PIK3R1 mutations cause syndromic insulin resistance with lipoatrophy.

Short stature, hyperextensibility of joints and/or inguinal hernia, ocular depression, Rieger anomaly, and teething delay (SHORT) syndrome is a developmental disorder with an unknown genetic cause and hallmarks that include insulin resistance and lack of subcutaneous fat. We ascertained two unrelated individuals with SHORT syndrome, hypothesized that the observed phenotype was most likely due to de novo mutations in the same gene, and performed whole-exome sequencing in the two probands and their unaffected parents. We then confirmed our initial observations in four other subjects with SHORT syndrome from three families, as well as 14 unrelated subjects presenting with syndromic insulin resistance and/or generalized lipoatrophy associated with dysmorphic features and growth retardation. Overall, we identified in nine affected individuals from eight families de novo or inherited PIK3R1 mutations, including a mutational hotspot (c.1945C>T [p.Arg649Trp]) present in four families. PIK3R1 encodes the p85α, p55α, and p50α regulatory subunits of class IA phosphatidylinositol 3 kinases (PI3Ks), which are known to play a key role in insulin signaling. Functional data from fibroblasts derived from individuals with PIK3R1 mutations showed severe insulin resistance for both proximal and distal PI3K-dependent signaling. Our findings extend the genetic causes of severe insulin-resistance syndromes and provide important information with respect to the function of PIK3R1 in normal development and its role in human diseases, including growth delay, Rieger anomaly and other ocular affections, insulin resistance, diabetes, paucity of fat, and ovarian cysts.

Peroxisome proliferator-activated receptor-γ mutations responsible for lipodystrophy with severe hypertension activate the cellular renin-angiotensin system.

OBJECTIVE: Inactivating peroxisome proliferator-activated receptor-γ (PPARγ) mutations lead to a syndrome of familial partial lipodystrophy (FPLD3) associated with early-onset severe hypertension. PPARγ can repress the vascular renin-angiotensin system (RAS) and angiotensin II receptor 1 expression. We evaluated the relationships between PPARγ inactivation and cellular RAS using FPLD3 patients' cells and human vascular smooth muscle cells expressing mutant or wild-type PPARγ. Approach and Results- We identified 2 novel PPARG mutations, R165T and L339X, located in the DNA and ligand-binding domains of PPARγ, respectively in 4 patients from 2 FPLD3 families. In cultured skin fibroblasts and peripheral blood mononuclear cells from the 4 patients and healthy controls, we compared markers of RAS activation, oxidative stress, and inflammation, and tested the effect of modulators of PPARγ and angiotensin II receptor 1. We studied the impact of the 2 mutations on the transcriptional activity of PPARγ and on the vascular RAS in transfected human vascular smooth muscle cells. Systemic RAS was not altered in patients. However, RAS markers were overexpressed in patients' fibroblasts and peripheral blood mononuclear cells, as in vascular cells expressing mutant PPARγ. Angiotensin II-mediated mitogen-activated protein kinase activity increased in patients' fibroblasts, consistent with RAS constitutive activation. Patients' cells also displayed oxidative stress and inflammation. PPARγ activation and angiotensin II receptor 1 mRNA silencing reversed RAS overactivation, oxidative stress, and inflammation, arguing for a role of angiotensin II receptor 1 in these processes. CONCLUSIONS: Two novel FPLD3-linked PPARG mutations are associated with a defective transrepression of cellular RAS leading to cellular dysfunction, which might contribute to the specific FPLD3-linked severe hypertension.

Premature senescence of vascular cells is induced by HIV protease inhibitors: implication of prelamin A and reversion by statin.

