Preethi Cherian

Higher plasma betatrophin/ANGPTL8 level in Type 2 Diabetes subjects does not correlate with blood glucose or insulin resistance

Betatrophin/ANGPTL8 is a newly identified hormone produced in liver and adipose tissue that has been shown to be induced as a result of insulin resistance and regulates lipid metabolism. Little is known about betatrophin level in humans and its association with T2D and metabolic risk factors. Plasma level of betatrophin was measured by ELISA in 1603 subjects: 1047 non-diabetic and 556 T2D subjects and its associations with metabolic risk factors in both non-diabetic and T2D were also studied. Our data show a significant difference in betatrophin levels between non-diabetic (731.3 (59.5–10625.0) pg/ml) and T2D (1710.5 (197.4–12361.1) p < 0.001. Betatrophin was positively correlated with age, BMI, waist/hip ratio, FBG, HbA1C, HOMA-IR and TG in the non-diabetic subjects. However, no association was observed with BMI, FBG, HbA1C or HOMA-IR in T2D subjects. TC and LDL showed negative association with betatrophin in T2D subjects. Multivariate analysis showed that subjects in the highest tertile of betatrophin had higher odds of having T2D (odd ratio [OR] = 6.15, 95% confidence interval [CI] = (3.15 – 12.01). Our data show strong positive associations between betatrophin and FBG and insulin resistance in non-diabetic subjects. However, correlations with FBG and insulin resistance were diminished in T2D subjects.

Proteomics Analysis of Human Obesity Reveals the Epigenetic Factor HDAC4 as a Potential Target for Obesity

Sedentary lifestyle and excessive energy intake are prominent contributors to obesity; a major risk factors for the development of insulin resistance, type 2 diabetes and cardiovascular diseases. Elucidating the molecular mechanisms underlying these chronic conditions is of relevant importance as it might lead to the identification of novel anti-obesity targets. The purpose of the current study is to investigate differentially expressed proteins between lean and obese subjects through a shot-gun quantitative proteomics approach using peripheral blood mononuclear cells (PBMCs) extracts as well as potential modulation of those proteins by physical exercise. Using this approach, a total of 47 proteins showed at least 1.5 fold change between lean and obese subjects. In obese, the proteomic profiling before and after 3 months of physical exercise showed differential expression of 38 proteins. Thrombospondin 1 (TSP1) was among the proteins that were upregulated in obese subjects and then decreased by physical exercise. Conversely, the histone deacetylase 4 (HDAC4) was downregulated in obese subjects and then induced by physical exercise. The proteomic data was further validated by qRT-PCR, Western blot and immunohistochemistry in both PBMCs and adipose tissue. We also showed that HDAC4 levels correlated positively with maximum oxygen consumption (VO2 Max) but negatively with body mass index, percent body fat, and the inflammatory chemokine RANTES. In functional assays, our data indicated that ectopic expression of HDAC4 significantly impaired TNF-α-dependent activation of NF-κB, establishing thus a link between HDAC4 and regulation of the immune system. Together, the expression pattern of HDAC4 in obese subjects before and after physical exercise, its correlation with various physical, clinical and metabolic parameters along with its inhibitory effect on NF-κB are suggestive of a protective role of HDAC4 against obesity. HDAC4 could therefore represent a potential therapeutic target for the control and management of obesity and presumably insulin resistance.

DNAJB3/HSP-40 cochaperone is downregulated in obese humans and is restored by physical exercise.

Obesity is a major risk factor for a myriad of disorders such as insulin resistance and diabetes. The mechanisms underlying these chronic conditions are complex but low grade inflammation and alteration of the endogenous stress defense system are well established. Previous studies indicated that impairment of HSP-25 and HSP-72 was linked to obesity, insulin resistance and diabetes in humans and animals while their induction was associated with improved clinical outcomes. In an attempt to identify additional components of the heat shock response that may be dysregulated by obesity, we used the RT2-Profiler PCR heat shock array, complemented with RT-PCR and validated by Western blot and immunohistochemistry. Using adipose tissue biopsies and PBMC of non-diabetic lean and obese subjects, we report the downregulation of DNAJB3 cochaperone mRNA and protein in obese that negatively correlated with percent body fat (P = 0.0001), triglycerides (P = 0.035) and the inflammatory chemokines IP-10 and RANTES (P = 0.036 and P = 0.02, respectively). DNAJB positively correlated with maximum oxygen consumption (P = 0.031). Based on the beneficial effect of physical exercise, we investigated its possible impact on DNAJB3 expression and indeed, we found that exercise restored the expression of DNAJB3 in obese subjects with a concomitant decrease of phosphorylated JNK. Using cell lines, DNAJB3 protein was reduced following treatment with palmitate and tunicamycin which is suggestive of the link between the expression of DNAJB3 and the activation of the endoplasmic reticulum stress. DNAJB3 was also shown to coimmunoprecipiate with JNK and IKKβ stress kinases along with HSP-72 and thus, suggesting its potential role in modulating their activities. Taken together, these data suggest that DNAJB3 can potentially play a protective role against obesity.

