CRISPR screens in cancer spheroids identify 3D growth-specific vulnerabilities.

Cancer genomics studies have identified thousands of putative cancer driver genes1. Development of high-throughput and accurate models to define the functions of these genes is a major challenge. Here we devised a scalable cancer-spheroid model and performed genome-wide CRISPR screens in 2D monolayers and 3D lung-cancer spheroids. CRISPR phenotypes in 3D more accurately recapitulated those of in vivo tumours, and genes with differential sensitivities between 2D and 3D conditions were highly enriched for genes that are mutated in lung cancers. These analyses also revealed drivers that are essential for cancer growth in 3D and in vivo, but not in 2D. Notably, we found that carboxypeptidase D is responsible for removal of a C-terminal RKRR motif2 from the α-chain of the insulin-like growth factor 1 receptor that is critical for receptor activity. Carboxypeptidase D expression correlates with patient outcomes in patients with lung cancer, and loss of carboxypeptidase D reduced tumour growth. Our results reveal key differences between 2D and 3D cancer models, and establish a generalizable strategy for performing CRISPR screens in spheroids to reveal cancer vulnerabilities.

Loss of the deglutamylase CCP5 perturbs multiple steps of spermatogenesis and leads to male infertility.

Sperm cells are highly specialized mammalian cells, and their biogenesis requires unique intracellular structures. Perturbation of spermatogenesis often leads to male infertility. Here, we assess the role of a post-translational modification of tubulin, glutamylation, in spermatogenesis. We show that mice lacking the tubulin deglutamylase CCP5 (also known as AGBL5) do not form functional sperm. In these mice, spermatids accumulate polyglutamylated tubulin, accompanied by the occurrence of disorganized microtubule arrays, in particular in the sperm manchette. Spermatids further fail to re-arrange their intracellular space and accumulate organelles and cytosol, while nuclei condense normally. Strikingly, spermatids lacking CCP5 show supernumerary centrioles, suggesting that glutamylation could control centriole duplication. We show that most of these observed defects are also present in mice in which CCP5 is deleted only in the male germ line, strongly suggesting that they are germ-cell autonomous. Our findings reveal that polyglutamylation is, beyond its known importance for sperm flagella, an essential regulator of several microtubule-based functions during spermatogenesis. This makes enzymes involved in glutamylation prime candidates for being genes involved in male sterility.

Loss of tubulin deglutamylase CCP1 causes infantile-onset neurodegeneration.

A set of glutamylases and deglutamylases controls levels of tubulin polyglutamylation, a prominent post-translational modification of neuronal microtubules. Defective tubulin polyglutamylation was first linked to neurodegeneration in the Purkinje cell degeneration (pcd) mouse, which lacks deglutamylase CCP1, displays massive cerebellar atrophy, and accumulates abnormally glutamylated tubulin in degenerating neurons. We found biallelic rare and damaging variants in the gene encoding CCP1 in 13 individuals with infantile-onset neurodegeneration and confirmed the absence of functional CCP1 along with dysregulated tubulin polyglutamylation. The human disease mainly affected the cerebellum, spinal motor neurons, and peripheral nerves. We also demonstrate previously unrecognized peripheral nerve and spinal motor neuron degeneration in pcd mice, which thus recapitulated key features of the human disease. Our findings link human neurodegeneration to tubulin polyglutamylation, entailing this post-translational modification as a potential target for drug development for neurodegenerative disorders.

Klf4 glutamylation is required for cell reprogramming and early embryonic development in mice.

