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Adipose tissue cellularity in human obesity
1 Adipose Tissue Cellularity in Human Obesity JULES HIRSCH BRUCE BATCHELOR
Clinical data suggest that human obesity may be meaningfully subdivided into several types. It is a common clinical observation that obesity with onset in childhood years is usually more severe and more intractable to treatment than obesity with onset in adult years; furthermore, various measures of behaviour are altered by weight reduction in those with obesity beginning in childhood, but not in adult years (Grinker, Hirsch and Levin, 1973). Methods have been developed for the measurement of adipocyte size in samples of human or animal adipose tissue and also for the estimation of the total number of adipocytes present in all adipose tissue depots (Hirseh and Gallian, 1968). The use of these methods has held promise for establishing morphological or anatomical correlates of the clinical subdivisions of obesity. The likelihood that there may be meaningful differences of adipose tissue morphology in human obesity has been suggested by a variety of studies of experimental animal obesity. Obesity produced by hypothalamic lesions in weanling or adult animals is reflected solely in an increase in the adipose cell size (Hirsch and Hart, 1969), but genetic obesities of rats and mice show either increases in adipose cell size or a combination of increase in adipose cell size and number (Johnson et al, 1971; Johnson and Hirsch, 1972). In addition, during the development of adipose depots in normal rats, adipose cell proliferation occurs early and can be influenced by early nutritional deficit or excess (Knittle and Hirsch, 1968). Previous studies of the cellularity of adipose tissue from obese humans, although suggesting marked differences between the cellularity of the moderately and severely obese, have not as yet provided us with a clear morphological classification of human obesity. Experimental obesity in adult man is characterised by increases in adipose cell size alone with no change in adipose cell number (Salans, Horton and Sims, 1971). Among individuals with spontaneously occurring obesity, those possessing a higher cell number are often found to be those with childhood or juvenile-onset obesity (Hirsch and Knittle, 1970). More recent studies have shown that moderate obesity is most often found in conjunction with only an enlargement in cell size, whereas severe obesity is associated with an increase in cell number as well as with an enlargement in cell size (SjSstrSm and BjSrntorp, 1974). At least one Clinics in E n d o c r i n o l o g y a n d M e t a b o l i s m - -
Vol. 5, No. 2, July 1976.
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study has specifically correlated the age of onset of obesity with hypercellularity (Salans, Cushman and Weismann, 1973). From the above observations, it has been reasonable to characterise obesity as being hypertrophic or hyperplastic. During the past few years, a large number of obese patients has been admitted to our clinical research centre for various studies along with weight reduction. The purpose of this chapter is to review the general and cellular characteristics of these individuals and to determine whether a morphological classification of human obesity can be meaningfully confirmed or clarified at this time. Specifically, the following questions are considered: 1. Can the adipose tissue of the obese be distinguished from that of normal weight individuals? 2. Are there subdivisions of this obese population based upon morphological findings? 3. Does examination of the data in terms of age of onset provide any conclusive classification of obesity? METHODS OF STUDYING ADIPOSE TISSUE CELLULARITY Upon completion of informed written consent, 106 obese individuals were admitted to the Clinical Research Center of the Rockefeller University Hospital. All individuals were selected so as to be within five per cent of their maximum weight by history and to be free of hyperlipidaemia, overt diabetes, endocrine disorders or severe psychiatric disturbances. They were placed on a diet with sufficient calories to maintain body weight, composed of either solid foods or a synthetic formula with 15 per cent of the calories from protein, 40 per cent from fat, and 45 per cent from carbohydrate. Following a routine medical history and physical examination, accurate determinations of height and weight were made. We also attempted to establish the age of onset of obesity as accurately as possible by asking all individuals to provide photographic or medical documentation of the age at which obesity began. In most cases, such confirming records were available. After an overnight fast, subcutaneous adipose tissue samples were obtained by needle aspiration from the mid-triceps region, the abdomen lateral to the umbilicus and the upper outer quadrant of the buttock (Hirsch and Goldrick, 1964). Samples were placed in Krebs--Ringer bicarbonate buffer, pH ---- 7.4, at 37°C and gassed with 95 per cent 02, five per cent CO2. Within the hour, some fragments of tissue were subjected to lipid extraction and other fragments were fixed in osmium tetroxide for determination of cell number by counting in a Coulter counter, as previously described (Hirsch and Gallian, 1968). The average cell size was considered to be the mean of the cell sizes determined from the three individual sites, i.e. triceps, abdomen, and buttock. Total body fat was estimated in each subject as the difference between body weight and lean body mass. The latter was determined from the space occupied by 75 to 100 ~Ci of tritiated water after four hours of equilibration, using the formula of Pace and Rathbun (1945). Total adipocyte number was estimated by dividing total body fat by the mean cell size.
ADIPOSE TISSUE CELLULARITY IN HUMAN OBESITY
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Comparative data were collected from 25 non-obese volunteers who were examined either as outpatients or as in-hospital members of the Rockefeller University Clinical Research Volunteer Program. Adipose cell sizes were determined as described above, but total body fat was estimated from measures of height and weight using formulae devised by Mellits and Cheek (1970). Calculations were then made of the total cell number, as described above. All data were placed on file in an IBM 360 computer and when retrieved treated by commonly used statistical tests, such as Student's 't' test, the Pearson product moment correlation coefficient and chi-square analysis. RESULTS Clinical data on the obese and non-obese populations are shown in Table 1. All are of similar age, but the obese population contains a higher female to male ratio, not necessarily due to a higher incidence of obesity in females but, more likely, the greater willingness of female obese to be hospitalised, studied and treated. Overall, the obese patients weighed almost twice as much as the non-obese and had an average of twice as much body fat (46 per cent vs. 23 per cent). Table 1. A comparison of non-obese and obese subjects
Non-obese (n = 25) Age in years Sex Weight in kg Body fat in kg (% body wt.)
