Satinath Mukhopadhyay

Fetuin-A acts as an endogenous ligand of TLR4 to promote lipid-induced insulin resistance.

Toll-like receptor 4 (TLR4) has a key role in innate immunity by activating an inflammatory signaling pathway. Free fatty acids (FFAs) stimulate adipose tissue inflammation through the TLR4 pathway, resulting in insulin resistance. However, current evidence suggests that FFAs do not directly bind to TLR4, but an endogenous ligand for TLR4 remains to be identified. Here we show that fetuin-A (FetA) could be this endogenous ligand and that it has a crucial role in regulating insulin sensitivity via Tlr4 signaling in mice. FetA (officially known as Ahsg) knockdown in mice with insulin resistance caused by a high-fat diet (HFD) resulted in downregulation of Tlr4-mediated inflammatory signaling in adipose tissue, whereas selective administration of FetA induced inflammatory signaling and insulin resistance. FFA-induced proinflammatory cytokine expression in adipocytes occurred only in the presence of both FetA and Tlr4; removing either of them prevented FFA-induced insulin resistance. We further found that FetA, through its terminal galactoside moiety, directly binds the residues of Leu100–Gly123 and Thr493–Thr516 in Tlr4. FFAs did not produce insulin resistance in adipocytes with mutated Tlr4 or galactoside-cleaved FetA. Taken together, our results suggest that FetA fulfills the requirement of an endogenous ligand for TLR4 through which lipids induce insulin resistance. This may position FetA as a new therapeutic target for managing insulin resistance and type 2 diabetes.

NF-κB mediates lipid-induced fetuin-A expression in hepatocytes that impairs adipocyte function effecting insulin resistance

Fetuin-A, a hepatic secretory protein, has recently been implicated in insulin resistance and Type 2 diabetes. It is an endogenous inhibitor of insulin receptor tyrosine kinase. However, regulation of fetuin-A synthesis in relation to insulin resistance is unclear. In the present paper, we report that both non-esterified (free) fatty acids and fetuin-A coexist at high levels in the serum of db/db mice, indicating an association between them. For an in-depth study, we incubated palmitate with HepG2 cells and rat primary hepatocytes, and found enhanced fetuin-A secretion to more than 4-fold over the control. Interestingly, cell lysates from these incubations showed overexpression and activity of NF-kappaB (nuclear factor kappaB). In NF-kappaB-knockout HepG2 cells, palmitate failed to increase fetuin-A secretion, whereas forced expression of NF-kappaB released fetuin-A massively in the absence of palmitate. Moreover, palmitate stimulated NF-kappaB binding to the fetuin-A promoter resulting in increased reporter activity. These results suggest NF-kappaB to be the mediator of the palmitate effect. Palmitate-induced robust expression of fetuin-A indicates the occurrence of additional targets, and we found that fetuin-A severely impaired adipocyte function leading to insulin resistance. Our results reveal a new dimension of lipid-induced insulin resistance and open another contemporary target for therapeutic intervention in Type 2 diabetes.

Adipocyte Fetuin-A Contributes to Macrophage Migration into Adipose Tissue and Polarization of Macrophages

Background: Obesity-induced inflammation is characterized by macrophage migration and polarization; signaling regulation therein remains poorly understood. Results: Lipid-induced fetuin-A from adipose tissue acts as chemoattractant for macrophage migration and also polarizes adipose tissue M2 macrophages to proinflammatory M1 subtype. Conclusion: Adipocyte fetuin-A is a novel signaling molecule in lipid-induced tissue inflammation. Significance: These findings have revealed an unseen area of inflammation. Macrophage infiltration into adipose tissue during obesity and their phenotypic conversion from anti-inflammatory M2 to proinflammatory M1 subtype significantly contributes to develop a link between inflammation and insulin resistance; signaling molecule(s) for these events, however, remains poorly understood. We demonstrate here that excess lipid in the adipose tissue environment may trigger one such signal. Adipose tissue from obese diabetic db/db mice, high fat diet-fed mice, and obese diabetic patients showed significantly elevated fetuin-A (FetA) levels in respect to their controls; partially hepatectomized high fat diet mice did not show noticeable alteration, indicating adipose tissue to be the source of this alteration. In adipocytes, fatty acid induces FetA gene and protein expressions, resulting in its copious release. We found that FetA could act as a chemoattractant for macrophages. To simulate lipid-induced inflammatory conditions when proinflammatory adipose tissue and macrophages create a niche of an altered microenvironment, we set up a transculture system of macrophages and adipocytes; the addition of fatty acid to adipocytes released FetA into the medium, which polarized M2 macrophages to M1. This was further confirmed by direct FetA addition to macrophages. Taken together, lipid-induced FetA from adipocytes is an efficient chemokine for macrophage migration and polarization. These findings open a new dimension for understanding obesity-induced inflammation.