Temporal and spatial-specific regulation of pluripotency networks is largely dependent on the precise modifications of core transcription factors. Misregulation of glutamylation is implicated in severe physiological abnormalities. However, how glutamylation regulates cell reprogramming and pluripotency networks remains elusive. Here we show that cytosolic carboxypeptidases 1 (CCP1) or CCP6 deficiency substantially promotes induced pluripotent cell (iPSC) induction and pluripotency of embryonic stem cells (ESCs). Klf4 polyglutamylation at Glu381 by tubulin tyrosine ligase-like 4 (TTLL4) and TTLL1 during cell reprogramming impedes its lysine 48-linked ubiquitination and sustains Klf4 stability. Klf4-E381A knockin mice display impaired blastocyst development and embryonic lethality. Deletion of TTLL4 or TTLL1 abrogates cell reprogramming and early embryogenesis. Thus, Klf4 polyglutamylation plays a critical role in the regulation of cell reprogramming and pluripotency maintenance.

Prolylcarboxypeptidase deficiency is associated with increased blood pressure, glomerular lesions, and cardiac dysfunction independent of altered circulating and cardiac angiotensin II.

Prolylcarboxypeptidase (PRCP) is a carboxypeptidase that cleaves angiotensin II (AngII) forming Ang(1-7). The impact of genetic PRCP deficiency on AngII metabolism, blood pressure (BP), kidney histology, and cardiac phenotype was investigated in two lines of PRCP-deficient mice: KST302 derived in C57BL/6 background and GST090 derived in FVB/N background. The GST090 line had increased mean arterial pressure (MAP) (113.7 ± 2.07 vs. WT 105.0 ± 1.23 mmHg; p < 0.01) and left ventricular hypertrophy (LVH) (ratio of diastolic left ventricular posterior wall dimension to left ventricular diameter 0.239 ± 0.0163 vs. WT 0.193 ± 0.0049; p < 0.05). Mice in the KST302 line also had mild hypertension and LVH. Cardiac defects, increased glomerular size, and glomerular mesangial expansion were also observed. After infusion of AngII to mice in the KST302 line, both MAP and LVH increased, but the constitutive differences between the gene trap mice and controls were no longer observed. Plasma and cardiac AngII and Ang(1-7) were not significantly different between PRCP-deficient mice and controls. Thus, PRCP deficiency is associated with elevated blood pressure and cardiac alterations including LVH and cardiac defects independently of systemic or cardiac AngII and Ang(1-7). An ex vivo assay showed that recombinant PRCP, unlike recombinant ACE2, did not degrade AngII to form Ang(1-7) in plasma at pH 7.4. PRCP was localized in α-intercalated cells of the kidney collecting tubule. The low pH prevailing at this site and the acidic pH preference of PRCP suggest a role of this enzyme in regulating AngII degradation in the collecting tubule where this peptide increases sodium reabsorption and therfore BP. However, there are other potential mechanisms for increased BP in this model that need to be considered as well. PRCP converts AngII to Ang(1-7) but only at an acidic pH. Global PRCP deficiency causes heart and kidney alterations and a moderate rise in BP. PRCP is abundant in the kidney collecting tubules, where the prevailing pH is low. In collecting tubules, PRCP deficiency could result in impaired AngII degradation. Increased AngII at this nephron site stimulates Na reabsorption and increases BP. KEY MESSAGE: Prolylcarboxypeptidase (PRCP) converts AngII to Ang (1-7) but only at an acidic pH. Global PRCP deficiency causes heart and kidney alterations and a moderate rise in BP. PRCP is abundant in the kidney collecting tubules, where the prevailing pH is low. In collecting tubules, PRCP deficiency could result in impaired AngII degradation. Increased AngII at this nephron site stimulates Na reabsorption and increases BP.

The Small Molecule Hyperphyllin Enhances Leaf Formation Rate and Mimics Shoot Meristem Integrity Defects Associated with AMP1 Deficiency.

