New insights in the mechanisms of weight-loss maintenance: Summary from a Pennington symposium.

Obesity is a chronic disease that affects more than 650 million adults worldwide. Obesity not only is a significant health concern on its own, but predisposes to cardiometabolic comorbidities, including coronary heart disease, dyslipidemia, hypertension, type 2 diabetes, and some cancers. Lifestyle interventions effectively promote weight loss of 5% to 10%, and pharmacological and surgical interventions even more, with some novel approved drugs inducing up to an average of 25% weight loss. Yet, maintaining weight loss over the long-term remains extremely challenging, and subsequent weight gain is typical. The mechanisms underlying weight regain remain to be fully elucidated. The purpose of this Pennington Biomedical Scientific Symposium was to review and highlight the complex interplay between the physiological, behavioral, and environmental systems controlling energy intake and expenditure. Each of these contributions were further discussed in the context of weight-loss maintenance, and systems-level viewpoints were highlighted to interpret gaps in current approaches. The invited speakers built upon the science of obesity and weight loss to collectively propose future research directions that will aid in revealing the complicated mechanisms involved in the weight-reduced state.

Describing the Weight-Reduced State: Physiology, Behavior, and Interventions.

Although many persons with obesity can lose weight by lifestyle (diet and physical activity) therapy, successful long-term weight loss is difficult to achieve, and most people who lose weight regain their lost weight over time. The neurohormonal, physiological, and behavioral factors that promote weight recidivism are unclear and complex. The National Institute of Diabetes and Digestive and Kidney Diseases convened a workshop in June 2019, titled "The Physiology of the Weight-Reduced State," to explore the mechanisms and integrative physiology of adaptations in appetite, energy expenditure, and thermogenesis that occur in the weight-reduced state and that may oppose weight-loss maintenance. The proceedings from the first session of this workshop are presented here. Drs. Michael Rosenbaum, Kevin Hall, and Rudolph Leibel discussed the physiological factors that contribute to weight regain; Dr. Michael Lowe discussed the biobehavioral issues involved in weight-loss maintenance; Dr. John Jakicic discussed the influence of physical activity on long-term weight-loss maintenance; and Dr. Louis Aronne discussed the ability of drug therapy to maintain weight loss.

P110α and P110δ catalytic subunits of PI3 kinase regulate lysophosphatidylcholine-induced TRPC6 externalization.

Lipid oxidation products, including lysophosphatidylcholine (lysoPC) inhibit endothelial cell (EC) migration in vitro and impair EC healing of arterial injuries in vivo, in part by activating phosphatidylinositol 3-kinase (PI3K), which increases the externalization of canonical transient receptor potential 6 (TRPC6) channels and the subsequent increase in intracellular calcium. Inhibition of PI3K is a potential method to decrease TRPC6 activation and restore migration, but PI3K is involved in multiple intracellular signaling pathways and has multiple downstream effectors. The goal of this study is to identify the specific p110 catalytic subunit isoforms responsible for lysoPC-induced TRPC6 externalization to identify a target for intervention while minimizing impact on alternative signaling pathways. Down-regulation of the p110α and p110δ isoforms, but not the p110ß or p110γ isoforms, with small interfering RNA significantly decreased phosphatidylinositol (3,4,5)-trisphosphate production and TRPC6 externalization, and significantly improved EC migration in the presence of lysoPC. These results identify an additional role of p110α in EC and reveal for the first time a specific role of p110δ in EC, providing a foundation for subsequent in vivo studies to investigate the impact of p110 isoform inhibition on arterial healing after injury.

Loss of MAGEL2 in Prader-Willi syndrome leads to decreased secretory granule and neuropeptide production.

