**2. Genetics of congenital generalized lipodystrophy**

There are four different genetic subtypes of CGL that result from different mutations.

## **2.1 Type 1 CGL (CGL1) and AGPAT2**

Mutations that are responsible for CGL1 (Online Mendelian Inheritance in Man [OMIM] #608594) occur in the region of 1-acylglycerol-3-phosphate O-acyltransferase 2 (*AGPAT2*) on chromosome 9q34. Only 4% of compound heterozygotes with a null and a missense mutation still maintain some residual enzymatic activity [7]. Also, homozygous missense mutations have been reported to be 2% of CGL1 patients. Interestingly, there is no strong correlation between the type of mutation and lipodystrophy phenotype (the level of adipocyte malfunction) [7, 11]. On the contrary, a founder mutation variant might exist in a particular population [11]. In fact, almost all patients that have African origin have the founder mutation in intron 4, c589-2A>G of one or both alleles [7, 12]. In the past 5 years, several novel variants of *AGPAT2* have been identified in CGL patients: c.144C>A, c.667\_705delinsCTGCG, c.268delC, and c.316+1G>T; c.134C>A and c.216C>G [13]; c.514G>A [14]; c.685G>T, and c.514G>A [15]. In addition, a very rare case of dual mutations (c.493-1G>C and c.299G>A) in *AGPAT2* was identified in two Chilean sisters [16].

AGPAT2 is a member of lysophosphatidic acid acyltransferases (LPAATs) including AGPAT family (AGPAT1–AGPAT11) and others such as CGI-58 and endophilin [17]. In fact, AGPAT2 was identified with AGPAT1 by searching an EST database for human homologs of yeast LPAAT in 1997 [18]. There are 11 isoforms of AGPATs that are involved in the de novo synthesis of phospholipids (PLs) and triacylglycerol from glycerol-3-phosphate (G3P). *AGPAT2* is predominantly expressed in adipose tissue, and its mRNA level increases by 30-fold during the differentiation of adipocytes [19]. Patients with CGL1 normally suffer extreme loss of all metabolically active adipose tissue in most subcutaneous areas, intra-abdominal areas, intrathoracic regions, and bone marrow; however, it is postulated that the redundancy of other AGPAT isoforms or the enhanced expression of other *AGPAT* genes helps preserve mechanical fat depots located in the palms, soles, under the scalp, retro-orbital, periarticular regions, perineum, vulva, and pericalyceal regions of the kidneys [7, 20].

#### *Lipodystrophy - A Rare Condition with Serious Metabolic Abnormalities DOI: http://dx.doi.org/10.5772/intechopen.88667*

In adipose tissue, the synthesis of PLs and TAG begins with the acylation of G3P with FA-CoA by glycerol phosphate acyltransferase (GPAT) at the SN1 position to form 1-acylglycerol-3-phosphate or lysophosphatidic acid (LPA). Then AGPAT2 catalyzes the conversion of LPA into phosphatidic acid (PA) via an acylation reaction at the SN2 position. PA is a pivotal intracellular signaling lipid for it sits at the branching point of de novo PL and TAG synthesis pathway and acts as a precursor for the lipin-mediated production of diacylglycerol (DAG), followed by phosphatidylcholine (PC), phosphatidylethanolamine (PE), and TAG, and as the substrate for the cytidine diphosphate synthase (CDS)-mediated generation of cytidine diphosphate diacylglycerol (CDP-DAG), followed by phosphatidylinositol (PI), phosphatidylglycerol (PG), and cardiolipin [21]. PA is a cone-shaped lipid that has the capacity to alter the curvature of the membranes, and it has been shown to mediate membrane fusion in both soluble N-ethylmaleimidesensitive factor attachment protein (NSF)-receptor (SNARE)-dependent and SNARE-independent fashions [22, 23]. It has been implicated in the fusion of multiple LDs to form a gigantic LD [24]. In addition, PA is believed to be an endogenous antagonist of peroxisome proliferator-activated receptor gamma (PPARγ) that is the master transcription factor in adipocyte differentiation [25]. The malfunctioned *AGPAT2* in CGL1 is assumed to cause dysregulated PL and TAG synthesis, leading to defective adipose tissue development [11, 19]. However, the exact mechanism of how AGPAT2 deficiency causes lipodystrophy remains unraveled. Intriguingly, the same effect cannot be achieved by knocking out other key enzymes in the TAG synthesis including glycerol-3-phosphate acyltransferase 1 (GPAT1), GPAT4, lipin, and diacylglycerol acyltransferase 1 (DGAT1) and DGAT2 [26–30].

