Simeon I. Taylor

Leptin reverses insulin resistance and hepatic steatosis in patients with severe lipodystrophy

Lipodystrophy is a rare disorder that is characterized by selective loss of subcutaneous and visceral fat and is associated with hypertriglyceridemia, hepatomegaly, and disordered glucose metabolism. It has recently been shown that chronic leptin treatment ameliorates these abnormalities. Here we show that chronic leptin treatment improves insulin-stimulated hepatic and peripheral glucose metabolism in severely insulin-resistant lipodystrophic patients. This improvement in insulin action was associated with a marked reduction in hepatic and muscle triglyceride content. These data suggest that leptin may represent an important new therapy to reverse the severe hepatic and muscle insulin resistance and associated hepatic steatosis in patients with lipodystrophy.

Development of a novel polygenic model of NIDDM in mice heterozygous for IR and IRS-1 null alleles.

NIDDM is a polygenic disease characterized by insulin resistance in muscle, fat, and liver, followed by a failure of pancreatic beta cells to adequately compensate for this resistance despite increased insulin secretion. Mice double heterozygous for null alleles in the insulin receptor and insulin receptor substrate-1 genes exhibit the expected approximately 50% reduction in expression of these two proteins, but a synergism at a level of insulin resistance with 5- to 50-fold elevated plasma insulin levels and comparable levels of beta cell hyperplasia. At 4-6 months of age, 40% of these double heterozygotes become overtly diabetic. This NIDDM mouse model in which diabetes arises in an age-dependent manner from the interaction between two genetically determined, subclinical defects in the insulin signaling cascade demonstrates the role of epistatic interactions in the pathogenesis of common diseases with non-Mendelian genetics.

AGPAT2 is mutated in congenital generalized lipodystrophy linked to chromosome 9q34.

Congenital generalized lipodystrophy is an autosomal recessive disorder characterized by marked paucity of adipose tissue, extreme insulin resistance, hypertriglyceridemia, hepatic steatosis and early onset of diabetes. We report several different mutations of the gene (AGPAT2) encoding 1-acylglycerol-3-phosphate O-acyltransferase 2 in 20 affected individuals from 11 pedigrees of diverse ethnicities showing linkage to chromosome 9q34. The AGPAT2 enzyme catalyzes the acylation of lysophosphatidic acid to form phosphatidic acid, a key intermediate in the biosynthesis of triacylglycerol and glycerophospholipids. AGPAT2 mRNA is highly expressed in adipose tissue. We conclude that mutations in AGPAT2 may cause congenital generalized lipodystrophy by inhibiting triacylglycerol synthesis and storage in adipocytes.

Point mutation causing a single amino acid substitution in the hormone binding domain of the glucocorticoid receptor in familial glucocorticoid resistance.

Familial glucocorticoid resistance is a hypertensive, hyperandrogenic disorder characterized by increased serum cortisol concentrations in the absence of stigmata of Cushings syndrome. Our previous studies of the first reported kindred showed a two- to threefold reduction in glucocorticoid receptor-ligand binding affinity in the propositus, and a lesser reduction in affinity in his mildly affected son and nephew. Glucocorticoid receptor cDNA from these three patients was amplified by polymerase chain reaction and sequenced. The cDNA nucleotide sequence was normal, except for nucleotide 2054, which substituted valine for aspartic acid at amino acid residue 641. The propositus was homozygous while the other relatives were heterozygous for the mutation. COS-7 monkey kidney cells were cotransfected with expression vectors for either wild type or Val 641-mutant receptors, together with the reporter plasmid pMMTV-CAT. Dexamethasone increased chloramphenicol acetyltransferase activity in cells expressing wild type receptor, but had no effect in cells expressing Val 641-mutant receptors, despite similar receptor concentrations, as indicated by Western blotting. The binding affinity for dexamethasone of the Val 641-mutant receptor was threefold lower than that of the wild type receptor. These results suggest that glucocorticoid resistance in this family is due to a point mutation in the steroid-binding domain of the glucocorticoid receptor.