32.6 9F, 68.8 15.5
_+ 3.0 16M _ 2.0 _+ 0.7 (23%)
Obese (n = 106) 34.1 -!-_0.9 68F, 38M 129.3 _ 3.6 58.9 _+ 2.2 (46%)
Adipose cellularity and severity of obesity Data on average adipocyte size expressed as /~g lipid per cell and total adipocyte number were plotted as a function of the degree of obesity. A variety of indices was used to estimate the severity of obesity, e.g. ponderal index, absolute amount of fat, percentage body fat, the ratio of fat to lean mass, and percentage ideal body weight. All indices gave qualitatively similar results. The findings using percentage ideal body weight are shown in Figures 1 and 2. Cell size increases sharply as percentage ideal body weight increases. Clearly, the major change in adipocyte size occurs with only moderate degrees of obesity. This is shown as Group I in Figure 1, which includes all subjects with body weight increases up to 170 per cent above normal. Beyond this level of obesity, there is little further increase in adipocyte size. The third group (III), containing subjects who might be termed the super-obese with weights in excess of 240 per cent of ideal, show greater variance of adipocyte size, but little increase in cell size. It must be emphasised that these three groups were arbitrarily devised on the basis of the cellular data obtained; thus, they cannot be used to predict the cellular events in the progression of
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obesity in a given individual, but can be a useful subdivision of obesity on morphological grounds. Cell number, in distinction to cell size, continues to m o u n t as the severity of obesity increases. The same division into three groups as shown in Figure 1, is also shown in Figure 2, relating cell n u m b e r to severity. It is evident that cell n u m b e r is least different from n o r m a l in the moderately obese, but increases most sharply in Groups II and III. 20
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Figure 1. Ideal body weight was determined from the new weight standards for men and women in the Statistical Bulletin of the Metropolitan Life Insurance Co., 40, 1-4 (1959). Cell size was determined as described in the text and represents the average of three subcutaneous sites. It would a p p e a r from these data that cell size reaches a m a x i m u m at an average of roughly 1.0 tag lipid per cell and this maximal cell size can be achieved with only moderately severe obesity. Cell number, on the other hand, correlates more closely with the degree of obesity. The correlation coefficient of cell n u m b e r and percentage ideal body weight is 0.74 for this group of subjects, but the correlation of cell size and percentage ideal body weight is only 0.42. Hence, obesity of any degree is 'hypertrophic' and cell size increase is the distinguishing morphological feature of h u m a n obesity; hyperplasia, on the other hand, is best correlated with severity and becomes most evident when body weight exceeds a level of 170 per cent above ideal. -
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It is of further interest to note that within the non-obese group, there is no evidence of an increase in cell number as percentage ideal weight differs within ± 5 per cent of normal; however, cell size (Figure 1) shows a suggestion of varying as a function of percentage ideal body weight even in the non-obese. The three high cell number points in the non-obese group in Figure 2 are difficult to explain on any clinical grounds. These subjects denied previous obesity or any problems which might have caused weight variation. These represent three of the four points under 'no' in Figure 1. If these three points were deleted, there would be an even sharper relationship of cell size to ideal body weight in the non-obese group, but no suggestion of such a relationship with cell number. These findings are summarised in Figure 3. Note the stepwise increases in body weight from group 'No', the non-obese, through Groups I, II and III. The major change in cell size is from non-obese to Group I, but changes in cell number are small when comparing the non-obese state to Group I and then cell number increases in large steps as obesity becomes more severe. Clearly, cell size alteration is a ' m a r k e r ' for obesity of any degree, but cell number is the better index of severity. Obese groups vs Non-obese Kg
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Using the same categorisation of obesity based on three levels of increase in percentage ideal body weight (I = 0 to 170 per cent; II ---- 160 to 240 per cent; I I I = >240 per cent), it is possible to examine the relationship of the severity of obesity to the age of onset. Ages of onset were arbitrarily divided into prepubertal (age one to eight), pubertal (age 9 to 12), adolescent (age 13 to 20) and adult (> age 20). From the first vertical column of Table 2, it can be seen that overall the highest numbers of subjects are found in those groups with early onset of obesity. Clearly, in this group of patients, the majority became obese at an
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early age. This observation emerges with greatest statistical validity (P <0.001) from the total analysis by chi-square. It is of interest, however, that even those with early ages of onset (the first horizontal row) are distributed in all three categories of severity. Examining those with most severe obesity (vertical column, Group III), it would appear that all ages of onset are represented but with some clustering of increased numbers of those with early ages of onset which does not achieve statistical significance in this type of analysis. In Group II, the same tendency of those with earlier ages to predominate just achieves significance at a level of P <0.01. Thus, it can be concluded that the population under study is heavily weighted with those having an early onset of obesity and there is some tendency for those with early onset to be found in the groups of greatest severity. Yet, even those with early onset can have a moderate type of obesity and, contrariwise, a few subjects with later onset can have marked obesity and may be found in Groups II and III. Table 2. Age of onset vs. groups Age of onset
1--8 9--12 13--20 >20
Group n
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II
III
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8 7 9 5
22 12 7 9
11 9 4 2
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3.33
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7.95 1.35 1.91 4.54
ap<0.01. Arrows point to the 'chi-square' determined for each column and each row. bp<0.001.
The next two tables (3 and 4) examine the relationship of cellularity to age of onset. In Table 3 there is a division of cell size into three arbitrary categories (<0.9 lag lipid per cell; 0.9 to 1.1 lag lipid per cell; >1.1 lag lipid per cell). This division was made to ensure reasonable numbers of subjects in each category. At all cell sizes there is a tendency to find increased numbers at early ages of onset, no doubt a reflection of the population studied, but an examination of the matrix horizontally, along rows, shows that any cell size can be found in obesities beginning at any age. Those with early or late onset do not tend to be more or less hypertrophic. Table 3. Age o f onset vs. cell s&e Age of onset
1--8 9--12 13--20 >20
n
<0.9 tag lipid/cell
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14 7 7 6
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13 9 3 6
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7.52
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0.05 1.35 1.71 0.49
Arros points to the 'chi-square' determined for each column and each row. None is significant.
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Table 4. Age of onset vs. cell number Age of onset 1--8 9--12 13--20 >20
n
<40 X 109
40-60 X 109
>60 X 109
41 28 20 16
6 7 4 2
12 8 10 9
23 13 6 5
3.91
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10.87a 2.21 2.81 4.62
ap<0.01" Arrows point to the 'chi-square' determined for each column and each row. bp<0.001.
Table 4 shows an analysis of cell number as a function of age of onset. The most hyperplastic group (>60 X 109 adipocytes) has the largest cluster of early onset obese. Of equal importance, those with early onset (first horizontal row) are significantly different in their pattern of cellularity, showing the highest number of individuals in the high cell number group. One can conclude, therefore, that: (1) obesity beginning at any age can be of any degree of severity, although it appears that those with early onset are more likely to be found in groups of greater severity, (2) cell size is similar in obesities beginning at any age, and (3) the most hypercellular varieties Of obesity are found in the early onset group. An absolutely clear classification of early onset and hyperplastic vs. late onset and hypertrophic obesity cannot be made, probably for two types of reasons: (a) Severity, age of onset and cell number are all interrelated. Many subjects with early onset can develop only moderate degrees of obesity, but in this population more of those with early onset developed marked obesity. Furthermore, severity (without regard to age of onset) has a high correlation with cell number. If one analyses this matter with partial correlation coefficients, the following is found: the partial correlation coefficient, 'r', of cell number vs. percentage ideal body weight (with age of onset held constant) = 0.74. The correlation of cell number vs. age of onset (with percentage ideal body weight held constant) = --0.17, an insignificant correlation. One reason for this low and insignificant correlation is the existence of many subjects with early onset who developed only mild obesity with little or no hypercellularity. An additional confounding feature is discussed next. (b) Some individuals with late onset obesity can be hypercellular. An analysis of individual subjects as to age of onset, severity and cellularity is made in the next three figures., Figure 4 is a graph of all subjects with cell number as ordinate and cell size as abscissa. The various levels of severity cluster together into bands covering broad areas of this: cell size vs. cell number graph. If each subject is to be examined as to the relative degree of hyperplasia or hypertrophy which comprises the obesity, a graph such as that of Figure 5 is useful. Here the relative increase in cell number, i.e. cell number divided by mean non-obese cell number, is plotted against the relative increase in cell size. The radiating triangular areas represent zones with various degrees of hyperplasia. The uppermost triangles, to the left,
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contain those subjects with most hyperplasia and least hypertrophy progressing down and to the right to those subjects with least hyperplasia and most cell enlargement. The curved lines enclose bands of equal severity. Thus, from each triangle or each set of curved lines, subjects can be examined who have either equal degrees of hyperplasia or equal severity.
