Dyslipidemia Is a Major Factor in Stem Cell Damage Induced by Uncontrolled Long-Term Type 2 Diabetes and Obesity in the Rat, as Suggested by the Effects on Stem Cell Culture

Abstract

Background. Previous work showed that muscle derived stem cells (MDSC) exposed long-term to the milieu of uncontrolled type 2 diabetes (UC-T2D) in male obese Zucker (OZ) rats, were unable to correct the associated erectile dysfunction and the underlying histopathology when implanted into the corpora cavernosa, and were also imprinted with a noxious gene global transcriptional signature (gene-GTS), suggesting that this may interfere with their use as autografts in stem cell therapy. Aim. To ascertain the respective contributions of dyslipidemia and hyperglycemia to this MDSC damage, clarify its mechanism, and design a bioassay to identify the damaged stem cells. Methods. Early diabetes (ED)-MDSC and late diabetes (LD)-MDSC were respectively isolated from nearly normal young OZ rats, and moderately hyperglycemic and severely dyslipidemic/obese aged rats with erectile dysfunction. Monolayer cultures of ED-MDSC were incubated 4 days in DMEM/10% fetal calf serum + or - aged OZ or LZ serum from non-diabetic lean Zucker rats (0.5–5%) or with soluble palmitic acid (PA) (0.5–2 mM), cholesterol (CHOL) (50–400 mg/dl), or glucose (10–25 mM). Outcomes. Fat infiltration was estimated by Oil red O, apoptosis by TUNEL, protein expression by western blots, and gene-GTS and microRNA (miR)-GTS were determined in these stem cells RNA. Results. Aged OZ serum caused fat infiltration, apoptosis, myostatin overexpression, and impaired differentiation. Some of these changes, and also proliferation decrease occurred with PA and CHOL. The gene-GTS changes by OZ serum did not resemble the in vivo changes, but some occurred with PA and CHOL. The miR-GTS changes by OZ serum, PA, and CHOL resembled most of the in vivo changes. Hyperglycemia did not replicate most alterations. Clinical Implications. MDSC may be damaged in long-term UC-T2D/obese patients and be ineffective in autologous human stem cell therapy, that may be prevented by excluding the damaged MDSC. Strengths and limitations. The in vitro test of MDSC is innovative and fast to define dyslipidemic factors inducing stem cell damage, its mechanism, prevention and counteraction. Confirmation is required in other T2D/obesity rat models and stem cells (including human), as well as miR-GTS biomarker validation as stem cell damage biomarker. Conclusion. Serum from long-term UC-T2D/obese rats or dyslipidemic factors induce a noxious phenotype and miR-GTS on normal MDSC, that may lead in vivo to the repair inefficacy of LD-MDSC. This suggests that autograft therapy with MDSC in long-term UT-T2D obese patients may be ineffective, albeit this may be predictable by prior stem cell miR-GTS tests.

Evaluation of the In Vitro Damage Caused by Lipid Factors on Stem Cells from a Female Rat Model of Type 2 Diabetes/Obesity and Stress Urinary Incontinence

“…As previously reported [ 8 ], 12 weeks old female Zucker Fatty (ZF) rats (ZUC-Lepr fa/fa 186), and their Zucker Lean (ZL) controls (ZUC-Lepr fa 185) of the same strain used for the study on male rats [ 5 , 6 , 7 ] were used here for isolating the female stem cells. The ZF rats are a model of metabolic syndrome, in the male evolving into frank diabetes and morbid obesity, whereas in the female leading to a more marginal hyperglycemia and moderate obesity, both identified as T2D/O.…”

“…Myostatin overexpression, detected by Western blot, is the feature that characterized the long-term in vivo exposure of MDSC male cells to the T2D/O milieu [ 5 , 6 ] and the in vitro exposure to dyslipidemic serum of both MDSC and the female ZF4-SC [ 7 , 8 ]. It was also investigated here for the lipid factors acting only on ZF4-SC, and not ZL4-SC.…”

“…Finally, we had previously reported that the miR-GTSs were considerably affected in both the male MDSC exposed in vivo to T2D/O and in vitro to dyslipidemic serum, and also in vitro in the female ZF4-SC exposed to this serum [ 5 , 7 , 8 ]. These miR-GTS alterations were also found in male MDSC incubated in vitro with cholesterol and palmitate, affecting many myostatin-related individual miRs [ 7 ].…”

“…Finally, we had previously reported that the miR-GTSs were considerably affected in both the male MDSC exposed in vivo to T2D/O and in vitro to dyslipidemic serum, and also in vitro in the female ZF4-SC exposed to this serum [ 5 , 7 , 8 ]. These miR-GTS alterations were also found in male MDSC incubated in vitro with cholesterol and palmitate, affecting many myostatin-related individual miRs [ 7 ]. Table 1 shows as reference (left columns for refs 7 and 8) all the myostatin-related miRs, previously reported for MDSC as downregulated in vivo over 2-fold (<0.5 as compared to controls) by the T2D/O milieu, and also downregulated in most cases in the female ZF4-SC to <0.5 by dyslipidemic OZ serum, but not by normal LZ serum.…”

“…Subsequently [ 7 ], our group demonstrated that some of these effects could be mimicked in cell culture. This was done by short-term incubation of the male MDSC from young OZ rats (virtually not exposed to T2D/O) with dyslipidemic serum from the aged, very obese, and mild hyperglycemic OZ rats.…”