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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2007-2356
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The Journal of Clinical Endocrinology & Metabolism Vol. 93, No. 11 4462-4470
Copyright © 2008 by The Endocrine Society

Fatty Acid Metabolism in Patients with PPAR{gamma} Mutations

Garry D. Tan, David B. Savage, Barbara A. Fielding, Jenny Collins, Leanne Hodson, Sandy M. Humphreys, Stephen O'Rahilly, Krishna Chatterjee, Keith N. Frayn and Fredrik Karpe

Oxford Centre for Diabetes, Endocrinology, and Metabolism (G.D.T., B.A.F., J.C., L.H., S.M.H., K.N.F., F.K.) and National Institute for Health and Research (NIHR) Oxford Biomedical Research Centre (F.K.), University of Oxford, Churchill Hospital, Oxford OX3 7LJ, United Kingdom; and Departments of Medicine and Clinical Biochemistry (D.B.S., S.O., K.C.), Addenbrooke’s Hospital, Cambridge CB2 2QQ, United Kingdom

Address all correspondence and requests for reprints to: Dr. Fredrik Karpe, Oxford Centre for Diabetes, Endocrinology, and Metabolism, Churchill Hospital, Oxford OX3 7LJ, United Kingdom. E-mail: fredrik.karpe{at}oxlip.ox.ac.uk.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Context: PPARG mutations may cause insulin resistance and dyslipidemia, but little is known about the mechanisms of the abnormalities of lipid metabolism.

Objective: We hypothesized that in PPARG mutations, abnormal adipose tissue triglyceride storage causes insulin resistance.

Design, Patients, and Main Outcome Measures: Whole-body and adipose tissue-specific metabolic phenotyping through arteriovenous blood sampling was made before and after a mixed meal including 13C-palmitic acid. Studies were performed in a 32-yr-old male with partial lipodystrophy and type 2 diabetes, heterozygous for the PPARG P467L mutation and in an apparently phenotypically normal 32-yr-old male heterozygous for the PPARG n.AAA553T mutation. Comparator groups were age- and sex-matched healthy participants (n = 10) and type 2 diabetes sex-matched participants (n = 6).

Results: The P467L patient had elevated unmodulated fasting and postprandial plasma nonesterified fatty acid (NEFA) concentrations, despite a low adipose tissue NEFA output. Instead, NEFA appeared to originate directly from triglyceride-rich lipoproteins: 13C-palmitic acid accumulated rapidly in the NEFA fraction, as a sign of impaired fatty acid trapping in tissues. In contrast to the Pparg haploinsufficient mouse, the patient with n.AAA553T mutation did not exhibit paradoxically insulin sensitive and showed a mostly normal metabolic pattern.

Conclusions: The lipodystrophic PPARG P467L phenotype include excessive and uncontrolled generation of NEFA directly from triglyceride-rich lipoproteins, explaining high systemic NEFA concentrations, whereas the human PPARG haploinsufficiency is metabolically almost normal.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
Both peroxisomal proliferator-activated receptor (PPAR)-{gamma} activation by thiazolidinediones and the Ala12 variant of the common Pro12Ala PPAR{gamma}2 polymorphism increase insulin sensitivity (1, 2, 3, 4, 5), whereas dominant-negative PPARG mutations cause severe insulin resistance (6, 7, 8). The precise mechanism by which PPARG influences insulin sensitivity is unclear, although it may involve changes in fatty acid metabolism. We and others have previously studied changes in fatty acid metabolism with thiazolidinedione-induced PPAR{gamma} activation (9, 10, 11) and PPAR{gamma} Pro12Ala polymorphism (12, 13, 14, 15), which may link the modulation of insulin sensitivity to PPARG.

Several rare dominant-negative mutations in PPARG have been described and are associated with partial lipodystrophy and severe insulin resistance including: PPAR{gamma} P467L (8), V290M (8), R425C (6) and F388L (7), R194W (16), C190S (17) and D424N (18), C114R (19), C131Y (19), C162W (19), R357X (19), [A935{Delta}C]fs.312[stop315] (19). The P467L and V290M mutations reduce the transcriptional activity of PPAR{gamma} in a dominant-negative fashion in vitro (20). We previously suggested that the sc adipose tissue (SCAT) metabolism of a subject with the PPAR{gamma} P467L mutation was abnormal compared with healthy controls demonstrating a reduced capacity to increase lipolysis in response to fasting and an inability to suppress fatty acid mobilization (20).