Lilly Lecture: Molecular Mechanisms of Insulin Resistance: Lessons From Patients With Mutations in the Insulin-Receptor Gene

Insulin resistance contributes to the pathogenesis of NIDDM. We have investigated the molecular mechanisms of insulin resistance in patients with genetic syndromes caused by mutations in the insulin-receptor gene. In general, patients with two mutant alleles of the insulin-receptor gene are more severly insulinresistant than are patients who are heterozygous for a single mutant allele. These mutations can be put into five classes, depending upon the mechanisms by which they impair receptor function. Some mutations lead to a decrease in the number of insulin receptors on the cell surface. For example, some mutations decrease the level of insulin receptor mRNA or impair receptor biosynthesis by introducing a premature chain termination codon (class 1). Class 2 mutations impair the transport of receptors through the endoplasmic reticulum and Golgi apparatus to the plasma membrane. Mutations that accelerate the rate of receptor degradation (class 5) also decrease the number of receptors on the cell surface. Other mutations cause insulin resistance by impairing receptor function—either by decreasing the affinity to bind insulin (class 3) or by impairing receptor tyrosine kinase activity (class 4). The prevalence of mutations in the insulin receptor gene is not known. However, theoretical calculations suggest that ∼0.1–1% of the general population are heterozygous for a mutation in the insulinreceptor gene; the prevalence is likely to be higher among people with NIDDM. Accordingly, it is likely that mutations in the insulin-receptor gene may be a con-tributory cause of insulin resistance in a subpopulation with NIDDM.

Deconstructing type 2 diabetes.

There is a natural temptation to extrapolate insights from these animal models to explain the pathophysiology of human diabetes. In some cases, the animal model closely resembles the human disease, as is the case with glucokinase knockout mice and MODY2. In contrast, insulin receptor knockout mice differ dramatically from patients with leprechaunism, the human disease resulting from absence of insulin receptors (9xElders, M., Schedewie, H., Olefsky, J., Givens, B., Char, F., Bier, D., Baldwin, D., Fiser, R., Seyedabadi, S., and Rubenstein, A. J. Natl. Med. Assoc. 1982; 74: 1195–1210PubMedSee all References, 20xTaylor, S.I. Diabetes. 1992; 41: 1473–1490Crossref | PubMedSee all References). While insulin receptor knockout mice develop a severe form of diabetes associated with marked hyperglycemia and ketoacidosis, patients with leprechaunism exhibit relatively mild hyperglycemia. Furthermore, intrauterine growth retardation, a key feature of the human disease, was not found in the knockout mouse. Why might the same mutation cause different phenotypes in different species? There may be differences in the genetic background; for example, parallel or redundant pathways may vary in importance in different species. Indeed, there can be striking differences in phenotype even when a specific mutation is introduced into different strains of mice. By mapping and ultimately cloning modifier genes that account for variations in phenotype, we will advance our understanding of polygenic diseases such as diabetes.The differences between insulin receptor knockout mice and the corresponding human disease invite the question: Are mice with tissue-specific insulin resistance good models for human disease?Insulin resistance in skeletal muscle. The phenotype of mice with insulin resistance at the level of muscle (16xMoller, D., Chang, P., Yaspelkis, B., Flier, J., Wallberg-Henriksson, H., and Ivy, J. Endocrinology. 1996; 137: 2397–2405PubMedSee all References, 6xBruning, J., Michael, M., Winnay, J., Hayashi, T., Horsch, D., Accili, D., Goodyear, L., and Kahn, C. Mol. Cell. 1998; 2: 559–569Abstract | Full Text | Full Text PDF | PubMedSee all References) closely resembles an early stage of human type 2 diabetes (DeFronzo 1997xDeFronzo, R. Diabetes Rev. 1997; 5: 177–269See all ReferencesDeFronzo 1997). Like “prediabetic” humans, these mice exhibit insulin resistance at the level of muscle, but maintain normal (or only minimally elevated) fasting plasma glucose levels. Unlike the animal models, the precise molecular cause of insulin resistance has not yet been elucidated in humans although it is known that there is a block at the level of glucose uptake/phosphorylation in muscle of patients with type 2 diabetes.Insulin resistance in pancreatic β cells. Because insulin resistance develops early in the natural history of type 2 diabetes, it has long been suspected that insulin resistance may play a causal role in the development of the defect in insulin secretion. Novel insights provided by βIRKO mice suggest a unified theory to explain the pathogenesis of type 2 diabetes. A genetic defect in the insulin action pathway may not only cause target cells to become insulin resistant, but also might directly cause β cell failure and insulin deficiency.However, Kulkarni et al. 1999xKulkarni, R., Bruning, J.C., Winnay, J., Postic, C., Magnuson, M., and Kahn, C. Cell. 1999; 96: 329–339Abstract | Full Text | Full Text PDF | PubMed | Scopus (770)See all ReferencesKulkarni et al. 1999 identified an important difference in phenotypes between βIRKO mice and human patients. Whereas insulin secretion is impaired in βIRKO mice, humans with mutations in the insulin receptor gene (e.g., patients with leprechaunism) exhibit striking hyperplasia of pancreatic islets and hypersecretion of insulin (Elders et al. 1982xElders, M., Schedewie, H., Olefsky, J., Givens, B., Char, F., Bier, D., Baldwin, D., Fiser, R., Seyedabadi, S., and Rubenstein, A. J. Natl. Med. Assoc. 1982; 74: 1195–1210PubMedSee all ReferencesElders et al. 1982). Furthermore, even in mice, a defect in insulin signaling does not necessarily cause deficiency in insulin secretion. Bruning et al. 1997xBruning, J.C., Winnay, J., Bonner-Weir, S., Taylor, S.I., Accili, D., and Kahn, C.R. Cell. 1997; 88: 561–572Abstract | Full Text | Full Text PDF | PubMed | Scopus (416)See all ReferencesBruning et al. 1997 engineered mice to become insulin resistant by introducing heterozygous null mutations into both the Insr gene and the Irs1 gene; these Insr+/−Irs1+/− mice developed striking hyperplasia of β cells. Interestingly, the phenotype of β cell hyperplasia observed in the Insr+/−Irs1+/− mice closely resembles the phenotype of patients with leprechaunism. Furthermore, Insr+/−Irs1+/− mice, like most patients with type 2 diabetes, exhibit generalized, partial insulin resistance rather than the total, tissue-specific insulin resistance of βIRKO mice. Thus, there appear to be complex interactions between insulin signaling and β cell function; depending upon the circumstances, insulin resistance may lead either to insulin deficiency (as in βIRKO mice) or to β cell hyperplasia plus hyperinsulinemia (as in humans with leprechaunism and Insr+/−Irs1+/− mice).Because type 2 diabetes is a complex heterogeneous disease with multiple genes contributing to the cause of this polygenic disorder, it is likely that these murine animal models will shed light on disease mechanisms that contribute to the pathophysiology of human diabetes. Defects in the insulin action pathway may be the cause of insulin deficiency in some patients, but there may also be patients in whom there are primary genetic defects that directly impair the machinery for insulin secretion. The approach of positional cloning, currently underway in several laboratories, holds great promise to resolve these controversies by directly identifying the genes that cause both insulin deficiency and insulin resistance.

