Abstracts

INTRODUCTION

Lipodystrophy syndromes (LS) are congenital and acquired disorders characterized by the generalized or partial absence of subcutaneous adipose tissue. Cases of lipodystrophy not associated with the use of highly active antiretroviral therapy (HAART) are rare conditions, and most determined by molecular defects in genes that regulate adipocyte differentiation, lipid metabolism, and lipid droplet morphology (¹1 Fiorenza CG, Chou SH, Mantzoros CS. Lipodystrophy: pathophysiology and advances in treatment. Nat Rev Endocrinol. 2011;7:137-50.). As the adipose tissue is an important endocrine organ that synthesizes hormones with cytokine-like actions (known as adipokines) (²2 Ahima RS, Flier JS. Adipose tissue as an endocrine organ. Trends Endocrinol Metab. 2000;11:327-32.,³3 Lima FB. Adipose tissue: a brief historical perspective and the present moment. Arq Bras Endocrinol Metabol. 2008;52:927-8.), its absence determines several metabolic defects, such as insulin resistance, diabetes, and hypertriglyceridemia, which can lead to the development of atherosclerosis, acute pancreatitis, and nonalcoholic fatty liver disease (NAFLD) (⁴4 Caldas D, Silva Júnior WS, Simonetti JP, Costa EV, Farias MLF. Biochemical, hormonal and genetic evaluation of the families of two Brazilian patients with type 2 familial partial lipodystrophy. Arq Bras Endocrinol Metabol. 2013;57:583-93.).

Leptin is one of the most abundant adipokines. It is an important metabolic regulator responsible for not only controlling food intake and energy expenditure, but also for maintaining glucose, insulin and lipid homeostasis (⁵5 Paz-Filho G, Mastronardi C, Delibasi T, Wong ML, Licinio J. Congenital leptin deficiency: diagnosis and effects of leptin replacement therapy. Arq Bras Endocrinol Metabol. 2010;54:690-7.,⁶6 Paz-Filho G, Mastronardi C, Franco CB, Wang KB, Wong ML, Licinio J. Leptin: molecular mechanisms, systemic pro-inflammatory effects, and clinical implications. Arq Bras Endocrinol Metabol. 2012;56:597-607.). Impaired leptin action, due to leptin resistance or leptin deficiency, results in several metabolic abnormalities such as insulin resistance, hyperinsulinemia, diabetes, and hypertriglyceridemia, which are similar to those that are observed in LS. In fact, LS and leptin deficiency have similar metabolic manifestations, with low leptin levels (7‐9).

Leptin replacement therapy is the treatment of choice for LS. Numerous animal models have shown that exogenous supplementation of leptin, in order to achieve physiological levels of the hormone, is effective in restoring normal glycemic control, normal serum lipid profile and liver function (¹⁰10 Asilmaz E, Cohen P, Miyazaki M, Dobrzyn P, Ueki K, Fayzikhodjaeva G, et al. Site and mechanism of leptin action in a rodent form of congenital lipodystrophy. J Clin Invest. 2004;113:414-24.,¹¹11 Nagao K, Inoue N, Ujino Y, Higa K, Shirouchi B, Wang YM, et al. Effect of leptin infusion on insulin sensitivity and lipid metabolism in diet-induced lipodystrophy model mice. Lipids Health Dis. 2008;7:8.). In humans, leptin replacement therapy (LRT) with recombinant methionyl human leptin (r-metHuLeptin, metreleptin) has been evaluated by several clinical trials (¹²12 Simha V. Metreleptin for metabolic disorders associated with generalized or partial lipodystrophy. Expert Rev Endocrinol Metab. 2014;9:205-12.), and has been recently approved by the FDA for the treatment of patients with generalized lipodystrophy (¹³13 Chou K, Perry CM. Metreleptin: first global approval. Drugs. 2014;73:989-97.). Administered as a subcutaneous injection once or twice daily, metreleptin reverses the metabolic abnormalities that are seen in LS, leading to significant improvements in overall health (14‐16).

Currently, there is no data that comprehensively and succinctly demonstrate the effect of LRT on metabolic and hepatic parameters in LS patients. Therefore, this meta-analysis sought to survey the literature in this area, and quantitatively assess the effect of LRT in patients with all forms of LS (generalized and partial, congenital and acquired; excluding HAART-related LS) on various clinical endpoints. We sought data from clinical trials and case series on blood concentrations of glucose, insulin and glycated hemoglobin (Hb1Ac) as markers of glycemic control; triglycerides (TG), total cholesterol (TC), high-density lipoprotein (HDL) cholesterol and low-density lipoprotein (LDL) cholesterol as markers of lipid control; and alanine aminotransferase (ALT), aspartate aminotransferase (AST) and albumin as markers of liver function. Also, total liver volume and liver fat were assessed as radiological findings of liver health in response to LRT.

MATERIALS AND METHODS

Literature search

We conducted a literature review of published work in accordance with the PRISMA guidelines (¹⁷17 Moher D, Liberati A, Tetzlaff J, Altman DG. Preferred reporting items for systematic reviews and meta-analyses: the PRISMA statement. PLoS Med. 2009;6:e1000097.). Relevant studies were retrieved on 9/26/2013 from the MEDLINE (January 1966 to September 2013) and Cochrane Library of Clinical Trials (January 1980 to September 2013) databases. In order to identify studies assessing the outcomes of LRT for the treatment of all forms of LS not related to HAART, we applied as search terms the following title/abstract phrases: [“leptin” AND (“lipodystrophy” OR “lipoatrophy”)] with no language restriction. Titles and abstracts of identified searches were screened. In addition, reference lists of all articles were manually scanned and the ‘related article tool’ provided following a term search was used to identify other potentially relevant studies. We termed these the “grey literature”. The full texts were assessed to determine eligibility for inclusion. Studies were eligible if they assessed the outcomes of systemically administered leptin as a treatment for LS. Specific exclusion criteria were: i) study population consisting of participants whose LS were a result of HAART for the treatment of HIV; ii) studies that did not report specific data on the outcome of glycemic, lipid or hepatic measures following leptin treatment; iii) studies evaluating an agent other than leptin or a derivative of leptin (e.g. r-metHu-leptin/metreleptin); iv) case-reports of a single individual patient; v) studies published in languages other than English.