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Figure 5. Cell size and number data for each obese subjects are shown as ratios, i.e. size or number divided by the average size or number of the non-obese group. The radiating lines, from number ----- 1.0, size = 1.0, encompass zones in which relative hypertrophy or hyperplasia predominate. A precisely horizontal line would cover those points for which hypertrophy was the exclusive finding, a line at 45 ° would cover points for which there was equal hypertrophy and hyperplasia, etc. The concave arcs are lines of equivalent fat mass. Along any such line, all subjects would have equal fat mass, although widely different degrees of hyperplasia and hypertrophy. The arcs and radiating lines enclose a larger number of four-sided figures which are 'linearised' in Figure 6.
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ADIPOSE TISSUE CELLULARITY IN HUMAN OBESITY
In Figure 6 the various curves and triangles are converted into more traditional rectilinear coordinates and each individual can be separately considered as to severity, age of onset and relative degree of hyperplasia. There can be little doubt that an occasional individual with adult onset, as well as can be determined by medical history and supportive photographs, can have both massive and hypercellular obesity. -
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Figure 6. Data from the four-sided figures in Figure S are shown as a matrix. The ordinate represents severity of obesity in terms of absolute fat mass present and the abscissa, relative hyperplasia. A 45 ° line from coordinates 1.0, 1.0 in Figure 5 are shown as double lines equal to 50:50 - - hyperplasia:hypertrophy. Clearly, hyperplasia and severity are related; yet, some adult onset cases can be found in boxes denoted as both hyperplasia and high severity. Also, childhood onset obesity is distributed rather equally throughout the matrix.
DISCUSSION It would be helpful to arrive at a simple classification of obesity relating age of onset to adipose tissue morphology. Ideally such a classification should be based not only on observations of the obese but also on firm knowledge of the cellular events that determine normal growth and development of the adipose mass. To some degree, this is now possible when considering experimental models of obesity in rodents. Recent data (Greenwood and Hirsch, 1974) suggest that adipocyte development in the Sprague Dawley rat begins with a stage of intense cellular replication in the early weeks of postnatal life. The rapidly proliferating cells or 'adipoblasts' are morphologically indistinguishable from other supportive cells in adipose tissue; however, by a measure of labelled thymidine incorporated into the nuclei of adipoblasts and later analysis of label incorporated into mature adipocytes, some reasonable guesses can be made as to the further development of the tissue. At roughly three to five weeks of age, most of the proliferation has ceased, but a bed of unfilled adipocytes exist; such cells migfit be termed 'pre-adipocytes.' The pre-adipocytes slowly fill over ensuing weeks and the full complement of adipocytes is achieved by 12 to 14 weeks of age. Thereafter no new adipocytes are added to the tissue and indeed there is no apparent turnover of adipocyte DNA.
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JULES HIRSCH AND BRUCE BATCHELOR
Under or over-nutrition during the time of adipoblast proliferation leads to a permanent change in both cell number and cell size; whereas, later nutritional alterations change only cell size. Thus, those obesities of rodents which are manifest at early ages such as the genetic obesity occurring in the Zucker rat are found to be a mixed hyperplastic--hypertrophic type. Obesities which occur later such as those produced by hypothalamic lesions or even by genetic influences acting at a later date, for example the Danforth or Yellow mouse obesity, are exclusively hypertrophic. A picture emerges, therefore, which is schematically shown in Figure 7. Normal development is shown in the top panel as an ordered sequence of cellular progression: a d i p o b l a s t - - ~ pre-adipocyte -~ adipocyte. The lower panel shows two types of known disorders of adipocyte development: early influences leading to permanent alterations in cell size and number and later influences provoking a more pure hypertrophic obesity. With our present understanding of this situation, it is easier to speculate on mechanisms whereby early nutritional effects can provoke alterations in cell number. The mechanisms which could lead to permanent change in cell size are unknown, yet it must be emphasised that cellular hypertrophy is a universal accompaniment of animal obesity, as is the case with h u m a n obesity.
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Figure 7. A schematic representation of cellular differentiation in rodent adipose tissue. The upper panel represents normal development. The lower panel shows two modes of abnormal development. The thick arrows indicate forces acting either early (left) to provoke a hyperplastic --hypertrophic obesity or later (right) leading to purely hypertrophic obesity.
A similar classification of obesity in man is made difficult by uncertainty as to the course of events in h u m a n adipose cellular development. There are, however, several helpful observations. It seems quite definite that purposeful overfeeding in adult man leads only to cellular enlargement. Furthermore, when both young and old obese subjects are reduced, the cell number does not decline. This gives at least some assurance that mature h u m a n adipocytes unlike erythrocytes or hepatocytes do not 'turn over' and that very likely in the adult state there is not a significant number of recruitable cells which can become adipocytes under conditions of increased caloric storage. There is as yet some reasonable debate as to whether or not the situation may be different in the hog (Widdowson and Shaw, 1973), but in man the best guess
ADIPOSE TISSUE CELLULARITY IN HUMAN OBESITY
309
is that there is a fixed adipocyte number that prevails in adult life. The other set of useful observations comes from the work of Salans, Cushman and Weismann (1973) and Sjostrom and Bjorntorp (1974), who have shown, as the data presented above support, that childhood onset obesity, severity and hypercellularity are all closely related. The subjects studied by Salans, Cushman and Weismann (1973) had a ~ar clearer relationship between hypercellularity and early onset than was demonstrable from our own data. Given the uncertainties imposed by patient selection and the difficulty in ascertaining age of onset with absolute precision, some of these differences may be reconciled. Yet it appears incontrovertible from our data that some patients with definite adult onset may be hyperplastic. This observation is strengthened by a recent publication of Bray and Gallagher (1974) in which they found at least one subject with onset of obesity in adult years related to hypothalamic dysfunction due to a lipoma in the interpeduncular fossa. This subject was found to have hypercellularity. It can, of course, be argued that in this patient or, for that matter, in any adult subject who becomes obese, the seeds were sown earlier and a hyperplastic bed of adipocytes were awaiting a nutritional environment permitting later filling and thereby the expression of obesity. A schema which summarises some of these possibilities as well as other unknowns of h u m a n adipose tissue development is shown in Figure 8. The uppermost panel shows the rapid progression of adipoblast to pre-adipocyte occurring early, perhaps in the first year of life, and the remaining growth of the tissue occurring more slowly as pre-adipocytes fill through childhood and adolescence. Were this the correct situation, then the notions of Brook (1972) Adipocyte Development-Man Cell size
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Figure 8. A schematic representation of possible modes of cellular differentiation in h u m a n adipose tissue. The uppermost panel assumes an early single phase of cellular replication (adipoblasts). The middle panel assumes a continued, although intermittent period of cellular
multiplication. The two lower panels show thickened arrows representing nutritional or other forces leading to hypertrophyand excessivehyperplasia. The lower left shows the effect of forces acting at different ages, assumingthat there is a single early phase of adipoblast multiplication. Hypertrophic or mixed hypertrophic--hyperplasfic obesity can occur. The lower right schema indicates the possibilitythat obesitywith onset after childhood or infancy can be hyperplasfic and hypertrophic, more in keeping with the developmentalsequence shown in the middle panel.
