In this study, skeletal muscle biopsies from male subjects with type 2 diabetes, first degree relatives, and healthy controls were investigated at the gene expression level using the microarray technology. The first degree relatives were slightly hyperinsulinemic in the fasting state and only mildly insulin resistant compared to type 2 diabetics, making them as close to the background population as possible. The same level of insulin resistance has previously been found in first degree relatives 
. The elevated fasting plasma insulin levels in the first degree relatives support the notion that they are in the pre-diabetic stage probably on their way to develop overt insulin resistance. The patients with type 2 diabetes were obese, and as expected they had elevated fasting glucose and plasma FFA levels compared to both controls and first degree relatives ().
The biopsies were taken after a 2 hour hyperinsulinemic euglycemic clamp thereby ensuring a constant and controlled metabolic environment. When analyzing the data it should be kept in mind that for genes regulated by insulin, any change in expression could simply be a direct consequence of insulin resistance, since the muscle tissue is subjected to high levels of insulin during the clamp. Nonetheless, all differences seen between groups are genuine differences since all groups were treated in the same way. Furthermore, we were unable to completely match subjects for advanced age and elevated BMI in this study, which are known characteristics of patients with overt type 2 diabetes. Accordingly, we cannot exclude the possibility that age and/or BMI per se contributed to the differences found in patients with type 2 diabetes. However, this does not change the overall finding and conclusion that genes involved in insulin signaling are upregulated in people at risk of – and prior to - type 2 diabetes development, and subsequently are downregulated in the diabetic state.
Overall, differences in expression were found to be modest with FCs ranging between 1.2 and 1.4 for most genes. However, even small changes in gene expression can have a major biological impact, and using pathway analysis tools we show that even small changes on an individual gene level can lead to highly significant changes when combined for an entire pathway.
None of the genes that have been linked to increased risk of type 2 diabetes development in GWA studies 
were found to have an altered expression in either group compared to the controls. This was validated by qRTPCR analysis for the gene TCF7L2
( and ), in which SNPs so far have shown the strongest link to increased risk of type 2 diabetes. Most speculatively, it seems logical that changes in a transcription factor like TCF7L2 will lead to altered expression of other genes and not TCF7L2
itself. However, it still needs to be verified that the SNPs associated to type 2 diabetes actually play a role in diabetes development and are not simply genetic markers for the disease.
Another general tendency was that genes and pathways found to be upregulated in the first degree relatives of type 2 diabetics were downregulated at the type 2 diabetic state. This phenomenon was found to be highly significant for the insulin signaling pathway.
Expression of insulin signaling molecules is upregulated in first degree relatives and downregulated in subjects with type 2 diabetes
The most striking finding in this study was the highly significant increase in expression of genes involved in insulin signaling in skeletal muscle from first degree relatives of type 2 diabetics, and the significant downregulation of the same pathway in type 2 diabetic skeletal muscle samples (, , and ). We hypothesize that the upregulation of the insulin signaling pathway at the gene expression level observed in the relatives could be an effective compensation for otherwise reduced insulin signaling activity. Since the first degree relatives are hyperinsulinemic they are most likely insulin resistant in a strictly molecular sense although not physiologically. Increased expression of insulin signaling molecules could possibly work in concert with increased levels of insulin protecting these individuals from insulin resistance and metabolic dysregulation. This compensation is later lost in type 2 diabetic muscle, and the insulin signaling pathways are at that state downregulated. Possible explanations for the loss of this compensatory mechanism in overt type 2 diabetes include glucose toxicity due to elevated plasma glucose levels, lipotoxicity due to elevated FFA levels, and/or failure of β-cell function. However, this remains speculative until specifically addressed in future studies.
