Regional similarities in the metabolic regulation of adipose tissue lipoprotein lipase

Regional

Similarities

in the Metabolic Regulation Lipoprotein Lipase

of Adipose Tissue

Trudy J. Yost and Robert H. Eckel Seven normal weight and 10 obese women were studied to determine the relative activities of adipose tissue lipoprotein lipase (ATLPL) in the gluteal and abdominal subcutaneous adipose tissue depots, both in the fasting state and in response to a B-hour insulin/glucose infusion. In normal weight women, fasting gluteal enzyme activity was greater than abdominal (P < .02). In the obese group, fasting levels of ATLPLwere higher in both the gluteal and abdominal depots than in the normal weight group, but similar between regions. The regulation of ATLPL by insulin/glucose was also similar between regions in each group. When both groups were considered together, there was a strong correlation between fasting ATLPL of both regions, and between the insulin responsiveness of gluteal ATLPL and abdominal ATLPL after a 6-hour infusion. Despite regional differences in fasting ATLPL in lean women, these studies indicate that the reaulation of ATLPL by insulin/glucose is largely similar in at least these two subcutaneous adipose tissue depots. Copyright 0 1992 by W.B. Saunders Company

L

IPOPROTEIN LIPASE (LPL) is bound on the capillary endothelium in many tissues where it acts to hydrolyze circulating triglyceride-rich lipoproteins (chylomicrons, very-low-density lipoproteins [VLDL]) into monoglycerides and free fatty acids (FFA). In adipose tissue, LPL is the rate-limiting step for the delivery of lipolysis products to adipocytes for triglyceride storage.’ Regional differences in adipose tissue lipoprotein lipase (ATLPL) activity may contribute to differences in regional body fat deposition.““ The goal of this study was to determine the relative activities of ATLPL in the gluteal and abdominal subcutaneous adipose tissue depots in fasting normal weight and obese women. Additionally, because ATLPL is subject to regulation by hormones,‘.’ responsiveness of ATLPL to insulin/glucose infusion was compared between regions and groups. METHODS Seven normal weight women and 10 obese women comprised the two study groups. The mean age of the normal weight group was 26 ? 1 years, whereas the obese group was 35 f 2 years. All study subjects were premenopausal. The normal weight subjects had never been obese, and the obese women were at their maximum weight and each had been weight-stable for at least 3 months. The mean body mass index (BMI; kg/m*) was 21.5 ? 0.7 in normal weight womenversus 31.7 2 1.2 in obese (mean + SEM). Individuals were excluded from study if they ingested medications known to affect lipid or carbohydrate metabolism, including oral contraceptives or hormone replacement. All subjects were free of acute and chronic illnesses as determined by physical examination and screening blood work. Thyroid-stimulating hormone, electrolytes, liver and renal panels, serum glucose, and complete blood cell counts were normal in all. Fasting serum triglyceride levels were also within the normal range for all subjects. All studies were performed on the Clinical Research Center at the University of Colorado Health Sciences Center after approval by the Human Subjects Committee and subsequent individual informed consent. Each subject consumed an isocaloric liquid formula composed of 45% carbohydrate, 40% fat, and 15% protein for 2 days before metabolic study. Insulin/glucose euglycemic clamp studies were performed in all subjects after a 12-hour overnight fast. All clamps were done at a 40 mUlmz/min insulin infusion rate and lasted for 6 hours as previously described.” A basal serum glucose concentration was determined in the fasting, unperturbed state on the morning of the Metaf~olism, Vol41, No 1 (January),