Data abstraction

The primary outcome of this review was to quantitatively assess and compare the impact of LRT on the blood concentrations of glucose, insulin, HbA1c, TG, TC, HDL, LDL, ALT, AST and albumin, and on liver fat and volume. Data capture was performed independently by two examiners (AJR, AGG). Data relating to the study design (including population, patient sampling, follow-up time and leptin replacement dosage), and baseline characteristics of study participants (including age, body mass index, LS etiology and other risk factors) were extracted. Further, we evaluated laboratory methods used to determine levels of systemic markers of leptin replacement outcomes. Additionally the quality of each study was assessed using a quality assessment questionnaire, which was modified from the previously validated QUADAS 2 tool as there is currently no accepted quality assessment tool for studies examining leptin to treat LS (¹⁸18 Whiting PF, Rutjes AW, Westwood ME, Mallett S, Deeks JJ, Reitsma JB, et al. QUADAS-2: a revised tool for the quality assessment of diagnostic accuracy studies. Ann Intern Med. 2011;155:529-36.). Our questionnaire contained eight points relating to study design, outcome measurement and analysis. Each point was marked as ‘yes’, ‘no’ or ‘unclear’, and the quality percentage score was based on the percentage of ‘yes’ responses that was obtained by each study. Sample copies of the data extraction form and quality assessment questionnaire are available upon request. Any questions or inconsistencies regarding these data were resolved through iteration and consensus. We included patients with acquired, congenital, partial and generalized forms of LS, and evaluated the effects of LRT collectively and separately by LS subtype.

Statistical methods

To mitigate against differences in the way marker concentrations were measured and reported, the absolute levels of the aforementioned markers were converted into a common unit by calculating a standardized mean difference (SMD). Standardized mean differences were derived from the reported p-values, by calculating the t-statistic and dividing by the square root of the population size. Standard errors were estimated as the inverse of the square root of the population size. We did not include data where significance tests were not performed (i.e. where p-values were not reported). Heterogeneity was determined by the I² index, which provides an indication into the inconsistencies between the studies (¹⁹19 Huedo-Medina TB, Sanchez-Meca J, Marin-Martinez F, Botella J. Assessing heterogeneity in meta-analysis: Q statistic or I2 index? Psychol Methods. 2006;11:193-206.). Results were presented as mean ± standard deviation (SD) or median and interquartile range (IQR). Meta-analysis was performed using an inverse-variance random effects model with RevMan v5.1 software (The Nordic Cochrane Centre, The Cochrane Collaboration, 2012). This software allows for the automated output of forest plots, I² index, test for overall effect, effect significance and a funnel plot as an indication into publication bias.

RESULTS

Included literature

The initial database search yielded 270 abstracts, of which 261 unique abstracts were reviewed for eligibility. Following abstract screening, 223 were excluded because they did not fit the inclusion criteria; mainly because they evaluated only HAART-associated LS (n=65), or did not include primary original data (n=56). Thirty-eight articles were eligible for full-text review. Of those, 26 were excluded mainly due to not being population-based data (case reports) or review articles and two of these studies were excluded as they represented data sets based on duplicated results from previous studies (²⁰20 Javor ED, Cochran EK, Musso C, Young JR, Depaoli AM, Gorden P. Long-term efficacy of leptin replacement in patients with generalized lipodystrophy. Diabetes. 2005;54:1994-2002.,²¹21 Oral EA, Ruiz E, Andewelt A, Sebring N, Wagner AJ, Depaoli AM, et al. Effect of leptin replacement on pituitary hormone regulation in patients with severe lipodystrophy. J Clin Endocrinol Metab. 2002;87(7):3110-7.). Overall, 12 studies were included for data analysis (14,16,22‐31). Figure 1 illustrates the flow diagram outlining literature search strategy and study selection.

Quality assessment

Quality percentage scores based on a modified QUADAS 2 tool ranged from 25% (³⁰30 Petersen KF, Oral EA, Dufour S, Befroy D, Ariyan C, Yu C, et al. Leptin reverses insulin resistance and hepatic steatosis in patients with severe lipodystrophy. J Clin Invest. 2002;109:1345-50.) to 75% (¹⁶16 Simha V, Subramanyam L, Szczepaniak L, Quittner C, Adams-Huet B, Snell P, et al. Comparison of efficacy and safety of leptin replacement therapy in moderately and severely hypoleptinemic patients with familial partial lipodystrophy of the Dunnigan variety. J Clin Endocrinol Metab. 2012;97:785-92.,²³23 Beltrand J, Lahlou N, Le Charpentier T, Sebag G, Leka S, Polak M, et al. Resistance to leptin-replacement therapy in Berardinelli-Seip congenital lipodystrophy: an immunological origin. Eur J Endocrinol. 2010;162:1083-91.), and the overall mean score was 56.3±14.6% (Table 1).

