secreted. This study suggests that TNF-alpha can upregulate the expression of bone formation genes that may be needed for tooth eruption.

Differences in early-phase insulin secretion and glucose disposition index between aged and middle-aged patients with newly diagnosed type 2 diabetes.

AIM: This cross-sectional study aimed to investigate the differences in ß-cell function and insulin sensitivity between newly diagnosed aged and middle-aged type 2 diabetes mellitus (T2DM) patients. METHODS: A total of 206 newly diagnosed T2DM patients aged ≥60 years (A-DM group) and 206 newly diagnosed sex- and glycated hemoglobin-matched T2DM patients aged <60 years (MA-DM group) were recruited. All patients underwent the 75-g oral glucose tolerance test. Plasma glucose, lipid profiles, liver and renal function, glycated hemoglobin, and insulin were measured. Homeostasis model assessment for insulin resistance, quantitative insulin sensitivity check index, area under the curve of glucose during 0-30 min (GluAUC30) × area under the curve of insulin during 0-30 min (InsAUC30) and ß-cell function indexes were calculated. RESULTS: The mean age of the total 412 patients (356 men and 56 women) was 59.76 ± 13.32 years. There were no significant differences in GluAUC120 between the two groups (106.89 ± 27.70 in A-DM vs 108.32 ± 27.58 in MA-DM; P = 0.6), but ΔI30/ΔG30, InsAUC30 and GluAUC30 × InsAUC30 levels were significantly higher in the A-DM group than in the MA-DM group (3.55 ± 4.54 vs 2.53 ± 3.83; P = 0.014, and 39.19 ± 32.19 vs 32.71 ± 28.81; P = 0.032, 675.05 ± 475.60 vs 584.56 ± 450.23; P = 0.048, respectively). The glucose disposition index (GDI) of the A-DM group was statistically higher than that of the MA-DM group (0.38 ± 0.40 vs 0.30 ± 0.35; P = 0.018). Age was positively associated with ΔI30/ΔG30 (r = 0.117; P = 0.017) and GDI (r = 0.147; P = 0.003), but had no correlation with InsAUC30, InsAUC120 or GluAUC30 × InsAUC30. After multiple adjustments for confounders, including sex, body mass index, glycated hemoglobin, triglyceride, total cholesterol and high-density lipoprotein cholesterol, age was positively associated with ΔI30/ΔG30, InsAUC30, InsAUC120, GluAUC30 × InsAUC30 and GDI. CONCLUSIONS: Aged patients have relatively higher early-phase insulin secretion and GDI than middle-aged patients in newly diagnosed T2DM. Geriatr Gerontol Int 2020; ••: ••-••.

An Integrated Analysis of mRNA and lncRNA Expression Profiles Indicates Their Potential Contribution to Brown Fat Dysfunction With Aging.

Brown adipose tissue (BAT) can convert fatty acids and glucose into heat, exhibiting the potential to combat obesity and diabetes. The mass and activity of BAT gradually diminishes with aging. As a newly found regulator of gene expression, long non-coding RNAs (lncRNAs) exhibit a wide range of functions in life processes. However, whether long non-coding RNA (lncRNA) involves in BAT dysfunction with aging is still unclear. Here, using RNA-sequencing technology, we identified 3237 messenger RNAs (mRNAs) and 1312 lncRNAs as differentially expressed in BAT of 10-months-old mice compared with 6- to 8-week-old. The protein-protein interaction network and k-score analysis revealed that the core mRNAs were associated with two important aging-related pathways, including cell cycle and p53 signaling pathway. Gene set enrichment analysis indicated that these mRNAs might participate in lipid metabolism and brown fat dysfunction. Functional enrichment analyses demonstrated that dysregulated lncRNAs were associated with mitochondria, regulation of cellular senescence, cell cycle, metabolic and p53 signaling pathways. Moreover, we revealed that two lncRNAs (NONMMUT024512 and n281160) may involve in the regulation of their adjacent gene peroxisome proliferator-activated receptor alpha (Pparα), a thermogenesis regulator. Collectively, these results lay a foundation for extensive studies on the role of lncRNAs in age-related thermogenic degradation.

Chronological gene expression of parathyroid hormone-related protein (PTHrP) in the stellate reticulum of the rat: implications for tooth eruption.