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as well as others that the first year of life is a particularly 'critical' period for ultimate adipocyte number would appear fully justified. The middle panel, on the other hand, seems an equally plausible schema of events. Man, unlike rodents, has rapid growth early, a long period of slower growth during childhood and then a pubertal and early adolescent burst of further growth. The middle schema assumes that the ultimate precursor cell or adipoblast can continue to divide in childhood and perhaps even later, for example under such influences as the hormonal alteration of pregnancy or other situations. Thus, puberty and early adolescence are 'critical' times since there is never a long period of already manufactured 'pre-adipocytes' as shown in the upper panel, but there could be critical periods at even later dates. Based on one or the other of the above possibilities, one can visualise the cellular sequence in obesity as shown in the lowest panel of this figure. Either the die is cast by early adipoblast proliferation and obesity becomes manifest by recruiting unfilled adipocytes at various ages, but always with over-filling and hence hypertrophy or, as shown at the lower right, forces can act at various later dates to provoke further cellular proliferation. In any event, the age of onset of obesity as correlated with hypercellularity need not always be found in earliest childhood nor even in puberty or adolescence. Yet adipoblast proliferation is greatest early in life and in most instances hypercellularity and early onset will be related. The investigation of the details of adipocyte development in man and animals remains an active area of investigation and hopefully the full facts will emerge shortly and permit a simpler classification of human obesity. The even larger unknown of the clinical significance of hypercellularity or hypertrophy and how such cellular changes modify the metabolic state of the obese is yet to be thoroughly studied.
SUMMARY All human obesity is characterised by adipocyte hypertrophy and when body weight exceeds 170 per cent of ideal, a maximum cell size of roughly twice normal is achieved. With greater severity, hyperplasia becomes increasingly manifest and when body weight exceeds 170 per cent of ideal, the degree of hyperplasia is well correlated with severity. Although severity and hypercellularity are often found in those with early onset of obesity, individuals can be found who are hypercellular but have had a later onset of obesity. Until the details of cellular development in man are more fully understood, the precise timing of 'critical' periods for cellular development must remain speculative.
ACKNOWLEDGEMENTS Work reported in this chapter was supported by Research Grants HD-03719 and RR-00102 from the National Institutes of Health. The superb technical assistance of Miss Florence Oetien is gratefully acknowledged.
ADIPOSE TISSUE CELLULARITY IN HUMAN OBESITY
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REFERENCES Bray, G. A. & Gallagher, R. F. (1974) Manifestations of hypothalamic obesity in man: A comprehensive investigation of eight patients and a review of the literature. Medicine, 54, 301-330. Brook, C. G. D. (1972) Evidence for a sensitive period in adipose cell replication in man. Lancet, ii, 624-627. Greenwood, M. R. C. & Hirsch, J. (1974) Postnatal development of adipocyte cellularity in the normal rat. Journal of Lipid Research, 15, 474-483. Grinker, J., Hirsch, J. & Levin, B. (1973) The affective responses of obese patients to weight reduction: A differentiation based on age at onset of obesity. Psychosomatic Medicine, 35, 57-63. Hirsch, J. & Gallian, E. (1968) Methods for the determination of adipose cell size in man and animals. Journal of Lipid Research, 9, 110-119. Hirsch, J. & Goldrick, R. B. (1964) Serial studies on the metabolism of human adipose tissue. I. Lipogenesis and free fatty acid uptake and release in small aspirated samples of subcutaneous fat. Journal of Clinical Investigation, 43, 1776-1792. Hirsch, J. & H a n , P. W. (1969) Cellularity of rat adipose tissue: Effects of growth, starvation and obesity. Journal of Lipid Research, 10, 77-82. Hirsch, J. & Knittle, J. L. (1970) Cellularity of obese and nonobese human adipose tissue. Federation Proceedings, 29, 1516-1521. Johnson, P. R. & Hirsch, J. (1972) Cellularity of adipose depots in six strains of genetically obese mice. Journal of Lipid Research, 13, 2-11. Johnson, P. R., Zucker, L. M., Cruee, J. A. F. & Hirsch, J. (1971) Cellularity of adipose depots in the genetically obese Zucker rat. Journal of Lipid Research, 12, 706-714. Knittle, J. L. & Hirsch, J. (1968) Effect of early nutrition on the development of rat epididymal fat pads: Cellularity and metabolism. Journal of Clinical Investigation, 47, 2091-2098. Mellits, E. D. & Cheek, D. B. (1970) The assessment of body water and fatness from infancy to adulthood. In Physical Growth and Body Composition: Papers from the Kyoto Symposium on Anthropological Aspects of Human Growth (Ed.) Brozek, Joseph. Vol. 35, no. 7, pp. 12-26. Chicago: University of Chicago Press. Pace, N. & Rathbun, E. N. (1945) Studies on body composition, III. The body water and claemically combined nitrogen content in relation to fat content. Journal of ]~'ological Chemistry, 158, 685-691. Salans, L. B., Cushman, S. & Weismann, R. E. (1973) Studies of human adipose tissue, adipose cell size and number in nonobese and obese patients. Journal of Clinical Investigation, 52, 929-941. Salans, L. B., Horton, E. S. & Sims, E. A. H. (1971) Experimental obesity in man: Cellular character of the adipose tissue. Journal of Clinical Investigation, 50, 1005-1011. Sj6str6m, L. & Bj6rntorp, P. (1974) Body composition and adipose tissue cellularity in human obesity. Acta Medica Scandinavica, 195, 201-211. Widdowson, E. M. & Shaw, W. T. (1973) Full and empty fat cells Lancet, ii, 905.