Most of the genes affected in the ‘R’ and the ‘D’ group are not overlapping. This is for example the case for SLC2A4
(Glucose transporter 4)), which is downregulated in the ‘D’ group and unaltered in the ‘R’ group. This observation can be explained by the fact that SLC2A4
expression is increased during a hyperinsulinemic clamp in healthy muscle but not in type 2 diabetic muscle 
. One of the few genes involved in insulin signaling found in this study to be upregulated in the type 2 diabetic muscle is VAMP2
(). This gene encodes a protein residing on the GLUT4 vesicle surface and plays an important role in the interaction between the vesicle and the plasma membrane target 
. An increase in the expression of proteins promoting efficient GLUT4 trafficking and fusion to the membrane (like VAMP2) could be a way to compensate for a decreased amount of GLUT4 protein.
Insulin signaling defects observed in muscle from people with type 2 diabetes has previously been reported to be specific for the metabolism regulating part of the pathway, thereby leaving the MAP kinase part of the pathway intact 
. However, we found that several of the MAP kinases were downregulated at the gene expression level (). The decreased amount of MAP kinase expression could lead to a decreased serine/threonine phosphorylation of for example the IRS proteins, ultimately increasing insulin signaling activity as part of a compensatory mechanism directed against insulin resistance.
We also found that several serine/threonine phosphatases had a decreased expression in diabetic muscle compared to controls (PPM1A, PPM1B, PPP1R9B, PPP2CB, and PPP2R5B) (). Possibly, this reduction in phosphatase expression will translate into an increased level of serine/threonine phosphorylation further worsening the intensity of insulin resistance in these patients.
OXPHOS genes and PGC1α/PGC1β
Oxidative phosphorylation (OXPHOS), which has previously been shown to be downregulated in both prediabetic relatives and people with type 2 diabetes 
, was not found to be significantly different in either group in this study. Several factors can partly explain this divergence in results. In the study of Patti et al., all subjects were Mexican-Americans, biopsies were taken at basal levels and from groups of mixed sexes. Additionally, HuGeneFL arrays from Affymetrix representing 7,129 sequences were used in that particular study 
. In comparison, the arrays used in the current study had more than 50,000 probesets representing approximately 47,000 transcripts. That fact alone is likely to result in different findings when it comes to pathway and functional analyses. In the study of Mootha et al., samples were taken after a hyperinsulinemic-euglycemic clamp, all subjects were of Caucasian origin, and the groups consisted of only males as in the present study. However, the arrays used (HG-U133A arrays from Affymetrix) covered only about half of the transcripts found on the arrays used in the current study 
Even though the OXPHOS genes as a group were not significantly changed at the expression level in first degree relatives or in type 2 diabetic patients, several individual genes involved in mitochondrial function and energy derivation had a decreased level of expression. NADH dehydrogenase 1 (NDUFS1), NADP transhydrogenase (NNT), 5-methyltetrahydrofolate-homocysteine methyltransferase reductase (MTRR), polymerase gamma (POLG), NADH dehydrogenase 2 (NDUFS2) were among others found to be down-regulated in the ‘D’ group ().
In this study, we could not detect any significant downregulation of PGC1α
in muscle biopsies from group ‘R’ or group ‘D’ ( and ). A significant decreased expression of these genes in pre-diabetic relatives and people with type 2 diabetes has previously been reported, contradicting the present results 
. However, a study of Karlsson et al. recently found that the mRNA expression of PGC1α and PGC1β in normo-glycemic first degree relatives was within the same range as for healthy controls, which supports the findings of the current study 
. To clarify this matter, we determined the protein expression of PGC1α in all three experimental groups, and found that PGC1α indeed looks like it is downregulated in some first degree relatives and diabetic patients, but not in others. Due to the high interpersonal variation the measured downregulation is not significant ().
Genes with altered expression levels in first degree relatives of type 2 diabetics
As previously mentioned, alterations in gene expression found in healthy first degree relatives of type 2 diabetics are good candidates when searching for underlying causes of the disease.
8 of the 11 genes found to be differentially expressed in muscle samples from first degree relatives had an increased level of expression compared to the controls. These genes include among others KIF1B and GDF8. Interestingly, the expression of KIF1B was downregulated in the ‘D’ group using both the microarray and the qRT-PCR approach. Both of these genes could turn out to play a crucial role in type 2 diabetes pathogenesis.