OBJECTIVE: To determine whether and how protease inhibitors (PIs) could affect vascular aging. METHODS AND RESULTS: HIV therapy with PIs is associated with an increased risk of premature cardiovascular disease. The effect of ritonavir and a combination of lopinavir and ritonavir (for 30 days) on senescence, oxidative stress, and inflammation was evaluated in human coronary artery endothelial cells (HCAECs). These HCAECs were either cotreated or not cotreated with pravastatin or farnesyl transferase inhibitor (FTI)-277 or with 2 antioxidants (manganese [III] tetrakis [4-benzoic acid] porphyrin [MnTBAP] and N-acetyl cysteine). Senescence markers were evaluated in peripheral blood mononuclear cells (PBMCs) from HIV-infected patients under PI treatment. PIs induced senescence markers, prelamin A accumulation, oxidative stress, and inflammation in HCAECs. Senescence markers and prelamin A were also observed in PBMCs from HIV-infected patients under ritonavir-boosted PIs. Pravastatin, FTI-277, and antioxidants improved PI adverse effects in HCAECs. Senescence markers were lower in PBMCs from PI-treated patients cotreated with statins. CONCLUSIONS: PIs triggered premature senescence in endothelial cells by a mechanism involving prelamin A accumulation. Accordingly, circulating cells from HIV-infected patients receiving PI therapy expressed senescence markers and prelamin A. Statin was associated with improved senescence in endothelial cells and patient PBMCs. Thus, PIs might promote vascular senescence in HIV-infected patients; and statins might exert beneficial effects in these patients.

Human lipodystrophies: genetic and acquired diseases of adipose tissue.

Human lipodystrophies represent a heterogeneous group of diseases characterized by generalized or partial fat loss, with fat hypertrophy in other depots when partial. Insulin resistance, dyslipidemia and diabetes are generally associated, leading to early complications. Genetic forms are uncommon: recessive generalized congenital lipodystrophies result in most cases from mutations in the genes encoding seipin or the 1-acyl-glycerol-3-phosphate-acyltransferase 2(AGPAT2). Dominant partial familial lipodystrophies result from mutations in genes encoding the nuclear protein lamin A/C or the adipose transcription factor PPARgamma. Importantly, lamin A/Cmutations are also responsible for metabolic laminopathies, resembling the metabolic syndrome and progeria, a syndrome of premature aging. A number of lipodystrophic patients remain undiagnosed at the genetic level. Acquired lipodystrophy can be generalized, resembling congenital forms, or partial, as the Barraquer-Simons syndrome, with loss of fat in the upper part of the body contrasting with accumulation in the lower part. Although their etiology is generally unknown, they could be associated with signs of autoimmunity. The most common forms of lipodystrophies are iatrogenic. In human immunodeficiency virus-infected patients, some first-generation antiretroviral drugs were strongly related with peripheral lipoatrophy and metabolic alterations. Partial lipodystrophy also characterize patients with endogenous or exogenous long-term corticoid excess. Treatment of fat redistribution can sometimes benefit from plastic surgery. Lipid and glucose alterations are difficult to control leading to early occurrence of diabetic, cardiovascular and hepatic complications.

Impact of CCR5, integrase and protease inhibitors on human endothelial cell function, stress, inflammation and senescence.

BACKGROUND: Ageing HIV-infected patients present an increased incidence of cardiovascular diseases, endothelial dysfunction being an early alteration. Some protease inhibitors (PIs) have been shown to increase the risk of cardiovascular disease. We evaluated here the effects of CCR5 or integrase inhibitors as compared to PIs on endothelial functions in vitro. METHODS: Human coronary artery endothelial cells (HCAEC) from adult and old non-HIV-infected donors were treated for 15 days with the CCR5 inhibitor maraviroc, the integrase inhibitors dolutegravir or raltegravir or the ritonavir-boosted PIs, darunavir (DRV/r) or atazanavir (ATV/r), all at Cmax concentrations. We evaluated endothelial function, secretion of adhesion molecules and cytokines, inflammation, oxidative stress and senescence. RESULTS: In endothelial cells from adult donors, we confirmed that ATV/r and DRV/r adversely affected all assessed endothelial functions and enhanced senescence, these effects being mild for DRV/r. Raltegravir had no effect and maraviroc a mild anti-inflammatory effect. Dolutegravir decreased inflammation, by inhibiting the NFκB pathway, and senescence, by repressing the p21 pathway. Moreover, HCAEC from an old donor presented, constitutively, a high level of senescence. Raltegravir mildly affected inflammation and senescence while maraviroc and dolutegravir decreased oxidative stress, inflammation and senescence and improved endothelial dysfunction. CONCLUSIONS: We report here that the integrase inhibitor dolutegravir and the CCR5 inhibitor maraviroc reduced inflammation of human adult endothelial cells to different extents while raltegravir was neutral. Dolutegravir also reduced senescence, while PI/r increased inflammation and senescence. It is important to address the clinical relevance of these results.

