Altered adipocyte differentiation associated with protease inhibitors and lipoatrophy
Simon Collins, HIV i-Base
The underlying mechanisms for lipoatrophy have not yet been explained although in vitro and animal studies reported at the annual lipodystrophy workshops have pointed to several possible theories.
These include the class action of either protease inhibitors through impaired adipocyte differentiation or reverse transcription inhibitors due to mitochondrial damage or indeed a cumulative effect of these two classes together. Importantly, a new French study helps confirm that lipodystrophy is not simply a matter of redistributed fat, or even absence or accumulation of fat at local sites, but involves structural changes to fat cells resulting from interference with normal cell differentiation.
The French national INSERM laboratories have produced important research into impaired adipocyte differentiation associated with protease inhibitors and Jean-Philippe Bastard and colleagues published their latest results in the Lancet of 23 March.  Previous studies from the group have shown in vitro effects of protease inhibitors on cell differentiation and insulin sensitivity, and preventative effect in vitro of rosiglitazone against cell differentiation, apoptosis and insulin resistance. 
The new study looked at the internal structure of fat cells together with chemical markers of differentiation of these cells from HIV-positive people with lipoatrophy and found significant differences to cells from a control group of HIV-negative individuals.
Adipocyte differentiation involves sequential activation of transcription factors that regulate the expression of adipocyte specific markers – many of which have already been shown to be directly inhibited by protease inhibitor in vitro. This network involves three major factors: CCAAT-Enhancer binding protein beta (C/EBP-b), peroxisome proliferator activated receptor gamma (PPAR–g) and C/EBP-alpha that are activated in cascade. Differentiation is enhanced by sterol-regulatory-element-binding-protein-1 (SREBP-1).
Twenty-six HIV-positive subjects (21 men, 5 women) with peripheral lipoatrophy (treated with both PIs and RTIs) were consecutively selected from a sample of patients undergoing autologous fat transfer at a single hospital. The control group contained eight women undergoing abdominal level plastic surgery and 10 men in a nutritional study that involved subcutaneous biopsies.
Subcutaneous abdominal fat was sampled in all cases, immediately fixed in 10% zinc formol, embedded in paraffin, cut into 5mm slices and stained with haemalun-phloxine-safron. The size of 100 adipocytes were measured on to separate slices for each sample. The concentration in fat-tissue RNA of transcription factors involved in adipocyte differentiation was measured by reverse transcription followed by competitive PCR amplification.
Fat cells from the control group were relatively homogenous in size and distribution whereas fat from HIV patients contained clusters of small cells together with normal cells. The percentage of cells < 50um and <70um was 6% vs 13% and 18% vs 32% in the control and HIV group respectively. Mean size of adipocytes (including > 90% cells) was 117um (SD 24) in the control and 94um (SD15) in the HIV-positive group (p=0.03).
Concentration of transcription factors and adipose markers was also consistently higher in the control group:
Median total mRNA concentration (amol/ug)
Concentrations of transcription factors and adipocyte markers correlated in most cases and often strongly (C/EBP-a and GLUT-4, and C/EBP-a and Leptin, both p <0.0001). Concentrations of TNF-a, a cytokine secreted by adipose tissue that has been reported to inhibit expression of adipogenic factors and induce adipocyte apoptosis was 2.9-fold higher in patients, and this correlated negatively with levels of SREBP-1, C/EBP-a and PPAR-g.
The researchers suggest that the larger proportion of smaller cells and clusters of small cells in the HIV-positive patients could be young regenerative cells replacing previous cells lost by apoptosis – rather than representing a general thinning of cells caused by triglyceride depletion – and that this would be supported by increased levels of TNF-a.
The alteration of adipocyte function at the level of SREBP-1, which has been shown in murine models to directly link to dyslipideamia, diabetes and insulin resistance, could enhance adipose tissue dysfunction, possibly through increased adipocyte expression and result in lipoatrophy through both loss of adipocytes and reduction in adipocyte size.
As SREBP-1 has already been shown to be targeted by protease inhibitors in vitro the study concludes that lipoatrophy may therefore be a protease effect. The researchers acknowledge that the role of RTIs, which fail to alter adipocyte differentiation in vitro, but which may work in synergy with protease inhibitors at the onset of lipoatrophy, remains to be determined.
It is disappointing that, while early results of thiazolidinediones, particularly the PPAP-g modulators rosiglitazone and piaglitazone have shown improvement in congenital and acquired lipoatrophy, they have shown disappointing results in early HIV studies. Notably, rosiglitazone failed to show benefit after 24 weeks dosed at 8mg/day at the recent Retrovirus conference. Perhaps these metabolic effects are particularly resistant to reversal in the setting of HIV-infection.
In relation to the above study researchers from Switzerland reported last year (Miserez AR. AIDS 2001 Oct 19;15(15):2045-9) that a single-nucleotide polymorphism in the sterol-regulatory element-binding protein 1c gene is predictive of HIV-related hyperlipoproteinaemia. SREBP-1c-3’322C/G was predictive of highly active antiretroviral therapy-related hyperlipoproteinaemia. Additionally Increases in cholesterol were less frequently associated with homozygous SREBP-1c-3’322G (genotype 22) than with heterozygous/homozygous SREBP-1c-3’322C (genotypes 11/12) and correlated with leptin and insulin increases, particularly in genotype 11/12 carriers.
Controlled trials initiating agents such as rosiglitazone at the same time as initiation of antiretrovirals may be useful to determine if any preventative action may be possible. Perhaps patient selection for such prophylactic use may also be guided by genotyping of the SREBP-1c gene.
- Bastard J-P, Caron M, Capeau J – Association between altered expression of adipogenic factor SREBP1 in lipoatrophic adipose tissue from HV-1-infected patients and abnormal adipocyte differentiation and insulin resistance. Lancet March 23 Vol359 1026-1031.
- Caron M, Auclair M, Capeau J et al – Differential in vitro effects of indinavir, nelfinavir and amprenavir on cell differentiation, insulin sensitivity and apoptosis in an adapted adipose cell model: preventative impact of rosiglitazone. Antiviral Therapy 2001; 6 (Supplement4):17.