Efficacy and Safety of Troglitazone in the Treatment of Lipodystrophy Syndromes

Obesity causes insulin resistance, a central feature in the pathogenesis of type 2 diabetes (1). Paradoxically, the absence of adipose tissue also causes insulin resistance and diabetes in humans (2, 3) and genetically engineered animal models (4-6). Lipoatrophy and lipodystrophy are features of a group of heterogeneous syndromes characterized by a paucity of fat, insulin resistance, and hypertriglyceridemia (7). If patients develop diabetes, the syndrome is referred to as lipoatrophic diabetes. The disease has several genetic forms, including face-sparing partial lipoatrophy (the Dunnigan syndrome or the KoberlingDunnigan syndrome, OMIM [Online Mendelian Inheritance in Man] 308980), an autosomal dominant form caused by mutations in the lamin A/C gene (8), and congenital generalized lipoatrophy (the SeipBerardinelli syndrome, OMIM 269700), an autosomal recessive form mapping to chromosome 9q34 in some pedigrees (9). These diseases are rare; reported estimated prevalences are less than 1 in 10 million (10), although our experience suggests that the actual prevalences may be somewhat higher. An association between lipoatrophy and autoimmune disease, such as juvenile dermatomyositis, has also been described (11), suggesting that autoimmune destruction of adipose tissue results in a form of lipoatrophy. Thiazolidinediones, a new class of antidiabetic drugs (12), are ligands for peroxisome proliferatoractivated receptor- (PPAR-), a nuclear receptor expressed predominantly in adipose tissue (13). Thiazolidinediones are believed to exert their primary actions in adipose tissue and to indirectly increase insulin sensitivity in other tissues (14). Because thiazolidinediones have been reported to both increase insulin sensitivity (15, 16) and promote adipocyte development (17), these drugs seemed ideally suited to treat lipoatrophic diabetes. Troglitazone, the first thiazolidinedione to be approved for therapeutic use in the United States, has been shown to improve glycemic control and ameliorate hypertriglyceridemia in patients with type 2 diabetes (18). However, the use of troglitazone is complicated by a rare form of severe, irreversible hepatotoxicity. Two additional thiazolidinediones, rosiglitazone and pioglitazone, were recently approved for use. These drugs are also effective in improving glycemic control in patients with type 2 diabetes (19). Although initial studies of rosiglitazone and pioglitazone suggested that they might not be toxic to the liver, recent reports have raised the possibility that rosiglitazone may rarely cause hepatotoxicity (19, 20). Because PPAR- ligands promote adipocyte differentiation in vitro (13), we hypothesized that troglitazone would promote adipocyte development in patients with various forms of lipoatrophy. This hypothesis implicitly assumes that some lipoatrophic patients possess pre-adipocytes that could be stimulated by troglitazone to complete adipocyte differentiation. In addition, we sought to determine whether troglitazone therapy would improve metabolic control in patients with various forms of lipoatrophy. In light of data suggesting that troglitazone exerts its primary effects on adipocytes, it was uncertain whether the drug would be effective in such patients. Methods Patients Potential study participants were referred by multiple physicians in the United States and Canada in response to advertisements placed in medical journals, notices on the Internet, or word-of-mouth. Some patients had been followed at the National Institutes of Health for varying periods of time (up to 20 years). Because of the rarity of the syndrome, it was not practical to conduct population-based recruitment. To be eligible for the study, patients had to have both insulin resistance and lipoatrophy. For our purposes, insulin resistance was defined as either a fasting plasma insulin level greater than 143 pmol/L or impaired response to intravenous insulin (0.15 U/kg). The latter criterion was defined as a decrease in plasma glucose of less than 50% in patients with fasting glucose levels of 11 mmol/L or less ( 200 mg/dL) or a decrease of 5.5 mmol/L or less (<100 mg/dL) in patients with fasting glucose levels greater than 11.1 mmol/L (>200 mg/dL). Of 33 patients screened for this study, 8 were excluded because serum aminotransferase concentrations were abnormal (range, 833 to 6666 nkat/L) and liver biopsies showed steatohepatitis with varying degrees of fibrosis. Five patients were excluded for various reasons, such as the inability to give informed consent or adhere to the study follow-up schedule. The remaining 20 patients were recruited into the study (Table). Table. Characteristics of the Study Patients Fat distribution was assessed by physical examination and magnetic resonance imaging (MRI). A region of the body was defined as affected if MRI showed a marked decrease in fat in that region. Four patients had generalized lipoatrophy, defined as