Study participants

Pooled together, the evaluated studies represented a composite population of 226 participants (45 males, 181 females). Studies had samples sizes ranging from 4 to 55, where the mean number of participants was 19.8±16.8. Follow-up times ranged from 3 to 100 months, where the mean time was 26.9±27.2 months. Table 1 summarizes the characteristics of the selected studies. The average age of the participants was 26.5±10.0 years. As shown in table 2, the average percentage of male participants was 22.4±25.7%, and four studies did not include males in the study population (27‐30). Where reported, the mean baseline BMI (body mass index) of study participants was 20.9±1.9 kg/m², mean baseline body fat percentage was 11.6±6.14%, and mean baseline fasting leptin concentration was 1.87±0.95 ng/mL. The number of diabetic patients was reported in only two studies (²⁸28 Oral EA, Simha V, Ruiz E, Andewelt A, Premkumar A, Snell P, et al. Leptin-replacement therapy for lipodystrophy. N Engl J Med. 2002;346:570-8.,³⁰30 Petersen KF, Oral EA, Dufour S, Befroy D, Ariyan C, Yu C, et al. Leptin reverses insulin resistance and hepatic steatosis in patients with severe lipodystrophy. J Clin Invest. 2002;109:1345-50.) (n=8/9 and 1/9 participants, respectively) (Table 2).

LRT details

All twelve studies included for review used r-metHuLeptin as the form of leptin supplementation. Administration method was uniform, as all patients received r-metHuLeptin via subcutaneous injections. Most studies (n=10) performed r-metHuLeptin administration twice daily (Table 3). Dosage of r-metHuLeptin ranged from 0.02 to 0.24 mg/kg/d for males and 0.04 to 0.24 mg/kg/d for females. For studies that did not stratify leptin dose according to gender, dosages ranged from 0.03 to 0.24 mg/kg/d (¹⁶16 Simha V, Subramanyam L, Szczepaniak L, Quittner C, Adams-Huet B, Snell P, et al. Comparison of efficacy and safety of leptin replacement therapy in moderately and severely hypoleptinemic patients with familial partial lipodystrophy of the Dunnigan variety. J Clin Endocrinol Metab. 2012;97:785-92.,²²22 Beltrand J, Beregszaszi M, Chevenne D, Sebag G, De Kerdanet M, Huet F, et al. Metabolic correction induced by leptin replacement treatment in young children with Berardinelli-Seip congenital lipoatrophy. Pediatrics. 2007;120:e291-6.,²³23 Beltrand J, Lahlou N, Le Charpentier T, Sebag G, Leka S, Polak M, et al. Resistance to leptin-replacement therapy in Berardinelli-Seip congenital lipodystrophy: an immunological origin. Eur J Endocrinol. 2010;162:1083-91.,²⁸28 Oral EA, Simha V, Ruiz E, Andewelt A, Premkumar A, Snell P, et al. Leptin-replacement therapy for lipodystrophy. N Engl J Med. 2002;346:570-8.,³¹31 Safar Zadeh E, Lungu AO, Cochran EK, Brown RJ, Ghany MG, Heller T, et al. The liver diseases of lipodystrophy: the long-term effect of leptin treatment. J Hepatol. 2013;59:131-7.). Instead of reporting daily leptin dosages per body weight, Chan and cols. reported the mean total dose received by patients, which was 2.75±0.43 mg for males and 5.55±0.33 mg for females (¹⁴14 Chan JL, Lutz K, Cochran E, Huang W, Peters Y, Weyer C, et al. Clinical effects of long-term metreleptin treatment in patients with lipodystrophy. Endocr Pract. 2011;17(6):922-32.). One study did not provide sufficient data on leptin administration dosages (³⁰30 Petersen KF, Oral EA, Dufour S, Befroy D, Ariyan C, Yu C, et al. Leptin reverses insulin resistance and hepatic steatosis in patients with severe lipodystrophy. J Clin Invest. 2002;109:1345-50.). These data are summarized in table 3.

Comparative data analysis

Glycemic control

Seven studies reported significant reductions in blood glucose (24‐26,28‐31). The largest reduction was seen in the study by Petersen and cols. (³⁰30 Petersen KF, Oral EA, Dufour S, Befroy D, Ariyan C, Yu C, et al. Leptin reverses insulin resistance and hepatic steatosis in patients with severe lipodystrophy. J Clin Invest. 2002;109:1345-50.), where patients with LS had an initial mean baseline fasting glucose concentration of 234±14 mg/dL. Following the three-month intervention, this declined to 122±21 mg/dL.

Three studies reported a reduction in fasting insulin following LRT but did not report whether this was significant (¹⁶16 Simha V, Subramanyam L, Szczepaniak L, Quittner C, Adams-Huet B, Snell P, et al. Comparison of efficacy and safety of leptin replacement therapy in moderately and severely hypoleptinemic patients with familial partial lipodystrophy of the Dunnigan variety. J Clin Endocrinol Metab. 2012;97:785-92.,²²22 Beltrand J, Beregszaszi M, Chevenne D, Sebag G, De Kerdanet M, Huet F, et al. Metabolic correction induced by leptin replacement treatment in young children with Berardinelli-Seip congenital lipoatrophy. Pediatrics. 2007;120:e291-6.,³⁰30 Petersen KF, Oral EA, Dufour S, Befroy D, Ariyan C, Yu C, et al. Leptin reverses insulin resistance and hepatic steatosis in patients with severe lipodystrophy. J Clin Invest. 2002;109:1345-50.). Four studies reported a significant reduction in HbA1c values at follow-up (²⁴24 Chong AY, Lupsa BC, Cochran EK, Gorden P. Efficacy of leptin therapy in the different forms of human lipodystrophy. Diabetologia. 2010;53:27-35.,²⁶26 Javor ED, Ghany MG, Cochran EK, Oral EA, DePaoli AM, Premkumar A, et al. Leptin reverses nonalcoholic steatohepatitis in patients with severe lipodystrophy. Hepatology. 2005;41:753-60.,²⁸28 Oral EA, Simha V, Ruiz E, Andewelt A, Premkumar A, Snell P, et al. Leptin-replacement therapy for lipodystrophy. N Engl J Med. 2002;346:570-8.,³¹31 Safar Zadeh E, Lungu AO, Cochran EK, Brown RJ, Ghany MG, Heller T, et al. The liver diseases of lipodystrophy: the long-term effect of leptin treatment. J Hepatol. 2013;59:131-7.), with the highest reduction being equal to 1.90% (²⁸28 Oral EA, Simha V, Ruiz E, Andewelt A, Premkumar A, Snell P, et al. Leptin-replacement therapy for lipodystrophy. N Engl J Med. 2002;346:570-8.). One study reported an increase of 1.77% from baseline mean HbA1c, but did not report whether this was significant (²³23 Beltrand J, Lahlou N, Le Charpentier T, Sebag G, Leka S, Polak M, et al. Resistance to leptin-replacement therapy in Berardinelli-Seip congenital lipodystrophy: an immunological origin. Eur J Endocrinol. 2010;162:1083-91.). Supplementary table 1 illustrates the changes in fasting plasma glucose, insulin and HbA1c.

