Fat Tissue: the Good, the Bad, and the Ugly

Websites

Journal of Lipid Research
The Journal of Lipid Research focuses on the science of lipids in health and disease.

Books

Awad A. Adipose tissue and inflammation. Boca Raton FL: Taylor & Francis; 2010.

Goss R. The physiology of growth. New York: Academic Press; 1978.

Levy-Marchal C. Adipose tissue development. From animal models to clinical conditions. Endocr. Dev. 2010;19:VII-IX.

Widmaier E. Vander, Sherman, & Luciano's human physiology: the mechanisms of body function. 9th ed. Boston: McGraw-Hill Higher Education; 2004.

Journal Articles

Hirsch

Hirsch J. The search for new ways to treat obesity. Proc. Natl. Acad. Sci. USA 2002;99(14):9096-9097.

Hirsch J, Leibel RL. The genetics of obesity. Hosp. Pract. (Minneap) 1998;33(3):55-59, 62-65, 69-70 passim.

Hudgins LC, Baday A, Hellerstein MK, et al. The effect of dietary carbohydrate on genes for fatty acid synthase and inflammatory cytokines in adipose tissues from lean and obese subjects. J. Nutr. Biochem. 2008;19(4):237-245.

Mayer LES, Klein DA, Black E, et al. Adipose tissue distribution after weight restoration and weight maintenance in women with anorexia nervosa. Am. J. Clin. Nutr. 2009;90(5):1132-1137.

Stern JS, Batchelor BR, Hollander N, Cohn CK, Hirsch J. Adipose-cell size and immunoreactive insulin levels in obese and normal-weight adults. Lancet 1972;2(7784):948-951.

Arner

Arner E, Westermark PO, Spalding KL, et al. Adipocyte turnover: relevance to human adipose tissue morphology. Diabetes 2010;59(1):105-109.

Arner E, Rydén M, Arner P. Tumor necrosis factor alpha and regulation of adipose tissue. N. Engl. J. Med. 2010;362(12):1151-1153.

Hoffstedt J, Arner E, Wahrenberg H, et al. Regional impact of adipose tissue morphology on the metabolic profile in morbid obesity. Diabetologia 2010;53(12):2496-2503.

Langin D, Dicker A, Tavernier G, et al. Adipocyte lipases and defect of lipolysis in human obesity. Diabetes 2005;54(11):3190-3197.

Spalding KL, Arner E, Westermark PO, et al. Dynamics of fat cell turnover in humans. Nature 2008;453(7196):783-787.

Hauguel-de Mouzon

Catalano PM, Hauguel-De Mouzon S. Is it time to revisit the Pedersen hypothesis in the face of the obesity epidemic? Am. J. Obstet. Gynecol. 2011.

Catalano PM, Presley L, Minium J, Hauguel-de Mouzon S. Fetuses of obese mothers develop insulin resistance in utero. Diabetes Care 2009;32(6):1076-1080.

Challier JC, Basu S, Bintein T, et al. Obesity in pregnancy stimulates macrophage accumulation and inflammation in the placenta. Placenta 2008;29(3):274-281.

Lattuada D, Colleoni F, Martinelli A, et al. Higher mitochondrial DNA content in human IUGR placenta. Placenta 2008;29(12):1029-1033.

Metzger BE, Lowe LP, Dyer AR, et al. Hyperglycemia and adverse pregnancy outcomes. N. Engl. J. Med. 2008;358(19):1991-2002.

Hausman

Dodson MV, Hausman GJ, Guan L, et al. Lipid metabolism, adipocyte depot physiology and utilization of meat animals as experimental models for metabolic research. Int. J. Biol. Sci. 2010;6(7):691-699.

Hausman GJ, Barb CR. Adipose tissue and the reproductive axis: biological aspects. Endocr. Dev. 2010;19:31-44.

Hausman GJ, Richardson LR. Histochemical and ultrastructural analysis of developing adipocytes in the fetal pig. Acta. Anat. (Basel) 1982;114(3):228-247.

Hausman GJ, Kasser TR, Martin RJ. The effect of maternal diabetes and fasting on fetal adipose tissue histochemistry in the pig. J. Anim. Sci. 1982;55(6):1343-1350.

Poulos SP, Dodson MV, Hausman GJ. Cell line models for differentiation: preadipocytes and adipocytes. Exp. Biol. Med. (Maywood) 2010;235(10):1185-1193.

Bartness

Bartness TJ, Song CK. Thematic review series: adipocyte biology. Sympathetic and sensory innervation of white adipose tissue. J. Lipid Res. 2007;48(8):1655-1672.

Bartness TJ, Shrestha YB, Vaughan CH, Schwartz GJ, Song CK. Sensory and sympathetic nervous system control of white adipose tissue lipolysis. Mol. Cell. Endocrinol. 2010;318(1-2):34-43.

Bartness TJ, Vaughan CH, Song CK. Sympathetic and sensory innervation of brown adipose tissue. Int. J. Obes. (Lond) 2010;34 Suppl 1:S36-42.

Bowers RR, Festuccia WTL, Song CK, et al. Sympathetic innervation of white adipose tissue and its regulation of fat cell number. Am. J. Physiol. Regul. Integr. Comp. Physiol. 2004;286(6):R1167-1175.