Ablation of *Agpat2* induces severe lipodystrophy in mice with the loss of both WAT and BAT during the first week of postnatal period, due to inflammatory infiltration to adipose tissue and massive adipocyte cell death [31]. In addition, both male and female mice have extreme insulin resistance, type 2 diabetes, hepatic steatosis, as well as organomegaly including hepatosplenomegaly, nephromegaly, and elongated small intestines as seen in human CGL [32]. Since almost all adipose tissues have disappeared, *Agpat2*<sup>−</sup>/<sup>−</sup> mice are not an ideal model to study the development of lipodystrophy; however, this type of animals can be particularly useful for the investigation of lipodystrophy-induced metabolic disorders in non-adipose tissues such as severe hepatic insulin resistance and steatosis in CGL [20]. Up to 90% of the total AGPAT activity is compromised in the liver tissue of *Agpat2*<sup>−</sup>/<sup>−</sup> mice and that can be restored by adenoviral delivery of *Agpat1* or *Agpat2*. However, overexpression of *Agpat1* or Agpat2 in *Agpat2*<sup>−</sup>/<sup>−</sup> liver failed to alleviate hepatic steatosis, indicating that the ectopic hepatic lipid accumulation was derived from insulin resistance and loss of body fat [33]. While reduced AGPAT activity is supposed to result in a reduction in PA synthesis, the PA level was seen to increase in the liver of male *Agpat2*<sup>−</sup>/<sup>−</sup> mice, which suggests an alternative pathway for PA synthesis with a compensatory activation of DAG kinase or phospholipase D [32, 34]. A high level of PA predisposes *Agpat2*<sup>−</sup>/<sup>−</sup> liver to elevated hepatic glucose production, which is attributed to insulin resistance [34]. PA accumulation was also found in differentiated *Agpat2*<sup>−</sup>/<sup>−</sup> murine embryonic fibroblast (MEF) adipocytes [31]. At day 6 of the differentiation, ultrastructural alterations in mitochondria, plasma membrane, and autophagosomes were found in *Apgat2*<sup>−</sup>/<sup>−</sup> MEF adipocytes, suggesting that in the absence of AGPAT2, cells can initiate adipogenesis, but a variety of cellular abnormalities eventually block the terminal phase of adipogenesis [31]. Enforcing adipocyte differentiation by overexpression of PPARγ, the master regulator of adipogenesis can also promote adipogenesis of *Agpat2*<sup>−</sup>/<sup>−</sup> MEFs; nevertheless, massive cell death occurred before they reached full differentiation [31]. In agreement with

this finding, preadipocytes from *Agpat2*<sup>−</sup>/<sup>−</sup> interscapular BAT underwent progressive cell death during the adipogenic induction with no activation of caspase 3 [35].

In addition, these BAT-derived preadipocytes exhibit an increased expression of autophagy-related proteins but a decreased autophagic flux [35]. In isolated musclederived multipotent cells (MDMCs) from CGL1 patients and 3T3-L1 preadipocyte cells with the knockdown of AGPAT2, cell death also proceeds during adipogenesis, which might be associated with defective Akt activation as a result of altered PI composition [36, 37]. The constitutively active Akt and PPARγ agonist pioglitazone partially rescued the adipogenic defect in the *Agpat2*<sup>−</sup>/<sup>−</sup> cells [37]. In addition, regarded as a therapy for CGL patients, leptin treatment normalizes the levels of plasma TAG, insulin, and glucose as well as improves hepatic steatosis in *Agpat2*<sup>−</sup>/<sup>−</sup> mice, which is independent of hepatic leptin receptor [38]. The role of AGPAT2 in LD biology has been rarely reported. In contrast to WT adipose tissue that manifests large unilocular LDs, *Apgat2*<sup>−</sup>/<sup>−</sup> adipocytes are featured by smaller multilocular LDs. Although LDs in *Apgat2*<sup>−</sup>/<sup>−</sup> MEFs are smaller than *Apgat2*+/+ cells, they are still normally coated with perilipin 1 (Plin1) [31].