In contrast, another PPARG mutation, a heterozygous frame shift, resulting in a premature stop mutation (n.AAA553T) does not seem to have an overt metabolic phenotype, except in the presence of an additional defect in an unrelated gene encoding the muscle-specific regulatory subunit of protein phosphatase (PPP1R3A) (21). The metabolic phenotype of the SCAT of n.AAA553T mutation has never been examined.

The mechanisms by which PPARG mutations cause insulin resistance are unclear (22). Because of the important role played by PPARG in adipocyte differentiation, Garg has suggested that decreased adipogenesis and decreased SCAT mass result in triglyceride deposition elsewhere in the body, such as in the liver (22), causing insulin resistance. However, little work has been done looking at lipid metabolism, and particularly at fatty acid metabolism, in these subjects. We have previously described plasma nonesterified fatty acid (NEFA) concentrations in the P467L subject (20).

We hypothesized that with disruptions to the PPARG gene, AT is unable to appropriately sequester fatty acids, exposing the body to high NEFA concentrations, thus causing severe insulin resistance. We used techniques of integrative physiology to investigate SCAT fatty acid handling in the subject with the dominant-negative P467L PPAR{gamma} mutation and the carrier of the n.AAA553T PPARG mutation, matched to appropriate comparator groups.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Subject selection

Two subjects with rare PPARG mutations were studied. The first was a 32-yr-old man heterozygous for the dominant-negative P467L PPARG mutation causing partial lipodystrophy and type 2 diabetes mellitus (T2DM) (patient A) (8, 20). It was difficult to select an appropriate control group: subjects of the same age, sex, and fat mass generally do not have T2DM, whereas subjects with T2DM with a similar degree of insulin resistance are usually older and obese. Thus, there was no ideal control group for patient A; therefore the metabolic phenotype was described in reference to two comparator groups: 1) healthy subjects matched for age, sex, and body mass index (BMI) (n = 10); and 2) T2DM subjects matched for sex and glycemic control (n = 6). Patient A was studied before and after 6 months of rosiglitazone treatment (8 mg/d). T2DM subjects were investigated after 3 months of treatment with rosiglitazone (8 mg/d) and after 3 months of placebo treatment (in a double-blind cross-over design); these diabetic subjects are part of a previously presented study (9).

The second subject was a physically very fit and healthy 32-yr-old man heterozygous for a 3-bp deletion (AAA) and a 1-bp insertion (T) at nucleotide 553 in PPARG, resulting in a premature stop codon (n.AAA553T) (patient B). In the presence of a mutation in PPP1R3A, the n.AAA553T PPARG mutation leads to severe insulin resistance; however, patient B was homozygous for the wild-type PPP1R3A. He had an apparently normal AT distribution. An age-, sex-, and BMI-matched comparator group was used for this subject (n = 10).

Study design

All subjects attended for a full day metabolic investigation after an overnight fast. The Research Ethics Committee approved the study. Subjects gave their written consent.

Metabolic investigation protocol

Tissue-specific arteriovenous techniques To assess in vivo metabolism of SCAT and skeletal muscle, we measured arteriovenous differences across abdominal SCAT and across forearm muscle; these measurements reflect that tissue’s metabolic activity. Inclusion of tissue blood flow into calculations allows quantification of net metabolic activity. On the day of the study, serial blood samples were taken in the fasting state, and for 6 h after a standardized mixed meal, containing [1,1,1-13C]tripalmitin.

Blood sampling Blood was sampled from three sites simultaneously as previously described (23): 1) arterialized blood from a vein draining a heated hand; 2) venous blood from SCAT from the superficial epigastric vein. This vein drains abdominal SCAT with negligible contribution from other tissues (24); and 3) venous blood representing muscle drainage was taken retrogradely from a vein draining deep structures of the forearm. Oxygen saturation and ultrasonography were used to confirm cannula position.

The mixed meal contained 40 g fat and 40 g carbohydrate as a fat emulsion [40 g olive oil, 600 mg [1,1,1-13C]tripalmitin (CK Gas Products Ltd., Wokingham, UK), 400 mg emulsifier, flavorings), skimmed milk (200 ml) and Rice Krispies (Kellogg Co., Manchester, UK)].

Tissue blood flow Abdominal SCAT blood flow was measured by 133Xe washout (25) and forearm muscle blood flow, by strain-gauge plethysmography (26).

Biochemical analyses

Plasma glucose, insulin, NEFA, triglyceride, 3-hydroxybutyrate, and blood glycerol concentrations were measured as described previously (27).