LMNA mutations resulting in lipodystrophy and HIV protease inhibitors trigger vascular smooth muscle cell senescence and calcification: Role of ZMPSTE24 downregulation.

BACKGROUND: Some LMNA mutations responsible for lipodystrophies, and some HIV-protease inhibitors (PIs) induce accumulation of farnesylated prelamin A and premature senescence in some cell types. Patients with LMNA mutations or under PI-based therapy suffer from early atherosclerosis. The metalloprotease ZMPSTE24 is the key enzyme in prelamin A maturation. AIM: We studied whether altered expression of ZMPSTE24 could contribute to vascular cell dysfunction in response to LMNA mutations or PI treatments. METHODS: Protein expression of prelamin A and ZMPSTE24 were evaluated in patients' cells and in human cultured VSMCs. Oxidative stress, inflammation, senescence and transdifferentiation/calcification were evaluated in VSMCs. RESULTS: Fibroblasts from LMNA-mutated lipodystrophic patients (mutations R482W, D47Y or R133L) and peripheral blood mononuclear cells from PI-treated-HIV-infected patients expressed increased prelamin A and decreased ZMPSTE24, which was also observed in VSMCs overexpressing mutant LMNA or treated with PIs. These alterations correlated with oxidative stress, inflammation, senescence and calcification (all p < 0.05). ZMPSTE24 silencing in native VSMCs recapitulated the mutant LMNA- and PI-induced accumulation of farnesylated prelamin A, oxidative stress, inflammation, senescence and calcification. A negative regulator of ZMPSTE24, miRNA-141-3p, was enhanced in LMNA-mutated or PI-treated VSMCs. The farnesylation inhibitors pravastatin and FTI-277, or the antioxidant N-acetyl cysteine, partly restored ZMPSTE24 expression, and concomitantly decreased oxidative stress, inflammation, senescence, and calcification of PI-treated VSCMs. CONCLUSIONS: ZMPSTE24 downregulation is a major contributor in VSMC dysfunctions resulting from LMNA mutations or PI treatments that could translate in early atherosclerosis at the clinical level. These novel pathophysiological mechanisms could open new therapeutic perspectives for cardiovascular aging.

Impact of darunavir, atazanavir and lopinavir boosted with ritonavir on cultured human endothelial cells: beneficial effect of pravastatin.

BACKGROUND: HIV-infected patients administered long-term ritonavir-boosted protease inhibitors (PIs) are at a greater risk for developing cardiovascular diseases. Endothelial dysfunction is an initiating event in HIV-associated atherosclerosis. Cultured endothelial cells can be used as a model to compare the endothelial toxicity of different PIs. METHODS: We compared the effect of darunavir (DRV), darunavir/ritonavir (DRV/r), lopinavir/ritonavir (LPV/r) and atazanavir/ritonavir (ATV/r), used at clinically relevant concentrations, on human coronary artery endothelial cell vascular function, oxidative stress, inflammation and senescence, and studied the effect of pravastatin on PI-induced alterations. RESULTS: Vascular endothelial cell function, evaluated by the expression of endothelial nitric oxide synthase and the production of nitric oxide and endothelin-1, was unaffected by DRV or DRV/r, but altered by LPV/r or ATV/r. DRV or DRV/r did not alter, or mildly induced oxidative stress and inflammation (phosphorylation of p65/RelA-NFκB, secretion of IL-6 and IL-8), while ATV/r and LPV/r induced a marked increase. Secretion of sICAM or sVCAM, indicative of altered cell integrity, was not or weakly altered by DRV or DRV/r, but increased by 2-3-fold by LPV/r or ATV/r. Similar results were observed regarding senescence markers: SA-ß-galactosidase activation and overexpression of phospho-p53, p16(ink4), p21(WAF-1) and prelamin A. Pravastatin could, in part, reverse PI-induced adverse effects. CONCLUSIONS: Ritonavir-boosted PIs differentially induced vascular endothelial cell dysfunction, reactive oxygen species production, inflammation and senescence with no effect or a mild effect of DRV/r, an intermediate effect of ATV/r, and a stronger effect of LPV/r. Statins could, in part, protect the cells from PI-induced endothelial dysfunction.

