This proof of concept study confirmed that in only 12 weeks, the MMPI, doxycycline, improved global measures of inflammation and muscle insulin sensitivity in people with DM2.
Global markers of inflammation, CRP and MPO levels in plasma, were significantly higher in obese participants with diabetes before treatment compared to controls, supporting the pro-inflammatory state expected in this population. CRP is a global measure of inflammation and oxidative stress even in apparently “healthy” populations
 and MPO is a lysosomal enzyme in neutrophils that has been associated with coronary plaque instability
 and therefore increased cardiovascular risk.
 As expected, MPO levels have been shown to be higher in people with diabetes compared to controls.
 MMPI treatment resulted in a significant decrease in both CRP and MPO by the end of the study whereas PL had no effect, supporting the anti-inflammatory properties of doxycycline. While medications such as HMG coA reductase inhibitors (“statins”) or aspirin are known to affect inflammatory markers (i.e. CRP, MPO), participants did not change medications during the study and so drug effects for our study participants should have been stable. Other than a significant decrease in the anti-inflammatory cytokine IL-10 in the PL group, there were no significant changes in cytokine levels in the PL or MMPI group. Changes in expression of cytokines after MMPI may be better measured in tissues rather than blood.
To test for an effect of MMPI on insulin sensitivity, three different kinases in the insulin signaling cascade in muscle (PDK1, Akt, GSK3β) were examined in muscle biopsies obtained directly after an OGTT (a relative insulin-stimulated state). In the MMPI treated group, there was a significant increase in the phosphorylation status of all of three kinases, indicating an improvement in the insulin sensitivity of the muscle, with no change in the PL group. Data are needed to link the global decrease in inflammation with improved insulin sensitivity in muscle.
Golub et al. first demonstrated inhibition of MMPs by tetracyclines in people with diabetes.
[14, 17, 32] The decision to use doxycycline in this study was based on these data, its broad spectrum MMPI
[9, 12, 13], and its successful MMPI in a number of pathological states
 including an animal model of diabetes.
 The literature provides additional evidence that doxycycline can inhibit the activity of other classes of proteases such as plasmin (a serine protease)
, and act as an oxygen radical scavenger.
 Doxycycline is also known to be relatively well tolerated with minor side-effects. This study resulted in one case of sun sensitivity with doxycycline use, two cases of nausea and one patient developed serum sickness. Serum sickness like syndrome is known to occur with the use of the tetracyclines, minocycline
 and doxycycline.
Dosing schemes of MMPI vary widely from 40 mg daily
 and upwards, including the usual adult antibiotic dose of 100 mg twice daily used in this proof of concept study, a dose reported to reduce CRP levels during inflammation associated with infection
. That doxycycline improved insulin sensitivity in muscle but not global glucose homeostasis in people with DM2 suggests the need for larger and longer clinical trials, as well as a dose finding study as lower amounts of doxycycline (40 mg daily) have been shown to significantly reduce CRP in people with cardiovascular disease over 6 months
The proposed mechanism by which doxycycline improves insulin sensitivity, is by inhibition of inflammatory MMPs that cleave the extracellular insulin receptor domain, thereby inactivating the receptor.
 Doxycycline and other MMPI have been shown to inhibit cleavage of other receptors including vascular endothelial growth factor receptor 2 (VEGFR-2).
 We did not find a difference in global MMP levels between control participants and those with DM2, and no change in global MMP levels after MMPI; this may be due to the relatively low sensitivity of this technique. Using a new and more sensitive technology to measure MMP 2/9 activity,
 we found higher MMP 2/9 activity levels in all obese study participants with diabetes compared to the control group prior to treatment, in agreement with others.
[2, 19, 41] This difference is likely due to the higher sensitivity of the electrophoretic zymographic technique and the fact that it does not require separation of blood cells from plasma.
 However, we found no significant effect of doxycycline on blood levels of MMP 2/9 activity at study completion using this new technique. We hypothesize that although we found improvements in insulin sensitivity, the time of the study was not long enough to see a reduction in these MMP levels in people with severe insulin resistance, and that gene expression of MMPs and TIMP levels need to be measured along with MMPs. During our study, it was reported that blood MMP 2/9 activity levels do not change in participants with DM2 during an OGTT.
 It appears that MMP blood activity, at present, could be more useful in screening for whether a patient should take MMPI (after observing elevated MMP activity), but isn’t as sensitive for monitoring the effect of treatment after a glucose load. It may require a stronger stimulus than glucose alone, such as a mixed meal, to induce measureable changes in MMP levels in the blood.
There are many factors that can affect MMP activity in blood, in addition to DM2. The development of more specific protease substrates that better differentiate the activity from individual types of MMPs and other proteases would be useful in studies such as these. From animal and in vitro studies, it may be that the best way to measure the effect of MMPI on MMP activity is not through blood samples but in tissues such as adipose tissue, where MMP activity is increased in obesity and tissue inhibitors of MMPs (TIMPs) are decreased.
 Studies are ongoing to test whether MMPI can affect MMP and TIMP levels, and insulin receptor integrity in humans.
Commonly measured clinical parameters of DM control were evaluated in this study including weight, A1c, triglycerides, and blood pressure which did not change in either the MMPI or PL groups. The early secretory response of insulin to an oral glucose load, however, was significantly lower after MMPI treatment as was the secretion of insulin measured by the HOMA-IS, and the AUCi was improving in the MMPI group but not the PL group. While there was a significant increase in activation of molecules in the insulin signaling pathways during this period, the time it takes to see a clinically significant effect on overall glucose control is likely longer. Improvements in global measures of glucose homeostasis by MMPI required 6 months in rats
 and may be even longer in a more genetically mixed population of people. DM2 participants in this trial were on antihypertensive, lipid lowering and hypoglycemic medications which may have affected changes in insulin sensitivity. Larger numbers of participants with both new onset DM or impaired glucose tolerance or impaired fasting glucose with minimal medication use and a longer trial will be important in determining if there are any benefits to MMPI use in the treatment of inflammation and reduced insulin sensitivity in humans. If doxycycline were to be used long-term, studies on development of resistance or changes in gastrointestinal flora would be helpful to understand the contribution of these changes to markers of inflammation and insulin sensitivity. Additional limitations of this study include the lack of assessment of tissue MMPs, and small study size.