Adipose tissue: Roles and function in diabetes and beyond. August 2015; SAGLB.DIA

Adipose tissue: Roles and function in diabetes and beyond August 2015; SAGLB.DIA.15.07.0398

Acknowledgement The following slides intend to summarise the key points presented during the Banting Medal for Scientific Achievement Award lecture by Professor Philip E. Scherer at the 75 th Scientific Sessions of the American Diabetes Association, June 5 9 2015, Boston, USA.

Summary of content Introduction: The biology of adipose tissue Overview of the roles of adipose tissue, adipocyte types and the importance of adipose tissue health What goes wrong in adipose tissue when we gain weight? Overview of the way in which adipose tissue expands to accommodate excess energy under normal and stress conditions What else is happening? The role of adipocytes Overview of how factors secreted from adipocytes, including adipokines, sphingolipids and uridine affect physiological processes Conclusions

Introduction: The biology of adipose tissue

Adipose tissue is a complex, metabolically active endocrine organ that is equipped for much more than energy storage 1,2 Fatty acids from hepatic de novo lipogenesis Fat Glucagon/ Epinephrine receptor To peripheral tissues Expanding adipose tissue stores excess energy in the form of triglycerides Insulin Triglycerides Triglycerides Glycerol to liver Reducing adipose tissue releases stored energy Glucose β-oxidation Lactate The primary role of adipose tissue is to store excess energy and release it when needed. However, it also expresses and secretes factors affecting many physiological processes 1,2 1. Kershaw EE, et al. J Clin Endrocinol Metab 2004;89:2548 56; 2. Rutkowski et al. J. Cell Biol 2015;208:501 12.

The primary cells in adipose tissue are white, brown and beige adipocytes 1,2 Dormant beige adipocytes Warmer conditions, exposure to high fat diet Cold conditions, exposure to β-agonists White adipocytes Primary function: Energy storage Represents the majority of cells in adipose tissue, and expand with obesity 1 Active beige adipocytes Primary functions: Energy storage/ homeostasis Dormant beige adipocytes are found in white adipose tissue and undergo reversible browning to under certain conditions 2 Brown adipocytes Primary function: Homeostasis Part of smaller brown fat pads, and become activated in response to certain stimuli (e.g. cold, exposure to β-agonists) 1,2 1. Rutkowski JM, et al. J. Cell Biol 2015;208:501 12; 2. Harms et al. Nature Medicine 2013;19:1252 63.

Insulin resistance Metabolic flexibility depends on the health, but not quantity, of adipose tissue 1 Metabolically unhealthy lean subjects Metabolically unhealthy obese subjects Metabolically healthy lean subjects Metabolically healthy obese subjects Fat mass The relationship between fat mass and insulin resistance is not strictly linear. Some obese subjects are metabolically healthy and some lean individuals are metabolically unhealthy. Dysfunctional, metabolically unfit cells display metabolic dysregulation and are associated with increased insulin resistance 1 1. Wernstedt Alsterholm I, et al. Drug Discov Today Dis Models 2007;4:17 24.

What goes wrong in adipose tissue when we gain weight?

A pro-inflammatory response allows adipocytes to rapidly expand to accommodate excess energy 1,2 Sufficient angiogenesis and vascular remodelling to supply oxygen 1,2 Extracellular matrix remodelling allows the cell to expand 1 Oxygen can no longer diffuse into the expanding cell quickly enough 2 Macrophages recruited to remove dead or unhealthy cells 2 Hypoxia due to limited oxygen 1,2 Inflammation 1,2 Fibrosis 2 A pro-inflammatory response allows healthy expansion to a mature adipocyte 1,2 Expansion limit Insulin resistance 2 Beyond the expansion limit, hypoxia, fibrosis, excess inflammation, and insulin resistance ensue 1,2. The unhealthy cell is removed by macrophages 2 1. Wernstedt Asterholm I, et al. Cell Press 2014;20:103 18; 2. Sun K, et al. J Clin Invest 2011;11:2094 2104.

In the obese state, adipose tissue expansion and remodelling is accelerated 1,2 Insufficient angiogenesis and vascular remodelling to supply oxygen leads to local hypoxia 2 Insulin resistance 2 Increased macrophage recruitment 2 Increased hypoxia 2 Expansion of the adipocyte is pathologically accelerated 2 Fibrosis 2 The expansion limit is quickly reached 2 Unfolding protein responses 1 Beyond the expansion limit, widespread cell stress induces chronic systemic inflammation and metabolic dysfunction 1,2 1. Wernstedt Asterholm I, et al. Cell Press 2014;20:103 18; 2. Sun K, et al. J Clin Invest 2011;11:2094 2104.

