Diabetes Care 1

Sabine Frank-Podlech,1–3 Leptin Replacement Reestablishes Julia von Schnurbein,4 Ralf Veit,1–3 Martin Heni,2,3 Jurgen¨ Machann,2,5 Brain Insulin Action in the Jaana M. Heinze,2,3 Stephanie Kullmann,2,3 Jaida Manzoor,6 Hypothalamus in Congenital Saqib Mahmood,7 Hans-Ulrich Haring,¨ 2,3 fi Hubert Preissl,2,3,8,9 Martin Wabitsch,4 Leptin De ciency and Andreas Fritsche2,3 https://doi.org/10.2337/dc17-1867

OBJECTIVE Human obesity is associated with impaired central insulin signaling, and in very rare cases, severe obesity can be caused by congenital leptin deficiency. In such patients, leptin replacement results in substantial weight loss and improvement in peripheral 1 metabolism. Institute for Medical Psychology and Behaviou- ral Neurobiology, University of Tubingen,¨ Tubingen,¨ Germany RESEARCH DESIGN AND METHODS 2 Institute for Diabetes Research and Metabolic In a leptin-deficient patient, we investigated the impact of leptin substitution on Diseases of the Helmholtz Center Munich at the central insulin action, as quantified by changes in neuronal activity after intranasal University of Tubingen,¨ German Center for Dia- insulin application. This was assessed before and during the 1st year of metreleptin betes Research, Tubingen,¨ Germany 3Department of Internal Medicine IV, University substitution. Hospital, Tubingen,¨ Germany 4Division of Pediatric Endocrinology and Diabe- RESULTS tes, Department of Pediatrics and Adolescent After only 1 year, treatment with metreleptin reestablishes brain insulin sensitivity, Medicine, University of Ulm, Ulm, Germany 5 particularly in the hypothalamus and, to a lesser degree, in the prefrontal cortex. Section on Experimental Radiology, Depart- ment of Diagnostic and Interventional Radiol- Results are depicted in comparison with a control group. In our patient, brain acti- ogy, University Hospital, Tubingen,¨ Germany DIABETES IN COMMUNICATIONS NOVEL vation changes were accompanied by substantial weight loss, reduced visceral adi- 6Children Hospital the Institute of Child Health, pose tissue, reduced intrahepatic lipid content, and improved whole-body insulin Lahore, Pakistan 7Department of Human Genetics and Molecular sensitivity. Biology, University of Health Sciences, Khayaban- e-Jamia Punjab, Lahore, Pakistan CONCLUSIONS 8Institute for Diabetes and Obesity, Helmholtz Leptin replacement and weight loss improved homeostatic insulin action in the Diabetes Center, Helmholtz Center Munich, Ger- patient in question. man Research Center for Environmental Health, Neuherberg, Germany 9Department Pharmacy and Biochemistry, Fac- ulty of Science, University of Tubingen,¨ Tubingen,¨ Patients affected by monogenetic leptin deficiency, whose leptin levels are therefore Germany practically indiscernible, suffer from severe obesity, hyperphagia, impaired satiety, and Corresponding Author: Sabine Frank-Podlech, metabolic impairments such as peripheral insulin resistance (1). Leptin replacement [email protected]. therapy is known to improve metabolism, normalize body weight, and introduce Received 7 September 2017 and accepted 26 changes in brain processes (2–5). In recent years, we and others have ascertained December 2017. that brain insulin action is crucial to various aspects of behavior and metabolism, S.F.-P. and J.v.S. contributed equally to this such as eating behavior, cognition, and whole-body metabolism (6,7). However, brain study. M.W. and A.F. contributed equally to this insulin action, which is quantified by changes in neuronal activity in the functional MRI, study. had never been assessed both before and after leptin replacement therapy in congen- © 2018 by the American Diabetes Association. ital leptin-deficient patients. On the basis of the close molecular interaction of brain Readers may use this article as long as the work fi is properly cited, the use is educational and not leptin and insulin signaling (8), we hypothesized that the untreated leptin-de cient for profit, and the work is not altered. More infor- patient will be brain insulin resistant, which can be reverted by substitution of leptin mation is available at http://www.diabetesjournals with metreleptin, a synthetic leptin analog. .org/content/license. Diabetes Care Publish Ahead of Print, published online January 24, 2018 2 Brain Insulin Sensitivity in Leptin Deficiency Diabetes Care

