Hi everybody. This is Dr. Swatinath Mukhopadhyay. I am professor and head at the Department of Endocrinology and Metabolism at the Institute of Postgraduate Medical Education and Research, Kolkata, India. At the outset, I would like to thank the Endocrine Society for having me for this webinar. I would also like to thank Jessica Brown for curating this program. Now the topic that I am going to walk you through over the next 45 minutes or so is lipid-induced insulin resistance, its molecular mechanisms, and what are the clinical implications. I have no disclosures for this particular presentation. Now if you look at the learning objectives, well, a surfeit of dietary lipids stimulates the production of Fetuin A, a pro-inflammatory hepatokine. Now Fetuin A, by acting as an adapter protein between free fatty acids and Tone-like receptor 4 that are present mainly on the surface of immune cells and adipocytes triggers the pro-inflammatory NF kappa B pathway, creating a pro-inflammatory state and insulin resistance. Lipid loading in metabolically unhealthy adipose tissue produces Kemerine, an adipokine, which drives inflammation in the adipose tissue through macrophage polarization. So hepatokines, adipokines, and myokines are taken together. They are now described as organokines, which represent potentially new targets to mitigate insulin resistance and prevent and treat type 2 diabetes. Way back in 1923, Elliot P. Jocelyn observed that from an excess of fat, diabetes begins, and from an excess of fat, diabetics die. Lipid abnormalities have been consistently shown to precede glucose abnormalities in the natural history of type 2 diabetes. And so some people wonder whether we should actually call the garden variety type 2 diabetes as diabetes mellitus or really diabetes lipidus. Now, upon exposure to a surfeit of nutrients in the form of free fatty acids and glucose, metabolically healthy white adipose tissue expands by hyperplasia. When adipose tissue hyperplasia occurs, there is a parallel increase in the vascularity of the adipose tissue. There is a recruitment of anti-inflammatory M2 adipose tissue macrophages and T-regulator cells. And if an anti-inflammatory state is created within the adipose tissue, which allows it to efficiently take up and store lipids and also glucose. A metabolically unhealthy white adipose tissue expansion, on the other hand, is characterized by adipose tissue hypertrophy and not hyperplasia. Now, when adipose tissue hypertrophy happens, you can see the individual adipocytes, they increase in size, they are loaded with fat, and the nuclei are pushed to the periphery. And adipocyte hypertrophy is not associated with a parallel increase in adipocyte vascularity. As a result, those hypertrophic adipocytes undergo hypoxia and this generates cellular stress, attracts pro-inflammatory macrophages into the adipose tissue and the natural killer cells, which together produce a host of pro-inflammatory cytokines that by mechanisms, which I'm going to share with you in a moment from now, causes insulin resistance. So adipose tissue is the seat of chronic low-grade inflammation and obesity that fuels insulin resistance and metabolic disorders arising out of chronic low-grade inflammation in the adipose tissue is now known as metaflammation. Now in this cartoon, actually we describe the mechanism by which hypertrophic inflamed adipocytes produce an adipokine known as kamerin that attracts plasma cytoid dendritic cells, the PDCs into the adipose tissue. And then kamerin binds to its cognate receptor, which is present on the cell surface of the plasma cytoid dendritic cells. Following this binding, there are downstream events that causes the appearance of toll-like receptor 9 within the PDC. Now, parallelly extracellular nucleic acids from damaged adipocytes, they, after binding to their binding protein, go on to bind, go on to act as a ligand and bind to the receptor for age. So this age-rage interaction then leads to internalization of this complex, which then goes on to bind TLR9. When that happens, the production of and release of type 1 interferon is triggered, which in turn causes a pro-inflammatory polarization of M2 macrophages in the adipose tissue into M1. Now M1 macrophages then produces a lot of pro-inflammatory cytokines, i.e. TNF alpha, interleukin 6, interleukin 1 beta, which by further downstream mechanisms produce insulin resistance that interferes with lipid and glucose uptake by the adipose tissue. So in this paper, actually, we described in detail the molecular pathways by which adipose recruitment and activation of plasma cytoid dendritic cells fuel metaflammation. And now we have a number of papers looking at the association between metabolic syndrome and a high k-merin level. And as you can see, there are intervention studies also