Circulating ANGPTL8/Betatrophin Is Increased in Obesity and Reduced after Exercise Training

Objective ANGPTL8 is a liver and adipose tissue produced protein that regulates the level of triglyceride in plasma as well as glucose homeostasis. This study was designed to evaluate the level of ANGPTL8 in obese and non-obese subjects before and after exercise training. Methods A total of 82 non-obese and 62 adult obese were enrolled in this study. Subjects underwent a three months of exercise training. Both full length and C-terminal 139–198 form of ANGPTL8 were measured by ELISA. Results Our data show that the full length ANGPTL8 level was increased in obese subjects (1150.04 ± 108.10 pg/mL) compared to non-obese (775.54 ± 46.12) pg/mL (p-Value = 0.002). C-terminal 139–198 form of ANGPTL8 was also increased in obese subjects 0.28 ± 0.04 ng/mL vs 0.20 ± 0.02 ng/mL in non-obese (p-value = 0.058). In obese subjects, the levels of both forms were reduced after three months of exercise training; full length was reduced from 1150.04 ± 108.10 pg/mL to 852.04 ± 51.95 pg/mL (p-Values 0.015) and c-terminal form was reduced from 0.28 ± 0.04 ng/mL to 0.19 ± 0.03 ng/mL (p-Value = 0.058). Interestingly, full length ANGPTL8 was positively associated with fasting blood glucose (FBG) in non-obese (r = 0.317, p-Value = 0.006) and obese subjects (r = 0.346, p-Value = 0.006) C-terminal 139–198 form of ANGPTL8 on the other hand, did not show any correlation in both groups. Conclusion In conclusion, our data demonstrate that ANGPTL8 was increased in obesity and reduced after exercise training supporting the potential therapeutic benefit of reducing ANGPTL8. The various forms of ANGPTL8 associated differently with FBG suggesting that they have different roles in glucose homeostasis.

Physical Exercise Reduces the Expression of RANTES and Its CCR5 Receptor in the Adipose Tissue of Obese Humans

RANTES and its CCR5 receptor trigger inflammation and its progression to insulin resistance in obese. In the present study, we investigated for the first time the effect of physical exercise on the expression of RANTES and CCR5 in obese humans. Fifty-seven adult nondiabetic subjects (17 lean and 40 obese) were enrolled in a 3-month supervised physical exercise. RANTES and CCR5 expressions were measured in PBMCs and subcutaneous adipose tissue before and after exercise. Circulating plasma levels of RANTES were also investigated. There was a significant increase in RANTES and CCR5 expression in the subcutaneous adipose tissue of obese compared to lean. In PBMCs, however, while the levels of RANTES mRNA and protein were comparable between both groups, CCR5 mRNA was downregulated in obese subjects (P < 0.05). Physical exercise significantly reduced the expression of both RANTES and CCR5 (P < 0.05) in the adipose tissue of obese individuals with a concomitant decrease in the levels of the inflammatory markers TNF-α, IL-6, and P-JNK. Circulating RANTES correlated negatively with anti-inflammatory IL-1ra (P = 0.001) and positively with proinflammatory IP-10 and TBARS levels (P < 0.05). Therefore, physical exercise may provide an effective approach for combating the deleterious effects associated with obesity through RANTES signaling in the adipose tissue.

Map kinase phosphatase dusp1 is overexpressed in obese humans and modulated by physical exercise