RESEARCH DESIGN AND METHODS z = 31) as a center with a sphere of CONCLUSIONS We performed resting functional MRI 6 mm for the PFC mask (9). Perfusion val- Congenital leptin deficiency is character- measurements on a leptin-deficient 31- ues were extracted for each voxel within ized by various metabolic impairments, in year-old Pakistani woman before therapy the masks for all measurements. Further- particular those associated with energy ho- (leptin-deficient untreated state), as well more, whole-brain mean perfusion was meostasis and peripheral insulin action. as 7 days after and 1 year after onset of calculated to standardize each extracted Earlier studies have shown not only weight substitution with 1.8 mg metreleptin. perfusion value for global perfusion dif- loss but also a marked improvement in Brain insulin sensitivity was assessed by ferences between measurements (rela- whole-body metabolic functions, e.g., insu- the quantification of cerebral blood flow tive perfusion values). Data were further lin sensitivity, reduction of visceral and liver (CBF) pre– and 30 min post–intranasal in- analyzed in SPSS 24 (IBM Corporation, fat, and behavioral changes once metre- sulin application (160 units) by a nasal Armonk, NY) using a sign test to investi- leptin substitution wasinitiated(12,13). spray (details in 9). Blood samples were gate the change in relative perfusion in In the current study, we show that brain taken before the first measurement to each voxel within each region of interest insulin action was impaired in a leptin- , determine peripheral glucose and insulin (P 0.05, Bonferroni corrected). deficient patient before, as well as 7 days fi levels. Fasting HOMA of insulin resistance For the quanti cation of visceral adi- after, metreleptin substitution. Interest- (HOMA-IR) was calculated for each mea- pose tissue (VAT) and intrahepatic lipids ingly, 1 year after metreleptin treatment, surement day [fasting insulin (pmol/L/ (IHL), abdominal MRI and localized pro- brain insulin action showed a marked re- ton MRS measurements were performed 6.945) * fasting glucose (mg/dL)/405] to duction in hypothalamic CBF but higher (12). estimate peripheral insulin sensitivity. PFC activity after insulin application. For interpretation of the results of the Scanning was performed on a 3T whole- Before and especially after 7 days patient from the perspective of “healthy” body scanner with a standard 20-channel of metreleptin substitution, our patient women, unpublished data of a female con- head coil (MAGNETOM Prisma, Siemens, showed an increase in hypothalamic CBF trol group from a previous study (9) are Erlangen, Germany). CBF was measured that exceeded even the highest increase included for results presentation (N =21; with a three-dimensional fast spin-echo se- of subjects of a healthy control group. By mean 6 SD BMI 26.5 6 5.5 kg/m2 and age quence using the FAIR QII perfusion mode contrast, after 1 year of treatment, our 26.1 6 3.6 years) (methods described with three-dimensional readout (3D patient showed the anticipated decrease in 9). GRASE). In addition, a high-resolution T1- in perfusion after intranasal insulin appli- weighted anatomical image was acquired. cation. This result corresponds to the con- CBF image processing was performed RESULTS trol subjects with the highest brain insulin using FSL (http://fsl.fmrib.ox.ac.uk) and Analyses showed increased relative sensitivity (and thus with reduced hypo- fi SPM12 (http://www. l.ion.ucl.ac.uk./spm). perfusion after intranasal insulin applica- thalamic activity after insulin application) Images from each session were realigned tion in the hypothalamus in the leptin- (Fig. 1C). It is well established that the two and resliced using FLIRT. Perfusion images deficient state and 7 days after initiation anorectic hormones insulin and leptin were calculated by pairwise subtraction of metreleptin treatment. After 1 year, closely interact with each