1992: pp 33-36

clamp and served as the euglycemic goal for the variable glucose infusion during the course of the 6-hour clamp. Paired adipose tissue biopsies from the gluteal and abdominal regions were performed for measurements of ATLPL in the fasting state and again at the 6-hour terminal timepoint of the euglycemic clamp. Both the biopsy technique and measurements of the lipase activity have been previously described.’ Abdominal wall biopsies of subcutaneous adipose tissue were performed inferior and lateral to the umbilicus using the same method. Briefly, ATLPL was eluted from adipose tissue pieces (40 to 50 mg) into Krebs-Ringer phosphate buffer (pH 7.4) containing heparin (2 pg/mL; Upjohn, Kalamazoo, MI; from beef lung). Enzyme activity was measured as hydrolyzed YYabeled fatty acids after incubation of 0.1 mL of eluted enzyme with 0.1 mL of substrate for 45 minutes. The substrate was prepared with 5 mg of unlabeled triolein (Sigma Chemical, St Louis, MO), 4 PCi [1-‘Qriolein (Amersham, Arlington Heights, IL), and 0.24 mg of egg lecithin (Applied Science Labs, State College, PA). Emulsification of the triolein and lecithin was performed with a mixture of 10% fatty acid-poor bovine serum albumin/normal human serum/2 mol/L Tris HCI buffer/distilled water (4:1.5:5:9.5) for 100 seconds of sonication (final vol. 4.0 mL). Fat cells were obtained according to the method of Rodbell: in which 100 mg of adipose tissue was digested in Krebs-Ringer bicarbonate buffer containing collagenase (3 mg/mL) and albumin (4 g/dL). After filtration through nylon mesh (250 km), centrifugation at 400 x g for 2 minutes, and washing twice in buffer, adipocyte size was determined with a calibrated microscope by the method of DiGirolamo et aL9 Plasma triglycerides were measured by the enzymatic method previously described.” Separation of high-density lipoprotein (HDL) from total cholesterol was accomplished by use of the two-step dextran sulfate-magnesium chloride precipitation method of Warnick et al.” Serum insulin was measured by radioimmunoassay.”

From the Department of Medicine, University of Colorado Health Sciences Center, Denver, CO. Supported by National Institutes of Health Grant No. DK-26356, a grant from Mead Johnson, Nuti’tional Division, and GCRC Grant No. RR-00051. Computation assistance was provided by the VRY computer system funded under the GCRC grant. Address reprint requests to Robert H. Eckel, MD, University of Colorado Health Sciences Center, 4200 E Ninth Ave, BI51, Denver, CO 80262. Copyright 0 I992 by W.B. Saunders Company 00260495/9214101-0007$03.00l0 33

34

YOST AND ECKEL

The waist to hip ratio (WHR) was calculated from minimum waist circumference (cm) and maximum hip circumference (cm) by tape measurement.” These measurements were done on each obese individual the day before metabolic study. Student’s t test for unpaired and paired differences. and Spearman-Rank correlation were used for statistical analyses.

Table 2. ATLPL Data Normal Weight (n _ 7) Fasting gluteal ATLPL (nmol/106cells/min)

1.48 ? 0.34

Baseline comparative data for the seven normal weight women and 10 obese women are shown in Table 1. The groups were significantly different in both mean weight and body mass index, with no overlap of these variables between the two groups. Although fasting serum insulin levels were generally higher in the obese than the normal weight subjects, this difference did not prove to be significant. However, fasting serum triglycerides and HDL cholesterol (HDL-C) levels were different between the two groups (triglycerides, P < .Ol; HDL-C, P = .02). As expected, fat cell size in both the gluteal region and the abdominal wall was significantly different in obese versus normal weight women. However, in the normal weight women, gluteal fat cell size was also greater than in the abdominal wall (mean + SEM, 239 ? 17 v 212 ? 14 pL; P < .Ol). In obese women, there was no difference in fat cell size between regions (gluteal, 602 f 98; abdominal, 594 + 140). The regional differences in fasting ATLPL in normal weight women are shown in Table 2. As shown, gluteal enzyme activity was greater than abdominal in the fasting state (1.48 ? 0.34 nmol FFA/lOh cellsimin v 0.74 ? 0.19; P < .02). In contrast, in the obese group (Table 2), the fasting levels of ATLPL were not different between regions (gluteal, 8.09 + 1.66; abdominal, 6.28 +- 1.45). However, ATLPL was higher in obese versus normal weight women in both the gluteal (P < .Ol) and abdominal (P < .Ol) depots. For all subjects, the fasting level of gluteal ATLPL was significantly correlated with fasting abdominal ATLPL (I = ,895, P = .OOOl) (Fig 1). Despite these group differences in regional fasting enzyme levels, the metabolic regulation of the enzyme was similar in both regions in each group (Table 2). In normal weight women, the responsiveness of gluteal ATLPL to a 6-hour insulin/glucose infusion at 40 mU/m’/min insulin was A3.05 + 0.73 nmol FFA/lO’ cells/min compared with the abdominal responsiveness, A3.50 * 0.73. In the obese