wherein disruption of the k-merin receptor gene is associated with reduced adiposity and glucose intolerance. Then k-merin has been known to exacerbate glucose intolerance in mouse models of obesity and diabetes. And a very interesting paper suggests that weight loss induced by bariatric surgery is actually associated with a reduction in k-merin level, and that actually correlates with the extent or degree of fatty liver in morbidly obese patients. So here is an evidence of a crosstalk between adipose tissue and the liver. So k-merin coming out of the adipose tissue actually induces fatty liver disease. And so following bariatric surgery, when the k-merin level goes down, you have an improvement in fatty liver disease also. Then our age-old antidiabetic agents, insulin and metformin, they are both known to regulate circulating and adipose tissue k-merin. And one of the mechanisms by which they reduce hyperglycemia and improve lipid control is by reducing the level of circulating k-merin. Now adiponectin, as you all know, is an anti-inflammatory molecule and insulin sensitive individuals have higher levels of adiponectin than insulin resistant individuals. And this paper identifies the inverse relationship between k-merin and adiponectin level, which actually contributes to metabolic syndrome. Now let me switch gear from here and pass on to the important role that liver plays in insulin resistance and type 2 diabetes. So fecuine, which is a pro-inflammatory hepatokine, is associated strongly with insulin resistance and type 2 diabetes. Fecuinae gene, as you can see, is located on chromosome 3q27, which is a known type 2 diabetes susceptibility locus. Fecuinae gene and protein expression in liver cells increases during insulin resistance and type 2 diabetes. Like the k-merin and adiponectin inverse relationship, serum levels of fecuinae and adiponectin are also inverse related. And interestingly, adiponectin gene is also located on the same chromosome, same position 3q27, just a few kilobases apart. So whether two organokines having opposing actions, their gene location on the same chromosome carries any clinical meaning is not currently known. We observed a couple of years ago that serum fecuinae concentration predicts glycemic outcomes in people with prediabetes, meaning people with prediabetes who progress to develop type 2 diabetes actually are shown in this study to have a higher serum fecuinae concentration. So people with prediabetes in the highest quartile of fecuinae had the most severe form of NAFLD. They also had higher BMI and a high level of this pro-inflammatory cytokine interleukin-1 beta. In this study, fecuinae level also correlated with fatty liver index scores. Now here in the center, you can see a blown up view of a lipid loaded hepatocyte. So this is a hepatocyte from representing a fatty liver. Now upon stimulation with saturated fatty acids and also with glucose, the glucose and saturated fatty acids, they work through different downstream molecular pathways, but both of them ultimately lead to the production of fecuinae. Now fecuinae induces insulin resistance by two major mechanisms, the first of which is that it's a powerful and direct inhibitor of insulin-mediated glucose uptake in the skeletal muscle and adipose tissue. And as you can recall, following a meal, around 70 to 80 percent of glucose uptake occurs in the skeletal muscles. So in an insulin-resistant state, when the fecuinae level is high, it actually prevents insulin-mediated glucose uptake in the skeletal muscle, thereby giving rise to hyperglycemia. Fecuinae also induces insulin resistance by another molecular mechanism, whereby it binds to toll-like receptor 4 present on the surface of immune cells and adipocytes and induces the release of pro-inflammatory cytokines that eventually causes insulin resistance. A few words about toll-like receptor 4, which was first isolated from drosophila flies and was recognized as a drosophila pattern recognition receptor. Now toll-like receptor 4 is actually intimately associated with immune function and mutation of toll gene in drosophila kills them by fungal infection. So what it tells us is that complete inhibition of toll-like receptor 4, although would be anti-inflammatory in nature, would be unlikely to be clinically meaningful because complete inhibition of TLR4 is bound to kill the host by widespread immune suppression. So what you need to focus on is that events downstream to TLR4, which can actually reduce the chronic low blood inflammation that is intimately associated with insulin resistance. Now here in this cartoon, you can see DBDB mice here who lack leptin receptor and become morbidly obese, as you can see. And on the right, you see high-fat diet obese mice. So in both the animal models, you can see with the rise in free fatty