Prader-Willi syndrome (PWS) is a developmental disorder caused by loss of maternally imprinted genes on 15q11-q13, including melanoma antigen gene family member L2 (MAGEL2). The clinical phenotypes of PWS suggest impaired hypothalamic neuroendocrine function; however, the exact cellular defects are unknown. Here, we report deficits in secretory granule (SG) abundance and bioactive neuropeptide production upon loss of MAGEL2 in humans and mice. Unbiased proteomic analysis of Magel2pΔ/m+ mice revealed a reduction in components of SG in the hypothalamus that was confirmed in 2 PWS patient-derived neuronal cell models. Mechanistically, we show that proper endosomal trafficking by the MAGEL2-regulated WASH complex is required to prevent aberrant lysosomal degradation of SG proteins and reduction of mature SG abundance. Importantly, loss of MAGEL2 in mice, NGN2-induced neurons, and human patients led to reduced neuropeptide production. Thus, MAGEL2 plays an important role in hypothalamic neuroendocrine function, and cellular defects in this pathway may contribute to PWS disease etiology. Moreover, these findings suggest unanticipated approaches for therapeutic intervention.

Hyperkeratotic and hypertrophic lichen nitidus.

Lichen nitidus typically presents as shiny pin-head sized papules on the trunk and extremities, often affecting children and young adults. In this prototypical form, it rarely presents a diagnostic challenge being characterized by distinctive clinical and histopathologic findings. We describe a rare variant of lichen nitidus, which we term "hyperkeratotic and hypertrophic lichen nitidus."

Deficiency in prohormone convertase PC1 impairs prohormone processing in Prader-Willi syndrome.

Prader-Willi syndrome (PWS) is caused by a loss of paternally expressed genes in an imprinted region of chromosome 15q. Among the canonical PWS phenotypes are hyperphagic obesity, central hypogonadism, and low growth hormone (GH). Rare microdeletions in PWS patients define a 91-kb minimum critical deletion region encompassing 3 genes, including the noncoding RNA gene SNORD116. Here, we found that protein and transcript levels of nescient helix loop helix 2 (NHLH2) and the prohormone convertase PC1 (encoded by PCSK1) were reduced in PWS patient induced pluripotent stem cell-derived (iPSC-derived) neurons. Moreover, Nhlh2 and Pcsk1 expression were reduced in hypothalami of fasted Snord116 paternal knockout (Snord116p-/m+) mice. Hypothalamic Agrp and Npy remained elevated following refeeding in association with relative hyperphagia in Snord116p-/m+ mice. Nhlh2-deficient mice display growth deficiencies as adolescents and hypogonadism, hyperphagia, and obesity as adults. Nhlh2 has also been shown to promote Pcsk1 expression. Humans and mice deficient in PC1 display hyperphagic obesity, hypogonadism, decreased GH, and hypoinsulinemic diabetes due to impaired prohormone processing. Here, we found that Snord116p-/m+ mice displayed in vivo functional defects in prohormone processing of proinsulin, pro-GH-releasing hormone, and proghrelin in association with reductions in islet, hypothalamic, and stomach PC1 content. Our findings suggest that the major neuroendocrine features of PWS are due to PC1 deficiency.

The subgingival microbiome, systemic inflammation and insulin resistance: The Oral Infections, Glucose Intolerance and Insulin Resistance Study.

BACKGROUND: Inflammation might link microbial exposures to insulin resistance. We investigated the cross-sectional association between periodontal microbiota, inflammation and insulin resistance. METHODS: The Oral Infections, Glucose Intolerance and Insulin Resistance Study (ORIGINS) enrolled 152 diabetes-free adults (77% female) aged 20-55 years (mean = 34 ± 10). Three hundred and four subgingival plaque samples were analysed using the Human Oral Microbe Identification Microarray to measure the relative abundances of 379 taxa. C-reactive protein, interleukin-6, tumour necrosis factor-α and adiponectin were assessed from venous blood and their z-scores were summed to create an inflammatory score (IS). Insulin resistance was defined via the HOMA-IR. Associations between the microbiota and both inflammation and HOMA-IR were explored using multivariable linear regressions; mediation analyses assessed the proportion of the association explained by inflammation. RESULTS: The IS was inversely associated with Actinobacteria and Proteobacteria and positively associated with Firmicutes and TM7 (p-values < 0.05). Proteobacteria levels were associated with insulin resistance (p < 0.05). Inflammation explained 30-98% of the observed associations between levels of Actinobacteria, Proteobacteria or Firmicutes and insulin resistance (p-values < 0.05). Eighteen individual taxa were associated with inflammation (p < 0.05) and 22 with insulin resistance (p < 0.05). No findings for individual taxa met Bonferroni-adjusted statistical significance. CONCLUSION: Bacterial measures were related to inflammation and insulin resistance among diabetes-free adults.