1

8.09 -r 1.66t

*

Fasting abdominal ATLPL (nmol/106cells/min)

RESULTS

Obese (n = 10)

0.74 2 0.19

6.28 _f 1.45

A O-6 h gluteal ATLPL

3.05 2 0.73

4.89 + 2.27

A O-6 h abdominal ATLPL

3.50 + 0.73

5.50 t 1.49

NOTE. Values are means % SE. *P < 0.02; tP < .Ol, obese v normal weight.

group, the ATLPL response to insulin/glucose in the gluteal region (A4.89 + 2.27) was similar to the response in the abdominal wall (AS.50 ? 1.49). Furthermore, there was a relationship between the responsiveness of ATLPL in both regions for all subjects (r = .693, P = .002) (Fig 2). There were no relationships between ATLPL and WHR in either adipose tissue region in obese subjects (WHR was not measured in normal weight subjects). DISCUSSION

Regional differences in ATLPL, and other processes which control FFA flux, eg, lipolysis and reesterification, all work together to control regional body fat deposition. In rats, regional differences in ATLPL have been found”~“; however, comparisons of ATLPL in two or more subcutaneous adipose tissue depots have not been reported. Although available data are also limited in humans, some evidence exists for regional differences in fasting ATLPL. Many of the documented differences are gender- and sex hormonedependent. In lean premenopausal women, ATLPL has been shown to be higher in the femoral than abdominal region.“,” Although these regional differences were re-

Table 1. Subject Data NormalWeight In = 7) Age

iv4

Weight (kg)

Obese (n = 10)

26 2 1.4

35 2 2.2*

62.6 ? 2.4

83.5 & 4.7$

BMI (kg/m21

21.5 2 0.7

31.7 2 1.29

Fasting insulin (pmol/L)

49.0 + 4.3

74.1 ? 13.4

Fasting triglycerides (mmol/L)

0.55 2 0.05

1.40 f 0.19t

Fasting HDL-C (mmol/L)

1.49 ? 0.06

1.23 ? 0.06t

Gluteal adipocyte size (pL)

239 2 17

602 2 98$

Abdominal adipocyte size (pL)

212 2 14

594 I? 1.40*

WHR

ND

0.78 + 0.03

NOTE. Values are means ? SE. lP < .05; tP 2 .02; SP < .Ol; §P < .OOl, obese v normal weight.

0

5 Fasting

15

10 Gluteal

ATLPL

(nmolll

20

O6 cellslmtn)

Fig 1. Fasting gluteal ATLPL activity vs fasting abdominal ATLPL activity in normal weight and obese woman (n = 17); r = .895, P = .OWl.

35

REGIONAL ADIPOSE TISSUE LIPOPROTEIN LIPASE

-5

1 -5

0 L\Gluteal

5 ATLPL

10

15

20

(nmoli 106 cells/mm)

Fig 2. Responsiveness of ATLPL to 6 h insulin/glucose infusion in normal weight and obese women (n = 17); gluteal v abdominal, r = .693, P = ,002.

