Shun Mei Liu

Phenotypes of Mouse diabetes and Rat fatty Due to Mutations in the OB (Leptin) Receptor

Mice harboring mutations in the obese (ob) and diabetes (db) genes display similar phenotypes, and it has been proposed that these genes encode the ligand and receptor, respectively, for a physiologic pathway that regulates body weight. The cloning of ob, and the demonstration that it encodes a secreted protein (leptin) that binds specifically to a receptor (OB-R) in the brain, have validated critical aspects of this hypothesis. Here it is shown by genetic mapping and genomic analysis that mouse db, rat fatty (a homolog of db), and the gene encoding the OB-R are the same gene.

Complete rescue of obesity, diabetes, and infertility in db/db mice by neuron-specific LEPR-B transgenes

We have generated mice that carry a neuron-specific leptin receptor (LEPR) transgene whose expression is driven by the rat synapsin I promoter synapsin-LEPR B (SYN-LEPR-B). We have also generated mice that are compound hemizygotes for the transgenes SYN-LEPR-B and neuron-specific enolase-LEPR B (NSE-LEPR-B). We observed a degree of correction in db/db mice that are hemizygous (Syn db/db) and homozygous (Syn/Syn db/db) for the SYN-LEPR-B transgene similar to that previously reported for the NSE-LEPR-B transgene. We also show complete correction of the obesity and related phenotypes of db/db mice that are hemizygous for both NSE-LEPR-B and SYN-LEPR-B transgenes (Nse+Syn db/db). Body composition, insulin sensitivity, and cold tolerance were completely normalized in Nse+Syn db/db mice at 12 weeks of age compared with lean controls. In situ hybridization for LEPR B isoform expression in Nse+Syn db/db mice showed robust expression in the energy homeostasis-relevant regions of the hypothalamus. Expression of 3 neuropeptide genes, agouti-related peptide (Agrp), neuropeptide Y (Npy), and proopiomelanocortin (Pomc), was fully normalized in dual transgenic db/db mice. The 2 transgenes in concert conferred normal fertility to male and female db/db mice. Male mice with partial peripheral deletion of Lepr, induced in the periweaning phase, did not show alterations in body composition or mass. In summary, we show that brain-specific leptin signaling is sufficient to reverse the obesity, diabetes, and infertility of db/db mice.

Phenotype of fatty due to Gln269Pro mutation in the leptin receptor (Lepr)

The rat fatty (fa) mutation produces profound obesity of early onset caused by hyperphagia, defective nonshivering thermogenesis, and preferential deposition of energy into adipose tissue. Genetic mapping studies indicate that fa and diabetes (db) are homologous loci in the rat and mouse genomes, respectively. It has been shown that db alleles carry mutations in the Lepr (leptin receptor) gene. This paper describes a point mutation in the fatty allele of Lepr. A nucleotide substitution at position 880 (A→C) causes an amino acid substitution at position 269 (Gln → Pro). The mutation generates a novel Msp I site that cosegregates with fa in 1,028 meioses examined in obese F2 progeny from two crosses (BN×13M and WKY×13M) and is still segregating in three rat colonies. PCR-based mutagenesis was used to introduce the fa mutation into the mouse Lepr cDNA. Transient transfection studies indicate that the mutant Lepr cDNA has greatly reduced binding of leptin (Lep) at the cell surface. These data are strong evidence that the single nucleotide substitution in the fa allele of Lepr (Leprfa) is responsible for the obese phenotype.

Phenotype of the Obese Koletsky (f) Rat Due to Tyr763Stop Mutation in the Extracellular Domain of the Leptin Receptor (Lepr): Evidence for Deficient Plasma-to-CSF Transport of Leptin in Both the Zucker and Koletsky Obese Rat

The obese phenotypes of the diabetes (db) mouse and fatty (fa) rat are due to functional null mutations of the leptin receptor (Lepr). The recessive mutation in the Koletsky (f) obese rat maps to the same genetic intervals as db and fa and fails to complement the fa mutation. Comparison of the sequence of brain Lepr cDNA from +/+ and f/f animals reveals a T2349A transversion resulting in a Tyr763Stop nonsense mutation in the gene just before the transmembrane domain. Virtual absence of Lepr mRNA in whole brain from f/f animals is consistent with the presence of a null mutation. The predicted reduced cerebrospinal fluid (CSF) transport of leptin in both f/f and fa/fa mutants is reflected in the ∼10-fold lower ratio of CSF/plasma leptin concentration in the obese versus lean animals. However, equivalent CSF leptin concentration between lean and obese rats (fa/fa, f/f) indicates that leptin can enter the CSF through a non–Lepr-mediated mechanism, which may be saturated at normal physiological plasma leptin concentration.