other in the hy- of the motion-corrected control and label intranasal insulin had effected a marked pothalamus and share molecular signaling images with ASL_FILE. The OXFORD_ASL reduction of relative perfusion in the pathways in neurons and nonneuronal moduleinFSLwasusedtocalculateresting- hypothalamus (Fig. 1A). No significant ef- cells (8). Furthermore, animal (8) and first state perfusion maps. Perfusion analysis fect of insulin application was observed human (6,7,14) data indicate a modula- was restricted by including only voxels for the PFC in either the leptin-deficient tion of peripheral insulin sensitivity by that were classified with a probability of state or 7 days after the onset of treat- the brain. Thus, the improvement in pe- at least 75% of being gray matter on the ment. After 1 year, however, insulin had ripheral insulin sensitivity after 1 year of basis of T1-weighted, anatomical images induced an increase in relative PFC perfu- metreleptin treatment observed in our (10). The perfusion maps of each session sion (Fig. 1B). patient might be due to restored brain and day were then individually coregistered As can be observed in Fig. 1C,hypotha- insulin action. However, improved insulin to the anatomical image and normalized to lamic activity increase was indeed high action in the brain was detected only af- Montreal Neurological Institute space. compared with a healthy female control ter 1 yeardnot after 7 days of metrelep- To investigate brain insulin sensitiv- group and showed reduced activity after tin treatment. We reported previously ity, we created masks to extract perfu- 1 year. Activity in the PFC was less prom- that saturated nonesterified fatty acids sion values of the hypothalamus and the inent, since the subject was within the (NEFAs) are associated with reduced ac- prefrontal cortex (PFC) within the right range of the control sample. tion of insulin in the brain (15). Soon after middle frontal gyrus, as a previous study After initiation of leptin replacement, initiation of metreleptin substitution, showed decreased hypothalamic activity the patient’s BMI was markedly reduced. systemic lipolysis is stimulated (12), as a but increased prefrontal activity 30 min She was, however, still severely obese consequence of which circulating NEFA after intranasal insulin application in an after 1 year. Peripheral insulin sensitivity, concentrations increase. Brain effects of obese group in comparison with a pla- assessed as HOMA-IR, also improved in these NEFAs might therefore mask en- cebo condition (9). The hypothalamic the course of leptin treatment (Fig. 1E). hanced insulin effects in the brain imme- mask was defined as the upper part of Furthermore, VAT and IHL were already diately after initiation of treatment (12). the hypothalamus (11). We used the reduced 7 days after leptin substitution Both visceral and intrahepatic fat were peak activation voxel (x = 39, y = 29, (Fig. 1F). reduced after only 7 days of treatment. care.diabetesjournals.org Frank-Podlech and Associates 3

Figure 1—A and B: Brain insulin sensitivity in the hypothalamus and prefrontal cortex before and after leptin substitution. Bar graphs show relative perfusion changes (30 min 2 baseline) for each measurement day. For illustration purposes, % mean relative perfusion (standardized on overall perfusion) was calculated over the number of voxels in the regions of interest, and the error bar represents SEM. An asterisk (*) indicates significantly different relative perfusion changes after intranasal insulin application. C and D: Results of the patient are depicted with data from a control group who also received intranasal insulin (on one measurement day only), and baseline-corrected values are shown for the hypothalamus and prefrontal cortex. For the leptin- deficient patient, all three time points are included in the graph. Empty circle = pretreatment initial measurement, triangle = 7 days measurement, and square = 1 year measurement. E and F: Change in BMI and HOMA-IR (left) as well as in VAT and IHL content (right) over the time course of 1 year, commencing in the leptin-deficient state. pre, pretreatment.