Apolipoprotein A-I mimetic peptide reverses impaired arterial healing after injury by reducing oxidative stress.

OBJECTIVE: Endothelial cell (EC) migration is essential for healing of arterial injuries caused by angioplasty, but a high cholesterol diet inhibits endothelial repair. In vivo studies suggest that apolipoprotein A-I (apoA-I), the major protein constituent of HDL, is essential for normal healing of arterial injuries. ApoA-I mimetics, including 4F, have been designed to mimic the amphipathic portion of the apoA-I molecule. This study was undertaken to determine if 4F improves endothelial migration and healing. METHODS: A razor scrape assay was used to analyze the effect of 4F on EC migration in vitro. Endothelial healing in vivo was assessed following electrical injury of carotid arteries in mice. Markers of oxidative stress were also examined. RESULTS: Lipid oxidation products inhibited EC migration in vitro, but preincubation with L-4F preserved EC migration. Endothelial healing of carotid arterial injuries in mice on a high cholesterol diet was delayed compared with mice on a chow diet with 27.8% vs. 48.2% healing, respectively, at 5 days. Administration of D-4F improved endothelial healing in mice on a high cholesterol diet to 43.4%. D-4F administration had no effect on lipid levels but decreased markers of oxidation. In vivo, there was a significant inverse correlation between endothelial healing and plasma markers of oxidative stress. CONCLUSION: These studies suggested that an apoA-I mimetic can improve endothelial healing of arterial injuries by decreasing oxidative stress.

Alanine transferase: An independent indicator of adiposity related comorbidity risk in youth.

BACKGROUND: Elevated levels of alanine aminotransferase (ALT) are associated with obesity and are often a consequence of non-alcoholic fatty liver disease (NAFLD). The aim of the present study was to assess the relationship between ALT and risk factors for adiposity-related co-morbidities in a diverse population of middle school children. METHODS: We measured height, weight, body fatness (bioelectrical impedance), waist circumference, insulin sensitivity, phase 1 insulin release (acute insulin response following intravenous glucose), beta-cell function (acute insulin response corrected for insulin sensitivity), ALT, lipid profiles, and circulating concentrations of interleukin-6 (IL-6), C-reactive protein, adiponectin, and tumor necrosis factor-α (TNF-α) in a multi-ethnic/racial population of 106 middle school students (aged 11-14 years, 45 female) of varying body mass indexes (BMI). RESULTS: Alanine aminotransferase was significantly correlated with BMI, % body fat, fat mass, waist circumference, fasting insulin, insulin resistance, triglycerides, and was inversely correlated with high-density lipoprotein cholesterol in children, even though all values of ALT were "normal" (range of 4.0-33.0 U/L). ALT was significantly higher in males than females even when corrected for body fatness. Significant correlations with lipids and insulin resistance persisted even when adjusted for age, gender, and body fatness. CONCLUSION: Even within the normative range, ALT levels were significantly correlated with anthropomorphic and biochemical risk factors for adiposity-related co-morbidities in youth. Therefore, because ALT is correlated with dyslipidemia, insulin resistance, and central fat distribution, it might also serve as a marker of risk for adiposity-related co-morbidities beyond NAFLD.

Hypercholesterolemia inhibits re-endothelialization of arterial injuries by TRPC channel activation.