Inflammation is not the only factor contributing to adipocyte health (AdipoChaser mouse model) 1 Lack of proinflammatory response prevents adipocyte expansion to accommodate excess energy 1 Limited expansion 1 Expansion limit not reached, yet ectopic lipid deposition, reduced metabolic function, systemic inflammation, impaired gut-barrier function and insulin resistance ensue 1 1. Wernstedt Asterholm I, et al. Cell Press 2014;20:103 18.

Glucose (mg/dl) Glucose (mg/dl) Glucose (mg/dl) Glucose (mg/dl) Modulation of the extracellular matrix plays an important role in fibrosis and remodelling 1 600 400 200 0 *** ** ** +/+ ob/ob col6koob/ob FPG 10 weeks 12 weeks 600 500 400 300 200 100 0 ** ** *** ob/ob col6koob/ob 0 30 60 120 180 Time (min) OGTT 1400 1200 1000 800 600 400 200 0 ** ** ** +/+ ob/ob col6koob/ob FPG FPG, fasting plasma glucose; OGTT, oral glucose tolerance test, WT, wild-type. **p<0.01, ***p<0.05. ** ** 1200 1000 800 600 400 200 0 ** ** OGTT ob/ob col6koob/ob *** 0 30 60 120 180 Time (min) In ob/ob mice lacking collagen VI (col6koob/ob), there is significantly less extracellular matrix accumulation in expanding adipose tissue (see photographs). This less Stable tissue is associated with improved glucose tolerance, as measured by FPG and OGTT 1 1. Figures adapted from Khan T, et al. Mol Cell Biol 2009;29:1575 91.

What else is happening? The role of adipocytes

The adipocyte is a professional secretory cell involved in inflammation and energy homeostasis 1,2 Adipokines e.g. adiponectin, leptin 1 Lipids e.g. sphingolipids 1 Metabolites e.g. uridine 2 1. Kershaw JM, et al. J Clin Endrocinol Metab 2004;89:2548 56; 2.Benjamin W, et al. J Lipid Res 1966;7:285 94.

Adipokines: focus on adiponectin Adipokines e.g. adiponectin, leptin 1 1. Kershaw JM, et al. J Clin Endrocinol Metab 2004;89:2548 56.

Adiponectin levels decrease as fat mass increases, suggesting that adiponectin levels correlate with insulin sensitivity 1 BMI, body mass index Metabolically healthy individuals display higher levels of adiponectin vs. metabolically unhealthy individuals with the same BMI. In contrast to other adipokines, adiponectin levels are inversely proportional to fat mass 1 1. Figures adapted from Turer AT, et al. Diabetologia 2011;54:2515 24.

In mice with high adiponectin levels, full metabolic flexibility was retained after a high fat diet 1 Genetic manipulation allowed adiponectin levels in mice with dramatically expanding fat pads to resemble those of a lean mouse. Their subcutaneous fat was still able to effectively absorb excess calories and spare other tissues from lipotoxicity 1 1. Kusminski CM, et al. Nat Med 2012;18:1539 49.

How were the metabolically healthy mice different to the metabolically unhealthy mice? 1 Subcutaneous white adipose tissue Lipid uptake and storage Insulin sensitivity Cell size Fibrosis Oxidative stress Immune cell filtration Inflammatory markers Liver Insulin sensitivity Improved lipid profile Ceramide levels Pancreas Islet numbers, with healthy morphology Gluconeogenesis & lipogenesis Gonadal white adipose tissue Lipid diversion Cell size Fat pad size The mice that retained metabolic flexibility after a high-fat diet had more but smaller and healthier adipocytes in subcutaneous white adipose tissue, compared with metabolically unhealthy mice. Positive effects were also observed in gonadal white adipose tissue, the liver and pancreas 1 1. Kusminski CM, et al. Nat Med 2012;18:1539 49.

% change in insulin sensitivity Some anti-diabetic drugs increase HMW adiponectin levels proportionally to the ability of the drug to improve insulin sensitivity 1 400 300 200 100-50 -100 50 100 150 200 200 % change in HMW:total adiponectin HMW, high molecular weight; PPAR, peroxisome proliferator-activated receptor Troglitazone, a thiazolidinedione that acts by agonism of PPAR-γ, affects the distribution of adiponectin by increasing the ratio of HMW adiponectin to total adiponectin. This occurs in parallel with increasing insulin sensitivity 1 1. Figure adapted from Pajvani UB, et al. J Bio Chem 2004;279:12152 62.