Hepatic outcomes

Three studies reported significant reductions in mean liver volume following leptin therapy (²²22 Beltrand J, Beregszaszi M, Chevenne D, Sebag G, De Kerdanet M, Huet F, et al. Metabolic correction induced by leptin replacement treatment in young children with Berardinelli-Seip congenital lipoatrophy. Pediatrics. 2007;120:e291-6.,²⁶26 Javor ED, Ghany MG, Cochran EK, Oral EA, DePaoli AM, Premkumar A, et al. Leptin reverses nonalcoholic steatohepatitis in patients with severe lipodystrophy. Hepatology. 2005;41:753-60.,²⁷27 Moran SA, Patten N, Young JR, Cochran E, Sebring N, Reynolds J, et al. Changes in body composition in patients with severe lipodystrophy after leptin replacement therapy. Metabolism. 2004;53:513-9.). Liver fat was only reported in a single study and after stratifying results according to the participants’ degree of hypoleptinemia, either severely or mildly hypoleptinemic, both groups of patients exhibited significant decreases in mean liver fat percentage (¹⁶16 Simha V, Subramanyam L, Szczepaniak L, Quittner C, Adams-Huet B, Snell P, et al. Comparison of efficacy and safety of leptin replacement therapy in moderately and severely hypoleptinemic patients with familial partial lipodystrophy of the Dunnigan variety. J Clin Endocrinol Metab. 2012;97:785-92.) (Supplementary Table 2).

Serum albumin concentrations were evaluated by a single study, which reported no significant changes after LRT (²⁷27 Moran SA, Patten N, Young JR, Cochran E, Sebring N, Reynolds J, et al. Changes in body composition in patients with severe lipodystrophy after leptin replacement therapy. Metabolism. 2004;53:513-9.). Two studies reported significant decreases in ALT and AST (²²22 Beltrand J, Beregszaszi M, Chevenne D, Sebag G, De Kerdanet M, Huet F, et al. Metabolic correction induced by leptin replacement treatment in young children with Berardinelli-Seip congenital lipoatrophy. Pediatrics. 2007;120:e291-6.,³¹31 Safar Zadeh E, Lungu AO, Cochran EK, Brown RJ, Ghany MG, Heller T, et al. The liver diseases of lipodystrophy: the long-term effect of leptin treatment. J Hepatol. 2013;59:131-7.). In the study by Simha and cols., neither cohort of participants showed significant reductions in ALT, but severely hypoleptinemic patients showed a significant reduction in AST levels (¹⁶16 Simha V, Subramanyam L, Szczepaniak L, Quittner C, Adams-Huet B, Snell P, et al. Comparison of efficacy and safety of leptin replacement therapy in moderately and severely hypoleptinemic patients with familial partial lipodystrophy of the Dunnigan variety. J Clin Endocrinol Metab. 2012;97:785-92.). Supplementary table 3 shows the changes in albumin and liver enzymes levels.