Enhanced ROS production and oxidative damage in subcutaneous white adipose tissue mitochondria in obese and type 2 diabetes subjects

Oxidative stress in the insulin target tissues has been implicated in the pathophysiology of type 2 diabetes. The study has examined the oxidative stress parameters in the mitochondria of subcutaneous white adipose tissue from obese and non-obese subjects with or without type 2 diabetes. An accumulation of protein carbonyls, fluorescent lipid peroxidation products, and malondialdehyde occurs in the adipose tissue mitochondria of obese type 2 diabetic, non-diabetic obese, and non-obese diabetic subjects with the maximum increase noticed in the obese type 2 diabetes patients and the minimum in non-obese type 2 diabetics. The mitochondria from obese type 2 diabetics, non-diabetic obese, and non-obese type 2 diabetics also produce significantly more reactive oxygen species (ROS) in vitro compared to those of controls, and apparently the mitochondrial ROS production rate in each group is proportional to the respective load of oxidative damage markers. Likewise, the mitochondrial antioxidant enzymes like superoxide dismutase and glutathione peroxidase show decreased activities most markedly in obese type 2 diabetes subjects and to a lesser degree in non-obese type 2 diabetes or non-diabetic obese subjects in comparison to control. The results imply that mitochondrial dysfunction with enhanced ROS production may contribute to the metabolic abnormality of adipose tissue in obesity and diabetes.

Mitochondrial bioenergetics is not impaired in nonobese subjects with type 2 diabetes mellitus

Although mitochondrial dysfunction has been well documented in obese people with type 2 diabetes mellitus, its presence or absence in nonobese subjects with type 2 diabetes mellitus has not been well studied so far. The aim of the present study was to assess the status of mitochondrial oxidative phosphorylation in subcutaneous adipose tissue of nonobese type 2 diabetes mellitus subjects in comparison to control, obese nondiabetic, and obese type 2 diabetes mellitus subjects. Mitochondria were isolated from subcutaneous white adipose tissue obtained from the abdominal region of control, obese nondiabetic, nonobese type 2 diabetes mellitus, and obese type 2 diabetes mellitus subjects. The activities of complex I, I to III, II to III, and IV; transmembrane potential; and inorganic phosphate utilization of mitochondria from different groups were measured. Mitochondrial transmembrane potential, inorganic phosphate utilization, and the activities of respiratory chain complexes were significantly reduced in obese nondiabetic and obese type 2 diabetes mellitus patients compared with those in control subjects. No detectable change in mitochondrial functional parameters was observed in case of nonobese type 2 diabetes mellitus subjects compared with control subjects. Furthermore, a significant difference was noticed in mitochondrial phosphate utilization and activities of respiratory complexes, for example, I, I to III, and II to III, between obese type 2 diabetes mellitus subjects and obese nondiabetic subjects. Obesity modulates mitochondrial dysfunction associated with type 2 diabetes mellitus.

Percutaneous ethanol ablation in managing predominantly cystic thyroid nodules: An eastern India perspective