involvement of the following nine regions: face, neck, upper trunk, abdominal subcutaneous fat, visceral fat, and all four extremities. Two of these patients (U1 and P1) had near-total absence of fat throughout their bodies; the other two (A1 and A2) had a generalized decrease in fat but retained some fat in their visceral abdomen. Sixteen patients, including 7 patients with the Dunnigan syndrome, had partial lipoatrophy affecting five to eight fat depots. Six patients had accompanying autoimmune disease or results on three or more laboratory tests that suggested autoimmunity (for example, antinuclear antibody, rheumatoid factor, and elevated erythrocyte sedimentation rate); these patients therefore were presumed to have an autoimmune cause of their lipoatrophy. The cause of lipoatrophy appeared to be genetic in 10 patients; lipoatrophy appeared shortly after birth in 1 patient, and 9 patients had several affected relatives. Seven of these 9 patients had Dunnigan partial lipodystrophy (21) (Table); the 7 patients were members of three pedigrees. After completion of the study, the diagnosis of the Dunnigan syndrome was confirmed by identifying the R482Q mutation in the lamin A/C gene in all three pedigrees (22). In 4 patients, the cause of disease was unknown. Of the 20 study patients (Table), 14 had diabetes and 2 had impaired glucose tolerance according to the 1997 American Diabetes Association criteria (23). Most diabetic patients were receiving pharmacotherapy before study entry. Five patients were receiving insulin (0.5 to 2 U/kg of body weight per day) and 5 were receiving sulfonylureas; patients continued to receive these therapies during the study. Two patients were receiving metformin, but this therapy was discontinued 6 weeks before initiation of troglitazone treatment. Syndromes of lipoatrophy are associated with substantial comorbid conditions. Of the 8 patients with triglyceride levels greater than 4.5 mmol/L (400 mg/dL), 6 had a history of pancreatitis. Seventeen patients had acanthosis nigricans, a dermatologic condition associated with insulin resistance. Twelve of the 18 female participants had histories of irregular menses and polycystic ovaries as documented by ultrasonography; 6 of these women had hirsutism. Of the 6 remaining female participants, 4 were postmenopausal, 1 was perimenopausal, and 1 was prepubertal. Fatty liver is another important feature sometimes associated with lipoatrophy. To be included in the study, patients had to have normal biochemical function of the liver (Table). Nevertheless, results of ultrasonography in 12 patients suggested fatty infiltration of the liver. Lipoatrophic diabetes was associated with chronic complications of diabetes in some patients. Six patients had albuminuria, seven had diabetic polyneuropathy, and three had diabetic retinopathy (one of whom had proliferative retinopathy). One patient had three-vessel coronary artery disease. Design Patients were treated with troglitazone in an open-label prospective trial in which each patient was compared with his or her own baseline state. Because of the rarity of lipoatrophy syndromes and the variability of the clinical features, it was not feasible to use a randomized, placebo-controlled design. The study was approved by the institutional review board of the National Institute of Diabetes and Digestive and Kidney Diseases. Informed consent was obtained from the patient or his or her legal guardian. The decision to analyze the data after 6 months of therapy was made before the study was begun. Patients were evaluated as inpatients at the Clinical Center of the National Institutes of Health before treatment with troglitazone was initiated. They were admitted again after 6 weeks, 3 months, and 6 months of treatment. Before starting troglitazone therapy, diabetic patients were followed for at least 6 weeks while receiving stable doses of medication. Patients receiving insulin or sulfonylureas continued therapy with these drugs; however, metformin therapy was discontinued before troglitazone therapy was initiated. In diabetic patients, troglitazone therapy was started at a dosage of 200 mg/d and was increased to 400 to 600 mg/d over the course of 6 to 12 weeks, with the goal of optimizing glycemic control. The slow titration was chosen to minimize the risk for hypoglycemia. Doses of insulin or sulfonylureas were decreased if this was necessary to prevent hypoglycemia. Patients received stable doses of lipid-lowering medication for at least 6 weeks before starting troglitazone therapy. In nondiabetic adult participants, troglitazone was prescribed at a dosage of 400 mg/d. In one 6-year-old child weighing 15 to 18 kg, the dosage was 100 mg/d. Liver function tests and blood counts were performed every 3 to 4 weeks. Patients completed weekly questionnaires about their symptoms to identify potential side effects. Patients were instructed not to change their diet and exercise habits during this study. Information about dietary habits was collected by using