Lipid profile

Seven studies reported significant reductions in serum TG (22,25,26,28‐31). The largest reduction was shown by Petersen and cols., where patients had a mean baseline TG level of 5,851±5,079 mg/dL, decreasing to 1,134±523 mg/dL following three months of LRT (³⁰30 Petersen KF, Oral EA, Dufour S, Befroy D, Ariyan C, Yu C, et al. Leptin reverses insulin resistance and hepatic steatosis in patients with severe lipodystrophy. J Clin Invest. 2002;109:1345-50.). When stratified by hypoleptinemic status, significant reductions in TG concentration were observed in participants with mild and severe hypoleptinemia (¹⁶16 Simha V, Subramanyam L, Szczepaniak L, Quittner C, Adams-Huet B, Snell P, et al. Comparison of efficacy and safety of leptin replacement therapy in moderately and severely hypoleptinemic patients with familial partial lipodystrophy of the Dunnigan variety. J Clin Endocrinol Metab. 2012;97:785-92.). Two studies showed significant reductions in TC concentrations (²⁴24 Chong AY, Lupsa BC, Cochran EK, Gorden P. Efficacy of leptin therapy in the different forms of human lipodystrophy. Diabetologia. 2010;53:27-35.,²⁹29 Park JY, Javor ED, Cochran EK, DePaoli AM, Gorden P. Long-term efficacy of leptin replacement in patients with Dunnigan-type familial partial lipodystrophy. Metabolism. 2007;56:508-16.), and one study reported a significant increase in TC levels following LRT (²²22 Beltrand J, Beregszaszi M, Chevenne D, Sebag G, De Kerdanet M, Huet F, et al. Metabolic correction induced by leptin replacement treatment in young children with Berardinelli-Seip congenital lipoatrophy. Pediatrics. 2007;120:e291-6.). Six studies recorded changes in HDL following LRT (⁷7 Chan JL, Oral EA. Clinical classification and treatment of congenital and acquired lipodystrophy. Endocr Pract. 2010;16:310-23.,²²22 Beltrand J, Beregszaszi M, Chevenne D, Sebag G, De Kerdanet M, Huet F, et al. Metabolic correction induced by leptin replacement treatment in young children with Berardinelli-Seip congenital lipoatrophy. Pediatrics. 2007;120:e291-6.,²⁴24 Chong AY, Lupsa BC, Cochran EK, Gorden P. Efficacy of leptin therapy in the different forms of human lipodystrophy. Diabetologia. 2010;53:27-35.,²⁶26 Javor ED, Ghany MG, Cochran EK, Oral EA, DePaoli AM, Premkumar A, et al. Leptin reverses nonalcoholic steatohepatitis in patients with severe lipodystrophy. Hepatology. 2005;41:753-60.,²⁸28 Oral EA, Simha V, Ruiz E, Andewelt A, Premkumar A, Snell P, et al. Leptin-replacement therapy for lipodystrophy. N Engl J Med. 2002;346:570-8.,³⁰30 Petersen KF, Oral EA, Dufour S, Befroy D, Ariyan C, Yu C, et al. Leptin reverses insulin resistance and hepatic steatosis in patients with severe lipodystrophy. J Clin Invest. 2002;109:1345-50.), but none of them reported significant changes. One study reported a significant decrease in LDL from a mean of 139±16 mg/dL at baseline and 85±7 mg/dL following 12 months of leptin replacement (²⁶26 Javor ED, Ghany MG, Cochran EK, Oral EA, DePaoli AM, Premkumar A, et al. Leptin reverses nonalcoholic steatohepatitis in patients with severe lipodystrophy. Hepatology. 2005;41:753-60.). Supplementary table 4 illustrates all the changes in blood lipids following LRT.

Sub-analysis of LRT on LS subtype

In order to assess whether the effects of LRT varied according to the type of LS, we compiled all available individual patient data from the included studies for each of the endpoints assessed. However, besides not being large enough, results from this data set were very heterogeneous within groups, not allowing meaningful comparative analyses. For example, liver fat data were available for 8 patients with generalized LS and for zero patients with partial LS. Briefly, in endpoints with available data, median changes in glycemic parameters were larger in patients with generalized LS (n=30), compared to patients with partial LS (n=7): fasting glucose decreased by a median 47.50 mg/dL in patients with generalized LS, and by 28.00 mg/dL in patients with partial LS; HbA1c was reduced by 1.85% in patients with generalized LS, compared to a reduction of 0.90% in patients with partial LS. However median reductions in TG were greater in patients with partial LS (332.5 mg/dL) compared to patients with generalized LS (306.3 mg/dL). When stratifying by acquired or congenital forms, where data is available, patients with acquired LS (n=5) showed greater median decreases in glucose (100.0 mg/dL vs. 57.0 mg/dL), HbA1c (1.30% vs. 0.90%), and TG (822.0 mg/dL vs. 238.0 mg/dL) relative to patients with congenital LS (n=24).

Meta-analysis

Leptin replacement therapy produced a reduction in fasting plasma glucose in patients with LS. The standardized mean difference was 0.75, with a 95% confidence interval ranging from 0.36 to 1.13, representing an overall significant decrease (p=0.0001). Heterogeneity for this test was high (I² =84%) (Supplementary Figure 1A). No study reported a significant alteration in fasting insulin following LRT, and this was reflected in a change in SMD of 0.00 (‐0.41, 0.41; p=1.00) (Supplementary Figure 1B). As this analysis showed a heterogeneity of 0%, meta-analysis was performed using a fixed-effects model. Patients on LRT experienced an overall decrease in HbA1c, with a SMD of 0.49 (0.17‐0.81). This effect was significant (p=0.003) and heterogeneity was high (I² =75%) (Supplementary Figure 1C).

Liver volume of LS patients significantly decreased following LRT. Three studies had significant reductions, and the overall SMD was 1.06 (0.51, 1.61), where p=0.0002. Heterogeneity was 64% (Supplementary Figure 2A). Liver transaminases were significantly decreased when comparing mean data in cases and controls; however only AST showed a significant reduction in meta-analysis 0.41 (0.10, 0.73) where p=0.01 and heterogeneity was 52%. For ALT, the SMD was a non-significant 0.36 (‐0.03, 0.75), p=0.07 and heterogeneity was 69% (Supplementary Figure 2E). Meta-analysis was not performed from results of liver fat or albumin as these data sets were based on data from single studies only.

Triglycerides and total cholesterol were significantly reduced. The SMD for TG was 1.00 (0.69, 1.31), where the heterogeneity was high (I² =64%) and p<0.00001 (Supplementary Figure 3A). The effect on TC was 0.62 (0.21, 1.02), where p=0.003 and I² =80% (Supplementary Figure 3B). There was no significant overall effect on HDL and LDL levels. The overall SMD for HDL was a non-significant 0.00 (‐0.16, 0.17) (Supplementary Figure 3C). Similarly, a single study reported a standardized mean reduction in LDL (²⁵25 Ebihara K, Kusakabe T, Hirata M, Masuzaki H, Miyanaga F, Kobayashi N, et al. Efficacy and safety of leptin-replacement therapy and possible mechanisms of leptin actions in patients with generalized lipodystrophy. J Clin Endocrinol Metab. 2007;92:532-41.). However, the overall effect of leptin on LDL levels was not significant [0.21 units (‐0.20, 0.62)] (Supplementary Figure 3D).