Adiponectin plays an important role in the function of many tissues 1 3 Adipose tissue Cardiovascular Endothelium Glucose uptake, fat storage & adipogenesis Injury, inflammation & apoptosis Angiogenesis & function Inflammation Vasodilatation Oxidative stress Macrophage β-cell Podocyte Insulin sensitivity Insulin secretion & viability Function & recovery Inflammation Apoptosis Oxidative stress & apoptosis Muscle Cancer Liver Inflammation Insulin sensitivity & fatty acid oxidation Cell growth (with exceptions) Insulin sensitivity Gluconeogenesis & lipogenesis 1. Ghantous CM, et al. Int J Endrocrinol 2015;534320; 2. Nigro E, et al. Biomed Res Int 2014:2104:658913; 3. Ye R, et al. Mol Metab. 2013;2:133 41.

ATTAC mouse models are used to probe the function of adiponectin 1 Caspase-8 FKBP domains bound to M FKBP FKBP Caspase-8 Caspase-8 in transgene carried by ATTAC mice Short-term treatment with AP20187 dimeriser M FKBP FKBP Caspase-8 AP20187 AP20187 M FKBP FKBP Caspase-8 Dimerization encodes adipocyte apoptosis ATTAC, Apoptosis Through Triggered Activation of Caspase-8; FKBP, FK506 binding protein ATTAC mice have a transgene that forces dimerisation of Caspase-8, which encodes apoptosis. The model can be used to investigate the ability of a cell to resist cell death, and, when the gene is de-activated, the ability of the cell to regenerate 1 1. Pajvani UB, et al. Nature Med 2005;11:797 803.

Live animals (% initial) HEART-ATTAC mice: In heart cells, higher adiponectin levels were associated with increased cell survival following Caspase-8 dimerisation 1 100 75 Adiponectin overexpressing (Tg/+) *** 50 25 Adiponectin deficient (-/+) Adiponectin WT (+/+) Tg, transgenic, WT, wild-type. ***p<0.05 vs. WT 0 14 16 18 20 24 Time (h) after dimerizer injection *** *** Adiponectin null (-/-) 1. Holland WL, et al. Nature Med 2011;17:55 63.

Glucose after OGTT* (mg/dl) Insulin (ng/ml) Insulin content (ng/mg pancreas) PANIC-ATTAC mice: Pancreatic β-cells were more susceptible to apoptosis in the absence of adiponectin 1 Drastically reduced levels of β-cells 700 600 500 400 300 200 100 *** *** *** WT *** 3 *** *** 2 1 0 0 0 30 60 120 180 0 15 30 WT P/P Time (mins) P/P * Time (mins) PANIC, pancreatic islet β-cell apoptosis; WT, wild-type; OGTT, oral glucose tolerance test, P/P, homozygous PANIC-ATTAC mice *p<0.001, ***p<0.05 Drastically reduced levels of β-cells can be observed in the PANIC-ATTAC mice, and subsequently insulin production was reduced 1 * P/P WT 300 250 200 150 100 50 *** 1. Figures adapted from Wang ZV, et al. Diabetes 2008;57:2137 48.

Glucose (mg/dl) PANIC-ATTAC mice: Pancreatic β-cell mass was restored more quickly with adiponectin overexpression, leading to restoration of euglycemia 600 500 400 PANIC-ATTAC Adiponectin null (-/-) PANIC-ATTAC Adiponectin WT (+/+) 300 200 PANIC-ATTAC Adiponectin Overexpresser (Tg/+) 100 * * * WT (not PANIC-ATTAC) 0 0 1 2 3 4 5 6 7 8 9 10 Time after dimerizer (weeks) PANIC, pancreatic islet β-cell apoptosis; WT, wild-type; Tg, transgenic *p<0.01 vs overexpresser mice Ye R, et al. elife 2014;3:e03851.

WT1+ nuclei/ glomerular area (µm 2 )x10000 POD-ATTAC mice: Adiponectin improves functional recovery in the kidney 1 30 25 20 15 Adiponectin WT Adiponectin null Adiponectin over-expressing * * * * ** * * ** ### # 10 5 0 0 2 7 28 60 0 2 7 28 60 0 2 7 28 60 Days post-dimerization WT, wild-type; WT1+, Wilms tumour gene 1-positive. **p<0.01, ***p<0.001 from Day 0; # p<0.001, ### p<0.05 from lowest count Adiponectin overexpressing POD-ATTAC mice showed enhanced kidney cell recovery, judged by WT1+ nuclei per glomerular area, compared with WT or adiponectin null mice 1 1. Rutkowski JM, et al. J Am Soc Nephrol 2013;24:268 82.