OBJECTIVE: Tooth eruption is a localized event that requires the expression of certain molecules at precise times to regulate bone resorption and bone formation. Parathyroid hormone-related protein (PTHrP) may be one of those molecules. Although PTHrP is produced in the stellate reticulum (SR) of the tooth and exerts its effect on the adjacent dental follicle, its expression pattern in the SR is unknown. Thus, it was the objectives of this study to determine the chronology of expression of PTHrP, and then to determine its effect on vascular endothelial growth factor (VEGF) expression for osteoclastogenesis and on bone morphogenetic protein-2 (BMP-2) for bone growth. DESIGN: Laser capture microdissection and RT-PCR were used to determine the chronological expression of PTHrP in vivo. In vitro, dental follicle cells were incubated with PTHrP and RT-PCR was conducted to determine its effect on VEGF and BMP-2 gene expression. RESULTS: PTHrP was maximally expressed at day 7 postnatally in the SR with the level of expression still high at day 9. In vitro, PTHrP upregulated VEGF120 and VEGF164 expression after 4h of incubation with a maximum effect at 6h. PTHrP upregulated BMP-2 gene expression with a maximal effect at 2h. CONCLUSIONS: Because the secondary burst of osteoclastogenesis needed for eruption occurs around day 10, it is possible that PTHrP is stimulating this osteoclastogenesis by upregulating VEGF. Concurrently, the upregulation of BMP-2 by PTHrP may stimulate bone growth at the base of the bony crypt to promote eruption.

Non-alcoholic fatty liver disease is an influencing factor for the association of SHBG with metabolic syndrome in diabetes patients.

Metabolic syndrome (MS) and non-alcoholic fatty liver disease (NAFLD) have been identified as risk factors affecting serum sex hormone binding globulin (SHBG) levels. We conducted this cross-sectional study to delineate whether MS or NAFLD has more impact on circulating SHBG levels in type 2 diabetes (T2D) patients. Anthropometric and biochemical parameters including serums SHBG, testosterone (TT), liver enzymes, lipids, insulin, C-peptide and plasma glucose were measured. Regardless of the MS status, SHBG level was significantly lower in NAFLD patients than in non-NAFLD patients (P < 0.001). In the multiple linear regression analysis, lower serum SHBG level was strongly correlated with a higher incidence of NAFLD, but not MS components. In logistic regression analyses, after adjusted for age, sex, duration of diabetes, smoking status, and alcohol use, the ORs and 95%CI for presence of MS was 2.26 (95%CI 1.91-2.68) and for presence of NAFLD was 6.36 (95%CI 4.87-8.31) with per one SD decrease in serum SHBG (both P < 0.001). In conclusion, lower serum SHBG is associated with a higher prevalence of NAFLD, compared with MS and other metabolic disorders, in T2D patients. NAFLD might be an important influencing factor for the association of circulating SHBG with MS in T2D patients.

Effect of vascular endothelial growth factor on RANK gene expression in osteoclast precursors and on osteoclastogenesis.

OBJECTIVE: The aim of this study was to determine if vascular endothelial growth factor (VEGF) can upregulate the gene expression of receptor activator of nuclear factor kappa B (RANK) in osteoclast precursors, as does CSF-1. A secondary aim was to determine if VEGF can promote osteoclastogenesis in vitro comparable to CSF-1. DESIGN: Osteoclast precursors (mononuclear cells) were incubated with different concentrations of VEGF, CSF-1, or a combination of the two, and the gene expression of RANK was determined by RT-PCR. A TRAP assay also was conducted to determine their effect on osteoclastogenesis. An Alamar blue assay was done to analyse the effect of the molecules on proliferation of the osteoclast precursors. RESULTS: VEGF upregulated RANK expression in osteoclast precursors as effectively as CSF-1. VEGF did not promote osteoclastogenesis, as did CSF-1. A combination of the two did. CSF-1 enhanced proliferation of the osteoclast precursors but VEGF did not. However, VEGF in combination with CSF-1 did increase proliferation. CONCLUSIONS: At the time of the secondary burst of osteoclastogenesis prior to tooth eruption, VEGF expression in the dental follicle is high but the expression of CSF-1 is low. This study demonstrates that VEGF can fully substitute for CSF-1 to upregulate the RANK expression in osteoclast precursors that is needed for osteoclastogenesis. However, VEGF alone neither can promote osteoclastogenesis nor stimulate proliferation of the osteoclast precursors in vitro. For proliferation and osteoclastogenesis, a low dose of CSF-1 in combination with VEGF is needed.

SGK1 inhibitor reverses hyperglycemia partly through decreasing glucose absorption.

This study investigates the effectiveness and mechanisms of a serum- and glucocorticoid-inducible kinase 1 (SGK1) inhibitor in counteracting hyperglycemia. In an in vivo experiment, we demonstrated that after an 8-week treatment with an SGK1 inhibitor, the fasting blood glucose and HbA1c level significantly decreased in db/db mice. RT-PCR and western blot analyses revealed that intestinal SGK1 and sodium glucose co-transporter 1 (SGLT1) expression were enhanced in db/db mice. Treatment with an SGK1 inhibitor decreased excessive SGLT1 expression in the intestine of db/db mice. In vitro experiments with intestinal IEC-6 cells showed that the co-administration of an SGK1 inhibitor partly reversed the SGLT1 expression and glucose absorption that were induced by dexamethasone. In conclusion, this study revealed that the favorable effect of an SGK1 inhibitor on hyperglycemia is partly due to decreased glucose absorption through SGLT1 in the small intestine. These data collectively suggest that SGK1 may be a potent target for the treatment of diabetes and other metabolic disorders.

