Translational studies confirmed key findings in man, especially up‐regulation of adipocyte LCFA uptake in patients with obesity ( 16 ). The present study extends these observations to patients classified as super‐obese participating in a two‐stage bariatric surgical protocol beginning with a sleeve gastrectomy ( 17 ). After major weight loss over the first post‐operative year, weight typically stabilized during year 2, leading to a second procedure, usually a biliopancreatic diversion with duodenal switch (BPD‐DS), in patients requiring further weight loss. Fat biopsies from these procedures facilitated studies of the effects of weight loss on multiple aspects of adipocyte biology.

Obesity is the accumulation of excess fat, principally triglycerides (TG), in adipocyte depots throughout the body. Excessive TG, typically within discrete droplets, also accumulates in the liver and heart, where it is responsible for clinical consequences such as nonalcoholic fatty liver disease ( 1 ) and obesity cardiomyopathy ( 2 ). In the late 1980s, skeptical of the concept that long chain fatty acids (LCFA) entered cells exclusively by diffusion, we postulated that the principal uptake process would prove to be regulatable, facilitated transport, and used studies of cellular LCFA uptake kinetics in rodents to prove that ( 3 - 7 ). We identified the first LCFA transporter ( 8 - 11 ) and showed that regulation of LCFA uptake in adipocytes was a control point for adiposity ( 12 - 14 ) and that upregulation of facilitated LCFA uptake in hepatocytes ( 1 ) and cardiomyocytes ( 2 , 15 ) was a key element in pathogenesis of obesity‐ and EtOH‐associated hepatic steatosis and cardiomyopathies in rodents.

Relationships between parameters were assessed by both linear and nonlinear correlations ( 28 ). For group comparisons, results are expressed as mean ± SE, with n = 10 per group. Each of the experimental groups was compared to the control group with two‐tailed Student's t tests. The other groups were also compared with each other by one way ANOVA as previously described ( 15 ). In addition, the effects on changes in LCFA uptake rates in response to weight loss, of age, gender, ethnicity, baseline weight, % body fat, metabolic status (as reflected in e.g., HbA1c and cholesterol), and the presence of specific co‐morbidities or medication use, were explored by effect adjustments in the ANOVA. In all statistical testing, significance was set at P ≤ 0.05.

Data fitting used the SAAM II version of the Simulation, Analysis and Modeling (SAAM) program of Berman and Weiss ( 25 , 26 ) to compute for each data set values of V max (pmol/sec/50,000 cells), K m (nM), and k (ml/sec/50,000 cells), and their variances and covariances. Prior studies showed that under the conditions employed, V 0 and derived parameters such as V max are measures of actual transmembrane transport ( 3 , 5 , 27 ). Further studies demonstrated that an increase in V max preceded an increase in adipocyte size early in the development of obesity ( 13 ), and a decrease in V max preceded a reduction in adipocyte size and body weight during leptin‐induced weight loss ( 14 ), showing that changes in V max do not simply reflect changes in cell size.

UT([OAu]) is the experimental measurement of uptake, in pmol/sec/50,000 cells, at the stated concentration of unbound OA ([OAu]); V max and K m are the maximal velocity of saturable OA uptake and the value of [OAu] at one‐half the maximal uptake velocity; k is the rate constant for nonsaturable uptake.

The unbound oleate concentration ([OAu]) in each test solution was calculated from ( 23 ), using the LCFA:BSA binding constants of Spector et al. ( 24 ). Measurements of initial OA uptake velocity ( V 0 ) at values of from 0.25‐2.0 were fitted to the sum of saturable and non‐saturable functions of the corresponding [OAu] ( 7 ) according to the equation: UT([OAu]) = V max • [OAu]/( K m + [OAu]) + k • [OAu].

Aliquots from each cell preparation were incubated at 37°C in Dulbecco's Modified Eagle's Medium (DMEM) containing 500 µM BSA and one of five different concentrations of OA, such that the OA:BSA molar ratio ( ) was 0.25, 0.5, 1.0, 1.5, or 2.0:1 ( 3 ). The initial velocity ( V 0 ) of cellular OA uptake from each test solution was determined by a rapid filtration technique from four samples obtained in triplicate over the initial 30 s of incubation, during which uptake was a linear function of time ( 3 , 5 , 7 , 10 , 14 , 16 , 22 ).