Shrestha YB, Vaughan CH, Smith BJ, et al. Central melanocortin stimulation increases phosphorylated perilipin A and hormone-sensitive lipase in adipose tissues. Am. J. Physiol. Regul. Integr. Comp. Physiol. 2010;299(1):R140-149.

Song CK, Enquist LW, Bartness TJ. New developments in tracing neural circuits with herpesviruses. Virus Res. 2005;111(2):235-249.

Song CK, Vaughan CH, Keen-Rhinehart E, et al. Melanocortin-4 receptor mRNA expressed in sympathetic outflow neurons to brown adipose tissue: neuroanatomical and functional evidence. Am. J. Physiol. Regul. Integr. Comp. Physiol. 2008;295(2):R417-428.

Shulman

Alves TC, Befroy DE, Kibbey RG, et al. Regulation of hepatic fat and glucose oxidation in rats with lipid-induced hepatic insulin resistance. Hepatology 2011;53(4):1175-1181.

Jornayvaz FR, Jurczak MJ, Lee H, et al. A high-fat, ketogenic diet causes hepatic insulin resistance in mice, despite increasing energy expenditure and preventing weight gain. Am. J. Physiol. Endocrinol. Metab. 2010;299(5):E808-815.

Olson DP, Pulinilkunnil T, Cline GW, Shulman GI, Lowell BB. Gene knockout of Acc2 has little effect on body weight, fat mass, or food intake. Proc. Natl. Acad. Sci. USA 2010;107(16):7598-7603.

Ferrante

He J, Xu X, Francisco A, Ferrante A, Krakoff J. Markers of adipose tissue macrophage content are negatively associated with serum HDL-C concentrations. Atherosclerosis 2011;215(1):243-246.

Kosteli A, Sugaru E, Haemmerle G, et al. Weight loss and lipolysis promote a dynamic immune response in murine adipose tissue. J. Clin. Invest. 2010;120(10):3466-3479.

Ortega Martinez de Victoria E, Xu X, Koska J, et al. Macrophage content in subcutaneous adipose tissue: associations with adiposity, age, inflammatory markers, and whole-body insulin action in healthy Pima Indians. Diabetes 2009;58(2):385-393.

Schenk S, Saberi M, Olefsky JM. Insulin sensitivity: modulation by nutrients and inflammation. J. Clin. Invest. 2008;118(9):2992-3002.

Subramanian V, Ferrante AW. Obesity, inflammation, and macrophages. Nestle Nutr. Workshop Ser. Pediatr. Program 2009;63:151-159; discussion 159-162, 259-268.

Sponsors

Speaker:
Jules Hirsch, Rockefeller University

Highlights

• Decades ago fat tissue was thought to be devoid of biochemical activity.
• The obese have a high number of large cells and when they lose weight those cells decrease in size, but not in number.
• The cause of obesity may be unrelated to the societal environment and to everyday behaviors.
• Reduced-obese individuals exhibit a need for fewer calories to maintain their body weight.

Introduction

Adipose tissue is now recognized as the largest endocrine organ in the human body with its multiple adipokines (signaling molecules secreted by adipose tissue), hormone receptors, growth factors, and complex functions affecting the vital processes of reproduction and energy balance as well as the pathology of prevalent chronic diseases. New technology has enabled re-examination of adipogenesis, fat storage, cellularity (the number, quality, and condition of fat cells), and the body's ability to monitor, regulate, and maintain the number of fat cells. This meeting assembled leaders in the field of adipose tissue research who provided a unique overview of, among other topics, a dominant factor in the inflammatory insulin-resistant syndrome often caused by chronic overnutrition: adiposity.

The production and deposition (otherwise called "accretion") of fat droplets in tissue from the fat people consume in their food is a complex process and one that is intimately connected to the etiology of obesity-related diseases, such as insulin-resistance. Although the adipose tissue of obese individuals clearly differs from that of their lower body-mass-index (BMI) counterparts, the relationship between cellular characteristics, such as adipocyte size and number, and the clinical markers used to determine research groups is by no means straightforward. In this field of research, which is so cognizant of its clinical implications, scientists aim to study phenomena that are both theoretically significant and clinically relevant. For this reason, they organize study participants into experimental groups on the basis of markers developed not from cytology, genetics, or the like, but from epidemiology. The terms "overweight" and "obesity" are the two most obvious, and possibly confusing, examples of this convention.

"Overweight" is a functional designation. Someone is overweight if he or she has excess fat that is associated with "a significant impairment of health" (Vander, Sherman, & Luciano's Human Physiology, 2004). But, since these "impairments" cannot be predicted and thus prevented for each individual before they occur, clinicians use statistical associations between an individual's body mass index (a weight measurement, scaled for height) and population-wide incidence of weight-related diseases to determine what weight is healthy for their patients. People are considered "obese" if they are extremely overweight. The difficulty here is both in determining what weight constitutes a health risk and in understanding what changes occur on the cellular level to cause that risk. It is important to note that although the categories "obese" and "non-obese" may seem dichotomous, they in fact describe ranges of clinical and cellular features, and moreover, the dividing line between "clinically obese" and "simply overweight" may not occur at the same BMI as the important transition for the cellular manifestations of obesity. These difficulties are just some of the many issues facing researchers in this area.