Clinical data suggest that human obesity may be meaningfully subdivided into several types. It is a common clinical observation that obesity with onset in childhood years is usually more severe and more intractable to treatment than obesity with onset in adult years; furthermore, various measures of behaviour are altered by weight reduction in those with obesity beginning in childhood, but not in adult years (Grinker, Hirsch and Levin, 1973). Methods have been developed for the measurement of adipocyte size in samples of human or animal adipose tissue and also for the estimation of the total number of adipocytes present in all adipose tissue depots (Hirseh and Gallian, 1968). The use of these methods has held promise for establishing morphological or anatomical correlates of the clinical subdivisions of obesity. The likelihood that there may be meaningful differences of adipose tissue morphology in human obesity has been suggested by a variety of studies of experimental animal obesity. Obesity produced by hypothalamic lesions in weanling or adult animals is reflected solely in an increase in the adipose cell size (Hirsch and Hart, 1969), but genetic obesities of rats and mice show either increases in adipose cell size or a combination of increase in adipose cell size and number (Johnson et al, 1971; Johnson and Hirsch, 1972). In addition, during the development of adipose depots in normal rats, adipose cell proliferation occurs early and can be influenced by early nutritional deficit or excess (Knittle and Hirsch, 1968). Previous studies of the cellularity of adipose tissue from obese humans, although suggesting marked differences between the cellularity of the moderately and severely obese, have not as yet provided us with a clear morphological classification of human obesity. Experimental obesity in adult man is characterised by increases in adipose cell size alone with no change in adipose cell number (Salans, Horton and Sims, 1971). Among individuals with spontaneously occurring obesity, those possessing a higher cell number are often found to be those with childhood or juvenile-onset obesity (Hirsch and Knittle, 1970). More recent studies have shown that moderate obesity is most often found in conjunction with only an enlargement in cell size, whereas severe obesity is associated with an increase in cell number as well as with an enlargement in cell size (SjSstrSm and BjSrntorp, 1974). At least one Clinics in E n d o c r i n o l o g y a n d M e t a b o l i s m - -
Vol. 5, No. 2, July 1976.
299
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study has specifically correlated the age of onset of obesity with hypercellularity (Salans, Cushman and Weismann, 1973). From the above observations, it has been reasonable to characterise obesity as being hypertrophic or hyperplastic. During the past few years, a large number of obese patients has been admitted to our clinical research centre for various studies along with weight reduction. The purpose of this chapter is to review the general and cellular characteristics of these individuals and to determine whether a morphological classification of human obesity can be meaningfully confirmed or clarified at this time. Specifically, the following questions are considered: 1. Can the adipose tissue of the obese be distinguished from that of normal weight individuals? 2. Are there subdivisions of this obese population based upon morphological findings? 3. Does examination of the data in terms of age of onset provide any conclusive classification of obesity? METHODS OF STUDYING ADIPOSE TISSUE CELLULARITY Upon completion of informed written consent, 106 obese individuals were admitted to the Clinical Research Center of the Rockefeller University Hospital. All individuals were selected so as to be within five per cent of their maximum weight by history and to be free of hyperlipidaemia, overt diabetes, endocrine disorders or severe psychiatric disturbances. They were placed on a diet with sufficient calories to maintain body weight, composed of either solid foods or a synthetic formula with 15 per cent of the calories from protein, 40 per cent from fat, and 45 per cent from carbohydrate. Following a routine medical history and physical examination, accurate determinations of height and weight were made. We also attempted to establish the age of onset of obesity as accurately as possible by asking all individuals to provide photographic or medical documentation of the age at which obesity began. In most cases, such confirming records were available. After an overnight fast, subcutaneous adipose tissue samples were obtained by needle aspiration from the mid-triceps region, the abdomen lateral to the umbilicus and the upper outer quadrant of the buttock (Hirsch and Goldrick, 1964). Samples were placed in Krebs--Ringer bicarbonate buffer, pH ---- 7.4, at 37°C and gassed with 95 per cent 02, five per cent CO2. Within the hour, some fragments of tissue were subjected to lipid extraction and other fragments were fixed in osmium tetroxide for determination of cell number by counting in a Coulter counter, as previously described (Hirsch and Gallian, 1968). The average cell size was considered to be the mean of the cell sizes determined from the three individual sites, i.e. triceps, abdomen, and buttock. Total body fat was estimated in each subject as the difference between body weight and lean body mass. The latter was determined from the space occupied by 75 to 100 ~Ci of tritiated water after four hours of equilibration, using the formula of Pace and Rathbun (1945). Total adipocyte number was estimated by dividing total body fat by the mean cell size.
ADIPOSE TISSUE CELLULARITY IN HUMAN OBESITY
30]
Comparative data were collected from 25 non-obese volunteers who were examined either as outpatients or as in-hospital members of the Rockefeller University Clinical Research Volunteer Program. Adipose cell sizes were determined as described above, but total body fat was estimated from measures of height and weight using formulae devised by Mellits and Cheek (1970). Calculations were then made of the total cell number, as described above. All data were placed on file in an IBM 360 computer and when retrieved treated by commonly used statistical tests, such as Student's 't' test, the Pearson product moment correlation coefficient and chi-square analysis. RESULTS Clinical data on the obese and non-obese populations are shown in Table 1. All are of similar age, but the obese population contains a higher female to male ratio, not necessarily due to a higher incidence of obesity in females but, more likely, the greater willingness of female obese to be hospitalised, studied and treated. Overall, the obese patients weighed almost twice as much as the non-obese and had an average of twice as much body fat (46 per cent vs. 23 per cent). Table 1. A comparison of non-obese and obese subjects
Non-obese (n = 25) Age in years Sex Weight in kg Body fat in kg (% body wt.)