We had already observed such a rapid by the leptin-induced stimulation of lipol- brain area or may be secondary to reduc- decrease in another leptin-deficient fe- ysis (12). Thus, the observed reestablish- tion of body fat. male patient after leptin substitution ment of brain insulin sensitivity in the In the PFC, intranasal insulin induced a (12). The rapid decrease of visceral fat hypothalamus may be triggered by the minimal increase in relative perfusion in and liver fat might again be explained restored leptin signaling directly in this the PFC, which did not change during the 4 Brain Insulin Sensitivity in Leptin Deficiency Diabetes Care

7 days after onset of treatment. However, by a grant of Else Kroner-Fresenius¨ Stiftung 5. Baicy K, London ED, Monterosso J, et al. Leptin 1 year after initiation of leptin replace- (2015_A28). replacement alters brain response to food cues in fl fi ment, the increase in relative perfusion Duality of Interest. No potential con icts of in- genetically leptin-de cient adults. Proc Natl Acad terest relevant to this article were reported. Sci U S A 2007;104:18276–18279 after insulin administration was found to Author Contributions. S.F.-P. and R.V. contrib- 6. Heni M, Kullmann S, Preissl H, Fritsche A, be further strengtheneddsimilar to the uted to the conception and design of the study, Haring¨ HU. Impaired insulin action in the human effect found in an obese control group performing the experiment, analyses, and interpre- brain: causes and metabolic consequences. Nat from our previous study (9). The direct tation and writing of the manuscript. J.v.S. contrib- Rev Endocrinol 2015;11:701–711 7. Kullmann S, Heni M, Hallschmid M, Fritsche A, comparison with the control group, how- uted to conception and design of the study, performing the experiment, and interpretation Preissl H, Haring¨ HU. Brain insulin resistance at ever, showed only a marginal effect in and critical revision of the manuscript. M.H., the crossroads of metabolic and cognitive dis- comparison with healthy subjects (Fig. H.-U.H., H.P., M.W., and A.F. contributed to con- orders in humans. Physiol Rev 2016;96:1169– 1D). Thus, impaired brain insulin sensitiv- ception and design of the study and interpretation 1209 ity might be particularly applicable to and critical revision of the manuscript. S.K. provided 8. Konner¨ AC, Bruning¨ JC. Selective insulin and leptin resistance in metabolic disorders. Cell homeostatic rather than to prefrontal re- data of the control group and contributed to the interpretationandcriticalrevisionofthemanuscript. Metab 2012;16:144–152 gionsinthispatient. J.Mac. contributed to performing the experiment, 9. Kullmann S, Heni M, Veit R, et al. Selective in- In conclusion, these data suggest that analyses, and interpretation and critical revision of sulin resistance in homeostatic and cognitive con- leptin contributes to the modulation of the manuscript. J.M.H. contributed to performing trol brain areas in overweight and obese adults. – insulin sensitivity in the human brain. Lep- the experiment and interpretation and critical re- Diabetes Care 2015;38:1044 1050 vision of the manuscript. J.Man. and S.M. contrib- 10. Li X, Sarkar SN, Purdy DE, Briggs RW. Quanti- tin replacement and subsequent changes uted to conception and design of the study, fying cerebellum grey matter and white matter per- in weight in this leptin-deficient patient performing the experiment, and critical revision of fusion using pulsed arterial spin labeling. BioMed improve insulin sensitivity in the periph- the manuscript. A.F. is the guarantor of this work Res Int 2014;2014:108691 ery and central homeostatic areas. and, as such, had full access to all the data in the 11.MatsudaM,LiuY,MahankaliS,etal.Altered study and takes responsibility for the integrity of the hypothalamic function in response to glucose data and the accuracy of the data analysis. ingestion in obese humans. Diabetes 1999;48: 1801–1806 Acknowledgments. The authors thank Shirley 12. von Schnurbein J, Heni M, Moss A, et al. Rapid Wurth¨ for language editing and proofreading of References improvement of hepatic steatosis after initiation the manuscript. 1. Montague CT, Farooqi IS, Whitehead JP, et al. of leptin substitution in a leptin-deficient girl. Funding. Initially AstraZeneca/Bristol-MyersSquibb Congenital leptin deficiency is associated with se- Horm Res Paediatr 2013;79:310–317 (New York) and then Aegerion Pharmaceuticals/ vere early-onset obesity in humans. Nature 1997; 13. Paz-Filho G, Mastronardi CA, Licinio J. 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Diabetes 2017;66: ter for Diabetes Research (DZD e.V.), by the ward, and food-related brain areas. J Clin Endo- 1797–1806 Helmholtz Alliance Imaging and Curing Environ- crinol Metab 2011;96:E1283–E1287 15. Tschritter O, Preissl H, Hennige AM, et al. The mental Metabolic Diseases (ICEMED), through 4. Farooqi IS, Bullmore E, Keogh J, Gillard J, insulin effect on cerebrocortical theta activity is the Initiative and Network Fund of the Helmholtz O’Rahilly S, Fletcher PC. Leptin regulates striatal associated with serum concentrations of satu- Association, by the Kompetenznetz Adipositas regions and human eating behavior. Science rated nonesterified Fatty acids. J Clin Endocrinol funded through the BMBF (01GIL22F), as well as 2007;317:1355 Metab 2009;94:4600–4607