Mutational and Haplotype Analyses of Families with Familial Partial Lipodystrophy (Dunnigan Variety) Reveal Recurrent Missense Mutations in the Globular C-Terminal Domain of Lamin A/C

Familial partial lipodystrophy (FPLD), Dunnigan variety, is an autosomal dominant disorder characterized by marked loss of subcutaneous adipose tissue from the extremities and trunk but by excess fat deposition in the head and neck. The disease is frequently associated with profound insulin resistance, dyslipidemia, and diabetes. We have localized a gene for FPLD to chromosome 1q21-q23, and it has recently been proposed that nuclear lamin A/C is altered in FPLD, on the basis of a novel missense mutation (R482Q) in five Canadian probands. This gene had previously been shown to be altered in autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD-AD) and in dilated cardiomyopathy and conduction-system disease. We examined 15 families with FPLD for mutations in lamin A/C. Five families harbored the R482Q alteration that segregated with the disease phenotype. Seven families harbored an R482W alteration, and one family harbored a G465D alteration. All these mutations lie within exon 8 of the lamin A/C gene-an exon that has also been shown to harbor different missense mutations that are responsible for EDMD-AD. Mutations could not be detected in lamin A/C in one FPLD family in which there was linkage to chromosome 1q21-q23. One family with atypical FPLD harbored an R582H alteration in exon 11 of lamin A. This exon does not comprise part of the lamin C coding region. All mutations in FPLD affect the globular C-terminal domain of the lamin A/C protein. In contrast, mutations responsible for dilated cardiomyopathy and conduction-system disease are observed in the rod domain of the protein. The FPLD mutations R482Q and R482W occurred on different haplotypes, indicating that they are likely to have arisen more than once.