SGK1 is regulated by metabolic-related factors in 3T3-L1 adipocytes and overexpressed in the adipose tissue of subjects with obesity and diabetes.

AIMS: The present study aimed to investigate the pathophysiological role of SGK1 in the development of metabolic syndrome by investigating the expression and regulation of serum and glucocorticoid-inducible kinase 1 (SGK1) in adipose tissues in obesity and diabetes. METHODS: SGK1 expression in adipose tissue was investigated using reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry. SGK1 regulation in differentiated 3T3-L1 adipocytes by metabolic-related factors was assessed using Northern blot analysis. Humans with obesity and type 2 diabetes and KKAy and db/db mice were used to evaluate SGK1 expression in the adipose tissue of subjects with obesity and diabetes using quantitative real-time PCR and Western blot analysis. RESULTS: SGK1 was expressed in white adipose tissue as shown by mRNA and protein levels. Aldosterone and glucocorticoids stimulated SGK1 expression in a time- and dose-dependent manner, whereas PPAR-γ agonists inhibited SGK1 expression in differentiated 3T3-L1 adipocytes. Furthermore, SGK1 mRNA and protein were overexpressed in the adipose tissue of mice and humans with obesity and type 2 diabetes. CONCLUSION: Aldosterone, glucocorticoids and other factors contribute to excessive SGK1 expression in adipose tissue. This excessive SGK1 expression may be related to adipose tissue dysfunction, which may contribute to the development of obesity, diabetes and metabolic syndrome.

Identification of the PGRMC1 protein complex as the putative sigma-2 receptor binding site.

The sigma-2 receptor, whose gene remains to be cloned, has been validated as a biomarker for tumour cell proliferation. Here we report the use of a novel photoaffinity probe, WC-21, to identify the sigma-2 receptor-binding site. WC-21, a sigma-2 ligand containing both a photoactive azide moiety and a fluorescein isothiocyanate group, irreversibly labels sigma-2 receptors in rat liver; the membrane-bound protein was identified as PGRMC1 (progesterone receptor membrane component 1). Immunocytochemistry reveals that both PGRMC1 and SW120, a fluorescent sigma-2 receptor ligand, colocalize with molecular markers of the endoplasmic reticulum and mitochondria in HeLa cells. Overexpression and knockdown of the PGRMC1 protein results in an increase and a decrease in binding of a sigma-2 selective radioligand, respectively. The identification of the putative sigma-2 receptor-binding site as PGRMC1 should stimulate the development of unique imaging agents and cancer therapeutics that target the sigma-2 receptor/PGRMC1 complex.

Evaluation of 5-ethynyl-2'-deoxyuridine staining as a sensitive and reliable method for studying cell proliferation in the adult nervous system.

Recently, a novel method for detection of DNA synthesis has been developed based on the incorporation of 5-ethynyl-2'-deoxyuridine (EdU), a thymidine analogue, into cellular DNA and the subsequent reaction of EdU with a fluorescent azide in a copper-catalyzed [3+2] cycloaddition ("Click" reaction). In the present study, we evaluated this method for studying cell proliferation in the adult central nervous system in comparison with the "gold standard" method of 5-bromo-2'-deoxyuridine (BrdU) staining using two behavioral paradigms, voluntary exercise and restraint stress. Our data demonstrate that the number of EdU-positive cells in the dentate gyrus of the hippocampus (DG) slightly increased in an EdU dose-dependent manner in both the control and voluntary exercise (running) mouse groups. The number of EdU-labeled cells was comparable to the number of BrdU-labeled cells in both the control and running mice. Furthermore, EdU and BrdU co-localized to the same cells within the DG. Voluntary exercise significantly increased the number of EdU- and BrdU-positive cells in the DG. In contrast, restraint stress significantly decreased the number of EdU-positive cells. The EdU-positive cells differentiated into mature neurons. EdU staining is compatible with immunohistochemical staining of other antigens. Moreover, our data demonstrated EdU staining can be combined with BrdU staining, providing a valuable tool of double labeling DNA synthesis, e.g., for tracking the two populations of neurons generated at different time points. In conclusion, our results suggest that EdU staining is a fast, sensitive and reproducible method to study cell proliferation in the central nervous system.