OBJECTIVE: After arterial injury, endothelial cell (EC) migration is essential for healing, but lipid oxidation products activate TRPC6 and TRPC5 ion channels, leading to increased intracellular calcium and inhibition of EC migration in vitro. The objective of this study was to further evaluate the role of TRPC channels in EC migration in vitro and to validate in vitro findings in an in vivo model. METHODS: Mouse aortic ECs were cultured, and the effect of lysophosphatidylcholine, the major lysophospholipid in oxidized low-density lipoprotein, on migration was assessed in a razor-scrape assay. EC healing after a carotid injury with electrocautery was evaluated in wild-type (WT), TRPC6(-/-), and TRPC5(-/-) mice receiving either a chow or high-cholesterol (HC) diet. RESULTS: Lysophosphatidylcholine inhibited EC migration of WT ECs to 22% of baseline and of TRPC5(-/-) ECs to 53% of baseline but had minimal effect on TRPC6(-/-) EC migration. Hypercholesterolemia severely impaired EC healing in vivo, with 51.4% ± 1.8% and 24.9% ± 2.0% of the injury resurfaced with ECs at 5 days in chow-fed and HC-fed WT mice, respectively (P < .001). Hypercholesterolemia did not impair healing in TRPC6(-/-) mice, with coverage of 48.4% ± 3.4% and 46.8% ± 1.6% in chow-fed and HC-fed TRPC6(-/-) mice, respectively. Hypercholesterolemia had a reduced inhibitory effect in TRPC5(-/-) mice, with EC coverage of 51.7% ± 3.0% and 37.% ± 1.4% in chow-fed and HC-fed TRPC5(-/-) mice, respectively. CONCLUSIONS: Results suggest that activation of TRPC6 and TRPC5 channels is the key contributor to impaired endothelial healing of arterial injuries in hypercholesterolemic mice.

The threshold shift paradigm of obesity: evidence from surgically induced weight loss.

BACKGROUND: The high prevalence of obesity has called attention to the near-intractable problem of sustained weight reduction and its underlying mechanisms. With diet-induced weight loss, achieved body weight is closely related to initial body weight. OBJECTIVE: The objective was to compare the relation between initial and achieved body mass index (BMI) in patients treated with diet-induced weight loss or bariatric surgery. DESIGN: We analyzed data from a cohort of 223 healthy individuals who lost a mean (±SD) of 5 ± 3 kg body weight over 3 y by diet (diet group) and data from 182 obese individuals [BMI (in kg/m(2)) ≥35] who had lost an average of 47 ± 17 kg 1 y after Roux-en-Y gastric bypass (a restrictive procedure; n = 71) or biliopancreatic diversion (a malabsorptive procedure; n = 111) (surgery group). RESULTS: In the diet group, final BMI was strongly related to initial BMI (r = 0.96, P < 0.0001). By multivariate analysis, the decrease in BMI at 3 y was age independent and was predicted only by initial BMI and sex (both P < 0.0001). Strikingly, final BMI was also strongly related to initial BMI (r = 0.67, P < 0.0001) in the surgery group, irrespective of the type of operation. The surgically induced decrease in BMI was predicted by age (P = 0.0002) and initial BMI (P < 0.0001). In 110 surgery patients, serum leptin concentrations decreased from 39 ± 16 to 10 ± 5 ng/mL after surgery (P < 0.0001) and were correlated with BMI both before and after surgery, but the slope of the relation was significantly (P < 0.01) flatter after surgery. CONCLUSION: The strong predictivity of initial BMI for achieved BMI observed even when voluntary control of energy intake is interfered with through diverse anatomical rearrangements of the gastrointestinal tract supports the concept of a weight "threshold" paradigm: in the obese, anabolic responses are triggered by adiposity-related signals at a higher threshold, which leads to defense of a higher body weight.

Racial/ethnic differences in clinical and biochemical type 2 diabetes mellitus risk factors in children.

OBJECTIVE: To examine whether periadolescent children demonstrate the significant racial/ethnic differences in body fatness relative to BMI and in the prevalence and relationship of body composition to risk factors for type 2 diabetes (T2DM) as in adults. DESIGN AND METHODS: Family history of obesity and T2DM, anthropometry, insulin sensitivity and secretory capacity, lipids, and cytokines (IL-6, CRP, TNF-α, and adiponectin) were examined in a cohort of 994 middle school students (47% male, 53%, female; 12% African American, 14% East Asian, 13% South Asian, 9% Caucasian, 44% Hispanic, and 8% other). RESULTS: Fractional body fat content was significantly greater at any BMI among South Asians. There were racial/ethnic specific differences in lipid profiles, insulin secretory capacity, insulin sensitivity, and inflammatory markers corrected for body fatness that are similar to those seen in adults. Family history of T2DM was associated with lower insulin secretory capacity while family history of obesity was more associated with insulin resistance. CONCLUSIONS: Children show some of the same racial/ethnic differences in risk factors for adiposity-related comorbidities as adults. BMI and waist circumference cutoffs to identify children at-risk for adiposity-related comorbidities should be adjusted by racial/ethnic group as well as other variables such as birthweight and family history.