Specific fatty acid measurements in triglyceride and NEFA fractions Lipids were extracted from arterialized plasma samples (28). Triglycerides and NEFAs were separated by solid-phase extraction (modified from Ref. 29), and fatty acids were methylated with methanolic sulfuric acid. Fatty acid composition was analyzed by gas chromatography (GC) (30). Fatty acid methyl esters were resolved in a BPX-70 fused silica capillary column (SGE Europe, Milton Keynes, UK) using a HP6890 GC (Hewlett Packard, Wokingham, UK) with an Orchid isotope ratio mass spectrometry (IRMS) interface (PDZ-Europa, Crewe, UK). Fatty acid methyl esters were converted to CO2 by heating at 860 C in the presence of PtCuO. The 13CO2/12CO2 ratio was determined using a 20:20 stable isotope analyzer (PDZ-Europa). Tricosanoic acid methyl ester was used as an isotope enrichment standard (1.134 atom percent). Fatty acids were identified by their retention times relative to standards. Individual fatty acid concentrations were calculated from their proportion of total fatty acids by multiplying fatty acid composition by the respective plasma concentration, as determined by enzymatic assay.

Measurement of plasma monoacyl (MAG) and diacyl glycerol (DAG) After extraction as described above and separation with thin-layer chromatography, MAG and DAG concentrations were measured using GC with flame ionization detection.

Stable isotopes [1-13C]palmitic acid enrichment in NEFA and triglyceride fractions were measured using GC-combustion-IRMS. Concentrations of NEFA-palmitate and triglyceride-palmitate were used to calculate tracer concentrations.

Fasting and postprandial 13C enrichment in expired breath CO2 samples were measured using IRMS.

Calculations

Absolute flux was calculated as the product of the arteriovenous or venoarterial metabolite concentration difference and tissue blood flow (23). Insulin sensitivity was calculated by homeostatic model assessment (HOMA %S) (31). Results from the two subjects with PPARG mutations (patients A and B) are described in relation to the 95% confidence intervals (CIs) of the comparator groups.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Baseline clinical data are in Table 1Go. Subjects with PPARG mutations were well matched to their comparator groups.


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  		TABLE 1. Subject characteristics

 
For each of the variables reported below, data are presented in the following order: patient A (P467L subject) relative to nondiabetic comparators and placebo-treated diabetic comparators; patient A on rosiglitazone, relative to rosiglitazone-treated diabetic comparators; patient B (n.AAA553T subject) relative to nondiabetic comparators. Values are described relative to the upper and lower 95% CIs of the comparator groups.

Plasma glucose

Patient A’s fasting glucose concentration (16.1 mmol/liter) was higher than the upper 95% CIs of both comparator groups [nondiabetic comparators: 5.4 mmol/liter (5.2–5.5) (mean (95% CIs); diabetic comparators: 9.0 mmol/liter (7.6–10.3)]. Postprandial glucose concentrations of patient A were also higher than nondiabetic and diabetic comparator groups (data not shown).

Rosiglitazone lowered fasting and postprandial glucose concentrations [patient A on rosiglitazone, fasting: 11.4 mmol/liter; diabetic comparators on rosiglitazone, fasting: 7.6 mmol/liter (6.5–8.8); postprandial data not shown].

Patient B’s fasting (5.3 mmol/liter) and postprandial plasma glucose concentrations were similar to those of the healthy comparators [5.4 mmol/liter (5.2–5.5)].

Plasma insulin (Fig. 1Go)

Patient A’s plasma insulin concentrations were similar to those of the nondiabetic group (compared with 95% CIs; Fig. 1AGo), lower than that of placebo-treated diabetic comparators in the fasting state (below lower 95% CI), but similar postprandially (Fig. 1BGo). Rosiglitazone lowered plasma insulin concentrations of the diabetic comparators but increased those of patient A (Fig. 1CGo).


Figure 1
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  		FIG. 1. A, Plasma insulin concentrations in patient A (solid triangles) and patient B (open circles) compared with healthy subjects (mean shown as dashed line; 95% CIs shown in gray shaded area around mean). B, Plasma insulin concentrations in untreated patient A (solid triangles) compared with diabetic subjects after placebo (mean shown as dashed line; 95% CIs shown in gray shaded area around mean). C, Plasma insulin concentrations in patient A after rosiglitazone treatment (solid triangles) compared with diabetic subjects after rosiglitazone treatment (mean shown as dashed line; 95% CIs shown in gray shaded area around mean).

 
Although patient B’s plasma glucose concentrations were normal, fasting and early postprandial plasma insulin concentrations were higher than the nondiabetic comparator subjects (Fig. 1AGo).

Plasma NEFA concentrations (Fig. 2Go)

Plasma NEFA concentrations of both subjects with PPARG mutations were markedly different from those of the comparator groups.