Diameter distributions in each adipocyte preparation were determined as reported by digital analysis of suspended cells using a Nikon Eclipse 80i microscope and Nikon Digital DXM 1200C camera. Digital images were analyzed using Nikon NIS‐Elements (NE) Br software, generating mean diameters (with distribution), in micrometers (µ), for each preparation. The mean cell surface area and cell volume, in µ 2 and pl, respectively, were computed from the diameters ( 20 , 21 ).

Biopsies of 5‐10 g were obtained from all enrolled patients from omental and anterior abdominal wall subcutaneous fat depots at each operation. Approximately 1/3 of each biopsy was frozen at −80°C in RNALater for subsequent qRT‐PCR gene expression and biochemical studies. Adipocyte single cell suspensions meeting established viability criteria were prepared with collagenase from the remainder of each biopsy and counted as described ( 10 , 16 , 18 , 19 ).

Of 34 O male and 49 O female bariatric surgery patients studied by our laboratory, 5 men and 5 women were similarly chosen on the basis of age for inclusion in this analysis. The minimum BMI of 35 in this group reflects the lower limits of BMI currently acceptable for bariatric surgery.

NO patients consented to donate a venous blood sample and omental and subcutaneous fat biopsies during a clinically indicated laparoscopic abdominal surgical procedure. Of 18 NO patients studied, 5 men and 5 women were selected to age‐match this cohort as closely as possible with the SO patients. Of the 10, 7 had undergone donor nephrectomy, 2 cholecystectomy, and 1 an inguinal herniorraphy.

We received omental and subcutaneous fat biopsies and a venous blood sample at both surgical procedures in order to examine the effects of surgical weight loss on aspects of adipocyte biology and adipocyte LCFA uptake. Power calculations indicated that at least 10 patients would have to complete both operations to reach our desired end‐points with appropriate statistical assurance. Accordingly, the study remained open to enrollment until 10 patients, now designated as SOr, had completed second stage surgery. By chance, those 10 patients consisted of 5 men and 5 women. As anticipated by prior experience, a total of 35 SO patients were enrolled to meet the study needs.

No significant effects of age, gender, ethnicity, baseline weight, metabolic status (e.g., HbA1c, cholesterol), presence of specific co‐morbidities or medication use on the observed changes in LCFA uptake rates in response to weight loss were detected by the effect adjustments in the ANOVA. However, this conclusion must be qualified because of the small group sizes available for these analyses.

Human serum leptin concentrations have a positive, nonlinear correlation with BMI; in contrast, Spexin concentrations and BMI are negatively c orrelated ( 29 ). These and others findings raise the possibility that leptin and Spexin are counter‐acting regulators of adipocyte LCFA uptake ( 29 ). In this study leptin concentrations in SO patients fell from 79.61 ± 22.05 to 37.72 ± 14.23 ng ml −1 (mean ± SE, P = 0.009) while serum Spexin increased from 1.65 ± 0.41 to 2.4 ± 0.36 ng ml −1 ( P = 0.043) between their two bariatric surgeries, consistent with the previously identified trend ( 29 ).

Contributions of adipocyte surface area and V max to V max′ . V max , the maximal rate of saturable LCFA uptake per cell, is in turn dependent on (i) V max′ , the maximal saturable LCFA uptake rate per u 2 of cell surface and (ii) the number of u 2 per cell. Ten SO patients in a two‐phase bariatric surgical protocol lost 113 ± 13 lbs in 16 ± 2 months between an initial sleeve gastrectomy and a second operation. Omental adipocyte LCFA uptake kinetics and cell surface area (SA) were studied at both surgeries. Ten NO and ten O surgical patients served as controls. The three panels illustrate mean ± SE in these patient groups for ( A ) mean LCFA uptake V max , ( B ) adipocyte surface area, and ( C ) V max′ , as calculated from the two measured variables. Dashed lines indicate changes in these parameters in SO patients between their initial and second (post weight loss) operations. Both V max and SA were decreased in the SOr patients. However, the proportional reduction in SA was greater than that in V max . Consequently, V max′ , defined as V max /SA, actually increased in these patients. In other settings V max′ may change in parallel with V max . These and other studies indicate that V max and SA are independently regulated.