Further complexity is introduced through the biological interconversion of dietary fats, blood serum lipids, and cellular fat deposits. Fat is ingested in two primary forms: cholesterol and triglycerides. The former is a precursor of cell membranes, steroid hormones, and other specialized molecules while the latter can serve as a metabolic fuel. Triglycerides from food are broken down into 1 monoglyceride and 2 fatty acids (with various levels of saturation) in the small intestine. From there the components are emulsified and combined with bile salts and phospholipids to form micelles, after which they can diffuse as component parts into epithelial cells lining the intestine. Once the lipids are in epithelial tissue, they go by various routes (some directly into the portal vein and some via the lymphatic system) to the blood and to adipocytes.

Whether these and other circulating lipids are metabolized for fuel or are stored in adipocytes for later use depends on which state, absorptive or post absorptive, the body is in. During the absorptive state ingested nutrients are entering the blood from the intestinal tract, and these nutrients are the body's primary source of energy. During the post absorptive state no nutrients are being absorbed, and the body must use fuels stored during the absorptive state for energy.

When food is abundant, during the absorptive state, both adipose tissue and the liver take up and catabolize glucose for energy. What the adipocytes do not need immediately gets converted into α-glycerol phosphate and fatty acids, which are ultimately linked together to form the triglycerides that make up adipocyte fatty deposits. The liver also synthesizes triglycerides from absorbed glucose, but most of these fats are packaged with specific proteins into aggregates called lipoproteins which circulate in the blood stream. Ultimately, most triglycerides synthesized from glucose in the liver end up being stored in adipose tissue.

The processes occurring during the absorptive phase are regulated by a set of very important hormones—the most influential of which is insulin. In order to understand how insulin resistance is related to weight gain and obesity, a topic on many speakers' minds at the symposium, researchers have spent years investigating insulin's role in many different aspects of metabolic regulation. Insulin, a pancreatic peptide hormone, is released during the absorptive phase, and it acts to stabilize blood glucose levels through a variety of mechanisms. Acting primarily on muscle, adipose, and hepatic tissue, insulin provokes net glucose uptake and utilization as well as many other energy storage processes. In adipose tissue specifically, insulin prompts net triglyceride synthesis, as described above.

By contrast, in the post absorptive state, when insulin levels are low, body tissues cease net glycogen, fat, and protein synthesis, and begin using fat (instead of glucose) to fuel cellular processes. Much of the transition from the absorptive to post absorptive state is mediated by the effects of insulin. One marker of this transition is the initiation of lipolysis in adipose tissue. Lipolysis, the catabolism of triglycerides in adipocyte fat deposits, is an important way for the body to maintain overall blood glucose levels. This process releases fatty acids and glycerol into the blood stream, and once the glycerol reaches the liver it can be used as a precursor in liver glucose synthesis.

Perhaps a more important marker of the post absorptive transition is the metabolic adjustment known as "glucose sparing" in which most organs and tissues reduce their glucose catabolism and begin using fat instead. The body thereby "spares" the glucose for tissues unable to use fat directly, such as the brain. Clinicians and researchers use serum lipid and glucose levels to monitor important metabolic changes over the short term and the long term—to track how obesity and related diseases develop.

A Look Back: 5 Decades of Adipose Tissue Research

Jules Hirsch, of Rockefeller University, kicked off the meeting with his presentation with a reflection on the global clinical importance of fat tissue research: "We are living through a veritable obesity epidemic, and it is one of the great public health menaces that we now have." But despite Herculean efforts on the part of physicians and public health experts to stop or slow the global rise in obesity prevalence there has been very little progress in abating the problem.

Querying the basis of these failures, Hirsch remarked that he has come to the conclusion that the two main factors that permit the epidemic to progress—indolence and ignorance—are not fundamental causes of obesity; they are just factors that make it possible to become obese. According to Hirsch, indolence (lack of behavior regulation) and ignorance (of the relationship between eating behavior and obesity), are necessary but insufficient conditions for the obesity epidemic. "I have absolutely convinced myself that the cause for the epidemic will be found by further study of biologic phenomenon," he said, "Perhaps adipose tissue and the way it functions or other scientific facts about the way we store and handle fat will offer up insight into how to battle the epidemic."

Hirsch began his career researching adipose tissue more than 50 years ago. Early on he was charged with learning about lipid metabolism and about the use of the then burgeoning field of chromatography. He also evaluated the effects of dietary fats on serum lipid content in obese individuals. From these investigations, Hirsch and other researchers in the field learned that unsaturated fats lower the quantity of lipids circulating in blood serum. The researchers also determined that obese and lean individuals can have the same lipid composition in their adipose tissue because whatever type of fat a person routinely eats (be it saturated, unsaturated or polyunsaturated) is the type of fat found in his or her tissue.

"I became thoroughly convinced that people do not become fat because of any different choice of macro-nutrients," said Hirsch. Instead it is related to the quantity of food consumed and the level of energy expenditure, he commented.

Further research on adipose tissue proved profoundly enlightening, Hirsch reflected. In particular, extensive research established that results obtained in rodent model studies could be applied usefully and relatively accurately to humans.

One confounding problem Hirsch faced at this time was how to understand the data in terms of clinical implications: from a cellular biology perspective, the clinically obese were like the clinically non-obese.