DISCUSSION

Lipodystrophy syndromes that are not associated with the use of HAART are rare conditions, and present several metabolic and hepatic abnormalities, caused by changes in circulating adipokines levels. The decrease of plasma leptin levels plays a key role in the pathophysiology of those abnormalities. Leptin replacement therapy is currently the therapy of choice for those conditions, and it was recently approved by the FDA for the treatment of patients with generalized lipodystrophy (¹³13 Chou K, Perry CM. Metreleptin: first global approval. Drugs. 2014;73:989-97.). Although several studies have investigated the safety and efficacy of LRT in patients with LS, no systematic meta-analysis of those results has been performed so far. This meta-analysis reports that LRT in non-HAART LS patients improves fasting plasma glucose and HbA1c values, reduces serum TG and TC levels, and reduces liver volume, and transaminases. Taken together, the results of this meta-analysis suggest that leptin is effective at ameliorating clinical and biochemical endpoints of undistinguished non-HAART LS patients, and at improving overall health in these patients.

Leptin is an adipokine with key roles in regulating food intake, energy balance and body weight. Leptin also plays important roles on lipid and glucose metabolism, the gonadal, adrenal, somatotropic and thyroid axes, sympathetic tone, biomarkers of cardiovascular disease, immunity, inflammation, and brain structure and function (5,6,32‐34). In humans, leptin deficiency is observed in cases of hypothalamic amenorrhea, anorexia nervosa, genetic deficiency due to mutations in the leptin gene, and LS. The metabolic abnormalities are similar in all cases of leptin deficiency: increased insulin resistance, hyperinsulinemia, altered fasting glucose or diabetes, hypertriglyceridemia, and hepatic steatosis (³⁵35 Moon HS, Dalamaga M, Kim SY, Polyzos SA, Hamnvik OP, Magkos F, et al. Leptin’s role in lipodystrophic and nonlipodystrophic insulin-resistant and diabetic individuals. Endocr Rev. 2013;34:377-412.). Leptin replacement therapy has been evaluated in those conditions, showing significant metabolic improvements. In humans with genetic leptin deficiency due to mutations in the leptin gene, our group has shown that LRT improves the glucose-insulin homeostasis, decreases serum triglycerides, reduces lipogenesis, and increases lipolysis (³⁴34 Paz-Filho G, Wong ML, Licinio J. Ten years of leptin replacement therapy. Obes Rev. 2011;12:e315-23.). In another patient, it has also been shown that LRT reduces circulating levels of transaminases, total cholesterol, LDL, insulin resistance, and liver fat content (³⁶36 von Schnurbein J, Heni M, Moss A, Nagel SA, Machann J, Muehleder H, et al. Rapid improvement of hepatic steatosis after initiation of leptin substitution in a leptin-deficient girl. Horm Res Paediatr. 2013;79:310-7.). Similarly, our previous research has shown that white adipose tissue transplantation from wild-type mice into genetically obese and leptin deficient ob/ob mice leads to the improvement of several metabolic parameters, and to the reversal of nonalcoholic fatty liver disease, due to the correction of the leptin-deficient state (³⁷37 Paz-Filho G, Mastronardi CA, Parker BJ, Khan A, Inserra A, Matthaei KI, et al. Molecular pathways involved in the improvement of nonalcoholic fatty liver disease. J Mol Endocrinol. 2013;51:167-79.).

The combined results of the studies included in this meta-analysis suggest that leptin administration is responsible for similar improvements. Analogous to the results in our mouse and human models of genetic leptin deficiency, the data presented here indicate that leptin influences the outcome of glucose and lipid metabolism in humans with LS. Fasting glucose and glycated hemoglobin, two important markers of glycemic control were decreased following intervention (p=0.0001 and p=0.003 respectively) (Supplementary Figure 1). Leptin therapy resulted in significant reductions in blood triglycerides (p<0.00001) and total cholesterol (p=0.003) (Supplementary Figure 3), which are predictive markers of cardiovascular disease. Moreover, liver volume and AST were significantly decreased (p=0.0002 and p=0.01) (Supplementary Figure 2), suggesting that LRT is effective in the improvement of hepatic pathology associated with LS. Liver fat was reduced in response to leptin therapy but meta-analysis was not possible as this data was based on results from a single study that reported the outcome of liver fat. However, recent studies do suggest that leptin therapy is effective at reducing hepatic steatosis associated with lipodystrophy (³⁸38 Casey SP, Lokan J, Testro A, Farquharson S, Connelly A, Proietto J, et al. Post-liver transplant leptin results in resolution of severe recurrence of lipodystrophy-associated nonalcoholic steatohepatitis. Am J Transplant. 2013;13(11):3031-4.).