In our model of LCFA transport ( 7 , 13 , 14 , 16 ), V max′ is defined as a measure of the density of LCFA transporters per unit of cell surface area ( V max′ = V max /cell surface area) (pmol/sec/µ 2 × 10 −8 cell surface) (Figure 6 C). V max′ , on average, increased with weight loss in omental SOr vs. SO adipocytes, and were elevated ∼5‐fold compared to the NO range and 3.7‐fold compared to that in O adipocytes (Figure 6 C). By contrast, in a few SOr patients, V max′ decreased rather than increasing with weight loss (Figure 7 ). Other than being a means of classifying adipocytes in terms of the response of their fatty acid uptake process to weight loss, the precise physiological implications of the nature of the change in V max′ require further exploration (see Supporting Information). By contrast to V max′ , k ′, a measure of non‐saturable (passive) uptake per unit surface area, was not significantly changed in any group, consistent with its previously ascribed role as a measure of cell membrane permeability to passive LCFA diffusion ( 14 , 16 ). The means of the 40 values of k ′ in the NO, O, SO, and SOr groups were 0.0034 ± 0.0005 × 10 −8 and 0.0033 ± 0.0004 × 10 −8 ml s −1 µ −2 cell surface area for omental and subcutaneous adipocytes, respectively.

( A ) Mean LCFA uptake V max ± 1 SE in adipocytes from omental and subcutaneous fat biopsies in the four patient groups of this study. While significantly reduced compared to SO, V max in SOr remained significantly greater than in O patients, who had comparable BMIs. ( B ) V max values in both omental and subcutaneous adipocytes from individual SO patients, each of whom was studied during both an initial sleeve gastrectomy (Study 1) and during a second bariatric procedure conducted after a mean weight loss of 113 lbs (Study 2). V max at the second study was decreased appreciably in both depots in 9 of the 10 patients. The exception was patient 1M, denoted by *, who was found, after the fact, to have drunk alcohol heavily between his two surgeries. Ethanol is known to increase LCFA uptake. Data in panels A and B are expressed on a per cell basis. ( C ) Adipocyte LCFA uptake V max′ . Data in this panel are expressed per unit of cell surface area. In contrast to panel A, because cell diameter and calculated cell surface area in the SOr patients decreased to an extent similar to or greater than the overall cellular expression of LCFA transport “machinery,” V max′ remained constant or even increased to some degree in association with weight loss. Statistical tests, Panels A and C: * P < 0.05, ** P < 0.01 compared to the control (NO) group; § P < 0.05, §§ P < 0.01 (O vs. SO); # P < 0.05, ## P < 0.01 (O vs. SOr); € P < 0.05, €€ P < 0.01 (SO vs. SOr).

Dimensions of isolated omental and subcutaneous adipocytes in NO, O, SO, and SOr patients. ( A ) Measured diameters. ( B ) Calculated cell surface areas. ( C ) Calculated cell volumes. Data are mean ± 1 SE. In the SOr group, values for all variables had fallen into the NO range although the BMIs for the SOr patients were similar to those of the O group. Statistical tests: ∗ P < 0.05, ∗∗ P < 0.01 compared to the control (NO) group; # P <0.05, ## P < 0.01 (O vs. SOr); € P < 0.05, €€ P < 0.01 (SO vs. SOr).

Effects of sleeve gastrectomy on BMI and on the V max for LCFA uptake by isolated ( A ) omental and ( B ) subcutaneous adipocytes. Exponentially increasing curves in both panels reflect the mean value ± 1 SE of the V max for LCFA uptake by isolated adipocytes in NO (▪), O (▲), and SO (•) patients, taken from Figure 2. A sleeve gastrectomy was performed as the initial bariatric surgical procedure in the super‐obese patients, after which both BMI and the V max for LCFA uptake decreased. After a mean of 16 months and loss of 113 lbs, the BMIs in the super‐obese patients (now designated SOr) had fallen to 44.4 kg m −2 , similar to that in the O group. However, V max (40.6 ± 11.5) in this weight‐reduced group remained almost twice that predicted from the BMI: V max regression among NO, O, and SO patients. A second bariatric surgical procedure was performed in the SOr patients, during which additional fat biopsies were obtained for further studies of LCFA uptake kinetics.