Turning their attention to fat tissue cellularity (the number and type of cells in adipose tissue), Hirsch and colleagues found new, more accurate methods for measuring the size of adipocytes in the late 1950s. Optical and other examinations of cell size demonstrated that the size of a person's adipocytes was dependent on his or her nutritional status: the more body fat the individual carried, the larger his or her cells.

Hirsch became intrigued by the cellular manifestation of clinical differences, and he wanted to estimate the number of cells in the tissue as well as the size of each cell. To do this he used a Coulter counter, which was new at the time. Hirsch noted that when he examined the adipocytes of obese, non-obese, and reduced-obese (formerly obese) individuals and discerned who had what kind of cells, it turned out that obese individuals had bigger cells—the expected outcome. Since adipose cells only enlarge to a certain degree, however, obese individuals must have more cells overall.

This slide is a heuristic for the relative size and number of adipocytes in "never obese," "obese," and "reduced-obese" individuals, respectively.

"But my great surprise was that after weight loss occurred in the reduced-obese subjects, the hypercellular situation persisted for long periods of time during the studies that we did." In other words, those who lost weight had a greater number of smaller cells but the same amount of fat than the never obese. "So the obese have a high number of large cells and when they lose weight, the number stays high but the cells shrink and are smaller than those of the non-obese controls," said Hirsh.

Hirsh added: "So what comes first, excess fat cells or overeating?" When non-obese humans are overfed, they gain weight, but only by enlarging existing cells rather than by producing more. When the overfeeding stops they spontaneously lose weight and shrink the same number of cells back to the size they were prior to the period of overfeeding. Is it possible that obesity is a cellular disorder of fat cell number and the 'bad' behaviors (i.e., overeating, under exercising etc.) are secondary phenomena? That is, secondary to having extra cells?"

His research team started looking for underlying biochemical alterations that might accompany the changes in cell size and number. In the early 1970s Hirsch and his colleagues, namely Judith Stern, discovered that cell size and levels of immunoreactive insulin were closely related to each other. "Now we had a biochemical alteration that changed with cell size," said Hirsch.

Until this period researchers had assumed that the state of being obese brought about hypercellularity, an excess of fat cells, but Hirsch and others helped show that this was not the case. Hirsch's particular perspective was influenced by Richard Goss's book The Physiology of Growth as well as by research demonstrating that animals undernourished early in life undergo changes in their cyto-architecture that affect the function and anatomy of their fat tissue permanently.

To close his presentation Hirsch left attendees with the story of one experiment with particular relevance to the question of the lasting impact of early nutritional status on later obesity. Typically, Osborne-Mendel rats, the type used in Hirsch's experiment, grow larger and larger when fed a high fat diet, noted Hirsch. By altering the fat status of a group of these rats very early in life by removing some fatty tissue from each individual via lipectomy, he could analyze the impact of the change in status over time.

After these rats matured, he compared them to non-lipectomized rats that had been fed the same diet, and interestingly, he observed no difference between the two groups of animals in the amount of cellular fat stored. However, animals with lipectomy had larger cells in their adipose tissue than did the rats without the lipectomy. When both adult rat groups were fed a high fat diet, the lipectomized rats showed a leveling off of body weight—at a certain point they no longer found the high fat diet palatable, and their fat cells reached a maximum size. By contrast, the group of rats that began with more adipose tissue cells continued to eat and grow larger.

Although many of the environmental factors and behaviors researchers and public health experts associate with obesity do indeed contribute to weight gain, trying to reduce obesity by changing behavior "may have no relevance to ... the central issue," he qualified. "There may be a central biological defect that ... makes for obesity that is totally unrelated to the societal environment," added Hirsch.

The evidence for this supposition, Hirsch explained, may be in the simple fact that the current modes of therapy are not working. Hirsch concluded with a plea for a focus on basic biological research. He also said that he very strongly believes that whatever obesity's contributing biological or environmental factors, its treatment has nothing to do with the fad diets that are forever waxing and waning in popularity.

Speakers:
Peter Arner, Karolinska Institutet
Sylvie Hauguel-de Mouzon, Case Western Reserve University
Gary Hausman, USDA-Agricultural Research Service

Highlights

• In adults, about 10% of fat cells are replenished and renewed every year.
• Twist1, a transcription factor that has recently been linked to fat cells, is important for regulating inflammatory factors.
• Hypertrophia (larger fat cells) in either lean or obese individuals is associated with decreased insulin sensitivity.
• Glucose is not the only contributing factor to fat growth in the developing human fetus. Lipids present in the mother's circulation also play a role.
• The placenta appears to play a role in adiposity of the fetus.

Adipogenesis: Understanding How Fat Is Formed

Peter Arner, of the Karolinska Institutet in Sweden, reiterated many of the important findings Hirsch discussed about the study of fat cells over the last 50 years. Before delving into his research findings about adipogenesis, Arner discussed his efforts to develop a method to determine the age of fat cells. Using the fact that cells only take up carbon 14 (¹⁴C, a radioactive isotope of carbon) during cell division, Arner and colleagues were able to relate the current level of radioactivity in fat cells' DNA to the change over time in atmospheric radioactivity, which allowed them to determine the age of each cell.