An allelic series for the leptin receptor gene generated by CRE and FLP recombinase.

Body weight regulation is mediated through several major signaling pathways, some of which have been delineated by positional cloning of spontaneous genetic mutations in mice. Leprdb/db mice are obese due to a defect in the signaling portion of the leptin receptor, which has led to extensive study of this highly conserved system over the past several years. We have created an allelic series at Lepr for the further examination of LEPR signaling phenotypes using both the FLP/frt and CRE/loxP systems. By inserting a frt-PGK-neo-frt sequence in Lepr intron 16, we have generated a conditional gene repair Lepr allele (Lepr-neo) that elicits morbid obesity, diabetes, and infertility in homozygous mice, recapitulating the obesity syndrome of Leprdb/db mice. Thus, in vivo excision of the PGK-neo cassette with a FLP recombinase transgene restores the lean and fertile phenotype to Leprflox/flox mice. In the same construct, we have also inserted loxP sites that flank Lepr coding exon 17, a region that encodes a JAK docking site required for STAT3 signaling. CRE-mediated excision of Lepr coding exon 17 from Lepr with a frameshift in subsequent exons results in a syndrome of obesity, diabetes, and infertility in LeprΔ17/Δ17 mice, which is indistinguishable from Leprneo/neo and Leprdb/db mice. We conclude that suppression of Lepr gene expression by PGK-neo is phenotypically equivalent to deletion of the Lepr signaling motifs, and therefore the Leprneo/neo mouse may be used to investigate conditional gene repair of Lepr signaling deficiency.

Effects of Leptin and Melanocortin Signaling Interactions on Pubertal Development and Reproduction

Leptin and melanocortin signaling control ingestive behavior, energy balance, and substrate utilization, but only leptin signaling defects cause hypothalamic hypogonadism and infertility. Although GnRH neurons do not express leptin receptors, leptin influences GnRH neuron activity via regulation of immediate downstream mediators including the neuropeptides neuropeptide Y and the melanocortin agonist and antagonist, α-MSH, agouti-related peptide, respectively. Here we show that modulation of melanocortin signaling in female db/db mice through ablation of agouti-related peptide, or heterozygosity of melanocortin 4 receptor, restores the timing of pubertal onset, fertility, and lactation. Additionally, melanocortin 4 receptor activation increases action potential firing and induces c-Fos expression in GnRH neurons, providing further evidence that melanocortin signaling influences GnRH neuron activity. These studies thus establish melanocortin signaling as an important component in the leptin-mediated regulation of GnRH neuron activity, initiation of puberty and fertility.

TXNIP in Agrp Neurons Regulates Adiposity, Energy Expenditure, and Central Leptin Sensitivity

Thioredoxin interacting protein (TXNIP) has recently been described as a key regulator of energy metabolism through pleiotropic actions that include nutrient sensing in the mediobasal hypothalamus (MBH). However, the role of TXNIP in neurochemically specific hypothalamic subpopulations and the circuits downstream from MBH TXNIP engaged to regulate energy homeostasis remain unexplored. To evaluate the metabolic role of TXNIP activity specifically within arcuate Agrp neurons, we generated Agrp-specific TXNIP gain-of-function and loss-of-function mouse models using Agrp-Ires-cre mice, TXNIP flox/flox mice, and a lentivector expressing the human TXNIP isoform conditionally in the presence of Cre recombinase. Overexpression of TXNIP in Agrp neurons predisposed to diet-induced obesity and adipose tissue storage by decreasing energy expenditure and spontaneous locomotion, without affecting food intake. Conversely, Agrp neuronal TXNIP deletion protected against diet-induced obesity and adipose tissue storage by increasing energy expenditure and spontaneous locomotion, also without affecting food intake. TXNIP overexpression in Agrp neurons did not primarily affect glycemic control, whereas deletion of TXNIP in Agrp neurons improved fasting glucose levels and glucose tolerance independently of its effects on body weight and adiposity. Bidirectional manipulation of TXNIP expression induced reciprocal changes in central leptin sensitivity and the neural regulation of lipolysis. Together, these results identify a critical role for TXNIP in Agrp neurons in mediating diet-induced obesity through the regulation of energy expenditure and adipose tissue metabolism, independently of food intake. They also reveal a previously unidentified role for Agrp neurons in the brain-adipose axis.

