Triglyceride Storage Disease: A Defect in Activation of Lipolysis in Adipose Tissue

ABSTRACT. A child, aged 6 years 3 months, with a triglyceride storage disorder in peripheral adipose tissue, niicrocephal@. and gross emaciation has been studied at autopsy. The mean triglyceride content of adipoc@tes in hand and foot was 0.17 ±0.02@sg/cel1 and 0.18 ±0.02@sg/ cell. Adipoc@tes from abdominal tissue were small, irregular,

and difficult to measure accurately, reflecting the degree of

cachexia. Basal tissue contents of cyclic AMP and release of glycerol and fatty acid from peripheral tissue of the child were in the same range as adult tissues. None of these Illeasurements, however, were increased by incubation with isoprenaline (10 M), compared to a three- to seven-fold increment in adult subcutaneous tissues and to a four- to ten fold increment of glycerol and cyclic AMP in peripheral adipose tissue of a control child aged 10 years. We postulate that the proband may have had a defect of adenyl cyclase or catecholamine receptor, which has a role in the abnormal storage of triglyceride in peripheral adipose tissue. Pediat

rl(.s, 59:442-447, 1977. TRIGLYCERIDE STORA(; E, AI)ENYL

CYCLASE, ADIPOSE TISSUE.

three variants have been considered: (1) defects at the adenyl cyclase level (type I); (2) defects of the activation of triglyceride lipase by protein kinase (type II); and (3) defects of the lipases (type III).

A child falling into the category of the type I disorder has been briefly reported with a failure of activation of adenyl cyclase by catecholamines associated with abnormal triglyceride deposits in peripheral adipose tissue.' We now wish to report more complete metabolic studies on autopsy

tissues of this child which are consistent with our

previous interpretation of a lipol@tic defect at the level of adenyl cyclase.

CASEREPORT

A microcephalic infant (F. A.; chromosolne pattern, 46,

XX), the second child of a healths' Nigerian family, was noted

from birth (birthweight, 2.4 kg, less than third percentile) to

Triglyceride storage disease is an uncommon group of disorders with excessive or abnormal accumulation of extralysosomal triglyceride asso ciated with a metabolic defect on the pathways of triglyceride mobilization (lipolysis). We have previously classified these disorders according to which stage of lipolysis is affected.I2 Briefly,

(Received May 17; revision accepted for pul)lication Jul@'14, 1976.)

Supported by grants froni the Medical Research Council, United Kingdom, and the Joint Research Board of St.

Bartholomew's Hospital.

ADDRESS FOR REPRINTS: (D.J.G.) Lipid Research Labo ratory, St. Bartholomew's Hospital, London, E.C.1, En gland.

442 PEDIATRICS Vol. 59 No. 3 March 1977

Triglyceride Storage Disease:A Defect in Activation of

Lipolysisin AdiposeTissue

DavidJ. Galton, M.D., Christine H. Gilbert, Ph.D., JamesJ. Lucey,M.R.C.Path., and John A. Walker-Smith,M.D.

Front the Lipid Research Laboratory and tII(' !)epart;nents of Child 11(01(11 (1)1(1 !Iistopathoh)gq. St. Bartholomew 5 Hospital, London

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FIG. 1. Top, F.A. at 5 years 3 months. Bottom, Appearance of right second finger at atLtopsv

showing deposits of adipose tissue despite gross emaciation.

have soft tissue swellings over arms, hands, legs, and feet. Subsequent development was markedly impaired; and she failed to reach any of the normal motor or social milestones.

B@'the age of 9 months severe feeding difficulties and intercurrent infection had led to emaciation 1@@t the abnormal peripheral limb deposits remained. At 5 years she was grossly emaciated (weight, 8.2 kg), the weight having increased only marginally over the previous four years and she la@ curled up with severe contractions at the joints associated with reduced voluntary movements (Fig. 1, top).

She died at 6 years 3 months of an extensive bronchopneu nionia.

80

60

40

20

In

a) E

Cell diameter (ii)

FIG. 2. A frequency distribution diagrani for adipocyte dianieter of tissue from abdomen and

hand of F.A. Isolated adipocvtes were prepared by collagenase digestion of small pieces of adipose tissue (20 to 30 mg) and the liberated cells were photographed with a Zeiss Jena @1icroscope with a Polaroid Camera attachment. Shaded area, cells from abdomen; open area,

cells from hand.

0

15

30

45

deposits of subcutaneous adipose tissue over the hands and feet (Fig. 1, bottom). The other major abnormalities were in the brain, liver, and lungs. The brain was imicrocephalic (430 gm) with gross dilatation of the lateral ventricles, but with no evidence of CSF obstruction. Cortical thickness varied from 0.2 to 0.7 cm. The midbrain and pons were normal. The liver weighed 400 gm and the spleen 15 gm. The endocrine system and kidneys were normal. The lungs showed a confluent

bronchopneumonia which was the cause of

death.

MICROSCOPIC FINDINGS

In the brain the normal layering and alignment of the cortical neurons was lost and abnormal aggregates of neurons were seen in many areas.