"We were quite surprised when we did these studies," noted Arner, explaining that according to conventional wisdom the fat cells should be very old. However the researchers discovered that in adults, about 10% of fat cells are replaced every year. This finding helped to dispel the old notion that adipocytes are not generated in adulthood, and further investigation revealed that obese individuals showed twice the rate of adipocyte turnover as non-obese individuals. Another interesting finding that Arner noted was the remarkable consistency in the number of fat cells over the course of an adult's lifetime. "It appears that although fat cell size and number are fairly constant, a small portion of fat cells are continually being renewed— but we still don't know why," he said.

An illustration of the process of adipocyte turnover.

When Arner and his team turned their attention to the relationship of fat cell size and fat cell number, the algorithm they developed to describe the relationship helped them to get a quantitative measure of cellularity (i.e., morphology) irrespective of body weight. A person with a given amount of body fat, would have an expected number of fat cells and fat cell size to account for that body fat, according to Arner's algorithm. If an individual had larger fat cells than expected, his or her tissue would be considered hypertrophic by this same algorithm. An individual with the same total amount of body fat but with smaller fat cells would need to have many more fat cells than the person with hypertrophic fat tissue. This second individual would be in the hyperplasia region of the graph shown in the figure below. "This is a way to study fat cell size without worrying about body weight," he said.

Relationship between fat cell size and body fat: an algorithm to obtain body fat independent classification.

Arner's group also learned that those who had hypertrophy have a much slower turnover of adipocytes and that these individuals' fat cells are therefore much older on average; on the other hand, those with hyperplasia were found to have faster turnover of adipocytes resulting in younger fat cells. Turnover affects two features of the adipose tissue, noted Arner: total mass and morphology.

Arner's next task was to try to identify a mechanism for the replacement of old cells with new ones: the driving force behind the turnover of adipose tissue. "The real breakthrough in this field came a few years ago when it was discovered that adipose tissue of obese people is infiltrated by macrophages," he said. It is now believed that inflammation is the culprit that links adipose tissue to diabetes, which often accompanies obesity.

According to Arner, it has been shown that both lean and obese individuals have similar levels of inflammatory factors so, perhaps, humans need inflammation to keep the adipose tissue turnover going. Of course, much more study is needed to understand the relationship between inflammation and adipocyte turnover, if one indeed exists.

Some of that study has already begun in Arner's lab. Arner's team has identified and begun to study a network of newly identified transcription factors that are associated with inflammatory proteins in human fat cells. In particular, he highlighted Twist1, a transcription factor that has recently been linked to fat cells. Biochemical experiments have demonstrated that Twist1 binds to the promoters of the three classical inflammatory genes—MCP-1, IL6, and TNF-α. "So it seems that Twist1 is important for regulating these inflammatory factors, and we think that there are many more transcription factors regulating inflammation and in turn the turnover of fat cells," explained Arner.

Arner concluded by noting that adipocyte turnover is important for obesity and adipose hypertrophy (fewer but larger fat cells), and he reiterated the finding that hypertrophy in either lean or obese individuals is associated with decreased insulin sensitivity. Arner added that he believes that local inflammation, governed by a network of transcription factors, is important for the morphology (cell number and size) of adipose tissue— it's "'a driving force' that perhaps acts as brake on the turnover process so it doesn't proceed too fast," he suggested.

Fetal Adiposity: Fat Accretion

Sylvie Hauguel-de Mouzon of Case Western Reserve University at MetroHealth Medical Center investigates the mechanisms that regulate the growth of the fetus in utero. In her talk Hauguel-de Mouzon was blunt about the formidable to-do list that faces researchers who hope to shed more light on this complex developmental process: "I am interested in fetal fat but the purpose of this talk is to show you that we don't know enough," she said. Investigating the ways in which the human fetus accrues fat cells is vital to understanding adiposity and obesity generally, she noted. The deposition of fat cells, known as adiposity, starts early in the neonatal phase and is associated with a cluster of metabolic functions that involve energy metabolism, energy intake and expenditure, and the immune system. When these functions become dysregulated the result is an increased in the risk of developing obesity later in life.

It is now well established that when a woman has diabetes during pregnancy (whether she has pre-gestational diabetes type 1 or gestational diabetes) the infant's birth weight is likely to be greater than that of an infant born to a woman who does not have diabetes. A similar pattern results in mothers-to-be with pregravid obesity compared to non-obese pregnant women: for the obese mothers there is an increase in the weight and fat mass of the newborn. In addition, obese mothers are also more likely to have an increase in the weight of the placenta.

Adiposity at birth is very different among different species. Rodents, for example are only born with 2–3% body fat, levels in newborn chimpanzees is about 3%–5% compared to human infants with 13%–15%. Low at birth total fat percentage suggests that fat develops after birth.

These phenomena have led Hauguel-de Mouzon and others to query the cause of fetal adiposity. Studies dating back several decades have shown that in early life almost 70% of oxidative metabolism goes to break down glucose while the remaining 30% metabolizes lipids and other substrates. In addition, early research has shown that when plasma glucose levels of a pregnant diabetic woman rise, the glucose levels of her fetus also rise, which produces an increase in fetal insulin and consequently in fetal fat accretion. The results of the "Hyperglycemia and Adverse Pregnancy Outcomes" study (N. Engl. J. Med., 2008; 258) support previous findings that the more glucose (i.e., energy substrate) a fetus receives, the more likely the fetus is to make fat out of it.