Identification of a Family of Sorting Nexin Molecules and Characterization of Their Association with Receptors

ABSTRACT Sorting nexin 1 (SNX1) is a protein that binds to the epidermal growth factor (EGF) receptor and is proposed to play a role in directing EGF receptors to lysosomes for degradation (R. C. Kurten, D. L. Cadena, and G. N. Gill, Science 272:1008–1010, 1996). We have obtained full-length cDNAs and deduced the amino acid sequences of three novel homologous proteins, which were denoted human sorting nexins (SNX2, SNX3, and SNX4). In addition, we identified a presumed splice variant isoform of SNX1 (SNX1A). These molecules contain a conserved domain of ∼100 amino acids, which was termed the phox homology (PX) domain. Human SNX1 (522 amino acids), SNX1A (457 amino acids), SNX2 (519 amino acids), SNX3 (162 amino acids), and SNX4 (450 amino acids) are part of a larger family of hydrophilic molecules including proteins identified in Caenorhabditis elegans andSaccharomyces cerevisiae. Despite their hydrophilic nature, the sorting nexins are found partially associated with cellular membranes. They are widely expressed, although the tissue distribution of each sorting nexin mRNA varies. When expressed in COS7 cells, epitope-tagged sorting nexins SNX1, SNX1A, SNX2, and SNX4 coimmunoprecipitated with receptor tyrosine kinases for EGF, platelet-derived growth factor, and insulin. These sorting nexins also associated with the long isoform of the leptin receptor but not with the short and medium isoforms. Interestingly, endogenous COS7 transferrin receptors associated exclusively with SNX1 and SNX1A, while SNX3 was not found to associate with any of the receptors studied. Our demonstration of a large conserved family of sorting nexins that interact with a variety of receptor types suggests that these proteins may be involved in several stages of intracellular trafficking in mammalian cells.

SGLT2 Inhibitors May Predispose to Ketoacidosis

CONTEXT Sodium glucose cotransporter 2 (SGLT2) inhibitors are antidiabetic drugs that increase urinary excretion of glucose, thereby improving glycemic control and promoting weight loss. Since approval of the first-in-class drug in 2013, data have emerged suggesting that these drugs increase the risk of diabetic ketoacidosis. In May 2015, the Food and Drug Administration issued a warning that SGLT2 inhibitors may lead to ketoacidosis. EVIDENCE ACQUISITION Using PubMed and Google, we conducted Boolean searches including terms related to ketone bodies or ketoacidosis with terms for SGLT2 inhibitors or phlorizin. Priority was assigned to publications that shed light on molecular mechanisms whereby SGLT2 inhibitors could affect ketone body metabolism. EVIDENCE SYNTHESIS SGLT2 inhibitors trigger multiple mechanisms that could predispose to diabetic ketoacidosis. When SGLT2 inhibitors are combined with insulin, it is often necessary to decrease the insulin dose to avoid hypoglycemia. The lower dose of insulin may be insufficient to suppress lipolysis and ketogenesis. Furthermore, SGLT2 is expressed in pancreatic α-cells, and SGLT2 inhibitors promote glucagon secretion. Finally, phlorizin, a nonselective inhibitor of SGLT family transporters decreases urinary excretion of ketone bodies. A decrease in the renal clearance of ketone bodies could also increase the plasma ketone body levels. CONCLUSIONS Based on the physiology of SGLT2 and the pharmacology of SGLT2 inhibitors, there are several biologically plausible mechanisms whereby this class of drugs has the potential to increase the risk of developing diabetic ketoacidosis. Future research should be directed toward identifying which patients are at greatest risk for this side effect and also to optimizing pharmacotherapy to minimize the risk to patients.