ALTERED MERISTEM PROGRAM1 (AMP1) is a member of the M28 family of carboxypeptidases with a pivotal role in plant development and stress adaptation. Its most prominent mutant defect is a unique hypertrophic shoot phenotype combining a strongly increased organ formation rate with enhanced meristem size and the formation of ectopic meristem poles. However, so far the role of AMP1 in shoot development could not be assigned to a specific molecular pathway nor is its biochemical function resolved. In this work we evaluated the level of functional conservation between AMP1 and its human homolog HsGCPII, a tumor marker of medical interest. We show that HsGCPII cannot substitute AMP1 in planta and that an HsGCPII-specific inhibitor does not evoke amp1-specific phenotypes. We used a chemical genetic approach to identify the drug hyperphyllin (HP), which specifically mimics the shoot defects of amp1, including plastochron reduction and enlargement and multiplication of the shoot meristem. We assessed the structural requirements of HP activity and excluded that it is a cytokinin analog. HP-treated wild-type plants showed amp1-related tissue-specific changes of various marker genes and a significant transcriptomic overlap with the mutant. HP was ineffective in amp1 and elevated the protein levels of PHAVOLUTA, consistent with the postulated role of AMP1 in miRNA-controlled translation, further supporting an AMP1-related mode of action. Our work suggests that plant and animal members of the M28 family of proteases adopted unrelated functions. With HP we provide a tool to characterize the plant-specific functions of this important class of proteins.

The Pseudomonas aeruginosa periplasmic protease CtpA can affect systems that impact its ability to mount both acute and chronic infections.

Proteases play important roles in the virulence of Pseudomonas aeruginosa. Some are exported to act on host targets and facilitate tissue destruction and bacterial dissemination. Others work within the bacterial cell to process virulence factors and regulate virulence gene expression. Relatively little is known about the role of one class of bacterial serine proteases known as the carboxyl-terminal processing proteases (CTPs). The P. aeruginosa genome encodes two CTPs annotated as PA3257/Prc and PA5134/CtpA in strain PAO1. Prc degrades mutant forms of the anti-sigma factor MucA to promote mucoidy in some cystic fibrosis lung isolates. However, nothing is known about the role or importance of CtpA. We have now found that endogenous CtpA is a soluble periplasmic protein and that a ctpA null mutant has specific phenotypes consistent with an altered cell envelope. Although a ctpA null mutation has no major effect on bacterial growth in the laboratory, CtpA is essential for the normal function of the type 3 secretion system (T3SS), for cytotoxicity toward host cells, and for virulence in a mouse model of acute pneumonia. Conversely, increasing the amount of CtpA above its endogenous level induces an uncharacterized extracytoplasmic function sigma factor regulon, an event that has been reported to attenuate P. aeruginosa in a rat model of chronic lung infection. Therefore, a normal level of CtpA activity is critical for T3SS function and acute virulence, whereas too much activity can trigger an apparent stress response that is detrimental to chronic virulence.

Early phase TGFß receptor signalling dynamics stabilised by the deubiquitinase UCH37 promotes cell migratory responses.

TGFß signals through serine/threonine kinase receptors and intracellular Smad transcription factors. An important regulatory step involves ubiquitination of Smads and/or TGFß receptors by specific ubiquitin ligases, in a process that can be reversed by the deubiquitinating enzyme UCH37. Here, to explore the physiological role of UCH37 in TGFß signalling we have generated stable and inducible HaCAT keratinocyte and Colo-357 pancreatic carcinoma cell lines mis-expressing UCH37. We show that UCH37 knockdown significantly inhibits the activity of a TGFß-dependent gene reporter and selectively decreases levels of some TGFß-dependent target genes, notably p21 and PAI-1, but only during the early phase of TGFß receptor activation. Interestingly, UCH37 knockdown in Colo-357 cells had no effect on TGFß-dependent cell proliferation and epithelial-mesenchymal transition, yet significantly impaired cell migration. Collectively, our data indicate that UCH37 sustains early TGFß pathway activation kinetics that determines threshold-specific gene expression patterns, and that opposing actions of ubiquitin ligases and deubiquitinases influences distinct biological TGFß-dependent biological responses. Moreover, we suggest that UCH37 could represent a viable target for novel and selective cancer therapeutics.

Aortic carboxypeptidase-like protein is expressed in fibrotic human lung and its absence protects against bleomycin-induced lung fibrosis.