Adiponectin: Conclusions 1 Brain Affects energy expenditure and encourages decreases in body weight Pancreas Decreases apoptosis in β-cells Promotes β-cell recovery Immune cells Promotes antiinflammatory macrophage phenotype Liver Reduces glycogen release Increases insulin sensitivity Increases fatty acid oxidation Cell signalling effects Adipocyte numbers Sphingolipid metabolism Lipid metabolism Reduction in inflammation Adiponectin is an important adipokine released by adipocytes Adiponectin levels are an indicator of adipose tissue health, a reflection of metabolic flexibility and systemic insulin sensitivity Adiponectin has potent anti-apoptotic function in many different cell types 1. Turer AT, et al. Diabetologia 2012;55:2319 26.

Adiponectin: Future research Adiponectin has highlighted a number of new potential therapeutic options 1 It is one of the best available biomarkers for existing metabolic disease 2 It is one of the best prospective biomarkers for cardiovascular and diabetes risk 2 A new AdipoChaser mouse has been generated to track new adipogenesis 3 A crystal structure of adiponectin has now been published to facilitate further research 4,5 1. Holland WL, et al. Science 2013;342:1460 61; 2. Trujillo ME, et al. J Int Med 2005;257:167 75; 3. Wernstedt Asterholm I, et al. Cell Press 2014;20:103 18; 4. Tanabe H, et al. Nature 2015;520:312 6; 5. Image sourced from Tanabe H, et al. Nature 2015;520:312 6 via RCSB Protein Data Bank, available at: http://www.rcsb.org/; last accessed: 21 Aug 2015.

The adipocyte is a professional secretory cell involved in inflammation and energy homeostasis 1,2 Adipokines e.g. adiponectin, leptin 1 Lipids e.g. sphingolipids 1 Metabolites e.g. uridine 2 1. Kershaw JM, et al. J Clin Endrocinol Metab 2004;89:2548 56; 2.Benjamin W, et al. J Lipid Res 1966;7:285 94.

Lipid release: focus on sphingolipids Lipids e.g. sphingolipids 1 1. Kershaw JM, et al. J Clin Endocrinol Metab 2004;89:2548 56.

The degradation of ceramides is metabolically important 1 CERAMIDE Ceramidase degradation SPHINGOSINE 1P Promotes apoptosis Promotes insulin resistance Increases inflammation Ceramide synthesis Promotes proliferation Promotes cell survival Adiponectin has been associated with the beneficial degradation of ceramides to sphingosphine phosphates, which signal cell survival and proliferation 1 1. Xia JY, et al. Cell Metab 2015;22:1 13.

Hepatic ceramide content (pmol/mg wt) Hepatic ceramide content (pmol/mg wt) Adiponectin decreases ceramide levels in the liver 1 400 300 *** 300 200 *** 200 100 ** 100 ** 0 Lean ob/ob mice ob/ob + adiponectin 0 Lean Dietinduced obese mice Dietinduced obese + adiponectin TZD, thiadolizinedione. **p<0.01 effect of adiponectin; ***p<0.05 compared with WT controls Therefore, anti-diabetic drugs that increase adiponectin levels, such as TZDs can induce decreases in hepatic ceramide levels, which leads to improved hepatic insulin sensitivity 2 1. Figures adapted from Holland WL, et al. Nature Med 2011;17:55 63; 2. Warshauer JT, et al. Diabetes Metab Res Rev 2015;ePub ahead of print.

Glucose infusion rate after HFD (mg/kg/min) Hepatic glucose production after HFD (mg/kg/min) Do ceramides direct hepatic insulin sensitivity or are they a by-product of insulin resistance? 40 50 30 ** 40 20 30 20 ** 10 10 0 WT Alb-AC 0 WT Alb-AC Alb-AC, inducible liver-specific acid ceramidase transgenic; HFD, high-fat diet; WT, wild-type; **p<0.05 This is still unclear at a cellular level; however, models have shown that reducing ceramides in the liver improves hepatic insulin sensitivity 1 1. Figures adapted from Xia JY, et al. Cell Metab 2015:22:266 78.