Figure 2
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  		FIG. 2. Plasma NEFA concentrations. A, Plasma NEFA concentrations in patient A (solid triangles) and patient B (open circles) compared with healthy subjects (mean shown as dashed line; 95% CIs shown in gray shaded area around mean). B, Plasma NEFA concentrations in untreated patient A (solid triangles) compared with diabetic subjects after placebo (mean shown as dashed line; 95% CIs shown in gray shaded area around mean). C, Plasma NEFA concentrations in patient A after rosiglitazone treatment (solid triangles) compared with diabetic subjects after rosiglitazone treatment (mean shown as dashed line; 95% CIs shown in gray shaded area around mean).

 
Patient A had higher NEFA concentrations compared with nondiabetic (Fig. 2AGo) and diabetic (Fig. 2BGo) comparators, with values above upper 95% CIs. Plasma NEFA concentrations decreased postprandially in all comparators (nondiabetic comparators, 61%; diabetic comparators, 55%). In contrast, patient A decreased plasma NEFA concentrations by only 26% from fasting-to-postprandial states (Fig. 2Go, A and B). Elevated plasma NEFA concentrations coincided with the postprandial rise in plasma triglyceride concentrations.

With rosiglitazone treatment, patient A’s fasting plasma NEFA concentrations normalized, although postprandial concentrations remained above upper 95% CIs (Fig. 2CGo).

Patient B’s fasting and early postprandial plasma NEFA concentrations were higher than the nondiabetic comparator group (above the upper 95% CI) (Fig. 2AGo).

NEFA generation (Fig. 3Go)

Role of sc abdominal adipose tissue (AT) To assess whether the lack of fasted-to-fed modulation of plasma NEFA concentrations in patient A was due to excess SCAT NEFA release, we measured SCAT NEFA output. Despite patient A’s high plasma NEFA concentrations, SCAT NEFA output was not elevated but was lower than nondiabetic (Fig. 3AGo) and similar to diabetic subjects (Fig. 3BGo). With rosiglitazone, patient A’s SCAT NEFA output remained lower than the rosiglitazone-treated diabetic comparator group (Fig. 3CGo). SCAT NEFA output of the nondiabetic comparator group (but not the diabetic comparator group) and patient A has already been reported (20). Note that SCAT NEFA output is expressed per unit mass and as such is higher per unit mass of tissue in the leaner nondiabetic comparator group compared with the heavier diabetic comparator group.


Figure 3
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  		FIG. 3. NEFA release from AT A, Output of NEFA from AT in patient A (solid triangles) and patient B (open circles) compared with healthy subjects (mean shown as dashed line; 95% CIs shown in gray shaded area around mean). B, Output of NEFA from AT in untreated patient A (solid triangles) compared with diabetic subjects after placebo (mean shown as dashed line; 95% CIs shown in gray shaded area around mean). C, Output of NEFA from AT in patient A after rosiglitazone treatment (solid triangles) compared with diabetic subjects after rosiglitazone treatment (mean shown as dashed line; 95% CIs shown in gray shaded area around mean). D, Plasma NEFA concentrations plotted against AT NEFA output (best fit lines shown). Groups shown are: patient A before (solid triangles) and after (open triangles)rosiglitazone, the diabetic subjects after placebo (solid squares) and after rosiglitazone (open squares), patient B (solid circles) and healthy subjects (solid diamonds). For each group studied, each line demonstrates the best fit relationship between time points (–30, 0, 30, 60, 90, 120, 180, 240, 300, and 360 min). Thus, each line reflects the relationship between the release of NEFA from sc abdominal adipose tissue and plasma NEFA concentrations. The figure is discussed further in the text.

 
In patient B, SCAT NEFA output was lower or similar to the nondiabetic comparator subjects (Fig. 3AGo).

To examine the relationship between plasma NEFA concentrations and upper-body SCAT NEFA output, plasma NEFA concentrations were plotted against SCAT NEFA output (Fig. 3DGo); data from the PPARG mutation subjects as well as that of the nondiabetic and diabetic comparators were included. In Fig. 3DGo, as SCAT NEFA output increased, plasma NEFA concentrations increased in nondiabetic and diabetic comparators (both after placebo and after rosiglitazone). A similar relationship also existed for patient B. In contrast, plasma NEFA concentrations were completely unrelated to SCAT NEFA output in patient A, even after rosiglitazone.