Effects of ritonavir-boosted darunavir, atazanavir and lopinavir on adipose functions and insulin sensitivity in murine and human adipocytes.

BACKGROUND: Ritonavir-boosted protease inhibitors (PIs) could adversely affect metabolism and adipose tissue to different extents, depending on the molecule. Using drugs with minimal adverse metabolic effects is an important consideration in at-risk HIV-infected patients. In vitro adipocyte models can be useful for comparing the effects of different PIs. METHODS: We compared the effects of darunavir, darunavir/ritonavir, atazanavir/ritonavir and lopinavir/ritonavir in murine and human adipocytes on differentiation, mitochondrial function, reactive oxygen species (ROS) production and insulin sensitivity. RESULTS: In human and murine adipocytes, differentiation evaluated by lipid content and protein expression of adipogenic markers, mitochondrial function evaluated by aggregation of the cationic dye JC-1 and by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide lysis, and mitochondrial mass evaluated by MitoTracker fluorescence and the expression of mitochondrial proteins were unaffected by darunavir, mildly affected by darunavir/ritonavir and further altered by atazanavir/ritonavir and lopinavir/ritonavir. ROS production was unaltered by darunavir and darunavir/ritonavir but was increased by lopinavir/ritonavir and atazanavir/ritonavir. Regarding insulin sensitivity, darunavir and darunavir/ritonavir had no significant effect on insulin activation of protein kinase B (Akt/PKB) and MAP kinase and of glucose transport, whereas lopinavir/ritonavir and atazanavir/ritonavir partly impaired the effect of insulin. The effect of atazanavir/ritonavir was generally milder than that of lopinavir/ritonavir. CONCLUSIONS: The various PIs differentially modified adipocyte functions. Darunavir alone did not affect adipocyte functions and only modestly altered differentiation and mitochondrial function when associated with ritonavir. Lopinavir/ritonavir adversely affected differentiation and lipid content, mitochondrial function, ROS production and insulin sensitivity, and the effect of atazanavir/ritonavir was intermediate. Thus, in vitro, darunavir/ritonavir presented a safer metabolic profile on adipocytes than atazanavir/ritonavir and lopinavir/ritonavir.

Molecular mechanisms of human lipodystrophies: from adipocyte lipid droplet to oxidative stress and lipotoxicity.

Adipose tissue is now recognized for its major role in the control of energy metabolism and insulin sensitivity. We review here the human lipodystrophies, that are rare conditions in which total or partial fat loss is associated with severe lipid and glucose abnormalities leading to diabetes with early cardiovascular and hepatic complications. The genetic origin of a number of human lipodystrophies has been recently unraveled, emphasizing the importance of proteins of previously unknown or unexpected functions. Major adipose functions were also illuminated when studying acquired forms of lipodystrophies linked to human immunodeficiency virus-antiretrovirals. Overall, most of the proteins or functions affected by mutations or antiretrovirals result in altered adipogenesis and insulin sensitivity, triglyceride storage and formation of the unique adipocyte lipid droplet, oxidative stress and fat remodeling. Some mutations or antiretrovirals could affect directly (peroxisome proliferator-activated receptor-γ, Akt2) or indirectly (lamin A/C, human immunodeficiency virus-protease inhibitors) adipogenesis, through the transcription factors peroxisome proliferator-activated receptor gamma-γ or sterol regulatory element binding protein 1c, and insulin signaling through Akt2 that controls adipocyte lipolysis. A number of proteins mutated in genetic lipodystrophies are involved in the control of triglyceride synthesis towards the lipid droplet (1-acylglycerol-3-phosphate-O-acyltransferase 2), or its functions (seipin, cell death-inducing DFF45-like effector C, perilipin, caveolin-1, cavin-1). Decreased triglyceride storage leads to adipocyte lipotoxicity, mitochondrial dysfunction and increased oxidative stress, which could also be induced by some thymidine analogue antiretrovirals. This results in production of inflammatory mediators and deregulated release of free fatty acids. Thus, the impaired ability of adipose tissue to safely store triglycerides inside the lipid droplet results in impaired insulin sensitivity and adverted liver, muscles and heart functions leading to early complications.