The pathological hallmarks of idiopathic pulmonary fibrosis include proliferating fibroblasts and myofibroblasts, as well as excessive collagen matrix deposition. In addition, both myofibroblast contraction and remodeling of the collagen-rich matrix contribute to the abnormal structure and function of the fibrotic lung. Little is known, however, about collagen-associated proteins that promote fibroblast and myofibroblast retention, as well as the proliferation of these cells on the extracellular matrix. In this study, we demonstrate that aortic carboxypeptidase-like protein (ACLP), a collagen-associated protein with a discoidin-like domain, is expressed at high levels in human fibrotic lung tissue and human fibroblasts, and that its expression increases markedly in the lungs of bleomycin-injured mice. Importantly, ACLP-deficient mice accumulated significantly fewer myofibroblasts and less collagen in the lung after bleomycin injury, as compared with wild-type controls, despite equivalent levels of bleomycin-induced inflammation. ACLP that is secreted by lung fibroblasts was retained on fibrillar collagen, and ACLP-deficient lung fibroblasts that were cultured on collagen exhibited changes in cell spreading, proliferation, and contraction of the collagen matrix. Finally, the addition of recombinant discoidin-like domain of ACLP to cultured ACLP-deficient lung fibroblasts restored cell spreading and increased the contraction of collagen gels. Therefore, both ACLP and its discoidin-like domain may be novel targets for anti-myofibroblast-based therapies for the treatment of pulmonary fibrosis.

Decrease of hirudin degradation by deleting the KEX1 gene in recombinant Pichia pastoris.

Our previous study on recombinant hirudin production in Pichia pastoris demonstrated that, although the total productivity of hirudin was fairly high, its degradation was still severe, even if many engineering methods were applied to improve cell viability and reduce the release of intracellular proteinases. In this work, a pop-in/pop-out method, replacing the auxotrophic marker ARG4 gene with the resistant marker sh ble gene, was used to delete the KEX1 gene to reduce hirudin degradation in P. pastoris GS115Hir. Using this strategy, hirudin degradation was greatly decreased. At the same wet cell weight and cell viability, the percentage of intact hirudin Hir65 in total hirudin in strain GS115HirDeltakex1 was always kept as high as 90% in the initial stage of the methanol fermentation phase and above 62% even in the later stage of the methanol fermentation phase, whereas the percentage for the undeleted strain GS115Hir was only about 40% in the whole methanol fermentation phase. As a result, the intact hirudin Hir65 concentration could maximally reach 2.4 g/l in GS115HirDeltakex1 while it was only 1.1 g/l in GS115Hir.

Angiotensin-converting enzyme 2 protects from severe acute lung failure.

Acute respiratory distress syndrome (ARDS), the most severe form of acute lung injury, is a devastating clinical syndrome with a high mortality rate (30-60%) (refs 1-3). Predisposing factors for ARDS are diverse and include sepsis, aspiration, pneumonias and infections with the severe acute respiratory syndrome (SARS) coronavirus. At present, there are no effective drugs for improving the clinical outcome of ARDS. Angiotensin-converting enzyme (ACE) and ACE2 are homologues with different key functions in the renin-angiotensin system. ACE cleaves angiotensin I to generate angiotensin II, whereas ACE2 inactivates angiotensin II and is a negative regulator of the system. ACE2 has also recently been identified as a potential SARS virus receptor and is expressed in lungs. Here we report that ACE2 and the angiotensin II type 2 receptor (AT2) protect mice from severe acute lung injury induced by acid aspiration or sepsis. However, other components of the renin-angiotensin system, including ACE, angiotensin II and the angiotensin II type 1a receptor (AT1a), promote disease pathogenesis, induce lung oedemas and impair lung function. We show that mice deficient for Ace show markedly improved disease, and also that recombinant ACE2 can protect mice from severe acute lung injury. Our data identify a critical function for ACE2 in acute lung injury, pointing to a possible therapy for a syndrome affecting millions of people worldwide every year.

Increase in macrophages in the testis of cathepsin a deficient mice suggests an important role for these cells in the interstitial space of this tissue.