acid level, there is a parallel increase in fetu-RNA levels, which you can see over the western blots there. Now, when FFA and fetu-RNA levels are high in the circulation, there is significant increase in the expression of TLR4 and phospho-NF kappa B, which is downstream to TLR4. And this is seen with serum samples obtained not only from mice, DBDB and HFD, but also from human serum samples obtained from people with insulin resistance. Now, fetu-RNA impairs adipocyte function in terms of lipid uptake by the adipocytes. And here on the right-hand panel, if you focus on the human adipocytes, see this is known as oil red O experiment, where red dots within the adipocyte actually indicates efficient lipid uptake by them. And on the second panel, you see when fetu-RNA is added to the media, there is complete, nearly complete abrogation of lipid uptake by those adipocytes. Now, rosiglitazone, you all know, is a powerful insulin sensitizer, and fetu-RNA induced abrogation of lipid uptake by the adipocytes is mitigated here to some extent by the addition of rosiglitazone in the media. Now, when you add fetu-RNA here into the adipocyte media and create a pro-inflammatory state, this is associated with a significant increase in serum levels of the pro-inflammatory cytokines or adipokines, interleukin-6, TNF-alpha, and interleukin-1-beta. Of note, anakinra, an interleukin-1 receptor antagonist, shows significant anti-inflammatory effects mitigating insulin resistance. And it's currently under consideration as an anti-inflammatory approach to treat insulin resistance and type 2 diabetes. Now, in this cartoon, you can see, in the control state in the adipocyte, the glucose transporter 4 that are responsible for feeding glucose from the cell membrane back into the cell, they are internalized within the cytoplasmic vesicle in the resting state. Now, following a meal or here following addition of nutrient and insulin. So, in the human physiology, when you eat a meal, there is an immediate release of the first phase insulin. So, insulin causes translocation of glucose transporter 4 from the cytoplasmic vesicles back onto the cell membrane, where they're in a position to ferry glucose efficiently back into the cells. Now, presence of fetu-RNA completely abrogates insulin-mediated glucose transporter 4 translocation to the cell membrane, as you can see over here. And when you add fetu-RNA on the left hand panel over here, you see that it creates a pro-inflammatory state where the p-per-gamma and adiponectin are down-regulated and there is an increase in the protein levels of TNF-alpha and interleukin-6. Now, molecular studies have shown that insulin inhibits the action of a cytoplasmic protein called AS160 that interferes with GLAD4 translocation. So, inter-cytoplasmic location of GLAD4 is actually ensured by AS160 in the control state. Insulin, by inhibiting AS160, actually disinhibits this cytoplasmic resting of GLAD4 and they are now translocated onto the cell membrane. Fetu-RNA, by blocking the insulin action on AS160, unmasks its activity, unmasks the activity of AS160, thereby preventing GLAD4 translocation from cytoplasmic vesicles to the cell membrane. So, to cut a long story short, excess of free fatty acids stimulates the production of fetu-RNA from the hepatocytes. The fetu-RNA then binds to the free fatty acids forming a dimer. This dimer then goes on to bind to TLR4 present on the surface of adipocytes and immune cells. Immune cells Following this trimer formation, there is downstream activation and phosphorylation of NF kappa B, which induces the pro-inflammatory cytokine genes. There is copious output of pro-inflammatory cytokines, which by further downstream mechanisms cause insulin resistance. So these are the two papers where we actually discuss how Fetuin A acts as an endogenous ligand of TLR4 to promote lipid-induced insulin resistance, and also how plasma Fetuin A triggers inflammatory changes in macrophages and adipocytes by acting as an adaptor protein between prefatty acids and TLR4. Now here is further evidence that Fetuin A actually causes pro-inflammatory polarization of macrophages. Over here you have the anti-inflammatory M2 macrophages at the top of the Boyden's chamber and in the bottom you have Fetuin A. Fetuin A permeates through the membrane here at the bottom and induces polarization of the macrophages. Once polarized, the M1 macrophages, they acquire amyloid movement and they pass through this membrane in the Boyden's chamber into the bottom, and here you can see the M1 macrophages lying into the bottom of the chamber. So this is clear evidence that Fetuin A induces macrophage polarization. Now we then went on to show that Fetuin A coming to the adipose tissue and causing macrophage polarization also induces a local production of Fetuin A from the