Brown remodeling of white adipose tissue by SirT1-dependent deacetylation of Pparγ.

Brown adipose tissue (BAT) can disperse stored energy as heat. Promoting BAT-like features in white adipose (WAT) is an attractive, if elusive, therapeutic approach to staunch the current obesity epidemic. Here we report that gain of function of the NAD-dependent deacetylase SirT1 or loss of function of its endogenous inhibitor Deleted in breast cancer-1 (Dbc1) promote "browning" of WAT by deacetylating peroxisome proliferator-activated receptor (Ppar)-γ on Lys268 and Lys293. SirT1-dependent deacetylation of Lys268 and Lys293 is required to recruit the BAT program coactivator Prdm16 to Pparγ, leading to selective induction of BAT genes and repression of visceral WAT genes associated with insulin resistance. An acetylation-defective Pparγ mutant induces a brown phenotype in white adipocytes, whereas an acetylated mimetic fails to induce "brown" genes but retains the ability to activate "white" genes. We propose that SirT1-dependent Pparγ deacetylation is a form of selective Pparγ modulation of potential therapeutic import.

Hypercholesterolemia and oxidative stress inhibit endothelial cell healing after arterial injury.

OBJECTIVE: Endothelial cell (EC) migration is essential for arterial healing after angioplasty. Oxidized low-density lipoproteins and oxidative stress decrease EC migration in vitro. The objective of this study was to determine the effect of hypercholesterolemia and oxidative stress on EC healing after an arterial injury. METHODS: C57BL/6 wild-type mice were placed in one of eight groups: chow diet (n = 11), high-cholesterol (HC) diet (n = 11), chow diet plus paraquat (n = 11), HC diet plus paraquat (n = 11), chow diet plus N-acetylcysteine (NAC) (n = 11), HC diet plus NAC (n = 11), chow diet plus paraquat and NAC (n = 11), and HC diet plus paraquat and NAC (n = 11). After 2 weeks on the assigned diet with or without NAC, the carotid artery was injured using electrocautery. Animals in the paraquat groups were given 1 mg/kg intraperitoneally to increase oxidative stress. After 120 hours, Evans Blue dye was infused intravenously to stain the area of the artery that remained deendothelialized. This was used to calculate the percentage of re-endothelialization. Plasma and tissue samples were analyzed for measures of oxidative stress. RESULTS: The HC diet increased oxidative stress and reduced EC healing compared with a chow diet, with EC covering 26.8% ± 2.8% and 48.1% ± 5.2% (P < .001) of the injured area, respectively. Administration of paraquat decreased healing in both chow and HC animals to 18.1% ± 3.5% (P < .001) and 9.8% ± 4.6% (P < .001), respectively. Pretreatment with NAC (120 mmol/L in drinking water) for 2 weeks prior to injury, to decrease oxidative stress, improved EC healing to 39.9% ± 5.7% (P < .001) in hypercholesterolemic mice and to 30.7% ± 3.6% (P < .001) in the paraquat group. NAC treatment improved healing to 24.6% ± 3.4% (P < .001) in hypercholesterolemic mice treated with paraquat. CONCLUSION: Re-endothelialization of arterial injuries is reduced in hypercholesterolemic mice and is inversely correlated with oxidative stress. An oral antioxidant decreases oxidative stress and improves EC healing. CLINICAL RELEVANCE: Vascular injury following cardiovascular intervention, including cardiac and peripheral arterial angioplasty and stenting, is associated with inflammation and oxidative stress. Hypercholesterolemia is also associated with increased oxidative stress. Oxidative stress, regardless of the source, induces cellular dysfunction in endothelial and smooth muscle cells that reduce healing after arterial injury. Decreasing oxidative stress with an exogenously administered antioxidant can improve endothelial cell healing, and this is important to control intimal hyperplasia and reduce the thrombogenicity of the vessel.

Effects of weight loss and leptin on skeletal muscle in human subjects.