Cathepsin A (PPCA) is a lysosomal carboxypeptidase that functions as a protective protein for alpha-neuraminidase and beta-galactosidase in a multienzyme complex. In the present study, the testes of PPCA -/- mice from 2 to 10 months of age were compared with those of their wild type counterparts. While germ and Sertoli cells appeared comparable in appearance and distribution, the mean profile area of seminiferous tubules showed a significant decrease between wild type and PPCA -/- mice, suggesting changes to the seminiferous tubules and their contents. In addition, macrophages in the interstitial space (IS) of PPCA -/- mice were large, spherical, and filled with pale lysosomes, unlike those seen in wild type mice, and a quantitative analysis of their frequency per unit area of IS in PPCA -/- mice revealed a significant increase compared to that of wild type mice; this was also the case for their mean profile area. Absence of mitotic figures, cycling cells, or degenerating figures in the IS suggests that the major recruitment of macrophages appears to be from the circulation. In the IS, Leydig cells also showed an accumulation of large pale lysosomes in PPCA -/- mice, and their frequency also increased significantly as compared to wild type mice. In the electron microscope, a close association of Leydig cell microvilli with the surface of macrophages was pronounced in PPCA -/- mice. Since macrophages and Leydig cells interact by secreting various factors between each other, and considering the fact that Leydig cells show an accumulation of large pale lysosomes in PPCA -/- mice, it is suggested that macrophages accumulate as a result of abnormalities occurring in Leydig cells. Taken together, the data on increase in frequency of macrophages suggests important functions for these cells in both wild type and PPCA -/- mice.

Developmental changes in opioid peptides and their receptors in Cpe(fat)/Cpe(fat) mice lacking peptide processing enzyme carboxypeptidase E.

Carboxypeptidase E (CPE) is involved in the biosynthesis of a number of neuropeptides including opioid peptides. A point mutation in this gene results in a loss of enzyme activity, decrease in mature neuroendocrine peptides, and development of late onset obesity as seen in Cpe(fat)/Cpe(fat) mice. In this study, we examined the processing of peptides derived from prodynorphin and proenkephalin in various brain regions of these mice during development. At 6 to 8 weeks, an age prior to the onset of obesity, levels of dynorphin peptides are decreased in all brain regions, whereas levels of ir-Met-enkephalin are differentially altered. There is an accumulation of C-terminally extended forms of all three opioid peptides in Cpe(fat)/Cpe(fat) mice, consistent with a lack of CPE activity. Thus, it appears that there is no direct correlation between the level of mature opioid peptides and the development of obesity in these mice. Since altered levels of peptides can influence the opioid receptor system, we examined the functional activity of mu and kappa opioid receptors using [35S]guanosine-5'-O-(gamma-thio)-triphosphate binding assays. We find no differences in kappa receptor activity in Cpe(fat)/Cpe(fat) compared with control littermate mice. In contrast, the mu receptor activity is differentially altered in select regions of Cpe(fat)/Cpe(fat) mice in response to a mu-specific ligand. Taken together, these results suggest that the lack of CPE activity leads to alterations in the level of opioid peptides during development and that changes in peptide levels differentially affect opioid receptor activity in vivo.

Deletion of the glutamate carboxypeptidase II gene in mice reveals a second enzyme activity that hydrolyzes N-acetylaspartylglutamate.