adipocytes. So here in this transculture system you can see adipocytes on the top, they produce Fetuin A upon treatment with free fatty acids and that Fetuin A comes into the bottom of the transculture system where it induces macrophage polarization. And here at the bottom you can see the M1 macrophages, they can be identified by their specific markers and macrophage polarization, like I said before, creates a pro-inflammatory state and here you can see in the transculture system, following macrophage polarization, there is an increase in TNF alpha IL-6 levels and a diminution in the anti-inflammatory PPAR gamma and interleukin-10. So this is the paper which talks about the contribution of adipocyte Fetuin A to macrophage migration and polarization into the adipose tissue and creation of a pro-inflammatory state. Now, as I said before, free fatty acid, Fetuin A, dimer, they go on to form a trimer with TLR4, then there is phospho-NF kappa B formation which induces the pro-inflammatory genes and the release of pro-inflammatory cytokines then causes insulin resistance, the question is how? Now here you can see there are two pathways by which the pro-inflammatory cytokines actually cause insulin resistance. Through one pathway here, they cause inflammasome activation and leading to high levels of caspase-1 which cleaves a protein known as sart1 and you know this sart1 is actually associated with high insulin sensitivity, exercise improves sart1 level and you can see even foods containing high sart1, they are sold over the counter as an insulin sensitizer, so caspase-1 by cleaving sart1 actually interferes with mitochondrial function because sart1 is essential for mitochondrial biogenesis and bioenergetics, so when sart1 is cleaved, you have mitochondrial dysfunction insulin resistance. Through a separate mechanism, Fetuin A by up-regulating Wnt3A actually down-regulates PPAR gamma adiponectin and MPK and MPK as you all know is the master metabolic sensor for the cell and is intimately associated with mitochondrial health, so when the Fetuin MPK level goes down, the PGC1 alpha and its target genes, they are not expressed, so you have impaired mitochondrial biogenesis, bioenergetics and a reduced mitochondrial ATP yield which causes cellular dysfunction, insulin resistance and type 2 diabetes. So in these papers, we actually describe how Fetuin A down-regulates adiponectin through the Wnt3A gamma pathway and how it again causes impairment of energy sensor sart1 and MPK in lipid-induced inflammatory adipocytes to cause insulin resistance. Now in two recent papers, we have demonstrated that Fetuin A also increases DPP4 expression in the pancreatic beta cells and when that happens, there is impaired insulin secretion from beta cells, but the conventional DPP4 inhibitors that are used as anti-hyperglycemic agents, whether they have some action here and can rescue insulin secretion from pancreatic beta cells by binding to DPP4 in the islets is not known at this point of time. In another recent paper, we have been able to show that Fetuin A through this molecular pathway of Ras-MEK and DRK causes inhibitory phosphorylation of P-per-gamma and thereby lowers adiponectin. So these are two additional mechanisms by which Fetuin A promotes insulin resistance. So this is the paper showing DPP4 expression in pancreatic beta cells upon treatment with Fetuin A and this paper talks about inhibitory phosphorylation of P-per-gamma by Fetuin A that causes insulin resistance. So this cartoon shows nicely the adipocyte and the myocyte crosstalk. Inhibitory phosphorylation of P-per-gamma leads to a low level of adiponectin. When adiponectin level is low, AMPK activation in the skeletal muscle suffers, which causes mitochondrial dysfunction, dwindling ATP generation by the mitochondria, there is skeletal muscle mitochondrial dysfunction and that leads to impaired insulin-mediated glucose uptake by the skeletal muscle, which causes insulin resistance and type 2 diabetes. So this cartoon actually nicely depicts the organokine crosstalks between liver, adipose tissue and skeletal muscle that under obese conditions causes disruption of metabolic homeostasis, creates a state of chronic low-grade inflammation and leads to metabolic syndrome and type 2 diabetes. So in this recent review actually we have covered all the molecular mechanisms by which inflammatory overtones of organokines give rise to metabolic syndrome and type 2 diabetes. So again switching gear from here in the next 10-15 minutes I'm going to walk you through the clinical implications of these molecular mechanisms underlying creation of a chronic low-grade inflammatory state. So Allison Goldfein's group from Jocelyn Diabetes Center and Harvard Medical School, they have recently published this novel therapeutic approaches targeting inflammation