Plasma glycerol concentrations and SCAT glycerol output.
Plasma glycerol concentrations and SCAT glycerol output also reflect SCAT metabolism. Fasting plasma glycerol was 80.0 µmol/liter (56.8–103.3) in nondiabetes comparators and 83.8 µmol/liter in patient B. In diabetes comparators rosiglitazone increased fasting plasma glycerol concentrations from 80.2 (71.8–88.6) µmol/liter (placebo) to 90.1 µ (62.0–118.2) mol/liter (rosigiltazone); but in patient A, rosiglitazone decreased fasting plasma glycerol concentrations from 46.0 µmol/liter before treatment to 42.1 µmol/liter.

In subjects with PPARG mutations, the relationship between SCAT glycerol-NEFA output was similar to that in the comparators (Fig. 4Go).


Figure 4
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  		FIG. 4. Glycerol and NEFA release from AT. Output of glycerol from AT plotted against output of NEFA from AT. For each group studied, consecutive time points (–30, 0, 30, 60, 90, 120, 180, 240, 300, and 360 min) are connected by lines on the graph. Groups shown are: patient A before (solid triangles) and after (open triangles) rosiglitazone, the diabetic subjects after placebo (solid squares) and after (open squares)rosiglitazone, patient B (solid circles with dotted lines) and healthy subjects (solid diamonds).

 
Appearance of meal-derived [13C]palmitate and unlabeled meal-derived specific fatty acids in plasma (Fig. 5Go) Meal-derived [13C]palmitate.
Because the meal contained [1,1,1-13C]tripalmitin, [13C]palmitic acid was incorporated into triglycerides in chylomicron particles postprandially. Lipoprotein lipase (LPL) lipolysis of labeled triglyceride-rich lipoproteins (TRLs) liberates [1-13C]palmitic acid, which has one of two fates: to be taken up and stored in the tissue or to spill over into the circulation. To assess whether the high unmodulated plasma NEFA concentrations of patient A were explained by increased TRL lipolysis and spillover, we measured plasma concentrations of both the substrate for the LPL ([13C]palmitic acid in the TRL fraction) and the product of lipolysis ([13C]palmitic acid in the NEFA fraction).


Figure 5
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  		FIG. 5. Meal-derived [13C]palmitate in plasma. A, Meal-derived [13C]palmitate in plasma triglyceride fraction of patient A (solid triangles) and patient B (solid circles) compared with healthy subjects (mean shown as dashed line; 95% CIs shown in gray shaded area around mean). B, Meal-derived [13C]palmitate in plasma triglyceride fraction of untreated patient A (solid triangles) compared with diabetic subjects after placebo (mean shown as dashed line; 95% CIs shown in gray shaded area around mean). C, Meal-derived [13C]palmitate in plasma triglyceride fraction of patient A after rosiglitazone treatment (solid triangles) compared with diabetic subjects after rosiglitazone treatment (mean shown as dashed line; 95% CIs shown in gray shaded area around mean). D, Meal-derived [13C]palmitate in plasma NEFA fraction of patient A (solid triangles) and patient B (solid circles) compared with healthy subjects (mean shown as dashed line; 95% CIs shown in gray shaded area around mean). E, Meal-derived [13C]palmitate in plasma NEFA fraction of untreated patient A (solid triangles) compared with diabetic subjects after placebo treatment (mean shown as dashed line; 95% CIs shown in gray shaded area around mean). F, Meal-derived [13C]palmitate in plasma NEFA fraction of patient A after rosiglitazone treatment (solid triangles) compared with diabetic subjects after rosiglitazone treatment (mean shown as dashed line; 95% CIs shown in gray shaded area around mean).

 
In patient A, the appearance of [13C]palmitic acid in the NEFA fraction was increased compared with both diabetes and nondiabetes comparators (on placebo and on rosiglitazone). This was not accompanied by an increased rate of disappearance of the substrate ([13C]palmitic acid in the TRL fraction); in fact, plasma [13C]palmitic acid concentrations in the triglyceride fraction remained higher in patient A than comparators (Fig. 5Go, A–C).

Meal-derived oleic acid.
The meal was rich in oleic acid. As another index of meal-derived TRL lipolysis, we measured the postprandial rise in the proportion of oleic acid in the plasma NEFA and triglyceride fractions. In the fasting state, the proportion of plasma oleic acid in the plasma of patient A (38% of plasma NEFAs) was similar to that of diabetic comparators [40% (37–43) (mean (95% CIs)]. Postprandially, plasma oleic acid concentrations rose more rapidly in patient A (48%) than the other subjects [43% (41–46); after rosiglitazone: P467L 48%; diabetic comparators: 44% (43–45)]. The proportion of oleic acid in the triglyceride fraction did not differ between patient A when fasting (40.1%) compared with diabetic comparators [39.0% (35.4–42.5%)] but did rise more rapidly postprandially [patient A, 50.1%; diabetes comparators, 45.1% (42.8–47.5)]. After rosiglitazone treatment, oleic acid in the TRL fraction was similar between patient A (fasting, 39.9%; postprandial, 49.7%) and the rosiglitazone-treated comparators [fasting, 40.3% (38.5–42.1); postprandial 46.8%(43.6–50.0)].