Type B insulin resistance syndrome associated with an immune reconstitution inflammatory syndrome in an HIV-infected woman.

CONTEXT: Type B insulin resistance syndrome is a rare condition characterized by the presence of autoantibodies directed against the insulin receptor. It has been reported in association with autoimmune diseases such as systemic lupus erythematosus. OBJECTIVE: We report a case of type B insulin resistance syndrome in a patient with HIV infection on highly active antiretroviral therapy (HAART). PATIENT AND METHODS: A 27-yr-old African woman with ketosis-prone diabetes and HIV infection developed severe insulin resistance after the initiation of HAART. Standard oral glucose tolerance tests using 75 g of glucose performed 1, 2, and 3 months after the initiation of HAART showed severe hyperinsulinemia and hypoglycemia. Six months later, she developed symptomatic hyperglycemia resistant to high-dose insulin therapy. To determine the cause of insulin resistance, we assayed the titer of insulin receptor autoantibodies in the serum of the patient. RESULTS: Plasma insulin receptor autoantibodies were present at the time of marked hyperglycemia and insulin resistance, confirming the diagnosis of type B insulin resistance syndrome. Simultaneously the diagnosis of immune reconstitution inflammatory syndrome was established according to increased CD4 T cell count, decreased plasma HIV1-RNA level, and tuberculosis reactivation, shortly after institution of HAART. Corticosteroid therapy improved insulin resistance and hyperglycemia. CONCLUSION: We report the first case of type B insulin resistance syndrome associated with immune reconstitution inflammatory syndrome in an HIV-infected patient.

HIV-associated lipodystrophy: from fat injury to premature aging.

Combination antiretroviral therapy (cART) against HIV infection dramatically reduces AIDS-related morbidity. However, many patients under cART display HIV-associated lipodystrophy. Moreover, some develop early age-related comorbidities. Thymidine analog reverse transcriptase inhibitors (tRNTIs) are mainly responsible for peripheral lipoatrophy, and protease inhibitors (PIs) for fat hypertrophy and metabolic complications. Long-term HIV infection probably also causes fat alterations. Severe mitochondrial toxicity and oxidative stress cause lipoatrophy, whereas the hypertrophy of upper body fat depots could result from mild oxidative stress, cortisol activation and inflammation. The metabolic complications associated with lipodystrophy are responsible for increased cardiovascular and hepatic risks and could also participate in premature aging. We propose that adipose tissue injury by HIV and cART induces fat hypertrophy or atrophy and contributes to premature aging.

Adipose tissue as a target of HIV-1 antiretroviral drugs. Potential consequences on metabolic regulations.

Adipose tissue redistribution occurred at first in HIV-infected patients about 15 years ago after initiation of combination antiretroviral treatment (ART) and the responsibility of drugs was rapidly considered. This lipodystrophic syndrome can associate lipoatrophy, affecting subcutaneous adipose tissue in priority with fat hypertrophy, in particular in the upper part of the body, and metabolic alterations, dyslipidemia and altered glucose tolerance with insulin resistance. The primary role of thymidine analogue reverse transcriptase inhibitors (tNRTI) in peripheral lipoatrophy has been clearly shown in vitro and in vivo, these drugs inducing a severe mitochondrial dysfunction and an increased oxidative stress together with fat inflammation leading to fat loss. In vitro and in vivo studies suggest that some protease inhibitors (PI) or non-NRTIs also exert adverse effects on adipocytes and could act in synergy to amplify the effect of tNRTI. While severe lipoatrophy is now less prevalent in HIV-infected patients, fat hypertrophy is frequently observed: a role for drugs from the different classes acting in synergy to induce fat hyperplasia and hypertrophy is suggested, with milder mitochondrial dysfunction but increased inflammation and activation of the cortisol system. In addition, it is now considered that long-term viral infection, even if controlled, could induce low-grade inflammation and prepare fat to the deleterious effect of ART. Both lipoatrophy and lipohypertrophy are involved in metabolic disorders and increased cardio-metabolic risk that likely participate to early aging reported in these patients. ART can also be directly responsible for metabolic alterations. Strategies to revert or reduce lipodystrophy are important to consider in these patients in addition to the required control of the metabolic disorders.