Neuropeptide Y and agouti-related peptide mediate complementary functions of hyperphagia and reduced energy expenditure in leptin receptor deficiency.

Neuropeptide Y (NPY) and agouti-related peptide (AGRP) can produce hyperphagia, reduce energy expenditure, and promote triglyceride deposition in adipose depots. As these two neuropeptides are coexpressed within the hypothalamic arcuate nucleus and mediate a major portion of the obesity caused by leptin signaling deficiency, we sought to determine whether the two neuropeptides mediated identical or complementary actions. Because of separate neuropeptide receptors and signal transduction mechanisms, there is a possibility of distinct encoding systems for the feeding and energy expenditure aspects of leptin-regulated metabolism. We have genetically added NPY deficiency and/or AGRP deficiency to LEPR deficiency isolated to AGRP cells. Our results indicate that the obesity of LEPR deficiency in AGRP/NPY neurons can produce obesity with either AGRP or NPY alone with AGRP producing hyperphagia while NPY promotes reduced energy expenditure. The absence of both NPY and AGRP prevents the development of obesity attributable to isolated LEPR deficiency in AGRP/NPY neurons. Operant behavioral testing indicated that there were no alterations in the reward for a food pellet from the AGRP-specific LEPR deficiency.

A susceptibility gene for kidney disease in an obese mouse model of type II diabetes maps to chromosome 8

Most mouse models of diabetes do not fully reproduce features of human diabetic nephropathy, limiting their utility in inferring mechanisms of human disease. Here we performed detailed phenotypic and genetic characterization of leptin-receptor (Lepr) deficient mice on the FVB/NJ background (FVB(db/db)), an obese model of type II diabetes, to determine their suitability to model human diabetic nephropathy. These mice have sustained hyperglycemia, significant albuminuria and characteristic diabetic renal findings including mesangial sclerosis and nodular glomerulosclerosis after 6 months of age. In contrast, equally obese, hyperglycemic Lepr/Sur1 deficient C57BL/6J (Sur1 has defective insulin secretion) mice have minimal evidence of nephropathy. A genome-wide scan in 165 Lepr deficient backcross progeny derived from FVB/NJ and C57BL/6J identified a major locus influencing nephropathy and albuminuria on chromosome 8B1-C5 (Dbnph1 locus, peak lod score 5.0). This locus was distinct from those contrasting susceptibility to beta cell hypertrophy and HIV-nephropathy between the same parental strains, indicating specificity to diabetic kidney disease. Genome-wide expression profiling showed that high and low risk Dbnph1 genotypes were associated with significant enrichment for oxidative phosphorylation and lipid clearance, respectively; molecular pathways shared with human diabetic nephropathy. Hence, we found that the FVB(db/db) mouse recapitulates many clinical, histopathological and molecular features of human diabetic nephropathy. Identifying underlying susceptibility gene(s) and downstream dysregulated pathways in these mice may provide insight into the disease pathogenesis in humans.

Identification of a Loss-of-Function Mutation in Ube2l6 Associated With Obesity Resistance

We previously mapped a locus on BALB/c chromosome 2 associated with protection from leptin-deficiency–induced obesity. Here, we generated the corresponding congenic mouse strain by introgression of a segment of C57BL/6J chromosome 2 to the BALB/c background to confirm the genotype–phenotype associations. We found that the BALB/c alleles decreased fat mass expansion by limiting adipocyte hyperplasia and adipocyte hypertrophy. This was concomitant to an increase in adipocyte triglyceride lipase (ATGL)-mediated triglyceride breakdown and prolongation of ATGL half-life in adipose tissue. In addition, BALB/c alleles on chromosome 2 exerted a cell-autonomous role in restraining the adipogenic potential of preadipocytes. Within a 9.8-Mb critical interval, we identified a nonsynonymous coding single nucleotide polymorphism in the gene coding for the ubiquitin-conjugating enzyme E2L6 (Ube2l6, also known as Ubch8) and showed that the BALB/c allele of Ube2l6 is a hypomorph leading to the lack of UBE2L6 protein expression. Ube2l6 knockdown in 3T3-L1 adipocytes repressed adipogenesis. Thus, altered adipogenic potential caused by Ube2l6 knockdown is likely critically involved in BALB/c obesity resistance by inhibiting adipogenesis and reducing adipocyte numbers. Overall, we have identified a loss-of-function mutation in Ube2l6 that contributes to the chromosome 2 obesity quantitative trait locus.