SOr patients exhibited appreciable reductions in the V max for LCFA uptake in both omental and subcutaneous adipocytes when compared to values at initial surgery. At their second surgery, BMIs had fallen from their initial 62.6 ± 2.8 kg m −2 to 44.4 ± 2.4 kg m −2 , a value not significantly different from the 50.1 ± 1.1 kg m −2 in the O group (Figures 4 A, 4 B). There were corresponding reductions in Body Fat % (Supporting Information Table 1). Both omental and subcutaneous adipocyte dimensions (Figure 5 A‐C) fell into the NO range in most SOr patients following initial bariatric surgery, and were reduced compared to those of steady‐state O adipocytes. However, V max's for facilitated adipocyte LCFA uptake (omental: 42.1 ± 6.4 pmol/sec/50,000 cells; subcutaneous: 37.7 ± 6.2 pmol/sec/50,000 cells) remained significantly increased to ∼2× that predicted for their BMI by the BMI: V max regression among NO, O, and SO patients (Figure 4 A, 4 B), 2× the value observed in the O patient group (Figure 6 A), and about fivefold compared to the NO range, indicating persistent upregulation of both omental and subcutaneous facilitated adipocyte LCFA uptake in SOr patients. As illustrated in Figure 6 B, the omental V max fell appreciably in the transition from SO to SOr status in 9 of 10 individual patients, the exception being a patient who—unknown to his surgeon at the time—began to drink heavily in the period between his two bariatric surgical procedures.

SOr participants who returned for a second operation 16.3 ± 2.2 mos after their initial surgery had lost a mean of 113 ± 13 lbs. As indicated by comparing SO with SOr results in Table 1 , weight loss was associated with modest changes in clinical laboratory parameters, including glucose, lipids, and liver tests. Medications for control of T2DM, hyperlipidemia, or cardiovascular disorders including hypertension, prescribed by referring physicians, were also little changed (Supporting Information Table 2).

Comparisons of V max for LCFA uptake by omental (abscissa) and subcutaneous (ordinate) adipocytes in individual non‐obese, obese, and super‐obese patients. ( A ) Results for V max from the two depots were very similar in each individual patient, so that the slope of the regression line (0.96) is close to unity and the correlation coefficient r = 0.96. ( B ) Data for the SOr group was added to those in panel A. Omental and subcutaneous V max values for 9 of the 10 super‐obese patients decreased to a similar amount in association with a mean weight loss of 113 lbs, so that the data points essentially moved downward along the initial regression line (solid line). Consequently, both the slope (0.89) and correlation coefficient ( r = 0.92) of the new regression line (dashed line) are very similar to those in panel A. Differences in these two parameters are entirely attributable to results in a single patient.

The V max for facilitated LCFA uptake by omental adipocytes increased exponentially as a function of BMI across the three patient groups (Figure 2 A). Mean values averaged 8.3 ± 0.6, 20.9 ± 1.4, and 68.7 ± 9.6 pmol/sec/50,000 cells in the NO, O, and SO patients, respectively. Data from subcutaneous adipocytes were very similar (Figure 2 B). The V max for omental and subcutaneous adipocytes in individual patients in each of the three patient groups were also very similar, and were linearly related ( r = 0.96) (Figure 3 A). Effects of surgical weight loss on V max (Figure 3 B) are examined below. Omental V max values in SO and O patients were highly correlated with BMI ( P ≪ 0.01) (Supporting Information Figure 1A), which in turn was highly correlated with Body Fat % ( P < 0.01) (Supporting Information Figure 1B). Results in subcutaneous adipocytes were virtually identical.