32.6 9F, 68.8 15.5
_+ 3.0 16M _ 2.0 _+ 0.7 (23%)
Obese (n = 106) 34.1 -!-_0.9 68F, 38M 129.3 _ 3.6 58.9 _+ 2.2 (46%)
Adipose cellularity and severity of obesity Data on average adipocyte size expressed as /~g lipid per cell and total adipocyte number were plotted as a function of the degree of obesity. A variety of indices was used to estimate the severity of obesity, e.g. ponderal index, absolute amount of fat, percentage body fat, the ratio of fat to lean mass, and percentage ideal body weight. All indices gave qualitatively similar results. The findings using percentage ideal body weight are shown in Figures 1 and 2. Cell size increases sharply as percentage ideal body weight increases. Clearly, the major change in adipocyte size occurs with only moderate degrees of obesity. This is shown as Group I in Figure 1, which includes all subjects with body weight increases up to 170 per cent above normal. Beyond this level of obesity, there is little further increase in adipocyte size. The third group (III), containing subjects who might be termed the super-obese with weights in excess of 240 per cent of ideal, show greater variance of adipocyte size, but little increase in cell size. It must be emphasised that these three groups were arbitrarily devised on the basis of the cellular data obtained; thus, they cannot be used to predict the cellular events in the progression of
302
JULES HIRSCH AND BRUCE BATCHELOR
obesity in a given individual, but can be a useful subdivision of obesity on morphological grounds. Cell number, in distinction to cell size, continues to m o u n t as the severity of obesity increases. The same division into three groups as shown in Figure 1, is also shown in Figure 2, relating cell n u m b e r to severity. It is evident that cell n u m b e r is least different from n o r m a l in the moderately obese, but increases most sharply in Groups II and III. 20
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Figure 1. Ideal body weight was determined from the new weight standards for men and women in the Statistical Bulletin of the Metropolitan Life Insurance Co., 40, 1-4 (1959). Cell size was determined as described in the text and represents the average of three subcutaneous sites. It would a p p e a r from these data that cell size reaches a m a x i m u m at an average of roughly 1.0 tag lipid per cell and this maximal cell size can be achieved with only moderately severe obesity. Cell number, on the other hand, correlates more closely with the degree of obesity. The correlation coefficient of cell n u m b e r and percentage ideal body weight is 0.74 for this group of subjects, but the correlation of cell size and percentage ideal body weight is only 0.42. Hence, obesity of any degree is 'hypertrophic' and cell size increase is the distinguishing morphological feature of h u m a n obesity; hyperplasia, on the other hand, is best correlated with severity and becomes most evident when body weight exceeds a level of 170 per cent above ideal. -
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ADIPOSE
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303
OBESITY
It is of further interest to note that within the non-obese group, there is no evidence of an increase in cell number as percentage ideal weight differs within ± 5 per cent of normal; however, cell size (Figure 1) shows a suggestion of varying as a function of percentage ideal body weight even in the non-obese. The three high cell number points in the non-obese group in Figure 2 are difficult to explain on any clinical grounds. These subjects denied previous obesity or any problems which might have caused weight variation. These represent three of the four points under 'no' in Figure 1. If these three points were deleted, there would be an even sharper relationship of cell size to ideal body weight in the non-obese group, but no suggestion of such a relationship with cell number. These findings are summarised in Figure 3. Note the stepwise increases in body weight from group 'No', the non-obese, through Groups I, II and III. The major change in cell size is from non-obese to Group I, but changes in cell number are small when comparing the non-obese state to Group I and then cell number increases in large steps as obesity becomes more severe. Clearly, cell size alteration is a ' m a r k e r ' for obesity of any degree, but cell number is the better index of severity. Obese groups vs Non-obese Kg
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Using the same categorisation of obesity based on three levels of increase in percentage ideal body weight (I = 0 to 170 per cent; II ---- 160 to 240 per cent; I I I = >240 per cent), it is possible to examine the relationship of the severity of obesity to the age of onset. Ages of onset were arbitrarily divided into prepubertal (age one to eight), pubertal (age 9 to 12), adolescent (age 13 to 20) and adult (> age 20). From the first vertical column of Table 2, it can be seen that overall the highest numbers of subjects are found in those groups with early onset of obesity. Clearly, in this group of patients, the majority became obese at an
304
JULES HIRSCH AND BRUCE BATCHELOR
early age. This observation emerges with greatest statistical validity (P <0.001) from the total analysis by chi-square. It is of interest, however, that even those with early ages of onset (the first horizontal row) are distributed in all three categories of severity. Examining those with most severe obesity (vertical column, Group III), it would appear that all ages of onset are represented but with some clustering of increased numbers of those with early ages of onset which does not achieve statistical significance in this type of analysis. In Group II, the same tendency of those with earlier ages to predominate just achieves significance at a level of P <0.01. Thus, it can be concluded that the population under study is heavily weighted with those having an early onset of obesity and there is some tendency for those with early onset to be found in the groups of greatest severity. Yet, even those with early onset can have a moderate type of obesity and, contrariwise, a few subjects with later onset can have marked obesity and may be found in Groups II and III. Table 2. Age of onset vs. groups Age of onset
1--8 9--12 13--20 >20
Group n
I
II
III
41 28 20 16
8 7 9 5
22 12 7 9
11 9 4 2
19.63 a
3.33
12.16 b
8.34
~ ~ ~ ~
7.95 1.35 1.91 4.54
ap<0.01. Arrows point to the 'chi-square' determined for each column and each row. bp<0.001.
The next two tables (3 and 4) examine the relationship of cellularity to age of onset. In Table 3 there is a division of cell size into three arbitrary categories (<0.9 lag lipid per cell; 0.9 to 1.1 lag lipid per cell; >1.1 lag lipid per cell). This division was made to ensure reasonable numbers of subjects in each category. At all cell sizes there is a tendency to find increased numbers at early ages of onset, no doubt a reflection of the population studied, but an examination of the matrix horizontally, along rows, shows that any cell size can be found in obesities beginning at any age. Those with early or late onset do not tend to be more or less hypertrophic. Table 3. Age o f onset vs. cell s&e Age of onset
1--8 9--12 13--20 >20
n
<0.9 tag lipid/cell
0.9--1.1
>1.1
41 28 20 16
14 7 7 6
14 12 10 4
13 9 3 6
6.46
7.52
7.88
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0.05 1.35 1.71 0.49
Arros points to the 'chi-square' determined for each column and each row. None is significant.
305
ADIPOSE TISSUE CELLULARITY IN HUMAN OBESITY
Table 4. Age of onset vs. cell number Age of onset 1--8 9--12 13--20 >20
n
<40 X 109
40-60 X 109
>60 X 109
41 28 20 16
6 7 4 2
12 8 10 9
23 13 6 5
3.91
3.92
17.10 b
~ --* --* ~
10.87a 2.21 2.81 4.62
ap<0.01" Arrows point to the 'chi-square' determined for each column and each row. bp<0.001.