2-Deoxyglucose uptake (mg/kg/min) Do ceramides in the liver direct adipocyte sensitivity? 5 4 2-DG uptake-hfd ** WT Alb-AC 3 2 ** ** 1 0 MWAT GWAT SWAT MWAT, mesenteric WAT; GWAT, gonadal WAT; SWAT, subcutaneous WAT; WAT, white adipose tissue; WT, wild-type. **p<0.05. Again, this is unclear, but models have also shown that reducing of ceramides in the liver improves adipocyte insulin sensitivity, as demonstrated by reduced glucose uptake of MWAT, GWAT and SWAT 1 1. Figure adapted from Xia JY, et al. Cell Metab 2015;22:266 78.

Sphingolipids: Conclusions Lipids e.g. sphingolipids 1 Adiponectin facilitates the degradation of ceramides to sphingolipids in the liver 2 Decreased liver ceramide is associated with improved hepatic and adipocyte insulin sensitivity 3,4 The mechanism for insulin sensitivity is not well understood, but there is evidence that sphingolipids direct insulin sensitivity, rather than being a by-product 2 4 1. Kershaw JM, et al. J Clin Endrocinol Metab 2004;89:2548 56; 2. Xia JY, et al. Cell Metab 2015;22:1 13; 3. Holland WL, et al. Nature Med 2011;17:55 63; 4. Xia JY, et al. Cell Metab 2015:22:266 78.

The adipocyte is a professional secretory cell involved in inflammation and energy homeostasis 1,2 Adipokines e.g. adiponectin, leptin 1 Lipids e.g. sphingolipids 1 Metabolites e.g. uridine 2 1. Kershaw JM, et al. J Clin Endrocinol Metab 2004;89:2548 56; 2.Benjamin W, et al. J Lipid Res 1966;7:285 94.

Metabolite release: focus on uridine Metabolites e.g. uridine 1 1. Benjamin W, et al. J Lipid Res 1966;7:285 94.

Uridine is important in many physiological reactions and pathways 1 RNA synthesis and cell proliferation Thermoregulation Cell growth Insulin signalling Reproduction URIDINE Glycemic control Cancer and infectious diseases Ischemia protection RNA, ribonucleic acid 1. Pizzorno G, et al. Biochim Biophys Acta 2002;1587:133 44.

Uridine is synthesised in the liver in the fed state, and in adipocytes in the fasted state 1 Suppressed uridine in liver High uridine in adipocytes High uridine in liver Suppressed uridine in adipocytes N Fasted state Fed state F E CAD, carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase; Xbp1s, X-box binding protein 1s In the fasted state, the liver switches it s role to release glucose. In parallel, Xbp1s induces activation of the CAD gene in the adipocytes, which elevates uridine synthesis in the adipocyte 1 1. Deng Y, et al. J Clin Invest 2013;123:455 68.

Uridine: conclusions Uridine biosynthesis switches from the liver to adipocytes in the fasted state. This switch is mediated by Xbps1 2 This allows the liver to focus on releasing glucose in the fasted state 2 Plasma uridine levels mediate core body temperature and subsequently metabolic energy requirements 3 Metabolites e.g. uridine 1 Xbp1s, X-box binding protein 1s 1. Benjamin W, et al. J Lipid Res 1966;7:285 94; 2. Deng Y, et al. J Clin Invest 2013;123:455 68; 3. Pizzorno G, et al. Biochim Biophys Acta 2002;1587:133 44.

Conclusions

Adipose tissue: What does the future hold? Metabolism Heart Liver Muscle Vascular Metabolism Endothelium Brain Pancreas Kidney It is important to keep adipocytes healthy in order to retain metabolic flexibility 1 Adipocytes play an important role in modulating insulin sensitivity 2 In addition to their key role in metabolic function, adipocytes have a role in many other pathways 3 and are associated with infectious disease and oncology 5 Infectious Disease Tumour Cell Interactions So far, research has only scratched the surface of the complex roles of adipocytes and many challenges and opportunities remain 6 1. Wernstedt Alsterholm I, et al. Drug Discov Today Dis Models 2007;4:17 24; 2. Turer AT, et al. Diabetologia 2011;54:2515 24; 3. Kusminski CM, et al. Nat Med 2012;18:1539 49; 4. Desruisseaux MS, et al. Infection and Immunity 2007;75:1066 78; 5. Hefetz-Sela S, et al. Pharmacol Ther 2013;138:197 210.

Further information To view the webcast of Professor Philip E. Scherer s Banting Award lecture, copy the following link into your browser: http://professional.diabetes.org/presentations_details.aspx? session=4707 To view an ADA TV interview with Professor Philip E. Scherer discussing his Banting Award lecture, copy the following link into your browser: https://www.youtube.com/watch?v=lskgnni2ugq Weblinks last accessed on 21 Aug 2015