Glutamate carboxypeptidase II (GCPII, EC 3.14.17.21) is a membrane-bound enzyme found on the extracellular face ofglia. The gene for this enzyme is designated FOLH1 in humans and Folh1 in mice. This enzyme has been proposed to be responsible for inactivation of the neurotransmitter N-acetylaspartylglutamate (NAAG) following synaptic release. Mice harboring a disruption of the gene for GCPII/Folh1 were generated by inserting into the genome a targeting cassette in which the intron-exon boundary sequences of exons 1 and 2 were removed and stop codons were inserted in exons 1 and 2. Messenger RNA for GCPII was not detected by northern blotting or RT-PCR analysis of RNA from the brains of -/- mutant mice nor was GCPII protein detected on western blots of this tissue. These GCPII null mutant mice developed normally to adulthood and exhibited a normal range of neurologic responses and behaviors including mating, open field activity and retention of position in rotorod tests. No significant differences were observed among responses of wild type, heterozygous mutant and homozygous mutant mice on tail flick and hot plate latency tests. Glutamate, NAAG and mRNA for metabotropic glutamate receptor type 3 levels were not significantly altered in response to the deletion of glutamate carboxypeptidase II. A novel membrane-bound NAAG peptidase activity was discovered in brain, spinal cord and kidney of the GCPII knock out mice. The kinetic values for brain NAAG peptidase activity in the wild type and GCPII nullmutant were Vmax = 45 and 3 pmol/mg/min and Km = 2650 nm and 2494 nm, respectively. With the exception of magnesium and copper, this novel peptidase activity had a similar requirement for metal ions as GCPII. Two potent inhibitors of GCPII, 4,4'-phosphinicobis-(butane-1,3 dicarboxilic acid) (FN6) and 2-(phosphonomethyl)pentanedioic acid (2-PMPA) inhibited the residual activity. The IC50 value for 2-PMPA was about 1 nm for wild-type brain membrane NAAG peptidase activity consistent with its activity against cloned ratand human GCPII, and 88 nm for the activity in brain membranes of the null mutants.

Quantitation of neuropeptides in Cpe(fat)/Cpe(fat) mice using differential isotopic tags and mass spectrometry.

Neuroendocrine peptides play important roles as intercellular messengers. We previously developed a technique to isolate and identify a large number of neuroendocrine peptides from Cpe(fat)/Cpe(fat) mice (Che, F.; et al. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 9971-6); these mice lack carboxypeptidase E activity and this defect causes an accumulation of neuropeptide intermediates that contain C-terminal Lys or Arg residues (Naggert, J. K.; et al. Nat. Genet. 1995, 10, 135-42). In the present study, we have developed a differential isotopic-labeling technique that can be used to quantitate changes in neuropeptide levels in Cpe(fat)/Cpe(fat) mouse tissues. Samples are treated with either the H6 or the D6 form of acetic anhydride, peptides that contain C-terminal basic amino acids are isolated by affinity chromatography on anhydrotrypsin agarose, and the isolated peptides are analyzed by mass spectrometry. Measurement of the regulation of pituitary peptides in response to dehydration showed a decrease in vasopressin. The general method described in this report should be widely applicable to a large number of neuroendocrine peptides, known and novel, in a variety of regulatory paradigms.

Identification of peptides from brain and pituitary of Cpe(fat)/Cpe(fat) mice.

Cpe(fat)/Cpe(fat) mice have a naturally occurring point mutation within the carboxypeptidase E gene that inactivates this enzyme, leading to an accumulation of many neuroendocrine peptides containing C-terminal basic residues. These processing intermediates can be readily purified on an anhydrotrypsin affinity resin. Using MS to obtain molecular mass and partial sequence information, more than 100 peptides have been identified. These peptides represent fragments of 16 known secretory pathway proteins, including proenkephalin, proopiomelanocortin, protachykinins A and B, chromogranin A and B, and secretogranin II. Many of the identified peptides represent previously uncharacterized fragments of the precursors. For example, 12 of the 13 chromogranin B-derived peptides found in the present study have not been previously reported. Of these 13 chromogranin B-derived peptides, only five contain consensus cleavage sites for prohormone convertases at both the C and N termini. Two distinct chromogranin B-derived peptides result from cleavage at Trp-Trp bonds, a site not typically associated with neuropeptide processing. An RIA was used to confirm that one of these peptides, designated WE-15, exists in wild-type mouse brain, thus validating the approach to identify peptides in Cpe(fat)/Cpe(fat) mice. These "orphan" peptides are candidate ligands for orphan G protein-coupled receptors. In addition, the general technique of using affinity chromatography to isolate endogenous substrates from a mutant organism lacking an enzyme should be applicable to a wide range of enzyme-substrate systems.