HIV protease inhibitors activate the adipocyte renin angiotensin system.

BACKGROUND: HIV-infected patients under antiretroviral therapy that includes HIV protease inhibitors (PIs) are prone to develop a complex metabolic syndrome including insulin resistance, lipodystrophy and hypertension. Whether hypertension and cardiovascular events could result from the adipocyte renin angiotensin system (RAS) overactivation has never been investigated. METHODS: Primary human adipocytes and 3T3-F442A murine adipocytes were incubated with lopinavir or atazanavir boosted with ritonavir, with or without the angiotensin II type-1 receptor (AT1R) blockers (ARBs), irbesartan or telmisartan, and the peroxysome proliferator-activated receptor-gamma (PPAR-gamma) regulators, rosiglitazone and GW9662. Adipose RAS activation and adipocyte functions were evaluated. RESULTS: The ritonavir-boosted PIs activated the adipose RAS in human and murine adipocytes as shown by the overexpression of AT1R protein, angiotensinogen messenger RNA and the amplified effect of angiotensin II on extracellular signal-regulated kinase 1/2 activity. ARBs prevented the PI effect on RAS activation (AT1R overexpression and signalling) and adipocyte functions (dedifferentiation, insulin resistance, oxidative stress and inflammation). Consistent with a role of PPAR-gamma signalling in PI-induced RAS activation, the PPAR-gamma agonist (rosiglitazone) normalized PI-induced AT1R overexpression and adipocyte dysfunction. Conversely, the PPAR-gamma antagonist (GW9662) induced AT1R overexpression and reduced the beneficial effect of telmisartan on PI toxicity. CONCLUSIONS: We report that two frequently prescribed PI combinations could activate the adipose RAS in cultured cells, in part through a PPAR-gamma-dependant signalling pathway. Our data suggest a role for the adipose RAS in the development of hypertension in HIV-infected patients under PI treatment, and point out the potential use of ARBs to decrease PI adverse effects.

Higher adiponectin levels in patients with Berardinelli-Seip congenital lipodystrophy due to seipin as compared with 1-acylglycerol-3-phosphate-o-acyltransferase-2 deficiency.

CONTEXT: Human lipodystrophies are characterized by loss of adipose tissue, insulin resistance, and metabolic complications. The mechanisms linking fat loss to severe insulin resistance remain unclear. Adipokines may have important roles as intermediary players in metabolism. OBJECTIVE: We sought to determine the plasma concentrations of leptin and adiponectin in patients with Berardinelli-Seip congenital lipodystrophy (BSCL) harboring mutations in the genes encoding either 1-acylglycerol-3-phosphate-O-acyltransferase-2 (AGPAT2) or BSCL2/seipin, in comparison with patients with other forms of inherited or acquired lipodystrophies or insulin receptor alterations. DESIGN: Leptin and total and high-molecular-weight adiponectin were measured in plasma of 16 BSCL1/AGPAT2 and 19 BSCL2/seipin patients and compared with heterozygous (n = 22) or nonmutated relatives (controls, n = 30); patients with Dunnigan-type partial lipodystrophy due to lamin A/C mutations (n = 23), HIV-related lipodystrophy (n = 124), and insulin receptor dysfunctions caused by mutations or autoantibodies (n = 17). RESULTS: Leptin was dramatically decreased in BSCL patients as compared with other subgroups. Adiponectin was decreased in BSCL as compared with controls and patients with altered insulin receptor but was discrepant between the two BSCL subgroups. Whereas total and high-molecular-weight adiponectin levels were almost undetectable in BSCL1/AGPAT2 patients, higher levels were detected in BSCL2/seipin patients, comparable with those of patients with partial lipodystrophy. Adiponectin greater than 1.6 mg/liter had a 100% negative predictive value for AGPAT2 mutations in inherited lipodystrophies. CONCLUSIONS: The presence of circulating adiponectin in BSCL2/seipin patients with near absence of adipose tissue outlines the complexity of adiponectin biology. Use of circulating adiponectin might be helpful to guide the genetic investigations in BSCL.