Chronic low-grade inflammation and dysregulation of the stress defense system are cardinal features of obesity, a major risk factor for the development of insulin resistance and diabetes. Dual-specificity protein phosphatase 1 (DUSP1), known also as MAP kinase phosphatase 1 (MKP1), is implicated in metabolism and energy expenditure. Mice lacking DUSP1 are resistant to high-fat diet-induced obesity. However, the expression of DUSP1 has not been investigated in human obesity. In the current study, we compared the expression pattern of DUSP1 between lean and obese nondiabetic human subjects using subcutaneous adipose tissue (SAT) and peripheral blood mononuclear cells (PBMCs). The levels of DUSP1 mRNA and protein were significantly increased in obese subjects with concomitant decrease in the phosphorylation of p38 MAPK (p-p38 MAPK) and PGC-1α and an increase in the levels of phospho-JNK (p-JNK) and phospho-ERK (p-ERK). Moreover, obese subjects had higher levels of circulating DUSP1 protein that correlated positively with various obesity indicators, triglycerides, glucagon, insulin, leptin, and PAI-1 (P < 0.05) but negatively with V̇O(2max) and high-density lipoprotein (P < 0.05). The observation that DUSP1 was overexpressed in obese subjects prompted us to investigate whether physical exercise could reduce its expression. In this study, we report for the first time that physical exercise significantly attenuated the expression of DUSP1 in both the SAT and PBMCs, with a parallel increase in the expression of PGC-1α and a reduction in the levels of p-JNK and p-ERK along with attenuated inflammatory response. Collectively, our data suggest that DUSP1 upregulation is strongly linked to adiposity and that physical exercise modulates its expression. This gives further evidence that exercise might be useful as a strategy for managing obesity and preventing its associated complications.

Increased ANGPTL3, 4 and ANGPTL8/betatrophin expression levels in obesity and T2D

BackgroundHypertriglyceridemia is associated with increased risk for cardiovascular diseases and type 2 diabetes (T2D). Angiopoietin like proteins particularly 3, 4 and recently 8 are well established regulators of plasma triglyceride level through regulating the activity of lipoprotein lipase. Plasma level and association between ANGPTL3, 4 and 8 is not well established in human subjects. This study was designed to establish the level of these proteins in plasma and adipose tissues and investigate the association between ANGPTL8 with ANGPTL3 and 4 in T2D and non-diabetics subjects.MethodsA total of 235 subjects were enrolled in this study, 144 non-diabetics and 91 T2D. ANGPTL 3, 4 and 8 levels were measured in plasma by ELISA and using real time RT-PCR in adipose tissues.ResultsIn this study, we showed that ANGPTL3, 4 and 8 were higher in T2D subjects. Dividing the non-diabetic subjects according to their BMI showed higher level of ANGPTL3, 4 and 8 in obese subjects compared to non-obese subjects. No significant difference was observed between the T2D subjects. ANGPTL8 was showed positive correlation with ANGPTL3 in the non-diabetic subjects in the non-obese (r = 0.2437, p-Value = 0.0543) and obese subjects (r = 0.418, p-Value = 0.0125). No association was observed in the T2D subjects. On the other hand, ANGPTL4 was positively associated with the obese subjects in both the non-diabetics (r = 0.3322, p-Value = 0.0316) and the obese T2D subjects (r = 0.3161, p-Value = 0.0211).ConclusionIn conclusion, our data shows that ANGPTL3, 4 and 8 are increased in obesity and T2D. ANGPTL8 associates with ANGPTL3 in the non-diabetic subjects while it associated more with ANGPTL4 in the obese and T2D subjects. Taken together, this data highlight the role of these proteins in metabolic diseases and how they interact with each other’s under different physiological and pathophysiological conditions.

Immunohistochemical profiling of the heat shock response in obese non-diabetic subjects revealed impaired expression of heat shock proteins in the adipose tissue

BackgroundObesity is characterized by a chronic low-grade inflammation and altered stress responses in key metabolic tissues. Impairment of heat shock response (HSR) has been already linked to diabetes and insulin resistance as reflected by decrease in heat shock proteins (HSPs) expression. However, the status of HSR in non-diabetic human obese has not yet been elucidated. The aim of the current study was to investigate whether obesity triggers a change in the HSR pattern and the impact of physical exercise on this pattern at protein and mRNA levels.MethodsTwo groups of adult non-diabetic human subjects consisting of lean and obese (n = 47 for each group) were enrolled in this study. The expression pattern of HSP-27, DNAJB3/HSP-40, HSP-60, HSC-70, HSP72, HSP-90 and GRP-94 in the adipose tissue was primarily investigated by immunohistochemistry and then complemented by western blot and qRT-PCR in Peripheral blood mononuclear cells (PBMCs). HSPs expression levels were correlated with various physical, clinical and biochemical parameters. We have also explored the effect of a 3-month moderate physical exercise on the HSPs expression pattern in obese subjects.ResultsObese subjects displayed increased expression of HSP-60, HSC-70, HSP-72, HSP-90 and GRP-94 and lower expression of DNAJB3/HSP-40 (P < 0.05). No differential expression was observed for HSP-27 between the two groups. Higher levels of HSP-72 and GRP-94 proteins correlated positively with the indices of obesity (body mass index and percent body fat) and circulating levels of IFN-gamma-inducible protein 10 (IP-10) and RANTES chemokines. This expression pattern was concomitant with increased inflammatory response in the adipose tissue as monitored by increased levels of Interleukin-6 (IL-6), Tumor necrosis factor-α (TNF-α), and RANTES (P < 0.05). Physical exercise reduced the expression of various HSPs in obese to normal levels observed in lean subjects with a parallel decrease in the endogenous levels of IL-6, TNF-α, and RANTES.ConclusionTaken together, these data indicate that obesity triggers differential regulation of various components of the HSR in non-diabetic subjects and a 3-month physical moderate exercise was sufficient to restore the normal expression of HSPs in the adipose tissue with concomitant attenuation in the inflammatory response.