Measured values for adipocyte cell diameters (left panels) and resulting calculated values for mean adipocyte surface areas (center panels) and cell volumes (right panels) in omental (above) and subcutaneous (below) adipocytes from NO (▪), O (▲), and SO (•) patients ( n = 10/group). Across the three patient groups, all of the parameters illustrated increased as statistically significant linear functions of BMI. Samples were obtained during bariatric surgery in the O and SO patients and during other clinically indicated abdominal surgical procedures in NO patients.

Diameters of both omental and subcutaneous adipocytes from the 30 NO, O and SO patients increased linearly with increasing BMI (omental: r = 0.55, P < 0.01; subcutaneous: r = 0.46, P < 0.05) (Figure 1 ). Computed adipocyte surface areas and volumes were similarly correlated with BMI. When averaged by group, the calculated omental adipocyte surface area increased from 23.2 ± 4.1 to 34.8 ± 2.0 to 39.1 ± 4.5 × 10 3 μ 2 per cell and cell volume from 437 ± 74 to 616 ± 53 to 749 ± 124 pl per cell in NO, O, and SO patients. Subcutaneous adipocytes showed very similar trends.

Additional data from the 10 individual SO/SOr patients that were, inter alia, the basis for our ANOVA effects adjustment testing are presented in Supporting Information Tables 1 and 2. Among these 10 SO patients, 8 were Caucasian‐non‐Hispanic, 1 Caucasian‐Hispanic, and 1 self‐described as a Caucasian/Hispanic/African American (Supporting Information Table 1). Initially 8 of the 10 SO patients had hypertension and 6 had diabetes. SO patients had a mean of three comorbidities each; one patient had only steatohepatitis, and only one patient had none.

Demographic and clinical laboratory data for the 10 participants in each of the NO, O, and SO groups are summarized in Table 1 , as are analogous data for the group designated as SOr, which were obtained from SO patients at the time of their second bariatric procedure. Mean ages, initial BMIs and clinical and laboratory data for the 10 SO patients who completed a second operation are very similar to corresponding data from all 35 SO patients enrolled in the study. Overall, the NO, O, and SO patient groups were similar in age (Table 1 ). O and SO weighed more than NO patients and had higher BMIs ( P < 0.001). While high‐density lipoprotein (HDL) values were lower and TG higher in the O and SO patients than in the NO controls (Table 1 ), there were no significant increases in glucose or cholesterol in these two groups of patients with obesity, possibly reflecting ongoing treatment for hyperglycemia and/or hypercholesterolemia. Albumin was marginally reduced and aspartate aminotransferase (AST) and/or alanine aminotransferase (ALT) marginally increased in the SO and O groups (Table 1 ). Overall, the abnormalities in aminotransferases were on the mild end of the spectrum observed in larger populations of SO patients ( 30 ). Gender differences were observed for several clinical and laboratory parameters summarized in Table 1 . However, as there were only 5 patients of each gender per group, these differences were not analyzed further.

Discussion

The complexity of changes in adipose gene expression was not appreciated (31) and Spexin (32) and its association with weight regulation (29) had not been discovered when the project began in 2006. Our initial expectations were that adipocyte size and saturable LCFA uptake would fall in parallel during bariatric surgery‐induced weight loss.

While omental adipocyte sizes (Figure 5) and the V max for LCFA (Figure 6A) uptake both decreased as SO patients' BMIs fell after initial bariatric surgery, they did not decrease in parallel. SOr cell sizes often fell into the normal range and were therefore small relative to BMI, but V max did not consistently fall even into the O range in these patients, whose BMIs remained in the 40s. Consequently, when expressed per unit surface area, V max′ of omental adipocytes actually increased 28%, from 3.6 ± 0.5 to 4.6 ± 0.9 pmol s−1 µ−2 × 10−8, a value nearly fourfold higher than the 1.2 ± 0.1 pmol s−1 µ−2 × 10−8 typical of O patients (Figure 6C). These findings are graphed (Figure 7) and their implications discussed in detail in Supporting Information. Our cell size and V max′ data are consistent with recent models (33) which propose that small for body weight adipocytes are an important stimulus to weight regain via several complex pathways.