These data support the 13C-labeled fatty acid results: fatty acids appear in the NEFA fraction in patient A to a much higher degree than the comparators.

NEFA removal

Forearm NEFA removal Forearm NEFA removal did not differ between patient A [33 nmol·min–1 per 100 g tissue (time averaged area under the curve)], and nondiabetic comparators (65 nmol·min–1 per 100 g tissue (30–101)] or diabetic comparators [20 (–3–43) nmol·min–1 per 100 g tissue]. With rosiglitazone treatment, patient A released NEFAs from his forearm (71 nmol·min–1 per 100 g tissue), in contrast to the uptake of NEFAs observed in the rosiglitazone-treated diabetic subjects [24 (0–48) nmol·min–1 per 100 g tissue]. Forearm NEFA uptake was unchanged in patient B (31 nmol·min–1 per 100 g tissue), compared with the nondiabetic comparators [65 (30–101) nmol·min–1 per 100 g tissue].

Fatty acid oxidation 3-Hydroxybutyrate.
To test whether patient A’s high plasma NEFA concentrations were due to decreased hepatic fatty acid oxidation, we measured plasma 3-hydroxybutyrate concentrations. In diabetic and nondiabetic comparator groups, 3-hydroxybutyrate concentrations decreased postprandially to a nadir at 120 min before increasing in the late postprandial period (data not shown). Patient A’s 3-hydroxybutyrate concentrations [87.9 µmol·liter–1 (time averaged area under the curve)] were not lower than either of the comparator groups [diabetic comparator group, 69.2 µmol·liter–1 (46.2–92.2); nondiabetic comparator group, 68.2 µmol·liter–1 (54.3–82.1)]. Patient B’s 3-hydroxybutyrate concentrations were 50.7 µmol·liter–1. Thus, plasma 3-hydroxybutyrate concentrations merely reflected plasma NEFA concentrations with no suggestion of decreased hepatic fatty acid oxidation in patient A.

Appearance of 13CO2 in breath.
To examine whether whole-body fatty acid oxidation was reduced, we measured 13CO2 concentrations in breath samples. Breath 13CO2 of patient A was not reduced compared with nondiabetic comparators (data not shown). Those of patient B were not reduced compared with the nondiabetic comparators (data not shown). Thus, whole-body NEFA catabolism again suggested no defect in NEFA catabolism to account for the abnormal plasma NEFA concentrations of patient A.

Triglycerides

Plasma triglycerides The stable isotope measurements above suggest that fatty acids produced from intravascular TRL lipolysis were not stored in SCAT. Thus, plasma triglyceride concentrations were of interest.

Patient A had higher fasting and postprandial triglyceride concentrations than both comparator groups [patient A, fasting: 5.5 mmol/liter, postprandial: 7.1 mmol/liter; nondiabetic comparators, fasting: 1.2 mmol/liter (0.8–1.7), postprandial: 1.6 mmol/liter (1.1–2.1); diabetic comparators, fasting: 2.3 mmol /liter (1.9–2.7), postprandial: 3.0 mmol/liter (2.5–3.4)].

Rosiglitazone lowered patient A’s plasma triglyceride concentrations in the fasting and early postprandial phases to concentrations close to the upper 95% CI of the rosiglitazone-treated diabetic subjects (data not shown).

Patient B’s plasma triglyceride concentrations (fasting: 1.5 mmol/liter, postprandial: 1.9 mmol/liter) were similar to the nondiabetic comparators (fasting: 1.2 (0.8–1.7) mmol/liter, postprandial: 1.6 (1.1–2.1) mmol/liter].

SCAT and skeletal muscle triglyceride removal

SCAT triglyceride removal was reduced in patients A and B (data not shown). Rosiglitazone normalized SCAT triglyceride removal in patient A (data not shown).

Forearm triglyceride removal was higher in patients A and B (data not shown). Rosiglitazone treatment did not normalize the removal of triglycerides by skeletal muscle despite normalizing the fasting and early postprandial triglyceride concentrations in patient A (data not shown).