Thrombin-activatable fibrinolysis inhibitor deficiency in cirrhosis is not associated with increased plasma fibrinolysis.

BACKGROUND AND AIMS: The bleeding tendency of patients suffering from cirrhosis is in part ascribed to accelerated fibrinolysis. In this study, the role of the recently discovered inhibitor of fibrinolysis, thrombin-activatable fibrinolysis inhibitor (TAFI) in cirrhosis was examined. METHODS: In 64 patients with cirrhosis of varying severity, TAFI antigen levels were measured by enzyme-linked immunosorbent assay and compared with TAFI levels in control subjects. Furthermore, a plasma-based fibrinolysis assay was performed in the presence and absence of a specific inhibitor of activated TAFI. RESULTS: TAFI levels were decreased in cirrhosis. Mean TAFI levels were 66% in Child's A, 55% in Child's B, 47% in Child's C cirrhosis, and 26% in acute liver failure. Decreased TAFI antigen levels were highly correlated with antithrombin and alpha(2)-antiplasmin activity levels. Clot lysis times and clot lysis ratio (defined as ratio between clot lysis time in the absence and presence of a specific inhibitor of activated TAFI) of cirrhotics were not significantly different from healthy controls. CONCLUSIONS: Despite decreased levels of TAFI and other components of the fibrinolytic system, no evidence of increased plasma fibrinolytic potential in cirrhosis is observed using the plasma-based assay of this study. The reduction of antifibrinolytic factors in cirrhosis is compensated by the concomitant reduction in profibrinolytics.

Attenuated processing of proglucagon and glucagon-like peptide-1 in carboxypeptidase E-deficient mice.

The maturation of many peptide hormones is attenuated in carboxypeptidase E (CPE)-deficient fat/fat mice, leading to a slowly developing, adult-onset obesity with mild diabetes. To determine the contribution of the hormones generated from the proglucagon precursor to this phenotype, we studied the tissue-specific processing of glucagon and glucagon-like peptide-1 (GLP-1) in these mice. In all tissues examined there was a great reduction in mature amidated GLP-1. Furthermore, a lack of CPE attenuates prohormone convertase processing of proglucagon in both the pancreas and the intestine. These findings suggest that defects in proglucagon processing together with other endocrine malfunctions could contribute to the diabetic and obesity phenotype in fat/fat mice.

Cholesterol, a cell size-dependent signal that regulates glucose metabolism and gene expression in adipocytes.

Enlarged fat cells exhibit modified metabolic capacities, which could be involved in the metabolic complications of obesity at the whole body level. We show here that sterol regulatory element-binding protein 2 (SREBP-2) and its target genes are induced in the adipose tissue of several models of rodent obesity, suggesting cholesterol imbalance in enlarged adipocytes. Within a particular fat pad, larger adipocytes have reduced membrane cholesterol concentrations compared with smaller fat cells, demonstrating that altered cholesterol distribution is characteristic of adipocyte hypertrophy per se. We show that treatment with methyl-beta-cyclodextrin, which mimics the membrane cholesterol reduction of hypertrophied adipocytes, induces insulin resistance. We also produced cholesterol depletion by mevastatin treatment, which activates SREBP-2 and its target genes. The analysis of 40 adipocyte genes showed that the response to cholesterol depletion implicated genes involved in cholesterol traffic (caveolin 2, scavenger receptor BI, and ATP binding cassette 1 genes) but also adipocyte-derived secretion products (tumor necrosis factor alpha, angiotensinogen, and interleukin-6) and proteins involved in energy metabolism (fatty acid synthase, GLUT 4, and UCP3). These data demonstrate that altering cholesterol balance profoundly modifies adipocyte metabolism in a way resembling that seen in hypertrophied fat cells from obese rodents or humans. This is the first evidence that intracellular cholesterol might serve as a link between fat cell size and adipocyte metabolic activity.