But glucose is not the only contributing factor to fat growth. Lipids present in the mother's circulation also play a role, as Hauguel-de Mouzon's review of the current research indicated. She summarized that fetal adiposity under hyperinsulinemia conditions (with excess insulin circulating in the blood) suggests that glucose is the substrate, but if lower fetal insulin levels are present, then fatty acids are the likely substrate for fat deposition. In other words, if fetal insulin is low, fat is more likely to be made out of lipids than out of glucose.

"It is important that, in addition to glucose, lipids from the mother can be used to make fat in the fetus because the obese mothers have more circulating lipids. Hence this would favor fat deposition in their fetuses," she said.

Hauguel-de Mouzon finished her talk by presenting data generated in her own lab that suggest that the placenta also plays a role in adiposity of the fetus. Her team has identified placental genes involved with lipid transfer and lipid synthesis. "In order for lipids to be transported from mother to fetus they need to be first metabolized within the placenta," said Hauguel-de Mouzon. "We have found that the placental genes responsible for lipid transport and synthesis are activated in the placenta of obese/diabetic mothers. This suggests that the placenta will facilitate the transport of lipids available to make fat in the fetus." In summary Hauguel-de Mouzon emphasized that the potential contributors for enhanced fetal fat accretion include maternal diet, the transport and metabolism of lipid substrates in the placenta, and the levels processing of fetal insulin.

Fetal Adipogenesis: Morphological and Molecular Aspects

Gary Hausman, from the USDA-Agricultural Research Service in Athens, Georgia, is also interested in the emergence and development of adipose tissue. Over the past 3 decades Hausman has helped uncover a wealth of valuable information about fetal pig adipogenesis. His results have characterized many features of the emergence of fetal adipose tissue, including the precocious expression of transcription factors of regulatory genes (notably C/EBPα, PPARγ, APO-A1, IGFBPs & TGFβ), the exceptionally early development of connective tissue and vasculature, and the mechanisms of hormonal regulation and the mediators of tissue formation.

Fortunately, the development of subcutaneous adipose layers in fetal pigs provides a good model for understanding the analogous development in humans. Hausman began his talk by introducing attendees to the various subcutaneous layers at different stages of fetal development. In the early 1990s, Hausman and colleagues identified anti-adipocyte monoclonal antibodies named AD-1 and AD-3 that have helped researchers since recognize preadipocytes in the very beginnings of adipose formation, even before lipid accretion begins.

Specifically, immunostaining in fetal and postnatal tissue showed that the adipocyte surface antigen is expressed by preadipocytes and capillary endothelial cells before overt adipogenesis occurs. Analysis of stromal-vascular cells from primary cultures showed preadipocytes before lipid accretion and type IV collagen and lamin expression—two important components of the eventual adipocyte structure.

The busiest time period for adipogenesis in fetal pigs seems to occur between 70–110 days during which period the expression of regulatory factors greatly increases, but signs of development begin to appear between 45 and 50 days. Hausman said, the overall process during these two phases can be characterized by a slight adipocyte size increase, not very much lipoprotein lipase (LPL) activity and scant lipogenesis. In general, very little lipogenesis is occurring during these phases of adipogenesis. Furthermore, adipocyte cluster development is very much dependent on the interaction of arteriolar and connective tissue development. The interaction of arteriolar and connective tissue development results in the physical nature and structure of the adipocyte cluster, noted Hausman. Development of dense connective tissue restricts fat cell cluster development and loose connective tissue permits cluster development.

Hausman went on to note that both fetal adipogenesis and angiogenesis are under central endocrine regulation. When he conducted a fetal hypophysectomy (surgical removal of the pituitary gland) on day 70 of gestation, there was no measurable effect on the animals' body weight, but there was a noticeable change in their specific adipose tissue morphology. The animals without pituitary glands showed a marked increase in adipocyte hypertrophy (the excessive size of adipocytes) and in AD-3 reactivity of the fat cells. They also experienced a 3-fold increase in adipose tissue lipogenesis—the formation of fatty acids and triglycerides—and a 50% decrease in adipocyte cluster development in vivo (in the outer subcutaneous layer) and in vitro.

Microarray studies of gene expression of 70–110 day fetal subcutaneous (SQ) adipose tissue revealed more than 50 expressed secreted factors including interleukins, growth factors, leptin, and many other cytokines, detected in vivo and in vitro and more than 150 expressed regulatory proteins detected in vivo and in vitro.

In his closing remarks Hausman told the group that his studies of tissue development indicate that connective tissue deposition, angiogenesis, and perhaps even blood flow could dictate adipogenesis early on. Furthermore, based on the correlation between the co-expression of important transcription factors, the beginnings of lipid accretion, and the proximity of preadipocytes to functional capillaries during lipid accretion, Hausman suspects that the formation of adipocyte clusters may be the crucial trigger for full formation of adipose tissue.

Speakers:
Tim Bartness, Georgia State University
Gerald Shulman, Yale University School of Medicine
Anthony Ferrante, Columbia University

Highlights

• Using a Pseudorabies virus, scientists identified parts of the brain that may be involved in directing and regulating lipolysis.
• Nearly all classes of immune cells in adipose tissue are quantitatively increased in obese individuals.
• Viewing ATMs (adipose tissue macrophage populations) through a prism of inflammation likely misses important changes induced by obesity in these populations.
• Following a fast or during the early period of weight loss, there is an increase in adipose tissue macrophages that is not associated with an increase in inflammatory gene expression.