Recently, the US Food and Drug Administration approved the use of metreleptin (the synthetic human leptin analogue used as the leptin supplement in all the trials presented here) to treat patients with only the generalized form of lipodystrophy, where the adipose tissue loss is systemic and usually subcutaneous. The FDA Endocrinologic and Metabolic Drugs Advisory Committee reviewed data from two NIH studies with a combined cohort of 72 participants: NIH Study 991265 (completed) and NIH Study 20010769 (ongoing), and from another study with a cohort of 28 participants, FHA101 Study (ongoing) (³⁹39 Endocrinologic and Metabolic Drugs Advisory Committee Briefing Document 2013 [30/01/2014]. Available on: http://www.fda.gov/downloads/advisorycommittees/committeesmeetingmaterials/drugs/endocrinologicandmetabolicdrugsadvisorycommittee/ucm377929.pdf.
http://www.fda.gov/downloads/advisorycom... ). Similar to the parameters assessed here, these studies evaluated the effects of metreleptin on HbA1c, fasting plasma glucose, TG and liver function. Combining data from these studies, after a 12 month trial period, the following parameters were reduced: HbA1c [‐1.4±0.2% (‐0.9, ‐1.8)], glucose [‐42±12 mg/dL (‐18, ‐65)], TG [‐673±223 mg/dL or ‐32±8% (‐17, ‐47)], liver volume (‐1001 mL); ALT (‐31.4 U/L) and AST (‐23.7 U/L). These reductions were more pronounced in patients with elevated baseline levels of these endpoints. For example, in patients with the generalized form with a baseline HbA1c ≥6%, a mean reduction of 2.3% was achieved following the 12-month trial period. However, in patients with a baseline HbA1c ≥8%, this reduction was even greater, at 2.7%. Similarly, in patients with partial lipodystrophy with baseline HbA1c ≥7%, the mean reduction in HbA1c was 0.8%, whereas mean HbA1c reduction of 1.4% was observed in patients with partial LS and baseline HbA1c ≥8%. Similar patterns were observed in all assessed parameters, which lead the investigators to suggest that in recovering a healthy phenotype, the effect of metreleptin is more pronounced in those patients with more severe underlying metabolic abnormalities. Furthermore, the investigators noted that the clinical efficacy of metreleptin was more related to the severity of the underlying metabolic abnormalities, rather than to the form of lipodystrophy (generalized or partial). To assess this claim, a further sub-analysis was performed by stratifying data for the two main outcomes (HbA1c and TG), according to lipodystrophy subtype (³⁹39 Endocrinologic and Metabolic Drugs Advisory Committee Briefing Document 2013 [30/01/2014]. Available on: http://www.fda.gov/downloads/advisorycommittees/committeesmeetingmaterials/drugs/endocrinologicandmetabolicdrugsadvisorycommittee/ucm377929.pdf.
http://www.fda.gov/downloads/advisorycom... ). This sub-analysis showed that more pronounced effects were seen in patients with the generalized form, with mean reductions in serum HbA1c and TG equal to 1.4% (SE =0.2) and 121 mg/dL (SE = not reported) respectively. Interestingly, similar results were seen across patients with congenital or acquired variants of each lipodystrophy subtype (³⁹39 Endocrinologic and Metabolic Drugs Advisory Committee Briefing Document 2013 [30/01/2014]. Available on: http://www.fda.gov/downloads/advisorycommittees/committeesmeetingmaterials/drugs/endocrinologicandmetabolicdrugsadvisorycommittee/ucm377929.pdf.
http://www.fda.gov/downloads/advisorycom... ). Our analysis showed a similar pattern of results where patients with generalized LS showed greater improvements in the endpoints of glucose and HbA1c relative to patients with partial LS. Similarly relative to patients with congenital LS, patients with acquired LS had greater reductions in blood glucose, HbA1c and TG. However, the small size and high heterogeneity of our data set makes statistical comparisons between subtypes of LS underpowered and not significant.

Our analysis suggests that, overall, metreleptin is more effective in ameliorating the condition of patients with the generalized forms of lipodystrophy, even though it has been suggested that the drug might be useful in partial lipodystrophy, who present a more heterogeneous disease profile. Overall, improvements with metreleptin have been related to the severity of the baseline metabolic abnormalities of the patient, and not necessarily dependent on severity of leptin deficiency or the type of LS. Although, the use of metreleptin to treat partial lipodystrophy was not entirely discouraged, but a thorough physical exam was advised by the authors of the NIH sponsored trials in determining further treatment (³⁹39 Endocrinologic and Metabolic Drugs Advisory Committee Briefing Document 2013 [30/01/2014]. Available on: http://www.fda.gov/downloads/advisorycommittees/committeesmeetingmaterials/drugs/endocrinologicandmetabolicdrugsadvisorycommittee/ucm377929.pdf.
http://www.fda.gov/downloads/advisorycom... ).

The results of this meta-analysis were influenced by several factors. Importantly, the sample sizes of each study varied significantly, from n=4 to n=55 (Table 1). Smaller studies represent important exploratory trials, providing data on clinical efficacy and safety, which are useful to direct regulatory bodies in developing guidelines for metreleptin use. However, from a statistical perspective, small trials may be prone to large variances in results, as individual differences have a significant impact in final data. To overcome this, we employed the standard error (SE) of the standardized mean difference (SMD) to be inversely proportional to the study population size. In this way, the smallest trials had the largest SE, which reflected into a lower weighting of those small trials in the final analysis. In the meta-analysis, heterogeneity was generally high, ranging from I² =84% for the outcome of glucose to 0% for HDL cholesterol and insulin (employed a fixed effects model in these instances). This means that a low percentage of all variability in effect size estimates was caused by sampling errors within the studies. In other words, most of the variability was caused by true heterogeneity between studies (¹⁹19 Huedo-Medina TB, Sanchez-Meca J, Marin-Martinez F, Botella J. Assessing heterogeneity in meta-analysis: Q statistic or I2 index? Psychol Methods. 2006;11:193-206.). In considering a consistent pattern of results seen in several parameters, large heterogeneity may be seen as strength, in that leptin improves these endpoints in several settings.

The dosage of r-metHu-leptin was uniform amongst the included trials. However, as only mean leptin dose was reported, it was not possible to determine if increases in leptin dosage may elicit greater changes. Interestingly Beltrand and cols. reported that some patients began not to respond to leptin treatment and developed resistance. This may represent some of the problems that clinicians will encounter in the future (²³23 Beltrand J, Lahlou N, Le Charpentier T, Sebag G, Leka S, Polak M, et al. Resistance to leptin-replacement therapy in Berardinelli-Seip congenital lipodystrophy: an immunological origin. Eur J Endocrinol. 2010;162:1083-91.).