Aims: Percutaneous aspiration and ethanol injection (PEI) is effective in managing predominantly cystic (>50% cystic) thyroid nodules with efficacy ranging from 38-85%. We aimed to evaluate efficacy, safety, and factors determining outcomes of PEI in managing simple cystic (purely cystic) vs. complex cystic (having both cystic and solid components) thyroid nodules. Materials and Methods: Predominantly cystic thyroid nodules, without any ultrasonography and/or fine needle aspiration, evidence of malignancy were aspirated ultrasonography guided. Sterile 100% ethanol (50-100% volume aspirated) was injected and reviewed monthly for 3 months and thereafter 3 monthly. Responders were defined as ≥ 50% reduction in nodule volume. Results: Sixty-five patients out of 152 considered underwent PEI. Sixty patients [simple cystic (42) and complex cystic (18)] with mean follow-up of 12.3 ± 2.88 months were analyzed. Response rate of PEI was 78.33% [simple cystic (92.86%) and complex cystic (44.44%) nodules; P < 0.001]. Also, 31.67% patients achieved remission at 1st month. And, 46.67% patients achieved remission between 1-6-months follow-up. Kaplan Meier analysis showed significantly improved outcomes in patients with simple cystic nodules (P < 0.001). Cox-regression revealed type of nodule (simple cystic vs. complex cystic) to be predictive of outcome (P = 0.034). Complex cystic nodules were 67.6% less likely to go into remission, compared to simple cystic nodules. Baseline nodule size, aspirate, or volume of ethanol injected did not predict outcome. Conclusions: PEI is safe and should be treatment of choice for simple cystic thyroid nodules. PEI for complex cystic thyroid nodules are associated with lower response, increased recurrence, and need for repeated PEI.

Vitamin D supplementation reduces thyroid peroxidase antibody levels in patients with autoimmune thyroid disease: An open-labeled randomized controlled trial

Background and Aims: Although Vitamin D deficiency has been linked to autoimmune thyroid disorders (AITD), the impact of Vitamin D supplementation on thyroid autoimmunity is not known. This study aimed to evaluate the impact of Vitamin D supplementation on thyroid autoimmunity (thyroid peroxidase antibody [TPO-Ab] titers) in patients with newly diagnosed AITD in a randomized controlled trial. Materials and Methods: One hundred two patients with newly diagnosed AITD (TPO-Ab > 34 kIU/L and/or sonographic evidence of thyroiditis) patients were randomized into Group-1 (intervention group) and Group-2 (control group). Group-1 received cholecalciferol 60,000 IU weekly and calcium 500 mg/day for 8 weeks; Group-2 received calcium 500 mg/day for 8 weeks. Responders were defined as ≥25% fall in TPO-Ab titers. Individuals with at least 3-month follow-up were analyzed. Trial is registered at ctri.nic.in (CTRI/2015/04/005713). Results: Data from 100 AITD patients (68 with thyroid stimulating hormone [TSH] ≤10 mIU/L, 32 with TSH > 10 mIU/L), 93% having Vitamin D insufficiency, were analyzed. TPO-Ab titers were highest among patients in the lowest 25-hydroxyvitamin D quartile (P = 0.084). At 3 months follow-up, there was significant fall in TPO-Ab in Group-1 (−46.73%) as compared to Group-2 (−16.6%) (P = 0.028). Sixty-eight percentage patients in Group-1 were responders compared to 44% in Group-2 (P = 0.015). Kaplan–Meier analysis revealed significantly higher response rate in Group-1 (P = 0.012). Significantly greater reduction in TPO-Ab titers was observed in AITD with TSH ≤ 10 mIU/L compared to TSH > 10 mIU/L. Cox regression revealed Group-1 followed by TPO-Ab and free tetraiodothyronine levels to be a good predictor of response to therapy (P = 0.042, 0.069, and 0.074, respectively). Conclusion: Vitamin D supplementation in AITD may have a beneficial effect on autoimmunity as evidence by significant reductions in TPO-Ab titers.

Challenges and controversies in diagnosis and management of gonadotropin dependent precocious puberty: An Indian perspective