Table 4 shows an analysis of cell number as a function of age of onset. The most hyperplastic group (>60 X 109 adipocytes) has the largest cluster of early onset obese. Of equal importance, those with early onset (first horizontal row) are significantly different in their pattern of cellularity, showing the highest number of individuals in the high cell number group. One can conclude, therefore, that: (1) obesity beginning at any age can be of any degree of severity, although it appears that those with early onset are more likely to be found in groups of greater severity, (2) cell size is similar in obesities beginning at any age, and (3) the most hypercellular varieties Of obesity are found in the early onset group. An absolutely clear classification of early onset and hyperplastic vs. late onset and hypertrophic obesity cannot be made, probably for two types of reasons: (a) Severity, age of onset and cell number are all interrelated. Many subjects with early onset can develop only moderate degrees of obesity, but in this population more of those with early onset developed marked obesity. Furthermore, severity (without regard to age of onset) has a high correlation with cell number. If one analyses this matter with partial correlation coefficients, the following is found: the partial correlation coefficient, 'r', of cell number vs. percentage ideal body weight (with age of onset held constant) = 0.74. The correlation of cell number vs. age of onset (with percentage ideal body weight held constant) = --0.17, an insignificant correlation. One reason for this low and insignificant correlation is the existence of many subjects with early onset who developed only mild obesity with little or no hypercellularity. An additional confounding feature is discussed next. (b) Some individuals with late onset obesity can be hypercellular. An analysis of individual subjects as to age of onset, severity and cellularity is made in the next three figures., Figure 4 is a graph of all subjects with cell number as ordinate and cell size as abscissa. The various levels of severity cluster together into bands covering broad areas of this: cell size vs. cell number graph. If each subject is to be examined as to the relative degree of hyperplasia or hypertrophy which comprises the obesity, a graph such as that of Figure 5 is useful. Here the relative increase in cell number, i.e. cell number divided by mean non-obese cell number, is plotted against the relative increase in cell size. The radiating triangular areas represent zones with various degrees of hyperplasia. The uppermost triangles, to the left,
306
JULES HIRSCH AND BRUCE BATCHELOR
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contain those subjects with most hyperplasia and least hypertrophy progressing down and to the right to those subjects with least hyperplasia and most cell enlargement. The curved lines enclose bands of equal severity. Thus, from each triangle or each set of curved lines, subjects can be examined who have either equal degrees of hyperplasia or equal severity.
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307
ADIPOSE TISSUE CELLULARITY IN HUMAN OBESITY
In Figure 6 the various curves and triangles are converted into more traditional rectilinear coordinates and each individual can be separately considered as to severity, age of onset and relative degree of hyperplasia. There can be little doubt that an occasional individual with adult onset, as well as can be determined by medical history and supportive photographs, can have both massive and hypercellular obesity. -
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Figure 6. Data from the four-sided figures in Figure S are shown as a matrix. The ordinate represents severity of obesity in terms of absolute fat mass present and the abscissa, relative hyperplasia. A 45 ° line from coordinates 1.0, 1.0 in Figure 5 are shown as double lines equal to 50:50 - - hyperplasia:hypertrophy. Clearly, hyperplasia and severity are related; yet, some adult onset cases can be found in boxes denoted as both hyperplasia and high severity. Also, childhood onset obesity is distributed rather equally throughout the matrix.
DISCUSSION It would be helpful to arrive at a simple classification of obesity relating age of onset to adipose tissue morphology. Ideally such a classification should be based not only on observations of the obese but also on firm knowledge of the cellular events that determine normal growth and development of the adipose mass. To some degree, this is now possible when considering experimental models of obesity in rodents. Recent data (Greenwood and Hirsch, 1974) suggest that adipocyte development in the Sprague Dawley rat begins with a stage of intense cellular replication in the early weeks of postnatal life. The rapidly proliferating cells or 'adipoblasts' are morphologically indistinguishable from other supportive cells in adipose tissue; however, by a measure of labelled thymidine incorporated into the nuclei of adipoblasts and later analysis of label incorporated into mature adipocytes, some reasonable guesses can be made as to the further development of the tissue. At roughly three to five weeks of age, most of the proliferation has ceased, but a bed of unfilled adipocytes exist; such cells migfit be termed 'pre-adipocytes.' The pre-adipocytes slowly fill over ensuing weeks and the full complement of adipocytes is achieved by 12 to 14 weeks of age. Thereafter no new adipocytes are added to the tissue and indeed there is no apparent turnover of adipocyte DNA.
308
JULES HIRSCH AND BRUCE BATCHELOR
Under or over-nutrition during the time of adipoblast proliferation leads to a permanent change in both cell number and cell size; whereas, later nutritional alterations change only cell size. Thus, those obesities of rodents which are manifest at early ages such as the genetic obesity occurring in the Zucker rat are found to be a mixed hyperplastic--hypertrophic type. Obesities which occur later such as those produced by hypothalamic lesions or even by genetic influences acting at a later date, for example the Danforth or Yellow mouse obesity, are exclusively hypertrophic. A picture emerges, therefore, which is schematically shown in Figure 7. Normal development is shown in the top panel as an ordered sequence of cellular progression: a d i p o b l a s t - - ~ pre-adipocyte -~ adipocyte. The lower panel shows two types of known disorders of adipocyte development: early influences leading to permanent alterations in cell size and number and later influences provoking a more pure hypertrophic obesity. With our present understanding of this situation, it is easier to speculate on mechanisms whereby early nutritional effects can provoke alterations in cell number. The mechanisms which could lead to permanent change in cell size are unknown, yet it must be emphasised that cellular hypertrophy is a universal accompaniment of animal obesity, as is the case with h u m a n obesity.
Adipocyte Development-Rot Ce~l size
~
~
Cell rtum!!r j ~ ~ . /~dipocyte / X Adipoblost Pre-odipocyte
&
Figure 7. A schematic representation of cellular differentiation in rodent adipose tissue. The upper panel represents normal development. The lower panel shows two modes of abnormal development. The thick arrows indicate forces acting either early (left) to provoke a hyperplastic --hypertrophic obesity or later (right) leading to purely hypertrophic obesity.
A similar classification of obesity in man is made difficult by uncertainty as to the course of events in h u m a n adipose cellular development. There are, however, several helpful observations. It seems quite definite that purposeful overfeeding in adult man leads only to cellular enlargement. Furthermore, when both young and old obese subjects are reduced, the cell number does not decline. This gives at least some assurance that mature h u m a n adipocytes unlike erythrocytes or hepatocytes do not 'turn over' and that very likely in the adult state there is not a significant number of recruitable cells which can become adipocytes under conditions of increased caloric storage. There is as yet some reasonable debate as to whether or not the situation may be different in the hog (Widdowson and Shaw, 1973), but in man the best guess
ADIPOSE TISSUE CELLULARITY IN HUMAN OBESITY
309
is that there is a fixed adipocyte number that prevails in adult life. The other set of useful observations comes from the work of Salans, Cushman and Weismann (1973) and Sjostrom and Bjorntorp (1974), who have shown, as the data presented above support, that childhood onset obesity, severity and hypercellularity are all closely related. The subjects studied by Salans, Cushman and Weismann (1973) had a ~ar clearer relationship between hypercellularity and early onset than was demonstrable from our own data. Given the uncertainties imposed by patient selection and the difficulty in ascertaining age of onset with absolute precision, some of these differences may be reconciled. Yet it appears incontrovertible from our data that some patients with definite adult onset may be hyperplastic. This observation is strengthened by a recent publication of Bray and Gallagher (1974) in which they found at least one subject with onset of obesity in adult years related to hypothalamic dysfunction due to a lipoma in the interpeduncular fossa. This subject was found to have hypercellularity. It can, of course, be argued that in this patient or, for that matter, in any adult subject who becomes obese, the seeds were sown earlier and a hyperplastic bed of adipocytes were awaiting a nutritional environment permitting later filling and thereby the expression of obesity. A schema which summarises some of these possibilities as well as other unknowns of h u m a n adipose tissue development is shown in Figure 8. The uppermost panel shows the rapid progression of adipoblast to pre-adipocyte occurring early, perhaps in the first year of life, and the remaining growth of the tissue occurring more slowly as pre-adipocytes fill through childhood and adolescence. Were this the correct situation, then the notions of Brook (1972) Adipocyte Development-Man Cell size
{
~
Cell humb e ~ A d i p o c y t e Adip/obiast
\ . Pre-edJpocyte
Figure 8. A schematic representation of possible modes of cellular differentiation in h u m a n adipose tissue. The uppermost panel assumes an early single phase of cellular replication (adipoblasts). The middle panel assumes a continued, although intermittent period of cellular
multiplication. The two lower panels show thickened arrows representing nutritional or other forces leading to hypertrophyand excessivehyperplasia. The lower left shows the effect of forces acting at different ages, assumingthat there is a single early phase of adipoblast multiplication. Hypertrophic or mixed hypertrophic--hyperplasfic obesity can occur. The lower right schema indicates the possibilitythat obesitywith onset after childhood or infancy can be hyperplasfic and hypertrophic, more in keeping with the developmentalsequence shown in the middle panel.