Maintenance of a 10% or greater reduced body weight results in decreases in the energy cost of low levels of physical activity beyond those attributable to the altered body weight. These changes in nonresting energy expenditure are due mainly to increased skeletal muscle work efficiency following weight loss and are reversed by the administration of the adipocyte-derived hormone leptin. We have also shown previously that the maintenance of a reduced weight is accompanied by a decrease in ratio of glycolytic (phosphofructokinase) to oxidative (cytochrome c oxidase) activity in vastus lateralis muscle that would suggest an increase in the relative expression of the myosin heavy chain I (MHC I) isoform. We performed analyses of vastus lateralis muscle needle biopsy samples to determine whether maintenance of an altered body weight was associated with changes in skeletal muscle metabolic properties as well as mRNA expression of different isoforms of the MHC and sarcoplasmic endoplasmic reticular Ca(2+)-dependent ATPase (SERCA) in subjects studied before weight loss and then again after losing 10% of their initial weight and receiving twice daily injections of either placebo or replacement leptin in a single blind crossover design. We found that the maintenance of a reduced body weight was associated with significant increases in the relative gene expression of MHC I mRNA that was reversed by the administration of leptin as well as an increase in the expression of SERCA2 that was not significantly affected by leptin. Leptin administration also resulted in a significant increase in the expression of the less MHC IIx isoform compared with subjects receiving placebo. These findings are consistent with the leptin-reversible increase in skeletal muscle chemomechanical work efficiency and decrease in the ratio of glycolytic/oxidative enzyme activities observed in subjects following dietary weight loss.

Endothelial function as measured by peripheral arterial tonometry increases during pubertal advancement.

BACKGROUND/AIMS: Sex steroids, such as estrogens, are known to influence endothelial function by their vasodilator action. The aim of this study was to study the relation of puberty and sex steroids with endothelial function using peripheral arterial tonometry (PAT). METHODS: In 89 healthy school boys and girls, we determined height, weight, waist circumference, percent body fat, BMI, BMI z-score, blood pressure (BP), BP percentiles, lipid profile, insulin, and glucose levels after overnight fast. Estrone (E(1)), estradiol (E(2)), DHEAS and E(1)-sulfate were measured using ultrasensitive assays. Participants were divided into 3 pubertal groups on the basis of their estrogen levels: group 1 (Tanner stage I), group 2 (Tanner stages II-III), and group 3 (Tanner stages IV-V). Endothelial function was measured by Endo-PAT 2000® and expressed as PAT index. A higher PAT index represents a higher reactive hyperemia response. RESULTS: The PAT index was lowest at 1.42 ± 0.44 (mean ± SD) in group 1 and significantly increased in group 2 at 1.71 ± 0.35 (p = 0.02) and group 3 at 1.92 ± 0.38 (p < 0.001). The PAT index correlated positively with E(2), DHEAS and age. CONCLUSION: Enhancement of the PAT index was associated with an increment in Tanner stages. The changes in E(2) and DHEAS levels may contribute to increasing endothelial response to shear stress or arterial blood flow.

Leptin administration does not prevent the bone mineral metabolism changes induced by weight loss.

The objective was to examine the effects of weight loss and leptin administration following weight loss on calciotropic hormones and bone turnover. This was a prospective, single-blinded study of 12 subjects (8 women, 4 men; 2 nonobese, 10 obese; age range, 19-46 years) who were studied on an inpatient basis while maintaining their usual weight [Wt(initial)] and during maintenance of 10% weight loss while receiving twice-daily injections of either a placebo [Wt(-10%P)] or replacement doses of leptin [Wt(-10%L)]. The main outcome measures were markers of bone formation (bone alkaline phosphatase and procollagen type 1 amino terminal propeptide) and resorption (N-telopeptide) as well as parathyroid hormone, calcium, and 25-hydroxy vitamin D measured from fasting morning serum. As expected, serum leptin declined with weight loss. Bone alkaline phosphatase decreased by 12.3% ± 3.9% between Wt(initial) and Wt(-10%P) and remained suppressed after leptin administration (both P < .01 compared with baseline). N-telopeptides increased by 37.2% ± 11.3% from Wt(initial) to Wt(-10%L) (P < .01). Procollagen type 1 amino terminal propeptide, parathyroid hormone, calcium, and 25-hydroxy vitamin D did not change. These results suggest that both decreased bone formation and increased bone resorption underlie bone loss associated with weight loss. Leptin administration did not prevent the uncoupling of bone remodeling that accompanies weight loss.