DNAJB3/HSP-40 cochaperone improves insulin signaling and enhances glucose uptake in vitro through JNK repression.

Heat shock response (HSR) is an essential host-defense mechanism that is dysregulated in obesity-induced insulin resistance and type 2 diabetes (T2D). Our recent data demonstrated that DNAJB3 was downregulated in obese human subjects and showed negative correlation with inflammatory markers. Nevertheless, DNAJB3 expression pattern in diabetic subjects and its mode of action are not yet known. In this study, we showed reduction in DNAJB3 transcript and protein levels in PBMC and subcutaneous adipose tissue of obese T2D compared to obese non-diabetic subjects. Overexpression of DNAJB3 in HEK293 and 3T3-L1 cells reduced JNK, IRS-1 Ser-307 phosphorylation and enhanced Tyr-612 phosphorylation suggesting an improvement in IRS-1 signaling. Furthermore, DNAJB3 mediated the PI3K/AKT pathway activation through increasing AKT and AS160 phosphorylation. AS160 mediates the mobilization of GLUT4 transporter to the cell membrane and thereby improves glucose uptake. Using pre-adipocytes cells we showed that DNAJB3 overexpression caused a significant increase in the glucose uptake, possibly through its phosphorylation of AS160. In summary, our results shed the light on the possible role of DNAJB3 in improving insulin sensitivity and glucose uptake through JNK repression and suggest that DNAJB3 could be a potential target for therapeutic treatment of obesity-induced insulin resistance.

Physical exercise alleviates ER stress in obese humans through reduction in the expression and release of GRP78 chaperone

BACKGROUND AND OBJECTIVES Perturbation of the endoplasmic reticulum (ER) homeostasis has emerged as one of the prominent features of obesity and diabetes. This occurs when the adaptive unfolded protein response (UPR) fails to restore ER function in key metabolic tissues. We previously reported increased inflammation and impaired heat shock response (HSR) in obese human subjects that were restored by physical exercise. Here, we investigated the status of ER stress chaperone; glucose-regulated protein 78 (GRP78) and its downstream UPR pathways in human obese, and their modulation by a supervised 3-month physical exercise. METHODS Subcutaneous adipose tissue (SAT) and blood samples were collected from non-diabetic adult human lean (n=40) and obese (n=40, at baseline and after 3months of physical exercise). Transcriptomic profiling was used as a primary screen to identify differentially expressed genes and it was carried out on SAT samples using the UPR RT(2) Profiler PCR Array. Conventional RT-PCR, immunohistochemistry, immunofluorescence, Western blot and ELISA were used to validate the transcriptomic data. Correlation analyses with the physical, clinical and biochemical outcomes were performed using Pearsons rank correlation coefficient. RESULTS Levels of GRP78 and its three downstream UPR arms; activating transcription factor-6 (ATF6), inositol-requiring enzyme-1α (IRE1α) and protein kinase RNA-like endoplasmic reticulum kinase (PERK) were increased in obese subjects. More interestingly, higher levels of circulating GRP78 protein were found in obese compared to lean subjects which correlated negatively with maximum oxygen uptake (VO2 Max) but positively with high-sensitivity C-reactive protein (hsCRP) and obesity indicators such as BMI, percentage body fat (PBF) and waist circumference. GRP78 increased secretion in obese was further confirmed in vitro using 3T3-L1 preadipocyte cells under ER stress. Finally, we showed that physical exercise significantly attenuated the expression and release of GRP78 with a concomitant reduction in the phosphorylation of IRE1α and eukaryotic initiation factor-2α (eIF2α). CONCLUSION Our results suggest that physical exercise alleviates ER stress in human obese through attenuation of GRP78 signaling network.