Fat Tissue: Innervation and Photoperiodism

Tim Bartness of Georgia State University began his presentation with a brief discussion on a critical biological function in the animal kingdom—the maintenance and loss of adipose tissue. It has long been observed that wild Siberian hamsters peak in body fat during in summer, when the days are longest, and then reduce body fat during winter. This phenomenon, the relationship between biological processes and daylight hours is known as photoperiodism. In the mid 1990s correlative evidence implied that the sympathetic nervous system (SNS) may play a role in this photoperiodic lipid fluctuation.

Further research by Bartness discovered that the SNS releases norepinephrine to innervate white adipose tissue (WAT), and this action is related to lipolysis, the breaking down of stored fat for eventual use as energy. Measurements of the norepinephine (NE) released into fat tissue revealed greater activity on shorter days. Destruction of the SNS nerves to WAT blocked short photoperiod-induced increases in lipid mobilization, he explained.

On a general level, this seasonal lipolysis is a result of increased SNS innervation of WAT, a relationship that establishes a link between fat function and the nervous system—a link beyond hormones.

Bartness and his colleagues then attempted to identify which neural pathways actually innervate WAT. In their experiment they introduce Pseudorabies virus (PRV) into the fat tissue, and the virus is taken up by the neurons that innervate it. That virus is then taken up by the connecting neurons all the way to the brain, thereby tracing the pathway to the brain and identifying parts of the brain that may be involved in regulating lipolysis.

Tim Bartness and his colleagues attempted to identify which neural pathways actually innervate adipose tissue by injecting Pseudorabies virus (PRV) and following its uptake to the brain. White adipose tissue (WAT) is innervated by postganglionic sympathetic nervous system (SNS) neurons, suggesting that lipid mobilization could be regulated by the SNS. When PRV was injected into WAT (EWAT and IWAT, respectively) of hamsters and IWAT of rats, PRV-infected neurons could be visualized by immunocytochemistry. Infected neurons were found in the spinal cord, brain stem midbrain (central gray), and several areas within the forebrain.

Ultimately, Bartness wanted to show that there are neurons in the brain that complete the circuit from the brain via the SNS to WAT. In order to understand the central control mechanisms of lipolysis better, Bartness hoped to identify any neurochemical phenotypes, including the specific neurotransmitters released by and other chemical features of SNS neurons, involved in the WAT pathway. His group also wondered whether it would be possible to trigger or inhibit lipolysis experimentally using particular neurotransmitters upstream of the direct interaction between neurons and fat cells.

His group looked at melanocortins, a group of hormones implicated in food intake and body fat control. The research team's studies revealed that indeed melanocortins activate the neurons that are linked to both WAT and BAT. They demonstrated that when melanocortins are injected in the hamster brain they can in fact induce lipolysis.

At this juncture, Bartness switched gears and spoke about his research team's efforts to understand how norepinephrine acts on adipocytes to cause lipid breakdown. The team was able to develop an in vivo assay to assess the phosphorylation of two proteins, perilipin A and hormone sensitive lipase. Norepinephrine released from the sympathetic nerve terminals innervating WAT triggers a cascade of intracellular signals ultimately resulting in the phosphorylation of these two proteins by protein kinase A (PKA). When perilipin A is phosphorylated, it relinquishes its job of protecting lipid droplets from being attacked by lipases that would cause the breakdown of the stored triacylglycerol (TAG). Perilipin A thus becomes, among other things, a scaffold for phosphorylated hormone-sensitive lipase as the lipase assist in the breakdown (lipolysis) of TAG from the lipid droplets (usually a single lipid droplet in each white adipocyte).

"The development of this assay for in vivo studies allows us ... the ability to know which WAT depots are undergoing lipolysis and to what extent," said Bartness. "Currently, people just measure the products of lipolysis in blood (glycerol and free fatty acids), but they cannot tell from which WAT depots or non-depots (liver, muscle) these products are derived," he added.

Much of the rest of his presentation involved unpublished research that is still in preparation. In short, the team is interested in knowing whether there is evidence that WAT sensory nerves monitor lipolysis. In other words, since the group successfully traced the pathway from brain neural activity to fat tissue, they are now eager to complete the circuit by investigating what chemical or neural signals are transmitted from the fat tissue to the brain. Bartness concluded that there does appear to be two-way neural communication between the brain and WAT.

Molecular Mechanisms of Insulin Resistance

The subject matter for the afternoon presentations then shifted to the mechanisms for insulin resistance at the cellular level. Understanding the molecular mechanisms of insulin resistance remains a major medical battle. Gerald Shulman of Yale University School of Medicine was one of the first in the field to study intramyocellular lipid levels (myocyte triglyceride and fatty acid content) as an important marker of, and perhaps even a player in, insulin resistance. Ultimately, Shulman hopes to shed light on a cellular mechanism responsible for the pathogenesis for type 2 diabetes. His efforts are focused on investigating glucose metabolism in the liver and in skeletal muscle.

Shulman has pioneered the use of magnetic resonance spectroscopy (MRS) to examine intracellular glucose and fat metabolism in humans non-invasively. This process has allowed him to obtain real-time biochemical measurements of intracellular metabolism in humans, which has led to several fundamental advances in the understanding of the regulation of glucose metabolism in the liver and muscles. These advances have particular significance for the understanding of dysregulated glucose metabolism in patients with type 2 diabetes mellitus (T2DM). His talk covered research into how fat and lipids relate to insulin resistance.