Fic. 3. Opposite page, Effect of isoprenaline (10 :‘NI) on

accumulation of cyclic AMP and release of glycerol and fatty acids from adipose tissue (hand and foot) of prohand. Adipose tissue (50 to 150 mg) was incubated in Earle's bicarbonate IMlifer (ph 7.4). Results of tissue cyclic AMP, glycerol, and fatty acid release are presented as individual measurements of six replicates in the presence and absence of isoprenaline

(10

@ NI). Assays of adult tissues under similar experimental

conditions are included to show the sensitivity of the methods. Top, Tissue accumulation of cyclic AMP. Center, Release of glycerol. Bottom, release of free fatty acid into the

nlcul)ation niedjuln.

LABORATORY FINDINGS

Hemoglobin was 12.8 gm/100 ml with heino globin A on electrophoresis; WBC was 15,000/cu iiiin with 71% neutrophils. The cerebrospinal fluid showed no cells or filaria and the culture was sterile; glucose was 80 ing/100 ml and protein 42

ing/10() 1111.

The BUN was 30 ing/100 nil and the

182 mg/100 ml; lipoprotein electrophoresis was normal. The skull X-ray film showed no bony thickening of the vault l)ut was microcephalic. No other abnormalities were seen on the X-ray film. The EEC recording was abnormal diffusely but there were no clear-cut focal features. The EGG was normal. Protein-bound iodine and agglutina tion tests for rubella and cytolmegalic virus were within normal limits; her plasma proteins and fasting glucose levels were also normal. A skin biopsy at 4 years 3 months from the abnormal deposits showed heavy racial pigmentation in the I)asal epidermis with increased focal extension of adipose tissue into the dermis. There was no evidence for formation of multiple lipomnata.

AUTOPSY FINDINGS

At autopsy, within two hours of death, the body was emaciated with marked contracture deformi ties of the arms and legs. There were abnormal

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PROBAND ADULTS

r—@Hand

@

r—@-

Foot

160

120

p.mol/105 cells/5mm.

80

40

0

PROBAND

I Hand

ADULTS

Foot

1000

800 n.moIIlO6 cells/h.

600

400

200

It:'.;

Nil 105M lsoprenalmne : Nml 105M Nil 105M

PROBAND ADULTS

Hand Foot

1200

1000

800 n.mol/106cells/h.

600

400

200

lsoprenaline : Nil 10GM

Isoprenaline : Nil 105M Nil 105M Nil 105M

There were no lipid inclusions in nerve cells although their number was decreased. Many of the neurons were poorly developed and showed short or absent dendritic processes. Due to the ininiaturity of the neurons only a thin layer of white matter was present. This showed many areas of spongiosis and demyelination. The midbrain and pons were normal.

Liver specimens were taken within four hours of death and showed no abnormal staining with PAS reagent. Oil-Red “¿0―staining of the liver revealed deposits of fat, predominantly in the centrilobular zones and also in the Kupffer cells. The lungs contained a confluent bronchopneu monia due to a gram-positive Diplococcus, probably D. pneumoniae.

METHODS

Within 40 minutes of death subcutaneous samples of adipose tissue were removed from the hand, foot, and abdomen of the child and trans ferred to the laboratory in 0.15 M saline for metabolic studies.

METABOLIC ASSAYS The Incubation System

Adipose tissue was preincubated for 30 minutes at 37 C in Earle's bicarbonate buffer (pH 7.4) containing crystalline bovine albumin (1 gni/100 Iml) under a mixture of oxygen and carbon dioxide (95:5). Tissue (50 to 100 img) was then transferred to 0.2 nil of fresh buffer and incubated for another hour at 37 C for release of glycerol and fatty acids, or for five minutes (with 10 mM of theo phylline) in the presence or absence of isopren aline (10

@ M) for accumulation of tissue cyclic

AMP.

Metabolite Assays

Release of glycerol into the medium was assayed by an enzylnic method and free fatty acids by an autolnated fluorimetric technique.

Cyclic AMP

Tissue was extracted in 2OO@il of ice-cold hydrochloric acid (0.7 M) and the fat removed with ether. The aqueous extract was neutralized with 50@d of 2.8 NI of potassium hydroxide and analyzed for cyclic AMP.@

Isolation of Adipocytes

Adipocytes were prepared by collagenase digestion of small pieces of adipose tissue (20 to 30 mmg)and the liberated cells were photographed with a Zeiss Jena Microscope with a Polaroid camera attachment.

@ Cell diameter and volume

were calculated from measurements of at least 300 cells.'

RESULTS

The general appearance of the child is shown in Figure 1, top. She was grossly emaciated (less than the third percentile for body weight) but retained peripheral adipose tissue over hands and feet. There were deposits of adipose tissue over the fingers (Fig. 1, bottom). Isolated adipocytes were prepared by the collagenase technique from this tissue and also from the foot. The imean trigly ceride content was 0.17 ±0.O2jtg/cell and 0.18 ±0.02@g/cell (compared to an average value of adult subcutaneous adipocytes of 0.47 ±0.04g/cell. Adipocytes prepared from abdominal tissue were small, irregular, and diffi cult to measure accurately. A frequency distribu tion diagram for adipocyte diameter of abdomen and hand (Fig. 2) shows a bimodal distribution.