for diabetes and associated cardiovascular risk and here you can see NF kappa B, which we have discussed a little while ago, which is a powerful pro-inflammatory mediator. NF kappa B is actually inhibited by salicylate and low-dose methotrexate and these two molecules are currently undergoing clinical trial as anti-inflammatory approaches to reduce insulin resistance. Then inflammasome formation, we have discussed how that impacts on SART1 cleavage and here you can see interleukin-1 receptor antagonists, then anti-interleukin-1 beta, so anakinra actually, and we also have humanized antibody against interleukin-1, they reduce inflammasome formation and improve insulin sensitivity. Then you have specific antibodies against interleukin-1 beta and then TNF alpha, which also mitigates chronic low-grade inflammation and improve insulin sensitivity. Then you all know that AMPK is the master metabolic regulator and then there are some endogenous activators of AMPK, which ameliorate insulin resistance and they are known as AMP and ICAR and as far as the exogenous activators of AMPK are concerned, important molecules are metformin, salicylate and low-dose methotrexate. Now can we utilize the known anti-inflammatory properties of diabetes drugs to mitigate insulin resistance and in fact, repurpose them to take care of the low-grade inflammation that provides additional anti-hyperglycemic benefits. So these are the available anti-hyperglycemic agents at our disposal and very quickly I will take you through their anti-inflammatory mechanisms. So here we actually discussed that human macrophages showed that exposure to SGLT2 inhibitors, they led to the inhibition of both NLRP3, which is inflammasome and interleukin-1 beta. So SGLT2 inhibitors have significant anti-inflammatory effects and that's the proposed mechanism by which they can actually improve insulin sensitivity beyond their known mechanism of action, which is by increasing renal glucose excretion or the glucuretic action. Now in a recent meta-analysis we have shown stroke benefits of GLP-1 receptor agonists and again the proposed mechanism by which these agents bring about stroke benefits are actually modulation of Haskell inflammation and also, you know, they confer neuroprotection to decrease production of pro-inflammatory cytokines and reactive oxygen species. So again significant anti-inflammatory effects beyond their actual mechanism of action of glucose-regulated insulin secretion from the pancreatic beta cells. Now pioglitazone significantly reduces serum fetu-RNA levels in patients with type 2 diabetes and therefore fetu-RNA still represents an attractive target for the development of type 2 diabetes treatments. Pioglitazone is one agent, several other agents are being explored to reduce levels of fetu-RNA and improve insulin sensitivity. Here in this paper exercise actually has been shown to lower fetu-RNA and ameliorate insulin resistance of the liver or in other words to improve hepatic insulin sensitivity. This cartoon shows nicely how with a reduction in delta-fetu-RNA there is a concomitant improvement in hepatic insulin sensitivity or amelioration of hepatic insulin resistance and here in the bottom right panel you can see how a reduction in fetu-RNA level is associated with an increase in anti-inflammatory and insulin sensitizing adiponectin. So the inverse relation is nicely depicted here, lower the level of fetu-RNA or greater is the level of adiponectin and there is amelioration of insulin resistance and type 2 diabetes. So given that overfeeding has been shown to increase fetu-RNA and impair systemic insulin sensitivity, lower fetu-RNA can be an important mechanism whereby exercise reduces type 2 diabetes risk and herein lies the importance of therapeutic lifestyle intervention in terms of restricted calorie intake and improved energy expenditure. Now coming to the last part of my presentation, vanadium compounds have been explored in the past as insulinomimetics and anti-hyperglycemics. So vanadium compounds have been used to treat diabetes since 1899 and they were the only drugs available to treat diabetes until the discovery of insulin. But the molecules did not see the light of the day because stability and toxicity issues. We recently developed dimethyl peroxyvanadate DMP as a non-toxic oral insulinomimetic agent. Now like I said before, fetu-RNA is a powerful inhibitor of insulin receptor tyrosine kinase and in presence of fetu-RNA, phosphotyrosine levels intracellularly are low and then they are further broken down by enzymes known as phosphotyrosine phosphatases. Now vanadium being a powerful inhibitor of phosphotyrosine phosphatase is able to maintain whatever phosphotyrosine formation occurs in presence of fetu-RNA. So vanadium, by rescuing phosphotyrosines, actually improves the metabolic