Recent studies measuring muscle triglyceride content by biopsy or intramyocellular lipid content by proton magnetic resonance spectroscopy have shown a strong relationship between intramuscular lipid content and insulin resistance in skeletal muscle. "The major reason our patients with type 2 diabetes are insulin resistant is that they can't [convert] glucose into muscle glycogen," said Shulman. In an effort to understand the rate controlling step in this process Shulman's team studied the insulin-regulated glucose transporter Glut4.

"What we found was that in the muscle of the diabetic patient G6P [a metabolic intermediate] is down and glucose is down compared to age-matched controls," said Shulman. The implications from these studies he noted, is that, most likely, the best target for fixing insulin resistance in skeletal muscle would be to find a way to activate Glut4.

The group also investigated whether or not the amount of fat located inside myocytes predicts insulin resistance, and they learned "that the more fat subjects had inside the muscle, the more insulin resistant they were."

Further investigation revealed that somehow fatty acids are directly interfering with insulin activation of Glut4. "We hypothesize that perhaps some intracellular lipid intermediate may be blocking the signaling cascade," he said. So far their research points to diacylglycerol (DAG)-mediated insulin resistance as the primary culprit. Shulman and colleagues believe there is an imbalance between the delivery and oxidation of fatty acids in liver and muscle cells that result in biochemical changes that block insulin signaling.

Shulman's work in rodent models of lipodystrophy similarly revealed DAG-mediated insulin resistance. "It doesn't matter how much fat we have," he said. "What matters ... is how the fat is distributed, and what's causing insulin resistance is the lipid in the myocyte and in the hepatocyte in the form of diacylglycerol. If we really want to fix insulin resistance, the best way to do it, in my mind, will be to melt fat away within the myocyte and hepatocyte," Shulman told the meeting attendees.

Inflammation and Metabolic Dysfunction

The conversation then moved from a discussion of the pathogenic buildup of metabolites in muscles to the detrimental effects of the buildup of inflammatory molecules as a consequence of metabolic dysfunction. Researchers have long understood that obesity and related metabolic disorders increase the concentration of inflammatory molecules found in the circulation and in key metabolic tissues. Studies conducted by Anthony W. Ferrante of Columbia University have shown that obesity-induced inflammation is part of a complex immune response in which macrophages, T-cells, and natural killer (NK) cells are recruited to metabolic organs and tissues during the development of metabolic disorders including obesity, diabetes, and non-alcoholic fatty liver disease.

During his talk, Ferrante focused on how his lab identifies and characterizes the immune cell populations that are altered by obesity and on how the immune system regulates metabolism. His hope is that any new information about these important questions will provide some insight not only into the seemingly pathological response to obesity but also into physiology.

Ferrante discussed an ongoing effort to catalogue the immune cells in adipose tissue, and he showed examples of myeloid cell populations with unusual morphology (very large lipid droplets) as a result of obesity. From this work Ferrante and colleagues conclude that nearly all classes of immune cells in adipose tissue, including all myeloid cell populations, are quantitatively increased by obesity. He also noted that the inflammatory phenotype of adipose tissue contributed by macrophages comes from their quantitative increase not from qualitative changes in overall adipose tissue macrophage (ATMs) populations. Ferrante's ongoing efforts to understand more about what functions ATMs serve has lead the him and his colleagues to investigate what happens to these populations during weight loss. "We know that after sustained weight loss ATMs are reduced, but little is known about the early events during weight loss or about the egress of cells from adipose tissue," he said.

Obesity-induced inflammation is part of a complex immune response in which macrophages, T-cells, and natural killer (NK) cells are recruited to metabolic organs and tissues during the development of metabolic disorders including obesity, diabetes and non-alcoholic fatty liver disease.

In closing Ferrante said he wanted to emphasize that he does not believe that ATMs are distinguishable primarily by their inflammatory profile: "There are alterations that occur that distinguish macrophages between lean and obese animals, a large portion of that reflects their ability to handle lipids," he said. He also noted that lipolysis, at least acute lipolysis, is a primary regulator of adipose tissue macrophage recruitment.

But, "there are many other questions to go," he added.

In many ways, Ferrante's closing remark was a major take away message from the entire day: There have been a lot of recent gains in understanding the adipose tissue, but much remains unknown. Today, it is hard to imagine any changes in fat's public perception anytime soon—the negative connotation of adipose tissue is probably here for a while. Despite this seemingly stable public perception, one day researchers may show that fat does more good than bad.

How does maternal nutrition influence adipogenesis?

What is the root cause of insulin resistance?

What are the effects of exercise on adipose function?

What factors released by adipose tissue macrophages alter adipocyte lipolysis?

What happens to whole adipose tissue metabolism if ATM number is altered?

How is lipolysis monitored?

Are there differences between lipolysis that occurs in obese individuals during a fast or in the early stages of weight loss compared to basal lipolysis that occurs in obese individuals?

What role does the placenta play in lipid transfer towards the fetus?

Which types of lipids are best transferred through the placenta to the fetus of obese mothers?

What are the consequences of placental inflammation for fetal fat synthesis?