versed during lactation, ATLPL activity in the abdominal wall remained unchanged. In the present study, normal weight premenopausal women demonstrated a level of fasting gluteal ATLPL activity which was greater than in the abdominal wall. The importance of female hormones to these regional differences in adipose tissue metabolism has been supported by the lack of differences in ATLPL between femoral and abdominal regions in men and postmenopausal women.‘” Moreover, treatment of postmenopausal women with a combination of estrogens and progestins increased ATLPL in the femoral, but not the abdominal region.‘” Although a lower level of ATLPL in the omental region of premenopausal women than in men has been reported, there were no regional differences in men or postmenopausal women between intraabdominal adipose tissue depots (retroperitoneal, mesenteric, omental) and abdominal subcutaneous adipose tissue.” In several studies, obese women have been shown to have higher levels of fasting ATLPL in the femoral and gluteal subcutaneous region than in the abdominal wa11.23However, this did not hold true in the subjects presented here. We found no significant difference in fasting enzyme activity between the gluteal and abdominal fat depots in premenopausal obese women. However, when obese and normal weight women were compared, fasting ATLPL was greater in both gluteal and abdominal subcutaneous depots in the obese group. This supports the results of Bose110 et al,” who showed increases in ATLPL in multiple regions in obese women when compared to lean. Fried and Kral looked at adipose tissue metabolism in four subcutaneous regions (femoral, gluteal, abdominal, epigastric) and two internal beds (mesenteric, omental) in men and women undergoing surgery for obesity.’ In 26 premenopausal women, femoral LPL activity was higher than in all other sites, but there was also much variability between all sites. When LPL activities in obese women for

subcutaneous sites (n = 4) and internal sites (n = 2) were averaged, there was little difference. Moreover, in the obese men, there was no difference in the ATLPL activity between all six sites. In recent studies by Raison et al: regional ATLPL related to both body fat distribution and menopausal status. In women with femoral obesity, femoral ATLPL was higher than that measured in abdominal subcutaneous tissue. This was true of both premenopausal and postmenopausal women. However, in women with abdominal obesity, ATLPL was similar between the regions. It is interesting to note that with increasing age, women demonstrate a proportionate increase in abdominal fat, particularly at the menopause.‘3,24When Raison et al expressed ATLPL as a ratio between abdominal and femoral fat, there was a moderately strong correlation with the WHR. In the premenopausal obese women studied here, there was no relationship between abdominal to gluteal ATLPL ratio and the WHR. In fact, we found no relationships at all between ATLPL and WHR in either region in obese subjects. Although often measured under fasting conditions, ATLPL is a metabolic enzyme that is responsive to regulation, eg, insulin/glucose.5 Studies of insulin infusion under conditions of euglycemia (the euglycemic clamp) have shown that insulin stimulates LPL activity in a dose- and time-dependent manner.’ Because high carbohydrate diets are also particularly effective in promoting ATLPL,25~2b insulin is thought to be an important hormonal regulator of the enzyme’s dynamics. Despite the aforementioned regional differences in fasting ATLPL, most of the available data indicate regulation of ATLPL by meals and insulin is similar between adipose tissue regions. Guy-Grand and Rebuffe-&rive” have found a similar response of ATLPL to meals in abdominal and gluteal depots. In the study presented here, the responsiveness of ATLPL to a 6-hour insulin/glucose infusion was similar in gluteal and abdominal sites, in both lean and obese women. In conclusion, regional differences in fasting ATLPL activity have been well documented and are supported by the results reported here. These differences are genderand sex hormone-dependent, but are also affected by the degree of relative adiposity. Certainly in normal weight, premenopausal women, the evidence is strong for significant divergence in ATLPL activity between subcutaneous adipose tissue depots. However, in obese women, investigations of regional adipose tissue LPL have yielded heterogeneous results. Regional differences in ATLPL in obesity are sometimes maintained, but not consistently. Although fasting ATLPL may differ between subcutaneous adipose tissue depots, the metabolic regulation of ATLPL by insulin/glucose appears to be similar between gluteal and abdominal regions.

ACKNOWLEDGMENT The authors thank Paul Awald laboratory of the General Clinical their technical assistance, Mary Lu and Linda Trefry for her secretarial

and members of the CORE Research Center (GCRC) for Jones for dietary consultation, skills.

36

YOST AND ECKEL

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