function of insulin through the downstream pathways. And here you can see fetu-RNA in high-fat diet giving rise to high fetu-RNA causes macrophage polarization. These are macrophage cell lines. This is rescued by DMP and when that happens, this is correlated with a reduction in fetu-RNA level. Then in the bottom, you can see how inhibition of macrophage polarization is associated with reduction in the levels of pro-inflammatory cytokines TNF alpha, IL-6 and IL-1 beta. And here you can see this is the CD11C is the M1 macrophage marker in presence of high-fat diet. The CD, the M1 marker, I mean the M1 macrophages increase, here you can see 34 percent and this is reduced to 20 percent with the addition of DMP. So DMP causes increase in SART1 level here, so it interferes with SART1 cleavage by the downstream mechanisms that are triggered by fetu-RNA. So DMP rescues SART1 cleavage. Here you can see SART1 mRNA levels increasing. This table summarizes the effect of DMP on macrophage polarization. You can see reducing macrophage population, lower number of pro-inflammatory M1 macrophages, rescue of SART1 activity and significant reduction in TNF alpha and IL-6 following addition of DMP here in presence of high-fat diet that induces insulin resistance. So here you can see how nicely DMP actually rescues the Wnt3A-mediated inhibition of p-per-gamma adiponectin and AMPK. Here you can see p-per-gamma levels being rescued. This is the fetu-RNA in this reduction in adiponectin level which is rescued with the addition of DMP. p-per-gamma is rescued and here you can see when AMPK is activated with the addition of DMP, there is improvement in mitochondrial function characterized by increased protein levels of NRF1 and TFRM which are markers of good mitochondrial function. So here you can see high-fat diet reducing mitochondrial density and this is being rescued by DMP and here this shows the mitochondrial DNA copy number that goes down with high-fat diet but is rescued by DMP and this is parallel by an increase in mitochondrial cytoplasmic oxidase activity. Addition of DMP actually rescues inhibition of fatty acid oxidation brought about by high-fat diet. When that happens, there is an increase in mitochondrial ATP production. Here you can see how nicely DMP actually causes translocation of glucose transporter four from the cytoplasmic vesicle onto the cell surface thereby significantly reducing the blood glucose levels over here and bringing diabetes under control in these mice and then it also reduces insulin resistance characterized by a decrease in HOMA-IR. So here in the L6 myotubes, again you can see DMP shows a significant insulin mimetic effect and causes a GLAD4 translocation to the myocyte membrane. A single oral administration of DMP in animals maintains plasma DMP levels in the therapeutic range over 24 hours. Here you can see how in streptozotocin-induced diabetic mice, DMP actually significantly reduces blood glucose levels and it also improves fatty acid uptake by the adipocytes in that mouse model. So DMP significantly improves insulin sensitivity also in DB mice. Here you can see the glucose tolerance test improving, there is improvement in insulin tolerance test and a significant decrease in HOMA-IR. So we perform detailed toxicity studies involving vanadium, the DMP and we're able to show lack of significant side effects with dimethyl peroxide vanadate and these are the papers showing insulin mimetic effects of vanadium and how macrophage accumulation and polarization is attenuated by DMP. So to conclude, Fetuin A acts as an adapter protein between free fatty acid and TLR4 to promote adipose tissue inflammation and insulin resistance. Fetuin A knockout mice are protected from lipid-induced insulin resistance. Free fatty acid Fetuin A dimer upon binding to TLR4 present on the surface of adipocytes and immune cells stimulates the production of pro-inflammatory cytokines TNF-alpha, interleukin-6 and IL-1-beta to the phospho-NF-kappa-B pathway. Exercise, metformin, thiazolidine dions, GLPM receptor agonists and SGLP2 inhibitors have significant anti-inflammatory effects which beyond their usual pathway of anti-hyperglycemic action, they also mitigate lipid-induced insulin resistance. And finally, there are molecules in the pipeline like salicylate, low-dose methotrexate, IL-1 receptor antagonists and arguably non-toxic vanadium compounds which offer promising anti-inflammatory approaches to mitigate lipid-induced insulin resistance and prevent and slash or treat type 2 diabetes. I think I'll stop here and would be happy to take questions or comments if there are any. Thank you for listening.

lipid-induced insulin resistance

fetuin A

inflammation

insulin sensitivity

pro-inflammatory cytokines

therapeutic approaches

GLP-1 receptor agonists

vanadium compounds