310
JULES HIRSCH AND BRUCE BATCHELOR
as well as others that the first year of life is a particularly 'critical' period for ultimate adipocyte number would appear fully justified. The middle panel, on the other hand, seems an equally plausible schema of events. Man, unlike rodents, has rapid growth early, a long period of slower growth during childhood and then a pubertal and early adolescent burst of further growth. The middle schema assumes that the ultimate precursor cell or adipoblast can continue to divide in childhood and perhaps even later, for example under such influences as the hormonal alteration of pregnancy or other situations. Thus, puberty and early adolescence are 'critical' times since there is never a long period of already manufactured 'pre-adipocytes' as shown in the upper panel, but there could be critical periods at even later dates. Based on one or the other of the above possibilities, one can visualise the cellular sequence in obesity as shown in the lowest panel of this figure. Either the die is cast by early adipoblast proliferation and obesity becomes manifest by recruiting unfilled adipocytes at various ages, but always with over-filling and hence hypertrophy or, as shown at the lower right, forces can act at various later dates to provoke further cellular proliferation. In any event, the age of onset of obesity as correlated with hypercellularity need not always be found in earliest childhood nor even in puberty or adolescence. Yet adipoblast proliferation is greatest early in life and in most instances hypercellularity and early onset will be related. The investigation of the details of adipocyte development in man and animals remains an active area of investigation and hopefully the full facts will emerge shortly and permit a simpler classification of human obesity. The even larger unknown of the clinical significance of hypercellularity or hypertrophy and how such cellular changes modify the metabolic state of the obese is yet to be thoroughly studied.
SUMMARY All human obesity is characterised by adipocyte hypertrophy and when body weight exceeds 170 per cent of ideal, a maximum cell size of roughly twice normal is achieved. With greater severity, hyperplasia becomes increasingly manifest and when body weight exceeds 170 per cent of ideal, the degree of hyperplasia is well correlated with severity. Although severity and hypercellularity are often found in those with early onset of obesity, individuals can be found who are hypercellular but have had a later onset of obesity. Until the details of cellular development in man are more fully understood, the precise timing of 'critical' periods for cellular development must remain speculative.
ACKNOWLEDGEMENTS Work reported in this chapter was supported by Research Grants HD-03719 and RR-00102 from the National Institutes of Health. The superb technical assistance of Miss Florence Oetien is gratefully acknowledged.
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REFERENCES Bray, G. A. & Gallagher, R. F. (1974) Manifestations of hypothalamic obesity in man: A comprehensive investigation of eight patients and a review of the literature. Medicine, 54, 301-330. Brook, C. G. D. (1972) Evidence for a sensitive period in adipose cell replication in man. Lancet, ii, 624-627. Greenwood, M. R. C. & Hirsch, J. (1974) Postnatal development of adipocyte cellularity in the normal rat. Journal of Lipid Research, 15, 474-483. Grinker, J., Hirsch, J. & Levin, B. (1973) The affective responses of obese patients to weight reduction: A differentiation based on age at onset of obesity. Psychosomatic Medicine, 35, 57-63. Hirsch, J. & Gallian, E. (1968) Methods for the determination of adipose cell size in man and animals. Journal of Lipid Research, 9, 110-119. Hirsch, J. & Goldrick, R. B. (1964) Serial studies on the metabolism of human adipose tissue. I. Lipogenesis and free fatty acid uptake and release in small aspirated samples of subcutaneous fat. Journal of Clinical Investigation, 43, 1776-1792. Hirsch, J. & H a n , P. W. (1969) Cellularity of rat adipose tissue: Effects of growth, starvation and obesity. Journal of Lipid Research, 10, 77-82. Hirsch, J. & Knittle, J. L. (1970) Cellularity of obese and nonobese human adipose tissue. Federation Proceedings, 29, 1516-1521. Johnson, P. R. & Hirsch, J. (1972) Cellularity of adipose depots in six strains of genetically obese mice. Journal of Lipid Research, 13, 2-11. Johnson, P. R., Zucker, L. M., Cruee, J. A. F. & Hirsch, J. (1971) Cellularity of adipose depots in the genetically obese Zucker rat. Journal of Lipid Research, 12, 706-714. Knittle, J. L. & Hirsch, J. (1968) Effect of early nutrition on the development of rat epididymal fat pads: Cellularity and metabolism. Journal of Clinical Investigation, 47, 2091-2098. Mellits, E. D. & Cheek, D. B. (1970) The assessment of body water and fatness from infancy to adulthood. In Physical Growth and Body Composition: Papers from the Kyoto Symposium on Anthropological Aspects of Human Growth (Ed.) Brozek, Joseph. Vol. 35, no. 7, pp. 12-26. Chicago: University of Chicago Press. Pace, N. & Rathbun, E. N. (1945) Studies on body composition, III. The body water and claemically combined nitrogen content in relation to fat content. Journal of ]~'ological Chemistry, 158, 685-691. Salans, L. B., Cushman, S. & Weismann, R. E. (1973) Studies of human adipose tissue, adipose cell size and number in nonobese and obese patients. Journal of Clinical Investigation, 52, 929-941. Salans, L. B., Horton, E. S. & Sims, E. A. H. (1971) Experimental obesity in man: Cellular character of the adipose tissue. Journal of Clinical Investigation, 50, 1005-1011. Sj6str6m, L. & Bj6rntorp, P. (1974) Body composition and adipose tissue cellularity in human obesity. Acta Medica Scandinavica, 195, 201-211. Widdowson, E. M. & Shaw, W. T. (1973) Full and empty fat cells Lancet, ii, 905.