Plasma MAG and DAG concentrations did not differ between groups (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
Using a combination of arteriovenous difference and stable isotope techniques, we studied the lipid metabolism phenotype of two subjects with PPARG disruptions: the dominant-negative P467L PPAR{gamma} mutation, which causes diabetes and partial lipodystrophy (patient A), and a physically fit and healthy, nondiabetic subject who had a PPARG frameshift mutation (n.AAA553T) (patient B). We compared the lipid metabolism of these subjects with that of appropriately matched comparator groups. We previously reported abnormally high plasma NEFA concentrations in patient A compared with nondiabetic subjects (9). In this paper, we examine SCAT and skeletal muscle NEFA metabolism in comparison both diabetic and nondiabetic comparators. We also explore potential mechanisms underlying the elevated plasma NEFA concentrations of patient A, including a novel mechanism: abnormal spillover of NEFAs from diet derived plasma triglycerides.

Plasma NEFA concentrations of patient A were elevated with little fasting-to-fed modulation. As SCAT NEFA output increased, plasma NEFA concentrations increased in the diabetic and healthy subjects; however, this was not true in the P467L patient. Instead, plasma NEFA concentrations remained high and unmodulated despite low-normal SCAT NEFA output. Further evidence of abnormal lipid metabolism in tissues other than SCAT is demonstrated by the abnormal ratio of plasma NEFA-plasma glycerol concentrations, compared with the normal ratio of SCAT NEFA output-glycerol output.

In all subjects, the ratio of NEFA and glycerol output from SCAT was 3:1, suggesting complete lipolysis of triglycerides with little fatty acid trapping. Glycerol output from SCAT appeared normal in patient A, but systemic plasma glycerol concentrations were inappropriately low for circulating NEFA concentrations when compared with that of all other subject groups. One explanation for the observed mismatch between plasma NEFA concentrations and plasma glycerol concentrations in patient A would be incomplete lipolysis of TRLs by LPL in the intravascular compartment. However, plasma MAG and DAG concentrations did not differ between groups, implying that hydrolysis of TRLs was identical across all subjects and incomplete hydrolysis did not contribute to the mismatch between plasma glycerol and NEFA concentrations in patient A. Differences in NEFA reesterification also did not account for the NEFA results as the ratio of AT glycerol-NEFA output was the same in all subjects.

The abnormal NEFA findings could also be explained by an induction of glyceroneogenesis by rosiglitazone (32). It has been previously assumed that because human AT lacks glycerol kinase, glycerol cannot be reesterified in AT and is therefore released. However, recent data support glycerol reesterification in rodent AT and ex vivo human AT (32). The authors state that they consider its contribution to be minor in their ex vivo studies. That this pathway is negligible in humans in vivo is supported by detailed in vivo characterization of AT glycerol metabolism, which suggests that glycerol reesterification in human AT is not significant (33).

We found no evidence of reduced NEFA catabolism to explain the abnormal plasma NEFA concentrations. Several indices of NEFA catabolism were normal: postprandial 3-hydroxybutyrate concentrations were not inappropriately suppressed, forearm NEFA removal was normal, and the generation of 13CO2 from plasma [1-13C]palmitic acid was not suppressed.

By administering stable isotope-labeled triglycerides, we could quantify fatty acid trapping from circulating TRL lipolysis by measuring stable isotope concentrations in plasma triglycerides and NEFAs. Triglyceride lipolysis was reduced, as demonstrated by the elevated [13C]palmitic acid in the triglyceride fraction. Despite the reduction in triglyceride lipolysis, the rate of appearance of [13C]palmitic acid in the NEFA fraction increased, suggesting that fatty acid trapping from TRL lipolysis was decreased in patient A, with increased fatty acid spillover. This increased spillover did not occur in the SCAT depot or in forearm muscle. Instead, we suggest that this increased spillover of fatty acids occurs in peripheral sc tissue, in which the fatty acids generated are unable to be stored. This lack of storage of fatty acids from the lipolysis of triglyceride TRL in peripheral tissue would be consistent with the observed lack of peripheral sc AT, accounting for partial lipodystrophy in patient A. With this mechanism, LPL acts on plasma lipoproteins, but tissues are unable to accommodate the products of lipolysis, resulting in the observed release of NEFAs from the forearm in lipodystrophy (as opposed to NEFA uptake in the other subjects). The stable isotope findings were supported by plasma oleic acid measurements, which reflected a relative increase in meal-derived fatty acids in patient A.