Because upregulation of adipocyte LCFA transport is closely associated with obesity in man (16), its upregulation predicts weight gain in multiple animal obesity models (1, 12, 13), and this study documents a significant correlation between LCFA uptake and BMI (Figure 2), it is tempting to speculate that persistent upregulation of LCFA uptake in our SOr patients following bariatric surgical weight loss is a harbinger of weight regain. Weight regain has become an increasingly important issue in obesity management, after both dietary weight loss (34) and bariatric surgery (35). The LABS consortium recently published the outcomes over 3 years following bariatric surgery in 2458 patients with obesity (36), of whom 1,738 underwent Roux‐en‐Y gastric bypass (RYGB), 610 laparoscopic placement of an adjustable gastric band (LAGB), and 110 other procedures including 59 sleeve gastrectomies. RYGB and LAGB patients experienced most of their total weight loss in the first post‐operative year. To evaluate weight patterns, five weight trajectory groups were identified for each procedure. The five RYGB trajectories all showed initial weight loss for 6 months after surgery, but by the third post‐operative year, trajectories for all five groups demonstrated weight regain, accompanied by some recurrences of co‐morbidites. Modest weight regain was reported during the second and third post‐operative years among patients who had undergone sleeve gastrectomy or RYGB in another trial comparing intensive medical therapy alone vs. intensive medical therapy plus bariatric surgery for treatment of diabetes (37). The prevalence of weight regain in these and earlier studies (35) became increasingly evident by the middle of the second post‐operative year, corresponding to when we noted persistent upregulation of adipocyte LCFA uptake in our SOr patients.

Persistence of weight gain‐promoting hormone patterns; increased insulin sensitivity, rates of glucose transport, and LPL activity; and a multiplicity of persistent metabolic abnormalities are factors believed to contribute to weight regain (33, 35, 38). Given our earlier demonstration of the association between weight gain and upregulation of adipocyte LCFA uptake, and of the present results, it is tempting to speculate that persistent upregulation of adipose tissue LCFA uptake is, at the least, another potential mechanism contributing to weight regain after initial weight loss induced by bariatric surgery. However, to prove that, it will be necessary to study it, other potential causes of weight regain, and weight regain per se in the same cohort. This was impossible in the current study because our protocol mandated a second omental fat biopsy, which could only be obtained during a second bariatric surgical procedure. This second procedure led to further weight loss, averaging 50 ± 13.5 lbs by a mean of 11 months post‐operatively, making any tendency to more modest weight regain from the first surgery undetectable. However, the finding that adipocyte dimensions and LCFA uptake kinetics in subcutaneous adipocytes are virtually identical to those in omental fat is important, since subcutaneous fat can be obtained by aspiration during routine outpatient visits. Correlating serial aspiration biopsies of subcutaneous fat and simultaneous weight determinations after a single bariatric surgical procedure in each patient will provide a much stronger assessment of the relationship between LCFA kinetics and weight regain, indicate whether any observed upregulation of adipocyte LCFA uptake persists with longer follow‐up or whether it eventually normalizes, and the extent to which it occurs with all bariatric surgical procedures or is a unique consequence of sleeve gastrectomy. Adaptive responses lead the majority of patients who previously had obesity, who then lost weight by dietary restriction, to later regain the lost weight (33, 34, 38). The role for persistently upregulated adipocyte LCFA uptake in that process is an open question.

Body weight and energy balance are principally regulated by integration of numerous signals, including concentrations of hormones released mainly from the gut and adipose tissues (39). The details of these processes are still being elucidated, as is the extent to which the persistent upregulation of LCFA uptake reflects abnormal hormonal patterns or a more complex pathogenesis.

Several large studies (35, 36, 40) show that bariatric surgery is the most effective current approach to short‐ and medium‐term weight reduction and, often, remission of co‐morbidities. However, its longer term efficacy and the role of weight regain in modulating its benefits are still uncertain. Because effective anti‐obesity drugs developed in response to improved understanding of obesity pathophysiology are likely to become available in the future, appropriate therapeutic choices for optimal weight management will require improved understanding of the underlying physiology. Accordingly, evaluating the impact of persistently increased LCFA uptake and other mechanisms on weight regain and long‐term obesity management should be actively pursued, and relevant processes, including increased adipocyte LCFA uptake, identified and studied in detail for their possible therapeutic benefits.