Managing precocious puberty (PP) has been a challenge due to lack of standardized definition, gonadotrophins assay, gonadotrophin stimulation, timings for blood sampling, and parameters for assessing outcomes. This review evaluated available literature to simplify the algorithm for managing gonadotrophin dependent/central PP (CPP), with an Indian perspective. CPP is one of the commonest forms of PP and mimics the normal course of puberty, at an age <8 and 9 years for girls and boys respectively. Basal and post gonadotrophin hormone releasing hormone analog (GnRHa) luteinizing hormone (LH) ≥0.3–0.6 IU/L and ≥4–5 IU/L (30–60 min after GnRH/GnRHa administration) respectively, using modern ultrasensitive automated chemiluminescence assays, can be considered positive for central puberty initiation. Uterine length of >3.5 cm and uterine volume of >1.8 ml are two most specific indicators for true CPP. Therapy is indicated in children with CPP with accelerated bone age, height advancement, or psychosocial stress. Treatment goal is to halt puberty progression to a socially acceptable age, allowing the child to attain optimal height potential. GnRHa is the treatment of choice, with best height outcomes when initiated <6 years age. Treatment is recommended till 11 years age. LH suppression to <3 U/L may be a reasonable target in patients on GnRHa therapy. Medroxyprogesterone acetate holds an important place in managing PP in India, cause of high costs associated with GnRHa therapy. There is an urgent need for clinical trials from India, for establishing Indian cut-off for diagnosis, treatment and follow-up of children with PP.

A Small Insulinomimetic Molecule Also Improves Insulin Sensitivity in Diabetic Mice

Dramatic increase of diabetes over the globe is in tandem with the increase in insulin requirement. This is because destruction and dysfunction of pancreatic β-cells are of common occurrence in both Type1 diabetes and Type2 diabetes, and insulin injection becomes a compulsion. Because of several problems associated with insulin injection, orally active insulin mimetic compounds would be ideal substitute. Here we report a small molecule, a peroxyvanadate compound i.e. DmpzH[VO(O2)2(dmpz)], henceforth referred as dmp, which specifically binds to insulin receptor with considerable affinity (KD-1.17μM) thus activating insulin receptor tyrosine kinase and its downstream signaling molecules resulting increased uptake of [14C] 2 Deoxy-glucose. Oral administration of dmp to streptozotocin treated BALB/c mice lowers blood glucose level and markedly stimulates glucose and fatty acid uptake by skeletal muscle and adipose tissue respectively. In db/db mice, it greatly improves insulin sensitivity through excess expression of PPARγ and its target genes i.e. adiponectin, CD36 and aP2. Study on the underlying mechanism demonstrated that excess expression of Wnt3a decreased PPARγ whereas dmp suppression of Wnt3a gene increased PPARγ expression which subsequently augmented adiponectin. Increased production of adiponectin in db/db mice due to dmp effected lowering of circulatory TG and FFA levels, activates AMPK in skeletal muscle and this stimulates mitochondrial biogenesis and bioenergetics. Decrease of lipid load along with increased mitochondrial activity greatly improves energy homeostasis which has been found to be correlated with the increased insulin sensitivity. The results obtained with dmp, therefore, strongly indicate that dmp could be a potential candidate for insulin replacement therapy.

Fetuin-A downregulates adiponectin through Wnt-PPARγ pathway in lipid induced inflamed adipocyte.

Adiponectin secreted from adipocytes is an anti-diabetic and anti-atherogenic adipokine. Adiponectin level is known to fall significantly in obesity induced type 2 diabetes which worsen insulin sensitivity because of aberrant lipid management. However, underlying mechanism of adiponectin decrease in obese diabetic condition is yet unclear. We report here that lowering of plasma adiponectin coincided with the higher Fetuin A (FetA) level in high fat diet (HFD) induced obese diabetic mice. Knock down of FetA gene (FetAKD) elevated adiponectin level markedly in HFD mice, while reinforcement of FetA into FetAKDHFD mice reduced its level again. These results indicate FetAs involvement in the lowering of adiponectin level in obesity induced diabetic mice. Our findings to understand how FetA could affect adiponectin decrease demonstrated that FetA could enhance Wnt3a expression in the adipocyte of HFD mice. FetA addition to 3T3L1 adipocyte incubation elevated Wnt3a expression in a dose dependent manner. Overexpression of Wnt3a by FetA inhibited PPARγ and adiponectin. FetA failed to reduce PPARγ and adiponectin in Wnt3a gene knocked down 3T3L1` adipocytes. All these suggest that FetA mediate its inhibitory effect on adiponectin through Wnt3a-PPARγ pathway. Inhibition of adiponectin expression through FetA and Wnt3a significantly compromised with the activation of AMPK and its downstream signalling molecules which adversely affected lipid management causing loss of insulin sensitivity. Downregulation of adiponectin in inflamed adipocyte by FetA through the mediation of Wnt3a and PPARγ is a new report.