The effect of isoprenaline (10

@ M) on tissue

levels of cyclic AMP, release of glycerol, and release of fatty acids into the incubation medium were measured (Fig. 3). Six replicates were performed for each condition and bars represent individual nieasurements. Basal levels of cyclic AMP are greater than in adult adipose tissue, perhaps due to contamination by other cell types

(e.g., mast cells, fibroblasts). However, no stimula

tion of levels of cyclic AMP were observed in tissue from the hand and foot of the proband. This compares to a six-fold increase in levels of cyclic AMP of adult subcutaneous adipose tissue using similar methods (Fig. 3, top). The increase in cyclic AMP in peripheral tissue (foot), incubated with isoprenaline (10

@ M) from a control child

(N. T.) aged 10 years undergoing routine ortho

pedic surgery was 8.4 ±3 pInol/10@ cells to 95 ±19 pmol/10@ cells at five minutes. The basal release of glycerol (Fig. 3, center) and fatty acids (Fig. 3, bottom) from hand and foot of proband is in the same range as release from adult adipose tissue. However, no stimulation of release of either mnetabolite from proband tissue was observed following incubation with isoprenaline

(10-@

M) comparedto a three-foldincrenientin

release from adult tissues. Antemortem adipose tissue from the foot of the control child N. T. (aged 10 years) stimulated with isoprenaline (10@ NI) showed an increase of glycerol from 9.5 ±2 to 31 ±5 iiniol/10@ cells/hr.

DISCUSSION

Impaired stimulation of cyclic AMP, glycerol release, and fatty acid from peripheral adipose

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tissue of the proband is compatible with a lipo lvtic defect at the level of catecholamine receptor or adenyl cyclase. The data are in accord with our previous antemortem findings in the proband' (1) reduced sensitivity of adenyl cyclase to stimu lation by noradrenaline (2 X 10

@ M); (2) im

paired response in tissue levels of cyclic AMP; and (3) impaired release of glycerol following stimulation with isoprenaline (10@

@ M). These

measurements were only of single or duplicate assays. The present data are based on an adequate nuiiil)er of replicates but suffer other limitations. The effects of profound emaciation (as observed in the proband) on lipolysis are not known because adipose tissue usually atrophies tinder

these conditions as it has over the trunk. For a

shorter period of weeks, lipolysis is activated by

fasting and still retains its full sensitivity to

catecholamines.' The anonialous feature of severe cachexia and peripheral deposits of triglyceride in adipose tissue of the proband is good clinical evidence for a storage disorder; and the only metabolic defect we have detected in the abnormal tissue is a defect of lipolysis. It was previously found that the pathways of lipogenesis from glucose and palmitate were intact.' Although the abnormal storage of triglyceride and the lipolytic defect in peripheral adipose tissue may be unrelated, it is possible that the metabolic defect is contributing to the storage disorder. Since adenyl cyclase is widely distrib

uted in brain, it is also possible that a defect of the enzyme in this organ could contribute to the neuronal abnormalities that were found at autopsy.

REFERENCES

1. Galton DJ, Gilbert CH, Reckless JPD, Kaye J: Trigl@

ceride storage disease: A group of inborn errors of triglyceride metabolism. Qu J Med 43:63, 1974. 2. Galton DJ, Reckless JPD, Gilbert CH: Triglyceride storage disease. In, Collip PJ (ed): Childhood Obes itv. Acton, Massachusetts, Publishing Sciences Group Inc, 1975, pp 149-160.

3. Reckless JPD, Gilbert CH, Galton DJ: Alpha adrenergic

receptor activity OIi cyclic AMP levels and lipolvsis in adipose tissue of hypothyroid man and rat.

J Endocrinol 68:419, 1976.

4. Brown BL, Albano JDM, Ekin RP, et al: .@simple and

sensitive saturation assay method for the measure nient of adenosine :3'S' cyclic monophosphate. Biochem J 121:561,1971.

5. Reckless JPD, Clifton-Bligh P. Galton DJ: The trigl@ ceride content of adipocvtes and plasma of patients with diabetes mellitus. Hormone Metab Res 7:407. 1975.

6. Di Giralamo NI, \lendlinger S, Fertig JW: A simple

method to determine fat cell size and number in

four mammalian species. Am J Phvsiol 221:850, 1971.

7. Reckless JPD: Thesis, London University, 1976. 8. Gilbert CH, Galton DJ: The effect of catecholamines

and fasting on cyclic AMP and release of glycerol human adipose tissue. Hormone Metal) Res 6:229,

1977;59;442

Pediatrics

David J. Galton, Christine H. Gilbert, James J. Lucey and John A. Walker-Smith

Triglyceride Storage Disease: A Defect in Activation of Lipolysis in Adipose Tissue

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1977;59;442

Pediatrics

David J. Galton, Christine H. Gilbert, James J. Lucey and John A. Walker-Smith

Triglyceride Storage Disease: A Defect in Activation of Lipolysis in Adipose Tissue

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