KIF1B has been shown to be highly involved in the transport of mitochondria and KIF1B
heterozygous mice have an impaired transport of synaptic vesicle precursors and suffer from a high degree of muscle weakness 
. Type 2 diabetes has been associated with a decreased mitochondrial level in skeletal muscle 
. An upregulation of mitochondrial transport by upregulation of KIF1B
could possibly be a way to compensate for a supposed decreased mitochondrial level. Interestingly, one of the gene regions recently found to associate with type 2 diabetes contains KIF11
– another kinesin family member 
GDF8 is also known as myostatin, which works as an inhibitor of skeletal muscle growth and is a member of the TGF-beta family. Myostatin has been suggested as a good candidate for therapeutic intervention in diseases with loss of muscle mass, including diabetes. Indeed, an increased expression of this gene has been reported in skeletal muscle from chronic muscle wasting conditions such as cachexia and aging in human and animal models 
. Finding GDF8
(myostatin) to be upregulated in healthy first degree relatives in this study suggests that this factor could play an initiating role in the muscle wasting observed in many diabetic patients and potentially in the development of insulin resistance in the prediabetic stage.
The only gene with a know function found to have altered expression levels in both the first degree relatives and the type 2 diabetics was LDHB
. LDHB catalyzes the conversion of pyruvate to lactate in the anaerobic glycolytic process and is therefore crucial for normal energy homeostasis. Mitochondrial ATP synthesis has been reported to be down in insulin resistant but non-diabetic offspring of parents with type 2 diabetes as well as in type 2 diabetic patients 
. The results of this study suggest that it is not only mitochondrial ATP production that is impaired in these individuals but also ATP generation via the anaerobic pathway. Since mitochondrial oxidative phosphorylation and LDHB in a way compete for same pool of pyruvate it is also a possibility that decreased levels of LDHB is a compensatory mechanism in response to impaired mitochondrial function as more pyruvate will be available for acetyl Coenzyme A conversion.
Additional genes with altered gene expression levels in type 2 diabetic skeletal muscle
Several interesting genes were found to be differentially expressed in the ‘D’ group compared to controls using the dChip program. One of the genes with the largest FCs is HK2
−2.75, ). This gene has previously been shown to have an impaired expression in type 2 diabetic skeletal muscle 
. Furthermore, it has been shown that HK2
expression is stimulated by insulin in healthy individuals but not in obese or type 2 diabetes patients 
. This can explain the decrease in expression of HK2
in the type 2 diabetics since subjects were submitted to a hyperinsulinemic clamp before samples were taken.
(histone deacetylase 7A) was also found to have a reduced expression in muscle from type 2 diabetes patients (FC
−1.54, ). It has previously been hypothesized that an abnormal acetylation/deacetylation pattern and thereby an altered regulation of gene expression could play a role in the pathogenesis of type 2 diabetes 
In summary, this study for the first time shows a striking difference in the gene expression of insulin signaling molecules between people with type 2 diabetes and first degree relatives in skeletal muscle. Insulin signaling was significantly upregulated in first degree relatives, and significantly downregulated in type 2 diabetes patients. We suggest that increased expression of insulin signaling molecules work in concert with increased levels of insulin protecting people in the pre-diabetic state from insulin resistance and metabolic dys-regulation. However, future studies are needed to clarify the molecular basis and clinical importance of this phenomenon, and it will be interesting to see if the same results will be obtained in other tissues like pancreatic islets and adipose tissue.
Furthermore, several potentially important genes regarding the underlying causes of insulin resistance and type 2 diabetes (for example KIF1B and GDF8) have been identified and shown to have different gene expression levels in healthy first degree relatives compared to controls. These new findings in first degree relatives could potentially be used as a diagnostic tool in the prediction of type 2 diabetes. Further investigations in the future will be imperative in clarifying specific possible roles of these results in type 2 diabetes pathogenesis.