Variation in effects may also be attributed to the differing follow-up times employed by the trials included in this meta-analysis. Some studies (¹⁶16 Simha V, Subramanyam L, Szczepaniak L, Quittner C, Adams-Huet B, Snell P, et al. Comparison of efficacy and safety of leptin replacement therapy in moderately and severely hypoleptinemic patients with familial partial lipodystrophy of the Dunnigan variety. J Clin Endocrinol Metab. 2012;97:785-92.,²²22 Beltrand J, Beregszaszi M, Chevenne D, Sebag G, De Kerdanet M, Huet F, et al. Metabolic correction induced by leptin replacement treatment in young children with Berardinelli-Seip congenital lipoatrophy. Pediatrics. 2007;120:e291-6.,³⁰30 Petersen KF, Oral EA, Dufour S, Befroy D, Ariyan C, Yu C, et al. Leptin reverses insulin resistance and hepatic steatosis in patients with severe lipodystrophy. J Clin Invest. 2002;109:1345-50.) involved follow-up times less than 12 months. However, longer trials, such as the one conducted by Chong and cols., arrived at similar conclusions, suggesting that leptin has similar immediate and long-term effects (²⁴24 Chong AY, Lupsa BC, Cochran EK, Gorden P. Efficacy of leptin therapy in the different forms of human lipodystrophy. Diabetologia. 2010;53:27-35.). Baseline mean levels of leptin varied significantly amongst the studies (range 0.7‐3.8 ng/mL). This may create an impact when comparing the effects of different r-metHu-leptin dosages in reaching target and physiological levels of leptin. However, a similar pattern of results is seen across all the studies, which would suggest that r-metHu-leptin is effective in patients with differing states of leptin-deficiency, paralleling the findings of the NIH-sponsored trials. Therefore, it would be reasonable to assume that leptin may be applicable for clinical use in other non-lipodystrophy leptin-deficient conditions. Indeed, leptin has previously been investigated in hypothalamic amenorrhea with positive results (⁴⁰40 Chou SH, Chamberland JP, Liu X, Matarese G, Gao C, Stefanakis R, et al. Leptin is an effective treatment for hypothalamic amenorrhea. Proc Natl Acad Sci U S A. 2011;108(16):6585-90.,⁴¹41 Musso C, Cochran E, Javor E, Young J, Depaoli AM, Gorden P. The long-term effect of recombinant methionyl human leptin therapy on hyperandrogenism and menstrual function in female and pituitary function in male and female hypoleptinemic lipodystrophic patients. Metabolism. 2005;54(2):255-63.). Finally, age- and sex-specific effects cannot be discounted as possible sources of variation and error, and future clinical trials should investigate these aspects, as some studies assessed here included only children, and others only women (Table 2).

This meta-analysis has some limitations. Most importantly, this analysis was restricted to clinical data from studies using leptin to treat forms of lipodystrophy that did not arise out of complications from HAART for HIV infection. This form of LS was excluded because it has a unique pathophysiological basis, and therefore may introduce an important confounding factor to the meta-analysis. As such, we have excluded a significant amount of data relating to the effect of leptin replacement in the treatment of lipodystrophy. Indeed, 65 articles were excluded, from the 261 abstracts eligible for inclusion from initial database searching (Figure 1). Secondly, in order to perform the meta-analysis, we pooled together data from prospective cohort studies and clinical case series that were conducted in a similar fashion. The exclusion of case reports [e.g. (21,22,24,27‐30)] may not reflect adequately the reality of the management of lipodystrophy, but this approach was necessary in order to increase the statistical quality and power of the analyzed data, as case reports are less powerful forms of clinical experimentation and observation, and are subject to increased bias compared to larger prospective studies. Thirdly, this review obtained data from publicly available literature, and as such, we did not have access to primary data. This hindered the completion of some observations and proper comparison of baseline and follow-up results. The outcome of the meta-analysis could be strengthened by including those missing data, including the significance levels of baseline and follow-up data. For example, Beltrand and cols. (²¹21 Oral EA, Ruiz E, Andewelt A, Sebring N, Wagner AJ, Depaoli AM, et al. Effect of leptin replacement on pituitary hormone regulation in patients with severe lipodystrophy. J Clin Endocrinol Metab. 2002;87(7):3110-7.) reported an increase in HbA1c, but did not provide a significance test, and therefore we could not include this as part of our data analysis. Those missing data also extend to other qualitative data such as reporting the use of other medications by the patients. The use of medications to treat the clinical manifestation of lipodystrophy, in particular glucose and lipid lowering drugs can significantly impact on interpreting the true effects of leptin on these endpoints. Finally, our quality assessment suggests that the studies included for meta-analysis can be improved in several aspects (range of quality scores 25‐75%), and that future studies would need to address the limitations outlined here and by the authors of the studies themselves.

To our knowledge, this paper represents the first comprehensive meta-analysis into the effects of leptin in patients with non-HAART associated LS, focusing on several metabolic parameters as well as classic liver parameters. In patients with undistinguished forms of LS (congenital/acquired, partial/generalized), leptin replacement therapy was effective in improving parameters of glucose-insulin and liver homeostasis, and blood lipids. Although data suggest that metreleptin may be clinically effective for patients with partial forms of lipodystrophy, a meta-analysis into this type of LS was not possible due to the small sample size and its heterogeneity. Our findings may guide future clinical research for expansion of the use of metreleptin for partial forms of LS, and for the development of leptin-based therapies for conditions such as metabolic syndrome, nonalcoholic fatty liver disease and other conditions where patients display similar metabolic abnormalities.

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