Pparg haploinsufficient mice appear to be healthy but on detailed phenotyping are more insulin sensitive with normal plasma NEFA concentrations compared with Pparg wild-type mice (34). Although ex vivo and ex vitro studies of the human PPARG frameshift mutation (n.AAA553T) suggests haploinsufficiency (the N terminal fragment has little activity), it is unclear whether the mutation results in haploinsufficiency in vivo. In contrast to haploinsufficient mice, our human subject with PPARG frameshift mutation had elevated NEFA and insulin concentrations, indicative of some degree of insulin resistance of fatty acid metabolism, although glucose concentrations were unchanged. Despite normal SCAT distribution, patient B also showed subtle abnormalities in his SCAT function. His SCAT was relatively quiescent, with little NEFA release or triglyceride uptake, despite the elevated fasting plasma NEFA concentrations. Thus, it would seem that human phenotype of the frameshift PPARG mutation does not share the increased insulin sensitivity seen in Pparg haploinsufficient mice.

The decreased triglyceride clearance in SCAT in patient A coupled with increased fatty acid spillover is consistent with the role of PPARG in AT regulation. PPARG activation induces AT LPL expression (35) and increases SCAT mass (36, 37). Thus, in the presence of PPARG mutations, LPL expression may be decreased in combination with a decreased SCAT mass. Why central AT depots are unaffected is not understood. This could be tested in future research by measuring the postheparin LPL mass and activity.

Rosiglitazone did not change just lipid metabolism but also plasma glucose and insulin concentrations. As rosiglitazone reduced plasma glucose in patient A, plasma insulin concentrations paradoxically increased, a finding consistent with rosiglitazone improving β-cell glucotoxicity. Rosiglitazone also decreased SCAT NEFA release in patient A, a finding most marked in the fasting state. It is possible that persistently elevated NEFA concentrations in patient A impair glucose-stimulated insulin secretion, thus explaining the increase in insulin concentrations as NEFA concentrations normalize with rosiglitazone.

The increase in insulin sensitivity with rosiglitazone in patient A is assessed by HOMA %S, and it is unclear whether this is a valid measurement of insulin sensitivity in this subject. However, rosiglitazone normalizes patient A’s glycosylated hemoglobin (HbA1c; despite raised fasting plasma glucoses). Significant improvement in HbA1c was observed, but fasting glucose on the day of the experiment was still rather high. Whether this reflects genuinely lowered fasting hyperglycemia or just an unusually high fasting glucose on the day is unknown.

This study does not take into account AT heterogeneity (38). Some techniques used here, e.g. mass balance, focus on one depot. In contrast, other techniques, such as labeled fatty acids, measure whole-body AT function. Thus, there are limitations to some of the techniques used. However, it has been previously demonstrated that the major contributor to plasma NEFA concentrations is upper body SCAT (39, 40), the depot studied here, rather than visceral AT.

The lipid metabolism of patients with HIV lipodystrophy syndrome has been carefully investigated by Sekhar et al. (41, 42), who identified defects in LPL activity and fatty acid entrapment, leading to increased very low-density lipoprotein-triglyceride production.

We suggest that lipodystrophy found in subjects with PPARG mutations share a final common pathway with the lipodystrophy of HIV lipodystrophy syndrome subjects: there is not simply an absence of peripheral SCAT; instead there is peripheral SCAT, which is lipolytically active but unable to store fatty acids. This would explain the apparent absence of peripheral SCAT, despite the apparently contradictory finding of increased NEFA spillover. This increased NEFA delivery to the liver then drives the overproduction of very low-density lipoprotein-triglycerides and explains the hypertriglyceridemia observed in lipodystrophic patients.


   Footnotes
 
Conflict of interest: G.D.T., D.B.S., B.A.F., J.C., L.H., S.M.H., K.C., K.N.F., and F.K. have nothing to declare. S.O. has received consulting fees by Merck Consultancy and Cambridge Antibody Technology and has equity interests in Prosidian.

First Published Online August 19, 2008

Abbreviations: AT, Adipose tissue; BMI, body mass index; CI, confidence interval; DAG, diacylglycerol; GC, gas chromatography; HbA1c, glycosylated hemoglobin; HOMA %S, insulin sensitivity calculated by homeostatic model assessment; IRMS, isotope ratio mass spectrometry; LPL, lipoprotein lipase; MAG, monoacylglycerol; NEFA, nonesterifed fatty acid; PPAR, peroxisome proliferator activated receptor; PPARG, peroxisome proliferator activated receptor-{gamma}; SCAT, sc adipose tissue; TRL, triglyceride-rich lipoprotein; T2DM, type 2 diabetes mellitus.

Received October 23, 2007.

Accepted August 11, 2008.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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